Genetics exam questions

what are mutations? what are the mechanisms? what are the main types? how do the effects on somatic cells differ from germline cells? what are the manifestations? how are they tested for? (56, 57)

A mutation is a change in genetic information . It is caused by various influences - most often considered to be mutagens: physical factors ( UV and ionizing radiation ); chemical factors (eg planar aromatic compounds, strong oxidants, radical initiators); biological factors ( viral infections , etc.). This is a random process, but it has also been shown that mutations in some areas of the genome are more common and are referred to as hot-spots . Mutations, if they occur (see below), can cause serious illness, whether various birth defects or neoplasia . However, it is also considered one of the mechanisms of evolution . Mutations are prevented by DNA repair processes , or so-called back mutations . An increased incidence of mutations occurs when there is a defect in genes encoding repair enzymes (mutator genes), which is the basis of various diseases (eg Fanconi's pancytopenia , xeroderma pigmentosum , Cockayn's syndrome Mechanism: I. Spontaneous mutations: they are the result from natural changes in DNA structure or DNA sequence. Spontaneous replication errors: even tough replication is very accurate; errors may be made in the course of DNA synthesis. Tautomer's (constitutional isomers of organic compounds enable different base pairing - Keto and Enol form) enable different base pairing other than the common Waston and Crick. The position of the protons in the Purine and Pyrimidine can alter, forming different constitutions of the bases. The two tautomeric forms of each base are in dynamic equilibrium, although, one form is more common than the other. The standard Watson and Crick base pairing - Adenine with Thymine, Cytosine with Guanine - are made between the common forms of the bases, but, if the bases are in their rare tautomeric forms, other base pairing may appear. Tautomer's has two forms - Keto and Enol: Keto means that the base has Ketone functional group (carbonyl) bonded with amine, while in the rare Enol for m - the hydrogen of the amine moved to the oxygen of the carbonyl - formed alcohol functional group (OH) and imine (N double bond with C). The flexibility of the DNA helical structure enable different base pairing which may lead to mispairing. After a mismatch of base pairs occurred during replication, in the next round of replication the two mismatched bases separate and each serves as template for the synthesis of a new nucleotide strand. One strand will form the original DNA, but the other will form a mutated DNA. Transponsable genetic elements - Transposons "Jumping Genes" - they are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. Class I - Copy & Paste themselves, Class II - Cut & Paste themselves to another place within the DNA strand and may induce mutations if introduced into coding area or promoter areas of other gene. II. Induced mutations: may be as a result of physical, chemical or biological mutagens. Physical mutagens: Induced mutations may be caused by physical mutagens -UV light which damage the nucleotide bases and causes incapability of base pairing, leads to cessation of the DNA polymerase during replication. Another physical mutation may be caused by ionizing radiation - X-rays, gamma rays, cosmic rays. They dislodge electrons from the atoms that they encounter, changing stable molecules into free radicals which are reactive ions. The radiation can strike the DNA molecule directly, ionizing and damaging (direct route), or can ionize water molecules, producing free radicals that react with the DNA molecules and damage it (indirect route). Chemical mutagens: Deamination - base changing as a result of removal of amine group. Base Analogs - they are chemical compounds that are sufficiently similar to the normal nitrogen bases of DNA; they are occasionally incorporated into DNA during replication in place of normal bases. Many of these analogs have pairing properties unlike those of the normal bases, and thus they can produce mutations by causing incorrect nucleotides to be inserted during replication. Alkylating agents are a class of chemotherapy drugs that bind to DNA and prevent proper DNA replication. They have chemical groups that can form permanent covalent bonds with nucleophilic substances in the DNA. These agents may lead to crosslinking between two or more molecules by a covalent bond - either in the same strand (intrastrand crosslink) or in the opposite strands of the DNA (interstrand crosslink). DNA replication is blocked by crosslinks, which causes replication arrest and cell death if the crosslink is not repaired. Biological mutagens Viruses - Virus DNA may be inserted into the genome and disrupts the genetics function Bacteria - some bacteria can cause inflammation, causing DNA damage and reducing efficiency of DNA repair system. 2. Effect on Structure: May be small-scale change: Point mutation - Transition (e.g. A becomes G [Purine changes to another Purine], C becomes T -Pyrimidine 🡪 Pyrimidine), Transversion (Purine 🡪 Pyrimidine) Missense mutation - replacement of single nucleotide results in different amino acid production into the protein sequence - may produce a malfunctioning protein; e.g. sickle cell anemia - caused by the change of one amino acid - Glutamic acid into Valine. Nonsense mutation - replacement of single nucleotide results in the formation of stop-codon, signaling the cell to shorten the protein. Silent point mutation - when the changed nucleotide doesn't have effect on the amino acid - it produces the same original one. Insertion and deletion of single nucleotide may cause frameshift mutation; one nucleotide that added, changed the amino acid sequence that follows. There are possible consequences of gene mutations outside of the coding sequence: Repeat expansion mutation - repeated trinucleotide adds a string of random amino acid to the protein. E.g. in Huntington disease - normal range of repeated sequence CAG is 9-37, a sick person has a range of 37-121 CAG repeats. at the promoter regulatory/operator site 5' UTR, 3' UTR splice recognition sequence large scale : indel, duplication, inversion, translocation Affected cells - somatic or germinal A mutation occurring in any cell that is not destined to become a germ cell (somatic cell) - the mutant cell continues to divide and the individual will come to contain a patch of tissue of genotype which is different from the cells of the rest of the body. A germline mutation is any detectable and heritable variation in the lineage of germ cells. Mutations in these cells are transmitted to offspring. A germline mutation gives rise to a constitutional mutation in the offspring, that is, a mutation that is present in virtually every cell in his body. Mutation by effect on function: Loss of function mutations - are the result of gene product having less or no function. Most often recessive. Gain of function mutations - change the gene product such that it gains a new and abnormal function, usually dominant phenotypes. Dominant negative mutations - have an altered gene product that acts antagonistically to the wild-type allele. These mutations usually result in an altered molecular function and are characterized by a dominant or semi-dominant phenotype. Lethal mutations - lead to phenotype incapable of effective reproduction. Mutagenicity testing: In industrial societies people are exposed to many artificial chemicals, some of them can be potential carcinogens. In order not to harm the population by introducing them to hazardous chemicals, there are several methods for detecting their mutagenicity. Testing the chemical on laboratory animals and compare the incidence of cancer in the treated animals with the control animals. It is not always accurate, since a mutagen may affect animals and humans differently. Ames test - based on the assumption that mutagenesis in bacteria could serve as an indicator of carcinogenesis in humans. The test uses 4 strains of the bacterium Salmonella typhimurium - one of the strains is used to detect base pair substitution mutation, and the others detect different type of frameshift mutations. The bacterium in the strains have gone through processes - their lipopolysaccharide coat which normally protects them from chemicals is damaged, the DNA repair system has been inactivated (increase susceptibility to mutagens), and they carry a mutation that renders the bacteria ability to synthesize the amino acid histidine, which is required for growth and proliferation. During the process different chemicals to be tested are added to the plates with the bacteria. After a while, the bacterial colonies in the tested plates are compared with that of the control group. Any chemical the significantly increases the number of colonies is mutagenic and probably also carcinogenic.

what is migration/ gene flow? how can this occur? how does this affect allelic frequencies? significance in sociology, zoology? (106)

Beyond natural selection and genetic drift there is another phenomena affecting allele frequencies - gene flow - the transfer of alleles into or out of a population due to the movement of fertile individuals or their gametes. Gene flow - also called migration - is the movement of genes between populations. This may happen through the migration of organisms or the movement of gametes (such as pollen blown to a new location) movement of alleles from one population to another aka migration The change in allelic frequency is a factor of: Extent of migration - how many? The difference in allelic frequencies between the 2 populations. Emigration - leaving a certain population Immigration - adding a new population to an already formed one. The deviation from Hardy-Weinberg equilibrium continues until the migrants have randomly interbred with natives. With each generation of migration - frequencies become more similar, until eventually they are equal. Equilibrium will be reached when both populations - immigrants and natives will have equal frequencies. For example, suppose that near our original hypothetical wildflower population from pervious example is another population consisting primarily of white-flowered individuals (Cw-Cw). Insects carrying pollen from these plants may fly to and pollinate plants in our original population. The introduced Cw alleles would modify our original population's allele frequencies in the next generation. Because alleles are transferred between populations, gene flow tends to reduce the genetic differences between populations. If it is extensive enough, gene flow can result in two populations combining into a single population with a common gene pool. Alleles transferred by gene flow can also affect how well populations are adapted to local environmental conditions. For example, gene flow has resulted in the worldwide spread of several insecticide-resistance alleles in the mosquito Culex pipiens, a vector of West Nile virus and other diseases. In their population of origin, these alleles increased because they provided insecticide resistance. These alleles were then transferred to new populations, where again, their frequencies increased as a result of natural selection. Gene flow has become an increasingly important agent of evolutionary change in human populations. Humans today move much more freely about the world than in the past. As a result, mating is more common between members of populations that previously had very little contact, leading to an exchange of alleles and fewer genetic differences between those populations. In sociology , migration, together with births and deaths, is a key element in the process of population development and significantly affects the social and cultural changes of the population at all levels. With economic development, the intensity of migration continues to increase. The division in this field is as follows: permanent - irreversible migration, migration; short-term - commuting to work, school, services, recreation; does not require a permanent change of residence. Dividing migration by theme: economic, political, religious. Further divisions: national, interstate (emigration, immigration), voluntary, forced. In zoology, it is perceived as the relocation of individuals or the entire population to another territory. The division is as follows: regular - eg seasonal bird movements irregular - eg leaving areas due to overgrowth and subsequent lack of food In parasitology , the term migration is used in connection with the transfer of the developmental stage of the parasite. A typical example is a steady decrease in the frequency of the D allele of the AB0 blood group system from approximately 0.6 in Western Europe to 0.3 in East Asia. Another example is the influx of "white" genes into the African American gene pool .

what are genetic consultations? why are they important? what are things that a genetics professional will not do and what should they do? (125)

Genetic counseling is the process by which the patients or relatives at risk of an inherited disorder are advised of the consequences and nature of the disorder, the probability of developing or transmitting it, and the options open to them in management and family planning. This complex process can be separated into diagnostic (the actual estimation of risk) and supportive aspects. Genetic counseling is a process of communication and education that addresses concerns relating to the development and/or transmission of hereditary disorder. An individual who seeks genetic counseling is known as a consultant. During the genetic counseling process, it is widely agreed that the counselor should try to ensure that the consultant is provided with information that enables him or her to understand: The medical diagnosis and its implications in terms of prognosis and possible treatment The mode of inheritance of the disorder and the risk of developing and or transmitting it The choices or options available for dealing with the risks. It is also agreed that genetic counseling should include a strong communicative and supportive element, so that those who seek information are able to reach their own fully informed decisions without pressure and stress. The most crucial step in genetic consultation is that of establishing the diagnosis - if it is incorrect, then inappropriate and totally misleading information could be given, with potentially tragic consequences. The fundamental steps of any medical consultation are: Diagnosis - which is based on taking full and accurate family and personal medical history. Carrying out an examination Undertaking appropriate investigations of the examination's results, there implications and consequences. Risk assessment - placing risks in context so patients able to decide for themselves whether a risk is high or low. Doctor-patient communication and discussion regarding the options Long-term contact and support Even when a firm diagnosis has been made, problem can arise if the disorder in question shows etiological heterogeneity. Common examples are disorders like hearing loss which may have either environmental or genetic factors. In the case of etiological heterogeneity - in which the disorder can be caused by more than one genetic mechanism, the counseling can be extremely difficult. Discussing the options- Having established the diagnosis and discussed the risk of occurrence or recurrence, the counselor is then obliged to ensure that the consultants are provided with all of the information necessary (tests, treatments etc.) for them to make their own informed decisions. Communication and support - readiness to listen is a key attribute for anyone involved in genetic counseling, as is an ability to present information in a clear, sympathetic, appropriate and if possible - optimistic manner. Genetic counselors need to take into account the complex psychological and emotional factors that can influence the counseling dialog. Support groups may also be an option for the patients. Families or individuals may choose to attend counseling or undergo prenatal testing for a number of reasons. Family history of a genetic condition or chromosome abnormality Molecular test for single gene disorder Increased maternal age (35 years and older) Increased paternal age (40 years and older) Abnormal maternal serum screening results or ultrasound findings Strong family history of cancer A genetics professional will NOT: tell a person which decision to make, advise a couple not to have children, recommend that a woman continue or end a pregnancy, tell someone whether to undergo testing for a genetic disorder. but WILL interpret and communicate complex medical information, help each person make informed, independent decisions about their health care and reproductive options, respect each person's individual beliefs, traditions, and feelings. importance about 2% of individuals become ill with monogenic disease (OMIM) at some point in their lives. Approximately 7 individuals out of 1000 born will have disorder in the number or structure of chromosomes. The largest part of human morbiditys o-called diseases with multifactorial etiology: genetic factors (polygenic inheritance), environmental factors ,interactions between them.

what is hemoglobinopathy? how can the formation of Hb be impaired? what are the relevant diseases? (63)

Hemoglobinopathy is a kind of genetic defect that results in abnormal structure of one of the globin chains of the hemoglobin molecule. More than 300 Hb variants have been found due to a variety of types of mutation - 200 of these are single amino acid substitutions resulting from a point mutation. The majority are rare and not associated with clinical disease, few are associated with disease and relatively prevalent in certain populations. Hemoglobinopatie is the one of the most common inherited diseases in the world in which the disease is inherited as single-gene disorders; in most cases, they are inherited as autosomal co-dominant traits (contributions of both alleles are visible in the phenotype). It is estimated that 7% of world's population are carriers for some hemoglobinopathy, with 60% of total carriers and 70% of pathological being are found in Africa. Hemoglobinopathies imply structural abnormalities in the globin proteins, such as in sickle-cell anemia. Thalassemia's, in contrast, usually result in underproduction of normal globin proteins, often through mutation in regulatory genes. The two conditions may overlap, since some conditions which cause abnormalities in globin proteins (hemoglobinopathy) also affect their production (thalassemia). Thus, some hemoglobinopathies (relate to hemoglobin structure) are also thalassemias (relate to the number of normal hemoglobin formed) Thus, hereditary diseases caused by impaired formation of hemoglobin can be divided into: Diseases with impaired structure chain of globin - causing hemolytic anemias (hemolysis of RBCs due to their structure) - Sickle cell anemia - unstable hemoglobin; reducing the ability of hemoglobin to carry oxygen (methemoglobinemia - abnormal amount of methemglobin is produced. The hemoglobin can carry oxygen, but is not able to release it) Diseases with impaired synthesis chain globin - Thalassemia. Either hemoglobinopathy or thalassemia may cause anemia. Hemoglobin variants not necessarily pathologic form of hemoglobin and do not cause anemia (e.g. embryonic and fetal hemoglobin). Mutations of hemoglobin Point mutation - substitution of one amino acid for another lead to altered hemoglobin - such as HbS, C, E - which are missense mutations (different amino acid synthesized). Deletion - There are number of Hb variants in which amino acids of one of the globin chains is missing or deleted (Hb Freiburg) Insertion - globin chains are longer than normal because of insertion (e.g. - Hb Grady) Frameshift - disruption of the normal triplet reading frame due to addition or removal of bases changes the triplets reading. The translation of the mRNA continues until a termination codon is read, thus, the variants can result in either an elongated or shortened globin chain. Sickle cell anemia This severe hereditary hemolytic anemia distorts the shape of RBCs under deoxygenated conditions - "sickling". RBC's with this type of hemoglobin has different mobility than HbA and this hemoglobin is called HbS (for sickle). SC disease following Autosomal Recessive inheritance, and is the most common hemoglobinopathy. The disease is especially prevalent in those areas of the world where malaria is endemic (heterozygote advantage). The parasite Plasmodium Falciparum (malaria) is disadvantaged because of red cells of sickle-cells heterozygotes are believes to express malarial cells self-antigens more effectively, resulting in more rapid removal of parasitized cells from the circulation, thus SC heterozygotes are relatively protected from malarial attacks - thus, reproductively, they are in higher fitness and the SC gene can be passed on to the next generation, resulting in high frequency in malarial-infested regions. The clinical manifestations (pleiotropic effect) include painful sickle cell crises - abdominal pain, splenic infraction, limb pain, bone tenderness, heart and renal failure, pneuomonia (inflammation of the alveoli), neurological motor and sensory disorders. All of these are the result of deformed, sickle-shaped red cells, which are less able to change shape and tend to obstruct small arteries, thus reducing oxygen supply to tissues. Sickled cells with damaged membrane are taken up by the reticuloendothelial system (monocytes). Short red cell survival times 🡪 rapid red cell turnover 🡪 anemia with increased amount of reticulocytes, jaundice and a tendency to the formation of gallstones. It is also causes reduced spleen due to ischemia and infraction. The mutational basis of Sickle-Cell disease lies by substitution of amino acid Valine in the betta-globin chain, by glutamic acid. The mutation is therefore a single base pair in the triplet code at this point, from GAG to GTG (identified by restriction enzyme MstII). Therapy: Early diagnosis is needed to effectively treat and minimize complications. Newborn screening is therefore in place in some Middle East and US countries. Antibiotic prophylaxis (greater vulnerability to infections due to spleen dysfunction). Blood transfusion. Hematopoietic stem cell transplantation (bone marrow). Pharmacologically hydroxycarbamide (hydroxyurea) - increases gene expression of HbF (fetal hemoglobin - α2γ2), which does not contain defective β-subunits. Gene therapy - was described (2017) using a lentiviral vector (however, its widespread implementation in areas of main occurrence in developing countries is unrealistic for financial reasons). Hemoglobinopathies affecting the ability of hemoglobin to carry oxygen Stability and hemoglobin synthesis is not affected by these mutations; rather the protein function is changed. Methemoglobinemia (HBM) - mutation in the gene coding for reductase enzyme - which regularly reduces methemoglobin to hemoglobin, leads to the ferric ion to stay in the ferric form (Fe3+) instead of the ferrous form (Fe2+). Thalassemias - disorders of reduced amount of hemoglobin synthesis The thalassemias are the commonest single group of inherited disorders in humans - Autosomal recessive inheritance. They are heterogeneous and classified according to the particular globin chain synthesized in reduced amount (e.g. - alpha, betta, gamma-beta etc). The imbalance of globin-chain production (mutated globin-gene produced less) results in the accumulation of free globin chains in the RBCs, which due to insolubility characteristic - precipitate and resulting in hemolysis of RBCs (hemolytic anemia). The consequence is compensatory hyperplasia (enlargement) of the bone marrow. Alpha-thalassemia - results from underproduction of the alpha-globin chains, most commonly occurs in Southeast Asia. Carrier frequency ranging from 15-30% in certain places. Two types of alpha-thalassemia with different severity - the severe form, in which no alpha chains are produced and these fetus not-compatible with life - also called Bart syndrome hydrops fetalis, characterized by excess fluid builds up in the body of the fetus before birth (edema) which is secondary to heart failure. In the milder forms of alpha-thalassemia - compatible with survival - some alpha chains are produced, but still there is a relative excess of betta chains. It results in production of beta-globin tetramer HbH - known as HbH disease. Both Hb Barts and HbH globin tetramers have an oygen affinity similar to that of myoglobin (do not release oxygen normally). The most common causes are deletions of some of the two genes codes for alpha chain. The genotype of the two genes (four alleles) reflects the severity and corresponds to the number of affected alleles. Betta Thalassemia - underproduction of the betta-globin chain of hemoglobin - reduced (insufficient - β+) or absent production (β0). It also causes anemia but only after the third month of life, when the synthesis of HbF (gamma chain) is replaced by the synthesis of HbA (betta-chain). Excess alpha chains damages RBCs and they disintegrate already in the bone marrow (ineffective erythropoiesis). Approximately 1:1000 northern Europeans are b-thalassemia carriers and in the UK, 22 babies with absent mode betta-thalassemia are born each year, and roughly 860 people live with the condition. The mutational basis of B-thalassemia is due to six main functional types: Transcription mutations - mutations in the TATA box or the promoter region of the B-globin gene 🡪 reduced transcription mRNA splicing mutations - abnormal splicing with consequent reduced level of betta-globin mRNA. Polyadenylation signal mutations - mutations in the 3' end of the untranslated region of the b-globin gene can lead to loss of the signal for cleave and polyadenylation of the betta-globin gene transcript. RNA modification mutations - mutations in the capping and polyadenylation of the mRNA - abnormal processing and transportation of the betta globin mRNA to the cytoplasm 🡪 reduced level of translation. Chain termination mutations - insertion, deletions, and points mutation - may generate a nonsense or chain termination codon, leading to premature termination of translation of the betta globin mRNA (shortened unstable mRNA) Missense mutations - highly unstable betta-globin Clinical aspects - anemia is microcytic, hypochromic, severe hepato (liver) and splenomegaly, bone marrow hyperplasia (enlargement). The treatment are required repeated transfusions of packed RBCs (reduce the stimulation of erythropoiesis and increase hemoglobin level). Excess iron 🡪 formation of oxygen radicals damaging various organs (cirrhosis, myocardium fibrotic changes). Hereditary Persistence of fetal hemoglobin - HbF production persists into childhood and beyond is included in the thalassemias. HbF may account for 20-30% of total Hb in heterozygotes and 100% in homozygotes. Individuals are usually symptom free

what are hereditary cancers? what are the characteristics of familial incidence of tumors? what are some examples? (115)

Hereditary cancers occur when a person is born with changes or mutations in one copy of proto-oncogenes or tumor-suppressor genes (include the genes that encode for the DNA repair proteins - mutator genes). In many types of cancers, beyond the environmental and sporadic causes that lead to DNA mutations, there are also hereditary factors which may induce carcinogenesis; about 10-15% of cancer has a hereditary character. Cancer is usually caused by gene mutations that occur randomly - somatic mutations - may arise as a natural consequence of aging or when a cell's DNA has been damaged. Acquired mutations are only present in some of the body's cells, and they are not passed on from parent to their children. However, in a small percentage of people with cancer, the disease is due to a different type of mutation called a hereditary mutation - germline mutation - inherited from the parents and are present in nearly every cell of the body (because it is present since the time the person was zygote). Because hereditary mutation are present in all body cells, they are also found in sperm and egg cells, thus can be passed down in families; people who carry such hereditary mutations do not necessarily get cancer, but their risk of developing cancer is higher than average. Familial incidence of tumors is characterized by: Frequent occurrence of more family members with the same type of tumor Early onset of the disease compared with the same type of tumor that occurs sporadically Multifocal (many sites of origin) or bilateral incidence Tumor multiplicity - the emergence of tumors in different organs in the same individual Examples for cancer with familial incidence: Neurofibromatosis - tumors grow in the peripheral nervous system; In half of the cases these are inherited from a person's parent (while the rest - occur during early development) AD - if only one parent has neurofibromatosis -offspring has 50% chance of being affected Tumors involve supporting cells (glial cells) rather than neurons Usually benign, some patients have an increased incidence of malignancies Li-Fraumeni syndrome - Germline mutations of the tp53 tumor suppressor gene - encodes p53 protein - regulates cell cycle, prevents genomic mutations and promotes apoptosis; Inherited in some cases, or can arise from de novo mutations during embryogenesis Also known as SBLA syndrome - sarcoma, breast, leukemia and adrenal gland syndrome. Hereditary breast cancer - Approximately 1 in 12 women in western societies will develop breast cancer - most common cancer in women between 40-55 years od age 15-20% of women who develop breast cancer have a family history of the disorder Mutations in the tumor suppressor gene BRCA1 and BRCA2 Linkage analysis (family studies) showed that in these families the tendency to develop breast cancer mapped to the long arm of chromosome 17 🡪 leading to identification of the BRCA1 gene. Families with early-onset breast cancer that didn't show linkage to this region, showed linkage to the long arm of chromosome 13 - resulting in identification of the BRCA2 gene. Prostate cancer - Most common cancer overall after breast cancer, most common affecting men Significant proportion - about 15% - to have a first-degree male relative with prostate cancer - 2 to 5 times the population risk of developing prostate cancer Familial adenomatous polyposis - FAP Colorectal cancer Caused by mutations in tumor suppressor gene APC (anaphase-promoting complex) located on the long arm of chromosome 5 Familial retinoblastoma - mutations in the tumor suppressor gene Rb1 (has negative regulator effect on the cell cycle arises from binding and inactivation of the transcription factor E2F thus repressing the transcription of genes which are required for the S phase) eye cancer Cowden - PTEN hamartoma tumor syndrome - AD macrocephaly, benign thyroid pathology, mucocutaneous hamartomas, colonic polyps breast cancer epithelial thyroid cancer endometrial cancer Peutz-Jeghers syndrome - harmartomatous intestinal polyposis - STK11 - can occur at any site of the GIT, severe GI bleeding Lynch syndrome - HPNCC MMR genes - MSH2, MLH1, PMS - colorectal cancer - extracolonic cancers - ovarian, endometrial)

what are inborn errors of metabolism? how can they be classified? what are some major examples of each type? (64)

Inborn errors of metabolism (IEMs) are genetic (=inherited) disorders, 700-800 disorders, in which the body cannot properly turn food into energy. The disorders are usually caused by defects (deficiency) in specific enzymes that help break down, i.e. metabolize, parts of food. Thus, IEM's occur mainly due to single genes defect that code for enzymes that facilitate conversion of various substrates into products. Dysfunction of transport protein or a disorder of another protein associated with metabolic pathway may also be a reason for IEMs. Incidence - 1:500 (1:15 frequency of heterozygotes). A food product that is not broken down into energy can build up in the body and cause a wide range of symptoms. Several inborn errors of metabolism cause developmental delays or other medical problems if they are not controlled. In most of the disorders, problems arise due to accumulation of substances which are toxic or interfere with normal function (due to improper metabolism), or to the effect of reduced ability to synthesize essential compounds. Most of the IEMs follow AR or X-linked recessive inheritance, with only a few being AD - this is because the defective protein, in most cases, is a diffusible enzyme, and there is usually sufficient residual activity in the heterozygous state for the enzyme to function normally in most situations. If, however, the reaction catalyzed by the enzyme is rate limiting (enzyme amount limiting) - sensitive to gene dosage and to normal levels of genes (only when two alleles are normal there is no mutation) - the disorder can manifest in the heterozygous state and follow dominant inheritance. Dozens of congenital metabolic diseases are now detectable by newborn screening tests - early treatment and diagnosis may prevent complications. Common treatments are diets, vitamins, gene therapy, dialysis, enzyme replacement, bone marrow or organ transplantation. IEM's can be classified to classes - Disorders of carbohydrate metabolism - glycogen storage disease, galactosemia Disorders of amino acid metabolism - PKU, maple syrup urine disease Urea cycle disorder Disorders of organic acid metabolism - Alcaptonuria Disorders of fatty acid oxidation and mitochondrial metabolism Lysosomal storage disorders Carbohydrate metabolism Glycogen-storage disease - mostly AR inheritance - sugar glucose is stored in muscle and liver as a polymer, acting as a reserve energy source. In the glycogen storage diseases - glycogen accumulates in excessive amounts in skeletal muscle, cardiac muscle and liver because of variety of inborn errors of the enzymes involved in synthesis and degradation of glycogen. In addition, because of the metabolic block, glycogen is unavailable as a normal glucose source 🡪 hypoglycemia. Galactosemia - AR disorder - resulting from a deficiency of the enzyme which necessary for the metabolism of galactose. If untreated, children may develop complications that include mental retardation, cataracts and liver cirrhosis. It may be treated by early diagnosis and feeding infants with milk substitutes that do not contain galactose or lactose (lactose is broken down into galactose). Amino acid metabolism - Phenylketonuria - PKU - 1/10000 incidence in western Europe - deficiency of the enzyme required for the conversion of phenylalanine to tyrosine - phenylalanine hydroxylase - causing a genetic block in the metabolic pathway. Deficiency occur due to mutations in the PAH gene (more than 450 different mutations have been identified). As a result of the enzyme defect, phenylalanine accumulates and is converted into phenylpyruvic acid and other metabolites that are excreted in the urine. The enzyme block leads to a deficiency of tyrosine, with a consequent reduction in melanin formation, and children therefore often have blond hair and blue eyes. In addition, areas of the brain that are usually pigmented, such as the substantia nigra, may also lack pigment. Main intellectual impairment seen in children is likely due to toxic levels of phenylalanine rather than a deficiency of tyrosine. Treatment is by control consumption of phenylalanine in the diet. Phenylalanine is an essential amino acid, thus cannot be removed entirely from the diet. After brain development is complete, dietary restriction can be relaxed. Guthrie test of the newborn - check for increased levels of phenylalanine in the blood, other method detects the presence of the phenylpyruvic acid in the urine by its reaction with ferric chloride. Children born to mothers with phenylketonuria have an increased risk of mental retardation due to the reduced ability of the mother with PKU to deliver an appropriate amount of tyrosine to her fetus in utero, which may cause reduced fetal brain growth. Maple syrup urine disease - The disease is named for the presence of sweet-smelling urine, an odor similar to that of maple syrup, The smell is also present and sometimes stronger in the ear wax. Mutations in four genes complex account for metabolism of amino acids Leucine, Isoleucine and Valine leads to their accumulation, which in high level has toxic effect on brain and other organs. Disorder of Lipid metabolism - Familial hypercholesterolemia is the most common AD single-gene disorder in Western society. Persons with FH have raised cholesterol levels with a significant risk of developing early coronary artery disease. Cells normally derive cholesterol from either endogenous synthesis or by dietary uptake from LDL receptors on the cell surface. Intracecllular cholesterol levels are maintained by a feedback system, with free cholesterol inhibiting LDL receptor synthesis as well as reducing the level of endogenous synthesis. High cholesterol levels in FH are due to deficient or defective function of the LDL receptors leading to increased levels of endogenous cholesterol synthesis. Lysosomal Storage Disorders - deficiency of a lysosomal enzyme involved in the degradation of complex macromolecules leads to their accumulation. Sphingolipidoses - lipid storage diseases - there is an inability to degrade sphingolipid, resulting in the progressive deposition of lipid or glycolipid, primarily in the brain, liver and spleen. CNS involvement results in progressive mental deterioration, often with seizures, leading to death in childhood. e.g. Tay-Sachs disease - 1:3600 in Ashkenazi Jews - infants usually present by 6 months of age with poor feeding. Developmental regression usually becomes apparent in late infancy, feeding becomes increasingly difficult and the infant progressively deteriorates, with deafness, visual impairment, and rigidity. Death usually occurs by the age of 3 years from respiratory infection.

What is Y-linked inheritance? (14)

Inheritance of genes on the Y chromosome, can only be transmitted from father to son. Y chromosome is acrocentric (centromere near the end), it is the smallest chromosome in human karyotype. The short arm of the chromosome include the SRY (sex region on chromosome Y) which responsible for male characteristics.

what is recombinant DNA? what are the applications? how does it work? what must be included in a vector? (55)

Definition: Recombinant DNA is artificially synthesized DNA that is produced by inserting the gene or parts of a gene of one organism into the genome of another. Recombinant DNA technology, also known as genetic engineering, is a set of molecular techniques for locating, isolating, altering and studying DNA segments. The term recombinant is used because frequently the goal is to combine DNA from two distinct sources: genes from two different bacteria might be joined, or a human gene might be inserted into a viral chromosome. Important field of recombinant DNA technology is biotechnology - creation of number of commercial products, including drugs, hormones and enzymes - useful for the humans in case of need (e.g. artifact insulin for diabetes patients). One of the aims of modern medical genetics is to characterize mutations that lead to genetic disease. Recent technology developments permit the detailed analysis of both normal and abnormal genes and the expression of thousands of genes in normal and disease states. Application of nucleic acid analysis and recombinant DNA: Analysis of gene structure, the mapping function Analysis of genetically determined diseases Prenatal diagnosis Detection of carriers of mutant alleles Diagnosis and Pathogenesis Biosynthesis - insulin , growth hormone, etc. Treatment of genetic diseases - Gene Therapy Molecular cloning and restriction enzymes- isolate a particular gene (or other DNA sequence) in large quantities for further studies, requires the transfer of DNA sequence of interest into a single cell of a microorganism (such as bacteria) - Cloning. The bacteria are grown in culture and reproduce the DNA sequence along with its own DNA. The key development that made recombinant DNA technology possible was the discovery of restriction enzymes - also called restriction endonucleases - that recognize and make double-stranded cuts in the sugar-phosphate backbone of DNA molecules at a specific nucleotide sequences. These enzymes are produced naturally by bacteria, where they are used in defense against viruses. The restriction enzymes recognize specific double-stranded sequences in DNA (usually from 4 to 8 bp long) and cleave both phosphodiester backbones of the DNA double helix near the recognition site. There are many types of restriction enzymes, each with its own recognition site. The sequences to be cleaved are usually palindromes - when read from 5' to 3' is the same on both strands. Well known is EcoRI. Cleave with this restriction enzyme generates two fragments, each with four-base, single-stranded overhang 5'-AATT-3' at the end. Cleavage of a DNA molecule with a particular restriction enzyme digests the DNA into a characteristic and reproducible collection of fragments whose length distribution reflects the frequency and the location of the enzyme's specific cleave sites. In the case of EcoRI enzyme, the length of each fragment is determined by how much DNA sits between two consecutive EcoRI sites. Because all the DNA molecules digested with EcoRI, regardless of their origin, have identical single-stranded sticky ends, any two DNA molecules that have been generated can be joined together in vitro by pairing of their complementary four-base pairs by a DNA ligase enzyme, creates a recombinant DNA molecule. Vector is a DNA molecule that can replicate in a host such as bacteria and achieve a high number of copies per cell. If a human DNA fragment is inserted into a vector (by ligase), the novel DNA molecule that results can be introduced into a bacterial host for the propagation of the inserted fragment along with the vector molecule. Plasmid is a widely used vector. It is circular double-stranded DNA molecule that exists separately from the bacterial chromosome and replicate independently. The plasmids carry antibiotic resistance genes thus could be passed easily from one bacterium to another, spreading antibiotic resistance rapidly. The plasmids designed for molecular cloning contain three critical components: Origin of replication Marker Restriction site that can be cut and used for the ligation (insertion by DNA ligase) of foreign DNA molecules

pre-conception prevention of heritable and inborn diseases what are the types of prevention? what are some common teratogens? what are some common infections? (131)

chromosomes are inherited from our parents. Outside influences are not as important however caution is recommended, such as increased intake of folic acid in pregnant women. It is active mainly against the development of neural tube disorders (NTD). Primary prevention is to minimize exposure to potentially pathogenic factors at work - biological, chemical, physical. It is said that the secret of national health lies in the homes of the people. Primary prevention is simply avoiding the causes of congenital abnormalities. It may operate on a community basis. Availability and acceptability of services are usually related to the overall distribution of resources in the community. Deprived groups have a limited access to preventive services - may result in a higher prevalence of retardation caused by inadequate immunization, inappropriate nutrition and poor contraceptive, prenatal examinations and obstetric care. Secondary prevention - More significant is screening of parents for early diagnosis. The examination is performed on blood samples using cytogenetic testing - especially in women over 35 years of age the risk is becoming to be significantly higher. Tertiary prevention is the complete recovery of congenital abnormalities by perform an early surgical intervention without residual defects or minimal after effects. E.g. surgical intervention in some types of congenital cardiovascular malformations (ventricular and atrial septal defects, patent ductus arteriosus), undescended testis etc. Preconception prevention contains protection against mutagens - such as trying to avoid from reproduction in late age. For women, increased age increases the risk of impaired fetal chromosomal aberrations, whereas for relatively old men (40+) the risk for point mutations increases. Common teratogens (agents that cause fetal malformation) include infections, drugs, and physical agents. Malformations are most likely to result if exposure occurs between the 2nd and 8th week after conception (the 4th to 10th week after the last menstrual period), when organs are forming. Other adverse pregnancy outcomes are also more likely. Pregnant women exposed to teratogens are counseled about increased risks and referred for detailed ultrasound evaluation to detect malformations. Common infections that may be teratogenic include herpes simplex, viral hepatitis, rubella, varicella, syphilis, toxoplasmosis etc. Commonly used drugs that may be teratogenic include alcohol, tobacco, and cocaine Pregnant and pre-pregnant mothers should keep a favorable health status. The mother creates an environment for the development of the fetus throughout pregnancy, and their diseases or treatment in the diseases may seriously impair fetal development. In addition, smoking and improper nutrition may also affect fetal development. Folic Acid - Coenzyme in DNA repair mechanism, Vitamin C - anti-oxidant - prevention of mutation and tertatogensis.

what does molecular cytogenetics focus on? what are the methods used? how to prepare them...different types of probes and their functions? advantages and disadvantages? (32)

Molecular cytogenetics is defined as a specific focus of biomedical sciences targeted at studying chromosomes at molecular resolutions and at all stages of the cell cycle. It comprises a set of the techniques that operate with either the entire genome or specific DNA sequences to analyze genomic structural and behavioral variations at chromosomal or sub-chromosmal level methods: FISH CGH array CGH

genetic aspects of immune system function related to cancer? how is cancer considered a genetic disease? what are the mechanisms of immune surveillance to tumor cells? how can tumors evade immune surveillance? (82)

Normal cells grow, divide, mature and die in response to a complex set of internal and external signals. A normal cell receives both stimulatory and inhibitory signals, and its growth and division are regulated by a delicate balance between these opposing forces. In a cancer cell, one or more of the signals has been disrupted - causes the cell to proliferate at an abnormally high rate. As losing their normal control, cancer cells gradually lose their regular shape and boundaries, eventually forming a distinct mass of abnormal cells - a tumor. If the cells of the tumor remain localized, the tumor is said to be benign; if the cells invade other tissues, the tumor is said to be malignant. Cells that travel to other sites in the body, where they establish secondary tumors, are said to be metastatic cells. Cancer as a genetic disease - cancer arises as a result of fundamental defects in the regulation of cell division; recent studies recognize that most, if not all, cancers arise from defects in DNA. Regarding aspects of immune system related to cancer, there are several canceric cells that possess antigens which elicit an immune response; these antigens can be classified according to their molecular structure and source. Antigens that are product of oncogenes and tumor suppressor genes - mutation in oncogenes (=gene which can transform a normal cell into a tumor cell) and tumor suppressor genes will result in canceric cell - a cell that divides frequently, forming solid tumours or flooding the blood with abnormal cells. The products of these canceric cells - oncoproteins and tumor suppressor proteins - are unique only for canceric cells. When they will be associated with MHC I molecules, the immune system won't recognize them as "self" and elicit immune response against them. Neoantigene - is a newly formed antigen that hasn't been previously recognized by the immune system and is often associated with tumor antigens, found in oncogenic cells. New antigens may be specific to particular types of tumors, and they are referred to as "tumor-specific transplantation antigens" (TSTA). Antigens which result in product of other mutated genes - The genetic instability of tumor cells results in high percentage of mutated genes in their DNA. Among these mutated genes are genes that code for proteins that have unknown function or potentially tumor antigens. These antigens are extremely diverse since the carcinogens that induce the tumors may randomly cause mutation to any host gene. These antigens may trigger an immune response, since there is no self-tolerance against them. Over or aberrantly expressed cellular proteins - tumor antigens may be normal cellular proteins that are abnormally expressed in tumor cells and elicit immune responses - may be over expressed in tumor cells. In melanoma, for example, one such antigen is tyrosinase - an enzyme involved in melanin biosynthesis. Normally, this enzyme is produced in such small amounts that aren't enough to be recognized by the immune system thus fails to induce self-tolerance. In tumor cells when it is over-expressed, the T-cells do not recognize this antigen as "self" and attack it. Oncofetal antigens - embryonic antigens is expressed only during embryogenesis. De-repression of the genes that encode these antigens causes their expression in colon and liver cancer. Antibodies can be arise against these antigens, and are useful for detection of oncofetal antigens. Tumor antigens produced by oncogenis viruses - some viruses are associated with cancers. These viruses produce proteins that are recognized as foreign by the immune system. Immune surveillance to tumor cells: Cell-mediated immunity is the dominant anti-tumor mechanism in vivo (immune response that doesn't involve antibodies, i.e. - involves activation of phagocytes and T-cytotoxic). Antibodies can also be made against tumors, but there is no evidence that they play a major protective role. CD8 T-cytotoxic cells - recognize non-self epitopes found on MHC I molecules, and may either release perforins that create holes in the cell membrane of the target cell, or attach to the cell and kill it by triggering mechanisms that induce apoptosis. NK - natural killers - can lyse a wide range of human tumors, even tumors that seem to be non-immunogenic for T-cells. Thus, T-cells and NK apparently provide complementary anti-tumor mechanisms. Tumors that fail to express MCH I antigens cannot be recognized by T cells, but may trigger NK cells: NK cells are unique, as they have the ability to recognize "stressed" cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. Macrophages - due to T and NK cells secretion of cytokines, macrophages activates and may kill tumors by production of reactive oxygen metabolites or secretion of tumor necrosis factor. Immune Evasion by tumors: Most cancers occur in people who don't suffer from any immunodeficiency. If immune surveillance exists, then cancer must have several mechanisms by which they can bypass the immune system: The immune system causes "natural selection" of the canceric cells - skipping harmful cells that may proliferate. Reduced or even loss expression of histocompatibility molecules enabling canceric cells to escape attack of CD8. But, may be attacked by NK cells (able to recognize cells without MHC). Immunosupression of the immune system - may be caused by the tumor or the tumor products. In addition, some canceric cells that recognized and elicited immune response may cause activation of T-reg cells which suppress the immune system. Many tumor cells produce a thick coat of external glycocalayx molecules which block the access of immune cells to their MHC - antigen masking mechanism. Downregulation of co-stimulatory molecules, which are required to initiate T-cell response. Tumor may reduce their expression. Immunosuppresion may be as a result of physical and chemical factors. TSA and TAA Any protein produced in a tumor cell that has an abnormal structure due to mutation can act as a tumor antigen. Such abnormal proteins are produced due to mutation of the concerned gene. Mutation of protooncogenes and tumor suppressors which lead to abnormal protein production are the cause of the tumor and thus such abnormal proteins are called tumor-specific antigens. In contrast, mutation of other genes unrelated to the tumor formation may lead to synthesis of abnormal proteins which are called tumor-associated antigens.

how is the cell cycle regulated? what are CDKs and what is p53? what are the possible disturbances? how does a cell deal with those? (22)

The cell cycle control system is based on oscillations in the activity of cyclin-independent kinases - CdK . These are protein kinases that form complexes with cyclins. They catalyze the phosphorylation of protein substrates, leading to changes in the enzymatic activity of the substrate and in its interaction with other proteins. We recognize a total of 9 Cdk. CDKs - cyclin-dependent-kinases are important regulators of the cell cycle. CDKs are kinases enzymes that activate or inactivate other proteins by adding phosphate groups to them. As their name implies, CDKs are functional only when they associate with another protein called a cyclin. The level of cyclin oscillates during the cell cycle; when bound to a CDK, cyclin specifies which proteins the CDK will phosphorylate. P53 - gene regulatory protein; it is activated by DNA-damage and stimulates transcription of several proteins that inhibit phase progress. Loss of P53 protein makes way for cancer mutation. - important regulator of both the G1/S and G2/M The main regulatory complexes include: complex cyclin E / CDK2 are : regulation of entry into the S phase; complex of cyclin B / CDK1 : regulation of mitosis. G 1 / S - cell cycle blockade if cell growth or environmental conditions are unfavorable for further division; similar manner, the same CDK is used, but it combines with different G1 cyclins - which cause the CDK to phosphorylate a different set of proteins needed for DNA replication in S phase. CDK-G1-cyclins activity inactivates retinoblastoma protein (Rb protein), which in turn, release E2F group of proteins that activate S phase gene expression. Level of CDK is constant, but levels of cyclin gradually increases during G1, till the CDK-G1-cyclins complex reaches a critical concentration 🡪 cell enters into S phase. G 2 / M - cell cycle arrest if DNA event replication is not completed. if the DNA is damaged; - G2/M checkpoint: Ensures that the initiation of mitosis won't occur until the last nucleotide of the genome was replicated properly. Cyclin B combines with CDK to form MPF - Mitosis Promoting Factor which is activated by the removal of a phosphate group from one of the amino acids of CDK. The amount of cyclin B changes throughout the cell cycle, but the amount of CDK remains constant. During G1, cyclin B levels are low; so the amount of MPF also is low. As more cyclin B is produced, it combines with CDK to form increasing amounts of MPF till the critical level of G2/M 🡪 commits the cell to divide, concentration of MPF reaching a peak in mitosis. The active form of MPF phosphorylates other proteins, which account to some mitotic events such as nuclear-membrane breakdown, spindle formation and chromosome condensation. At the end of metaphase, cyclin B degraded 🡪 lowers MPF 🡪 initiating anaphase. High level of MPF - initiates mitosis, low levels - returns to interphase. M / G 1 - at the metaphase / anaphase transition, stopping if the chromosomes are not properly attached to the mitotic spindle. Many cancers are caused by defects in the cell cycle's regulatory machinery; for example, mutation in the gene that encodes cyclin D (G1/S checkpoint) contributes to the rise of B-cell lymphoma. The overexpression of this gene is associated with both breast and esophageal cancer. Likewise, P53 - tumor suppressor gene - when mutated (75% of colon cancers) - doesn't regulate a potent inhibitor of CDK activity. Generally, the activity of CDK-cyclin complexes is regulated by CDKIs - CDK inhibitors - which are activated when a damaged DNA or chromosome has been recognized, preventing the cell from completing replication. Checkpoint activation delays the cell cycle and triggers DNA repair mechanisms; if DNA damage is too severe to be repaired - the cells are eliminated by apoptosis or enter to non-replicative state of G0, primarily through P53 dependent mechanism.

details about mapping the human genome? what was the method used? what is the Human Genome Project? what method was used? what were the results and what is the significance? (69)

The ultimate goal of structural genomics is to determine the ordered nucleotide sequences of entire genome of organism. The main obstacle is the huge amount of nucleotides (billions of base pairs), and the fact the only small fragments of DNA (usually 500-700 nucleotides) can be sequenced at one time 🡪 there is a need of breaking the DNA into millions of smaller fragments, and to put them back in the correct order. The first draft of the human genome was completed in June 2000. The first method for assembling short, sequenced fragments into a whole-genome sequence called a "map-based sequencing" - used the "shotgun sequencing method", but instead of randomly sequence fragments and look for overlapping (the entire genome is sheared randomly into small fragments and then reassembled), large fragments are ordered with the use of genetic and physical maps - thus it requires the initial creation of detailed genetic and physical maps of the genome, which provide known locations of genetic markers along each chromosome - with this method, a minimal number of fragments that cover the entire chromosome are selected for sequencing. These markers are used to help align the short, sequenced fragments into their correct order. After the genetic and physical maps are available, chromosomes are separated by their size gel electrophoresis or flow cytometry - which separates chromosome optically by their size. The chromosomes first sheared into larger pieces - only partial digestion - which means that the restriction enzymes are allowed to act for only a limited time so that not all restriction sites in every DNA molecule are cut. Then, the fragments are cloned 🡪 the partial digestions and the cloning is used to produce overlap pieces that will help in the correct order sequencing of the fragments. The method for sequencing relies on the presence of a high-density map of genetic markers - complementary DNA probe is made for each marker, and it hybridizes with any fragment contain the marker. Because the clones are much larger than a one marker - in some of the fragments there will be more than one marker. E.g. Clone A might have markers M1 and M2, clone B M2 M3 and M4, and clone C markers M4 and M5 - such a result would indicate that these clones contain areas of overlapping fragments 🡪 determine the order of clones. A set of two or more overlapping DNA fragments is called a Contig. Because it involves first creating a genetic map of the genome, the map-based sequencing is slower than whole-genome shotgun sequencing, and relies less heavily on computer algorithms than whole-genome shotgun sequencing which produce much more fragments. Due to the speed and cost efficiency of whole-genome shotgun sequencing, it is sometimes preferred method for genome sequencing. The Human Genome Project The project started in the Laboratory of Los Alamos and Lawrence Livermore Laboratory in 1983, when it began to create a library of DNA clones. The basis of the research was the random sequencing of the genomes of volunteers. It was necessary to find an ethnically diverse group. Individual sequences obtained were then propagated in the appropriate vector (E.coli) to several million copies - and formed the so-called "bacterial artificial chromosomes". HUGO - Human Genome Organization, established in April 1988. An international collaboration was planned to undertake the human genome project - initial estimates suggested that 15 years and 3 billion dollars would be required to accomplish the task. The project included obtaining the sequence, identifying the genes, and describing their function. As a part of the effort, the genomes of several model organisms such as Escherichia coli and Drosophila Melangoster (fruit fly) were to be sequenced as well to help develop methods that could then be applied to the sequencing of the human genome and to provide sequenced genomes with which to compare the organization and structure of the human genome. The human genome project official started in October 1990 (even tough in 1983, laboratory in Los Alamos New Mexico began creating libraries of individual DNA clones). By 1993, large-scale physical maps were completed for all 23 pairs of human chromosomes. At the same time, automated sequencing techniques had been developed that made large-scale sequencing feasible. The first sequenced human chromosomes were chromosome 16 and 19 by 1995.The project was described on 80,000 human genes. Two competing teams tried to sequence the human genome - an international public association (collaboration of 20 research groups and hundreds of individual researches) and a private effort of company called Celera Genomics, managed by Craig Venter. Each team used different approaches: The public consortium (association) used a map-based approach - many copies of the human genome were cut into fragments of about 150,000 bp each (Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing), inserted into bacterial artificial chromosomes which were sheared into smaller overlapping fragments and sequenced - the whole genome was assembled by putting together the sequence of the BAC clones. Celera Genomic used a whole-genome shotgun approach - in which overlap between the sequences of small fragments is compared by computers. Results and Significance Both teams announced the completion of a draft that included most of the sequence of the human genome in the summer of 2000 - 5 years ahead of schedule. Analysis of this sequence was published 6 months later - the project declared completed in the spring of 2003. For most chromosomes, the finished sequence is 99.999% accurate. The availability of the complete sequence of the human genome is proving to be of enormous benefit - it has made it easier to identify and isolate genes that contribute to many human diseases and to create probes that can be used in genetic testing, diagnosis and drug development. The sequence is also providing important information about many basic cellular processes. Comparisons of the human genome with those of other organisms are adding to our understanding of evolution and the history of life. Moreover, there is better identification in forensic medicine using DNA. The human genome is the manual needed to make the human body - it is used to discover the genetic basis for health and the pathology of human disease. It will eventually enable medical science to develop highly effective diagnostic tools, to better understand the health needs of people based on their individual genetic make-ups, and to design new and highly effective treatment for disease - development of pharmacogenomics, prepare individual treatments for each patient, prevent harming the patient. Through genetic research, medicine will look more into the fundamental causes of diseases rather than concentration on treating symptoms. The project also elicit some legal and social issues - such as how this information would be interpreted and used, who would have access to it, and how can society prevent harm from improper use of genetic information. Genetic engineering issue - the map enable us to diagnose and eventually treat many diseases, as well as enable us to determine the genetic basis of numerous physical and psychological traits, which raises the possibility of altering those traits through genetic intervention. methods: In primer walking, we divide the long sequence into several consecutive short ones. We sequence the first fragment as it was shorter fragment, around 1000 bp are sequenced. We use the end of this fragment - final 20 bases, as a primer for the next part of the long DNA sequence. That way, the short part of the long DNA that is sequenced keeps walking along the sequence. In shotgun sequencing, DNA is broken up randomly, with partial digestion (means that not all sites are cleaved) into small segments which are sequenced using the Sanger method. We perform several round of this fragmentation and sequencing, each time we obtain different fragments because of different restriction points. Multiple overlapping reads for the target DNA is produced, and computer programs use the overlapping ends of different reads to assemble them into Contig - according their order.

what is translation? what are the components? (45)

Translation is the process of protein synthesis, when mRNA is translated from 5' 🡪 3', producing a polypeptide chain of amino acids to produce protein molecule. The process occurs in the cytosolic ribosomes (proteins required in the cytosol itself or destined for the nucleus, mitochondria or peroxisomes) or the RER membrane (proteins to be exported from the cell, or proteins that are destined to become incorporated into plasma, ER or Golgi membranes, as well as lysosomal proteins; translation is always starts at the cytosolic ribosomes and in case of protein to be exported - targeting signal found at the beginning of the mRNA transcript signals to the regulatory element that mediates the transport to the RER Amino acids - all the amino acids that eventually appear in the protein must be present at the time of protein synthesis, otherwise the translation process stops. It demonstrates the importance of having all the essential amino acids in sufficient quantities in the diet. tRNA - transfer RNA - at least one specific type of tRNA is required for each amino acid. There are at least 50 species of tRNA. Because there are only 20 different amino acids, some amino acids have more than one specific tRNA molecule. Each tRNA molecule has an amino acid attachment site for a specific amino acid at its 3'-end. The carboxyl group of the amino acid is in an ester linkage with 3'-hydroxyl of the ribose portion of the adenine (A) nucleotide in the - CCA sequence at the 3'-end of the tRNA. When tRNA is covalently attached amino acid, it is said to be charged. Each tRNA molecule contains a three-base nucleotide sequence, the anticodon, which pairs with a specific codon on the mRNA. This codon specifies the insertion into the growing peptide chain of the amino acid carried by that tRNA. Aminoacyl-tRNA synthetases - this family of enzymes is required for attachment of amino acids to their corresponding tRNAs. Each member of this family (20 different enzymes - one for each AA) recognizes a specific amino acid and all the tRNAs that correspond to that amino acid. The overall reaction of attaching the carboxyl group of an amino acid to the 3'-end of its corresponding tRNA requires ATP. It starts by recruiting the Amino acid into the enzyme active site, utilize ATP and attach adenosine-mono-phosphate to the Amino acid. Then, tRNA molecule arrives to the other active site of the enzyme, and the Adenosine-phosphate released while the tRNA and matched AA are attached - forming Aminoacyl tRNA. In addition to their synthetic activity, the aminoacyl-tRNA synthetases have a "proof-reading" (or editing) activity that can remove an incorrect amino acid from the enzyme or the tRNA molecule. mRNA - messenger RNA - specific mRNA required as a template for the desired protein synthesis. Functionally competent ribosomes - ribosomes are large complexes of proteins and rRNA. The rRNA part, 60% of the ribosome, consist of two subunits - large subunit (which accounts for the peptide bond formation - act as catalyzer - "ribozyme") 60S and small subunit 40S - whose relative sizes are given in terms of their sedimentation coefficients - S values (Svedberg). The small subunit binds mRNA and is responsible for the accuracy of translation by ensuring correct base-pairing between the codon in the mRNA and the anticodon in the tRNA. The large ribosomal subunit catalyzes formation of the peptide bonds that link amino acid residues in a protein. Each ribosome has A, P, and E binding sites for tRNA molecules; each of them extends over both subunits. Together, they cover 3 neighboring codons. During translation, the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This codon specifies the next amino acid to be added to the growing peptide chain. The P-site codon is occupied by peptidyl-tRNA. This tRNA carries the chain of amino acids that has already been synthesized. The E-site is occupied by the empty tRNA as it is about to exit the ribosome. Initiation, elongation, and termination (Release) protein factors are required for peptide synthesis (eIF, eEF, eTF - e for Eurkaryoic, I - initation, E - elongation, T - termination). ATP and GTP are required as sources of energy - cleavage of four high-energy bonds is required for the addition of one amino acid to the growing polypeptide chain.

what are the repair mechanisms of a cell ? why are they needed? what are the four main types and how do they work? (58,59)

Despite the effect of damaging agents, the rate of mutation remains remarkably low, thanks to the efficiency with which DNA is repaired. Less than one in a thousand DNA lesions is estimated to become a mutation. DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. Four general mechanisms of single strand DNA repair: mismatch repair, direct repair, base-excision repair and nucleotide-excision repair. Mismatch repair: Some of the mismatches of base-pairing corrections are done by DNA polymerase proofreading activity. But many incorrectly inserted nucleotides that escape detection by proofreading are corrected by mismatch repair. The mismatch repair enzymes are able to distinguish between the old and the new strands; in E-coli: after replications, adenine nucleotides in the sequence GATC are methylated. The process of methylation is delayed. Thus, immediately after replication, the old strand is methylated while the new strand is not, which is identified by the repairing enzymes. Incorrectly paired bases distort the 3D structure of the DNA and mismatch repair enzymes detect these distortions - Mut ("mutator"). After the error has been recognized by MutS, MutL arrives and mediate connection with MutH which arrives to the non-methylated strand. Looping mechanism follows, and MutH nicks the non-methylated strand. The mismatch repair exonucleases cut out the distorted section of the newly synthesized strand, and Polymerase fill the gap with new nucleotides, by using the original DNA strand as a template and DNA ligase seals the nicks produced by the exonucleases. Direct Repair - (Direct Reversal) Most damage to DNA is repaired by removal of the damaged bases followed by re-synthesis of the excised region. Some lesions in DNA, however, can be repaired by direct reversal of the damage, which may be a more efficient way of dealing with specific types of DNA damage that occur frequently. Only a few types of DNA damage are repaired in this way, particularly pyrimidine dimers resulting from exposure to ultraviolet (UV) light and alkylated guanine residues. UV light is one of the major sources of damage to DNA - exposure to solar UV irradiation is the cause of almost all skin cancer in humans. The major type of damage induced by UV light is the formation of pyrimidine dimers, in which adjacent pyrimidines on the same strand of DNA are joined. The formation of such dimers distorts the structure of the DNA chain and blocks transcription or replication past the site of damage, so their repair is closely correlated with the ability of cells to survive UV irradiation. Direct repair of UV light damage utilizes Photoreactivation which perform dimerization reaction: The process is called photoreactivation because energy derived from visible light is utilized to break the ring structure formed. Enzyme called - DNA Photolyase - light-depended enzyme, upon photoactivation it transfers electrons to thymidine's and removes the dimer. Another mechanism for direct repair is done when spontaneously methylation (alkylation) occurs to guanine Base excision repair: Spontaneous mutation such as oxygenation or deamination yield modified base. Modified base is first excised, and then the entire nucleotide is replaced. Common mutation is spontaneous deamination - Uracil and Cytosine differ from each other by the presence of amine group. Deamination of Cytosine results in Uracil, which then creates mutations. The excision of modified bases is catalyzed by a set of enzymes called DNA glycosylases, each of which recognizes and removes a specific type of modified base by cleaving the bond that links the base to the 1'-carbon atom of the deoxyribose (cleaves the N-glycosidic bond), creates an AP region (Apyrimidine). After the base has been removed, an enzyme called AP endonuclease cuts the phosphodiester bond, and deoxyribose phosphate lyase removes the deoxyribose sugar. DNA polymerase then adds new nucleotides to the exposed 3'-OH group, replacing a section of nucleotides on the damaged strand. The nick in the phosphodiester backbone is sealed by DNA ligase. Nucleotide excision repair: a more serious damage may be caused by UV light that cannot be repaired by direct reversal. It requires the reparation of longer strand. It is versatile and can repair many different types of DNA damage. The nucleotide excision repair process uses a set of proteins called UvrA, B, C, D (Ultraviolet light repair). The process begins with loading of UvrA and UvrB, scanning for damage that distorts the double helix. When the complex recognizes the distorted dimer, UvrA released, while UvrB stays, recruit UvrC ("Uvr Cleave") which binds to the site and makes two cuts on the damaged strands, several bases away from the damage site. UvrD with helicase activity, binds to the site and separate the segment of damage strand from the rest of the DNA molecule. The gap is filled by DNA polymerase, sealed by ligase.

genetic mechanisms of evolution? why does evolution occur? what are the sources of evolution? how can evolution be assessed? what can alter gene frequencies? (118)

Evolution occurs due to genetic variation. Individuals within a species vary in their specific characteristics - genetic variations. Some phenotypic variation doesn't result from genetic differences among individuals - e.g. body builders alter their phenotypes dramatically but do not pass their muscles on to the next generation. Thus, only genetically determined part of phenotypic variation can have evolutionary consequences - without genetic variation, evolution cannot occur. The genetic variation on which evolution depends originates when mutation, gene duplication, genetic drift, as well as natural selection - cause the production of new alleles in a population (and the possible elimination of the others). Selection hypothesis: the cause of evolution is the pursuit of balance and positive selection. Neutralistic hypothesis: emphasizing the effect of mutations, random genetic drift, and negative selection. Mutation hypothesis: emphasizes the effect of mutation pressure and accidental drift. Mutations - a change in the nucleotide sequence of an organism's DNA. In multicellular organisms, only mutations in cell lines that produce gametes can be passed to offspring. Organisms reflect many generations of past selection, and hence their phenotypes tend to be well matched to their environments. As a result, most new mutations that alter a phenotype are at least slightly harmful. In some cases, natural selection quickly removes such alleles. "Heterozygote protection" - effects of mutated gene are masked by a favorable dominant allele - thus maintains a huge pool of alleles. While many mutations are harmful, many others are not and on occasions actually make the bearer better suited to the environment, enhancing reproductive success. Altering gene number of position - when chromosomes changes are deletion or other disruption - it is usually harmful, however, when such large-scale changes leave genes intact, they may not affect the phenotype. In some cases, chromosomal rearrangements may even be beneficial - e.g. translocation of part of one chromosome to different chromosome could link genes in a way that produces a positive effect. A key potential source of variation is the gene duplication - duplication of genes due to errors in meiosis (unequal crossing over) or due to slippage during DNA replication etc. Duplications of large chromosome segments are often harmful, but the duplication of smaller pieces of DNA may not be and may persist over generation, allowing mutations to accumulate. The result is an expanded genome with new genes that may take on new functions, plays a major role in evolution. For example, the remote ancestors of mammals had a single gene for detecting odors, which has since been duplicated many times. As a result, humans today have about 350 functional olfactory receptors genes, and mice have 1000. These dramatic proliferations of olfactory genes enable the early mammals to detect faint odors and to distinguish among many different smells. Rapid reproduction - in prokaryotes, which have many more generations per unit of time than eukaryotes, mutations can quickly generate genetic variation in their population (medical application of these concept - HIV has generation spam of about 2 days, and has RNA genome which has a much higher mutation rate than a typical DNA genome - thus, single-drug treatments are unlikely to be effective against HIV - the most effective AIDS treatments are "cocktails" drug, that combine several medication. It is less likely that a set of mutations that together confer resistance too all the drugs will occur). Sexual Reproduction - In sexually reproductive organisms, three mechanisms contribute to the alleles shuffling, providing much of the genetic variation that makes evolution possible - crossing over, independent assortment of chromosomes and fertilization. During meiosis, homologous chromosomes of individual, one inherited from each parent, trade some of their alleles by crossing over. These chromosomes are then distributed at random into the individual gametes (many genetically different variations of gametes occur) and the variety of possible mating combinations exists, fertilization brings together gametes that have different genetic backgrounds Hardy-Weinberg equation can be used to test whether a population is evolving In short - one way to assess whether natural selection or other factors are causing evolution at a particular locus is to determine what the genetic makeup of population would be if it were not evolving at that locus. We can then compare that scenario with the data we actually observed for the population. If there are no differences - we can conclude that the population isn't evolving. If there are differences, this suggests that the population may be evolving. In a population that isn't evolving, allele and genotype frequencies will remain constant from generation to generation - such a population is said to be in in Hardy-Weinberg equilibrium. Conditions for hardy Weinberg equilibrium: No mutations, There is random mating, there is no natural selection and differences in the survival and reproductive success of individuals carrying different genotypes, the population is large (smaller population 🡪 more likely that allele frequencies will fluctuate by chance)), No gene flow. Any deviation from these conditions is a potential cause of evolution. Natural selection, genetic drift and gene flow can alter allele frequencies in population 🡪 cause to evolution Natural selection - based on differential success in survival and reproduction: individuals in a population exhibit variations in their heritable traits, and those with traits that are better suited to their environment tend to produce more offspring than those with traits that aren't as well suited. In genetic terms, selection results in alleles being passed to the next generation in proportions that differ from those in the present generation (DDT fruit fly example). By consistently favoring some alleles over others, natural selection can cause adaptive evolution (better match between organisms and their environment). Genetic drift - Chance events can cause allele frequencies to fluctuate unpredictably from one generation to the next, and to effect on evolution. Key points in genetic drift - significant in small populations, may cause allele frequencies to change at random and can lead to a loss of genetic variation within populations. Certain circumstances can result in genetic drift having a significant impact on a population - two examples are the founder effect (individuals become isolated from a larger population - smaller group may establish a new population whose gene pool differs from the source population) and the Bottleneck effect (sudden change in environment such as fire or flood 🡪 reduce the size of population 🡪 by chance alone, certain alleles may be overrepresented among the survivors and others may be underrepresented or absent 🡪 changing the gene pool and the genetic variation). Gene Flow - the transfer of alleles into or out of a population due to the movement of fertile individuals or their gametes. Because alleles are transferred between populations, gene flow tends to reduce the genetic differences between populations. Selection the classical evolutionary mechanism of Darwinism selection types: normalizing selection - maintaining the current state of the population by eliminating deviations from the norm (eg hereditary diseases) balancing selection - maintains a certain degree of polymorphism in the population, eg preferences of heterozygotes directional selection - applied when changing external conditions, when the best adapted phenotype survives. It is a natural choice in the sense of classical Darwinism. An example is the industrial melanism of an insect. Haldane's dilemma: selection reduces the number of offspring. When selected against several different genes, these losses are already not negligible, which would lead to the extinction of the population. However, if the selection were not strong enough to lead to extinction, evolution would proceed much more slowly than it actually is. Fisher's fundamental theorem = "The rate of increase in the relative fertility of any organism at any given time is equal to the genetic variance of relative fertility at that time." Better: "The greater the genetic variability that selections toward greater fitness can affect, the greater the progress in fitness." or "The rate of change of a trait depends solely on the additive genetic variation in the fitness of that trait." (1930) Many scholars have criticized the purely linear model of relative fertility inheritance.

what are the genetics and clinical importance of blood group systems? what are the determinants? what is the Bombay phenotype? (72)

Blood groups are determined by antigens on the red blood cell membrane . Glycolipids , carbohydrates , glycoproteins or proteins can be used as antigens . There are currently around 30 different blood group systems. The most important and well-known are the AB0 system , Rh factor (maternal negativity in terms of the so-called D antigen - ie Rh factor - and baby positivity can cause hemolytic disease of newborns ) and MNS system , where the presence of anti-S antibodies in the recipient can cause post-transfusion hemolysis . There is also, for example, the Kell antigen system (incompatibility can cause autoimmune hemolytic anemia and hemolytic disease of the newborn ). The presence of plasma antibodies against antigens other than the AB0 and Rh systems is rare, but if present, it can cause severe post-transfusion reactions . These antibodies we refer to as irregular antibodies. ABO blood group There are four major groups in the ABO system - A, B, AB and O. Those with blood group A possess the antigen A on the surface of their RBCs, B has antigen B, AB has both antigens, and O have neither. People with A blood group have naturally anti-B antibodies (aggultinins), B - have anti A and O have both antibodies. The alleles at the ABO blood group locus are inherited in a co-dominant manner, but are both dominant to the gene for the O antigen; thus - there are six possible genotypes. Blood group AB individuals do not produce A or B antibodies, so they can receive a blood transfusion from all the other groups, and referred to as universal recipients. Group O individuals do not express A and B antigens - therefore, they won't be attacked by any of the antibodies and referred to as universal donors. Landsteiner's Rule - If an antigen is present on a patients RBCs (e.g. A antigen), the corresponding antibody (e.g. A antibody) won't be present in the patients plasma under normal conditions. HH Blood group, also known as Oh or the Bombay blood group is a rare blood type - the RBCs appeared to lack all of the ABO blood group antigens. Individuals with the Bombay phenotype do not express H antigen, the antigen which is present in blood group O. As a result, they cannot make A or B antigen on their RBCs, whatever alleles they may have of the A and B blood group genes, because A and B antigens are made from H antigen. People with Bombay phenotype can donate RBCs to any member of the ABO blood group, but they cannot receive blood from any member, except of people with Bombay phenotype. Rhesus blood group The Rh blood group system involves three sets of closely linked antigens - Cc, Dd, and Ee. D is very strongly antigenic and persons are either Rh positive (possessing the D antigen) or Rh negative (lacking the D antigen). There are two types of Rh red cell membrane polypeptide - one corresponds to the D antigen, and the other to the C and E series of antigens. Two genes code for the Rh system - one for D and d, and a second for both C and c and E and e. The D locus is present in most persons and codes for the major D antigen present in those who are Rh-positive. Rh-negative individuals are homozygous for a deletion of the D gene - therefore, an antibody has never been raised to d. DNA analysis of Rh-negative persons, who were homozygous for C\c or E\e groups, allowed identification of the genomic DNA sequences responsible for the different antigenic variants at this locus - revealing that they are produced by alternative splicing of the mRNA transcript. Erythroblastosis fetalis (also known as hemolytic disease of the newborn) is a disease of the fetus\newborn which his mom possesses Rh-negative whereas the fetus is Rh-positive. When Rh-positive blood is given to persons who are Rh-negative, the majority will develop anti-Rh antibodies, even due to exposure of very small quantities of blood. In the case of an Rh-negative mother carrying an Rh-positive fetus, fetal red cells that cross to the mother's circulation can induce the formation of maternal Rh antibodies. In a subsequent pregnancy, these antibodies can cross the placenta from the mother to the fetus, leading to hemolysis and severe anemia. After a woman has been sensitized (exposed to Rh-positive blood), there is a significantly greater risk that a child in a subsequent pregnancy, if Rh-positive, will be more severely affected. By giving the mother an injection of Rh antibodies - Anti-D - fetal cells in the maternal circulation are destroyed before the mother can become sensitized. MNS antigen system This human blood group system based upon two genes (Glycophorin A for MN and glycophorin B for S U) on chromosome 4. There are currently 46 antigens in the system, but the five most important are called M, N, S, s and U. This system can be thought of as two separate groups - the M and N antigens are one, and the S, s, and U are the second. The two groups are very closely located together on chromosome 4 and are inherited as a haplotype (groups of genes that are inherited together from a single parent). The MN blood group in humans is under the control of a pair of co-dominant alleles - LM and LN. The blood type is due to a glycoprotein present on the surface of RBCs, which behaves as a native antigen. Phenotypic expression at this locus is codominant because an individual may exhibit either one or both antigenic substances. The S antigen is relatively common (55% of the population), and the s antigen is very common (89%). Anti-S and anti-s can cause hemolytic transfusion reaction. The U antigen is a high incidence antigen, occurring in 99.9% of the population - thus, "U" is "Universal" (almost). P Antigen system Another human blood group system, based upon genes on chromosome 22. The P antigens are carbohydrate antigens that include P1 , P , and PK. P phenotypes are defined by reactivity to antibodies anti-P1, anti-P, anti-PK and anti-PP1PK. P1 Phenotype: anti-P1 (+), anti-P (+) and anti-PP1Pk (+) and anti-Pk (-). Seen in 95% of Blacks and 80% of Whites. P2 Phenotype: anti-P1 (-), anti-P (+), antiPP1Pk (+), and anti-Pk (-). Seen in 5% of Blacks and 20% of Whites. Rare p phenotype (absence of P antigens): anti-P1 (-), anti-P (-), anti-PP1Pk (-), and anti-Pk (-). These individuals have a very strong anti-PP1Pk which can be associated with delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn Kell Antigen System The Kell blood group system is complex and contains many antigens that are highly immunogenic. These antigens are the third most potent, after those of the ABO and Rh blood groups, at triggering an immune reaction. KEL gene is located on the 7th chromosome and is associated with X-linked gene XK. Antibodies that target Kell antigens can cause transfusion reactions and hemolytic disease of the newborn (HDN). In the case of HDN, ABO and Rh incompatibility are more common causes. The infrequent cases of HDN caused by Kell immunization tend to result in severe fetal anemia because maternal anti-Kell target fetal red blood cell (RBC) precursors, suppressing the fetal production of RBCs. These antibodies are classes IgG and occur as a result of immunization. Kidd System The Kidd antigen system (also known as Jk antigen) is present on the membranes of red blood cells and the kidney and helps determine a person's blood type. The Jk antigen is found on a protein responsible for urea transport in the red blood cells and the kidney. The gene encoding this protein is found on chromosome 18. Three Jk alleles are Jk(a), Jk(b)and Jk3. Antibodies are not common but are very dangerous, usually formed after immunization. More blood groups - Duffy system, Lewis system, Lutheran system.

what is cancer? characteristics of cancer cells? types of cancer cells? what are the common traits (SARCOMA)? key processes that are affected in tumor cells? (108)

Cancer kills one of every five people in the US. It is not a single disease; rather, it is a heterogeneous group of disorders characterized by the presence of cells that do not respond to the normal control on division. Cancer cells divide rapidly and continuously, creating tumors that rob healthy tissues of nutrients. Cancer cells can then separate from the tumor and travel to distant sites in the body, where they may develop new tumors. Nature of cancer cells Cancer is a genetic disorder caused by DNA mutations that can either be acquired spontaneously or induced by environmental factors. Cancer is fundamentally a genetic disease. Mutations in several genes are usually required to produce cancer. Normal cells grow, divide, mature and die in response to internal and external signals; a normal cell receive both stimulatory and inhibitory signals, and its growth and division are regulated by their balance. In cancer cell, one or more of the signals has been disrupted - the cell proliferates at high rate (defects in homeostasis), lose its regular shape and boundaries and forming a distinct mass of abnormal cell called tumor. Benign cancer cell - the cell remains localized in the tissue of origin Malignant cancer cell - the cells invade other tissues Metastasis - cells travel to other sites in the body where they established secondary tumors. Important to note that tumor is not synonymous with cancer - while cancer is by definition malignant, a tumor can be benign, precancerous or malignant. Self-sufficiency in growth signals - "Accelerator pedal stuck on" In order to grow and divide, cells require hormones and other molecules; cancer cells have the ability to grow without these external signals. They may produce these signals by themselves - Autocrine signaling, they may destroy "off switches" that prevents excessive growth from these signals (prevents negative feedback), and importantly - the cell-cycle, which is typically highly controlled by many proteins and check-points becomes deregulated - the proteins controllers are altered 🡪 Increased growth and cell division within the tumor. Insensitivity to anti-growth signals - "brakes don't work" Tumor suppressor genes encode for proteins that control cell growth and division - halt division if the cell is damaged or not ready to divide. In cancer, these proteins altered and lost their effectiveness. In addition, normal cells have mechanism of "contact inhibition" which prevents over-division - when the cells fill up the space they are and touch other cells, there is no further growth. Cancer cells do not have contact inhibition - continue to grow and divide regardless their surroundings. Evading programmed death - aren't eager to die Cancer cells bypass programmed cell death by alter the mechanisms that detect damage or abnormalities in the cells, thus prevent apoptosis. Limitless replicative potential - Infinite generations of descendants Normal cells have an intrinsic programs - "Hayflick limit" - that limit their multiplication to about 60-70 doubling. Each division, the telomeres of chromosomes shorten every cell division, till certain limit (2.5kb) which stops cell dividing. Cancer cells bypass this barrier by manipulating enzymes that increase the length of telomeres - thus, divide indefinitely. Sustained angiogenesis - developing blood vessels, takes blood supply from other tissues Cancer cells have the ability to increase the production of factors that promote blood vessels formation (angiogenesis) in order to deliver oxygen and nutrient to cancer cells. Tissue invasion and metastasis - migration and spreading to other tissues One of the most well-known properties of cancer cells is their ability to invade neighboring tissues - metastasis (malignant tumor). In metastasis the cancer cells travel through the blood or lymph system and form new tumors (metastatic tumors) in other parts of the body. Other hallmarks of cancer cells: Deregulated metabolism - they use abnormal metabolic pathways to generate energy; they turn-on glycolysis for rapid dividing. Evading the immune system - invisible to the body's immune system Genome instability - cancer cells have chromosomal abnormalities, which worsen with disease progresses. Inflammation - local chronic inflammation has role in induce man types of cancer The body naturally prevents cancer cells Although humans surrounded by environmental agents that are mutagenic - cancer are relatively rare outcomes of these encounters due to the ability of cells to repair DNA damage; DNA repair mechanism maintains the integrity and stability of the genome. Tumor suppressor genes are capable of detecting mutations and repairing them, in case repairing isn't possible - they induce apoptosis. The cell cycle that promotes division is highly regulated by these proteins. Mutations in these are usually inherited and greatly increase the risk for development of cancers. key processes: mitotic hyperstimulation WNT-beta-cateninin signaling - promitotic hyperstimulation degregulation of cell cycle and DNA repair processes deregulation of apoptosis

ecology and ecogenetics what is ecology? what is dispersion? what is population density? what is ecogenetics? what are the factors? (133)

Ecology is the study of interactions among organisms and their environment - interconnects biology, geography and earth science. Ecogenetics is a branch of genetics that studies genetic traits related to the response to environmental substances - it deals with effects of pre-existing genetically determined variability on the response to environmental agents. Population ecology examines the influence of environmental factors on population. The word environmental is defined broadly to include the physical, chemical, biological, atmospheric, and climate agents - all of them are agents which the hereditary differences in reactions of people affected by. Pharmacogenetics and modern medicine based on it. Example: Physical influences may be, for example - UN radiation - which from one hand necessary for vitamins formations, but from the other hand, may act as a mutagen, induces mutations. Protection of the human body appeared by pigmentation, which is polygenic hereditary level of protection. The influence of UV irradiation on individuals varies. There are genetic diseases associated with sensitivity to this physical environmental factor, e.g. Xeroderma Pigmentosum - which is an AR disease - the skin is extremely sensitive to sunlight, and exposed places tends to form carcinomas. It occurs due to defect endonucleases and mutation in roughly 6 genes. population A population is a group of individuals of the same species inhabiting a certain area in a given period of time. Population ecology examines the influence of ecological factors on populations (democology). In a population, individuals are distributed so that everyone is likely to participate in the reproduction of the population's offspring. It is therefore a living system in which not only the biological properties of individuals but also the biological properties of the whole group are manifested. Thus, the population can: grow, age, differentiate, maintain, have a certain structure, birth rate, mortality, dispersion,... Population ecology allows you to study these characteristics, thus contributing to a better understanding of the gene pool of nature. Dispersion Dispersion - distribution of individuals in the population, informs about the location of individuals in the living space. Divided into: linear ; flat; spatial. Population density Population density is related to the distribution of individuals - it is given by the number of individuals per unit area - abundance . Population density is subject to species changes: Population age structure Another characteristic of the population is its age structure. From this point of view, we can divide the population into 3 categories: preproductive ; productive ; postproductive (old individuals). Natality is directly dependent on metabolic rate and indirectly dependent on size. Mortality . The animal population is subject to a kind of spatial activity, either during movement due to the expansion of the species, or also under heat, due to reproduction, behind food, Vertical activity (chamois) is interesting. An important feature is the form of population growth: closed growth (S-curve) - the maximum population density fluctuates around the so-called carrying capacity of the environment; open growth (curve of the letter J) - initially a gradual increase, then a sharp rise and finally a steep decline Ecogenetics _ It studies inherited differences in people's response to physical, biological and chemical environmental influences. Follows the pharmacogenetics and development of modern medicine. Physical effects UV radiation - a powerful mutagen for single-celled organisms. In humans in small amounts needed for the production of vitamins. The protection is provided by the pigment. Pigmentation is polygenically inherited, the degree of protection is determined by genotype . UV radiation causes mutations , which are removed by the action of repair enzymes . People with repair disorders have an increased risk of malignancies. Diseases associated with UV sensitivity: Xeroderma pigmentosum is an autosomal recessive disease. The skin is extremely sensitive to sunlight, and carcinomas develop in exposed areas at an early age. Endonuclease defect + mutations of about 6 other genes , individuals with a combination of Aa alleles are less affected. Ataxia telangiectasia . Bloom's syndrome (erythema congenitale telangiectaticum Bloom) is an autosomal recessive syndrome of chromosomal instability. Food Fats Hyperlipemia with subsequent atherosclerosis, coronary heart disease , MI . Individual risk is conditioned not only by lifestyle, but also by genetic predisposition. Fat metabolism depends on its transport in the blood, its binding to cell receptors and the breakdown of fats in cells. The amount of fat can be affected by diet and medication. Salt the sensitivity of sensors to salty taste is influenced by the sensitivity threshold of salty taste and gender habits; genetically modified, modified in childhood; persons with a dominant inherited disorder of Na + transport from cells and K + to cells (Na-K pump); arterial hypertension. Milk decreased lactase activity → undigested lactose → GIT problems; AR hereditary lactase deficiency - atrophic enteritis ; degenerative changes in the renal tubules. Flour People with celiac disease (gluten enteropathy) - this is an inability to break down gluten in flour, nutrient resorption disorders, digestive problems. Heredity is irregularly dominant. The condition is adjustable by diet (gluten-free). Proteins Toxic to children with congenital disorders of amino acid metabolism (eg PKU , diet low in phenylalanine). Alcohol Alcoholism is conditioned by social factors and genetically. Alcohol cohydrogenase (ADH) - alcohol is metabolized in the liver to acetaldehyde (absorption already in the stomach). Alcohol tolerance is also affected by acetaldehyde dehydrogenase (ALDH) activity. Inhalants dust (antitrypsin deficiency → pulmonary emphysema); smoking (lung cancer); allergens (changes in the immune response, bronchial asthma). diseases Insulin dependent DM (type I DM): manifestations in childhood; inherited antigenic equipment (HLA haplotypes, DR3 and 4) - in 95% of those affected; Immune disorders: agama globulinemia ( GR ); thymic aplasia ; AIDS, EB-virus mononucleosis; Gastric or peptic ulcer: Helicobacter pylori. Jaundice, TBC ,...

what is meiosis? what are the stages? what are the outcomes? draw a schematic (25)

Mitosis produce only genetically identical progeny - if only mitosis occurred, all the people around us would be clones - copies of one another, except of occasional mutations that would introduce genetic variability; that's exactly what happened till 2 billion years ago - till cells started to produce genetically variable offspring through sexual reproduction - which shuffling the genetic information from two parents and greatly increases the amount of genetic variation - allows for accelerated evolution. Meiosis is a type of cell division that reduces the number of chromosomes in the parent cell by half, and produces four haploid (23 chromosomes) gamete cells for each parent cell; this process is required to produce egg and sperm cells for sexual reproduction. Like mitosis, meiosis is preceded by an interphase stage that includes G1, S and G2 phases. M-phase consists of two distinct processes: meiosis I and meiosis II, each includes a cell division: meiosis I is termed the reduction division (number of chromosomes is reduced by half) and the second division is termed the equational division. stages: Interphase - during interphase, the chromosomes are relaxed and visible as diffuse chromatin; they replicate, as well as cellular organelles replication and synthesis of the enzymes and proteins which are needed to the M-phase. Prophase I - elongated phase - composed of 5 different stages - "Lazy Zebras Ponder Dire Disasters" Leptotene - the chromosomes condense, become visible Zygotene - the chromosomes continue to condense, homologous chromosomes pair up and begin synapsis - a very close pairing association. Each homologous pair of synapsed chromosomes consists of four chromatids called "Bivalent" or "Tetrad". , synaptonemal complex develops between homologous chromosomes - it mediates chromosome pairing, synapsis and recombination. Pachytene - the chromosomes become shorter and thicker; in this stage - crossing over takes place, in which homologous chromosomes exchange genetic information. Diplotene - centromeres of the paired chromosomes move apart; synaptonemal complex dissociates - the homologous chromosomes remain attached only in the Chiasma - which is the result of crossing over. Diakinesis - chromosome condensation continues and the chiasmata move toward the ends of the chromosomes - the homologs remained paired only at the tips. Near the end of prophase I - the nuclear membrane breaks down and the spindle forms. Metaphase I - homologous pairs of chromosomes align along the metaphase plate - a microtubule from one pole attaches to one chromosome of a homologous pair, and a microtubule from the other pole attaches to the other member of the pair. Anaphase I - separation of homologous chromosomes to opposite poles; although the homologous chromosomes are paired - the sister chromatids remain attached. Telophase I - the chromosomes arrive at the spindle poles 🡪 Cytokinesis - cytoplasm divides Results at the end of Meiosis I - 2 separated cells, each contain 23 pairs of chromosomes, each pair consist of two sister chromatids (total - 46 sister chromatids in each cell). The period between Meiosis I and Meiosis II termed Interkinesis - in which the nuclear membrane re-forms around the chromosomes, spindle breaks, chromosomes relax. These cells then pass to meiosis II: Prophase II - chromosomes re-condense, spindle re-forms, nuclear envelope breaks down again (in some cells - the chromosomes skip the interkinesis part and move directly to metaphase II) Metaphase II - similar to metaphase of mitosis, individual chromosomes line up on the metaphase plate - with the sister chromatids facing opposite poles. Anaphase II - kinetochores of the sister chromatids separate and he chromatids are pulled to opposite poles; each chromatid is now a separated distinct chromosome. Telophase II - chromosomes arrive at the spindle poles, nuclear envelope re-forms around the chromosomes, cytoplasm divides in cytokinesis. Chromosomes relax and no longer visible. Important to remember that ovulated eggs become arrested in metaphase II until fertilization triggers the second meiotic division. The pairs of sister chromatids resulting from the separation of bivalents in meiosis I are further separated in anaphase of meiosis II. In oocyte, one sister chromatid is segregated into the second polar body, while the other stays inside the egg. During spermatogenesis, each meiotic division is symmetric such that each primary spermatocyte gives rise to 2 secondary spermatocytes after meiosis I, and eventually 4 spermatids after meiosis II. Each original cell produces four haploid cells Chromosome number is reduced by half - cells produced by meiosis are haploid. Cells produced by meiosis are genetically different from one another and from the parental cell - genetic differences among cells result from crossing over between non-sister chromatids (which takes place I prophase I) which is the basis for intra-chromosomal recombination - creating new combinations of alleles on chromatid. The two homologous chromosomes separate, each going into a different cell; in meiosis II - the two chromatids of each chromosome separate, and thus each of the four cells resulting from meiosis carries a different combination of alleles. The second process of meiosis that contributes to genetic variation is the random distribution of chromosomes in anaphase I of meiosis after their random alignment in metaphase I. How each pair of homologs aligns and separates is random and independent of how other pairs of chromosomes align and separate. In summary, crossing over shuffles alleles on the same chromosome into new combinations, whereas the random distribution of maternal and paternal chromosomes shuffles alleles on different chromosomes into new combinations. Together, these two processes are capable of producing genetic variation among the cells resulting from meiosis.

what is the molecular basis of genetic diseases, how are they caused? what are the different types of pathologies? how can the structure of a chromosome be affected? what are some major diseases? (60)

Molecular reason of a genetic disease is a mutation - either inherited or acquired. The same disease, such as some forms of cancers, may be caused by inherited genetic condition in some people, by new mutations in other people, and by environmental factors in others. The definition of a genetic disease is a disease which occurs when an alteration in the DNA of an essential gene changes the amount or function or both, of the gene product - mRNA, which leads to dysfunctional protein. It may occur as a result of chromosomal disorders (whole or parts of chromosome are missing or changed), as a single gene disorders (mutation affects one gene, usually alter the function of a protein, e.g. sickle cell anemia), or multifunctional polygenic disorders (complex disorder, associated with the effect of multiple mutation in two or more genes, may be in combination with lifestyle and environmental factors) There are two different types of pathologies regarding genetic diseases: Mutation at the control genes - the rate of protein synthesis changes but the phenotype is normal Mutations at the structural genes - causes genetic disease - the amounts and functions of gene products changes. The mutations at the structural genes cause: Loss of function of a protein - most common effect on proteins, may result from alteration of gene coding due to nucleotide substitutions, deletions, insertions, rearrangements. E.g. Thalassemias (deletion of globin gene) Retinoblastoma (loss of function to tumor suppressor gene). These include the introduction of a premature stop codon or of a missense mutation that impairs protein function. Gain a new function - can alter the biochemical phenotype by increasing the function of a protein (more is not necessarily better), e.g. increase in the level of protein's expression (Trisomy 21). Gain of function mutation may be due to either an increase in the abundance of the protein (increase in its expression) or an increase in the ability of each protein molecule to perform one or more normal functions. New property mutation - change in the amino acid sequence causes disease by conferring a new property on the protein, without necessarily altering its normal function. E.g. Sickle cell anemia - one base pair substitution causes amino acid substitution. It has no effect on ability of sickle cells RBCs to transport oxygen, but the sickle cells hemoglobin tends to aggregate in the deoxygenated state and causes deformation - sickle shape - of RBC. Expression of a gene at the wrong time or place. There are mutations that associated with altered regulatory regions (mutation of control genes) of genes and cause inappropriate gene expression. E.g. oncogene normally promotes cell proliferation, but if isn't normally expressed - may cause cancer. General diagram showing the path of a gene (or of the DNA) to a protein in the molecular biology known as the central dogma: Normal state: gene (DNA) 🡪 mRNA standard sequence 🡪 normal functioning protein Pathological state: a mutated gene (DNA) 🡪 mRNA sequences which aren't standard 🡪 nonfunctional protein or impaired function 🡪 may lead to present or future disease. diseases: Enzyme defect - amino acids (PKU - phenyl alanine hydroxylase AR disease), complex lipid (Tay sachs disease), Porphyria (any one of the enzymes of heme synthesis) Receptor defect - Hypercholesterolemia Defect in molecular transport - Thalassemia (defect in hemoglobin globin chains), Cystic fibrosis Defect cell structure - Muscular dystrophy, sickle cell anemia Defect in regulation of growth and differentiation - tumor suppressors, sex determination, X-chromosome inactivation Defect intercellular communication - e.g. diabetes type II causes intolerance to insulin, growth hormone.

genetics and biology of bacteria - how can they be classified - what are the structural components? - how do they reproduce? - DNA recombination? importance in medicine - what can they be used for? (86)

Morphology and classification of bacteria: Bacteria are much smaller than eukaryotic cells, with an average size of 1-5 µm. Three categories of bacteria according their shape: 1. Rod shaped - large surface area 🡪 better metabolic exchanges with surrounding. 2. Spherical (coccus) 3. Spiral Bacteria belonged to the evolving chain of life on Earth. They originated about 3 billion years ago and affected both the development of the environment and the development of other species, as infections are important factors in selection . More than 2000 species of bacteria have been described . They do not have a nuclear membrane or nucleolus . Transcription and translation take place almost simultaneously in the cytoplasm. They have an irreplaceable role in ecosystems : degrade organic matter and recycle nutrients ( saprophytes ) some are able to capture atmospheric nitrogen they are extremely adaptable - they show a huge diversity of metabolism and the ability to use different energy sources Bacterial chromosome is called nucleoid - a specific region in the prokaryote cell. Usually has size of 200kb. It lacks nuclear membrane. The nucleoid (bacterial chromosome) comprises a closed circle of dsDNA that is highly supercoiled to form a compact structure. The A and C bases of bacteria are methylated, helping the endonucleases to recognize the bacterial DNA from a viral DNA. Bacteria DNA contains transponsons which are DNA sequences coding for enzymes that enable them to be translocated into different areas in the bacterial genome. Some bacteria contain additional DNA in the form of small self-replicating extrachromosomal elements called plasmids. Plasmids do not contain essential gene for growth and reproduction, thus the cell may survive without them. However, some plasmids may give a survival advantage to the bacteria since it may encode for toxins or resistance to antibiotics. Plasmids can be passed from cell to cell - used as a vector in molecular engineering. The cell itself is not able to create a plasmid - it can obtain it: By conjugation through fimbriae from another bacterial cell - spread of antibiotic resistance , horizontal transmission of genetic information. Transduction - through the bactriophage. Transformation - the transfer of free DNA from the environment to the cell ( Griffith's experiments , evidence of DNA as a carrier of genetic information). Granular ribosomes: Ribosomes are composed of a complex of protein and RNA, and are the site of protein synthesis in the cell. Prokaryotic ribosomes are 70S, with sub units of 50S and 30S. Inclusion bodies are granular structures found in the cytoplasm and act as food reserves. They may contain organic compounds such as starch, glycogen or lipid. Endospores: An endospore is a dormant, tough, and non-reproductive structure produced by certain bacteria from the Firmicute phylum. They are a dormant form to which the bacterium can reduce itself. Production of endospores is limited to only certain types of bacteria and usually takes place when the surrounding becomes incompatible for living, such as: extreme pH or temperature, shortage of nutrients, etc. In endospore formation, the bacterium divides within its cell wall. It can survive almost every environmental condition, and when the environment returns to be favorable, they are revived and the bacteria "come back to life". Plasma membrane: The cytoplasm and its contents are surrounded by a plasma membrane, composed of lipid bi-layer and proteins. Difference between eukaryotic plasma membrane and prokaryotic plasma membrane: I. Whereas eukaryotes possesses mitochondria, in prokaryotes the proteins found on the plasma membrane contain enzymes associated with these functions. II. Eukaryotes membranes have cholesterol as part of their lipid components, while the majority of bacterial membranes do not. However, many do contain molecules called Hopanoids that are derived from the same precursors, and assist in maintaining membrane stability. Cell wall: Bacteria have a thick, rigid cell wall, which maintains the integrity of the cell and determines its shape. The major component of the cell wall is peptidoglycan. Precursor molecules for peptidoglycan are synthesized inside the cell, and transported across the plasma membrane. There are two distinct structural types of cell wall: I. Gram-positive cell walls - are relatively simple in structure, comprising several layers of peptidoglycan connected to each other by cross-linkages to form a strong, rigid scaffolding. The negative charge of the cell wall comes from the phosphate groups in the teichoic acid, which is a component in the cell wall. II. Gram-negative cells - have an inner thin layer of peptidoglycan and an outer layer named as the outer membrane. The outer membrane consists of two layers: an inner layer of phospholipids and an outer layer of lipopolysaccharides. Proteins called porins incorporated into the outer membrane and penetrating its entire thickness to form channels, which allow the passage of water and small molecules to enter the cell. Some bacteria, have a periplasmic space between the plasma membrane and the cell wall. This is the site of metabolic activity, and contains a number of enzymes and transport proteins. Extra cellular structures: Involved either with locomotion of the cell or its attachment to a suitable surface. Flagella is a thin hair-like structures, often much longer than the cell itself, and used for locomotion in many bacteria. There can be a single flagellum, one at each end, or many. Each flagellum is a hollow but rigid cylindrical filament made of the protein flagellin and attached to a basal body, which secures it to the cell wall and plasma membrane. Rotation of the flagellum, is an energy-dependent process driven by the basal body. Pili are Resemble short flagella, but they do not penetrate to the plasma membrane, and not associated with motility. Their function is to anchor the bacterium to an appropriate surface. Glycocalyx: Can be found in two forms: Loosely bound slim layer - helps protect against dryness, and is important in the attachment of bacteria to the layers of other organisms tissue or Thick capsule - offer protection to certain pathogenic bacteria against the phagocytic cells of the immune system. Function Adherence - function of hosts, coaggregation of bacteria. The formation of biofilms . Resistance to phagocytosis. Resistance to antibiotics. types HousingClearly separated from the environment, it clings firmly to the cell wall .Structural integrity (well condensed polymer).Antigenic properties, virulence and invasiveness factor . Mucus - loose amorphous mass. S-layer - a squamous glycoprotein on the cell wall surface . Reproduction of bacteria Prokaryotes do not reproduce sexually. The bacterium E-coli, for example, reproduces by binary fission in a human intestine, one of its natural environments. After repeated rounds of division, most of the offspring cells are genetically identical to the original parent cell. However, if errors occur during DNA replication, some of the offspring cells may differ. Binary fission: is a kind of asexual reproduction. After replicating its genetic material, the cell divides into two nearly equal sized daughter cells. The genetic material is also equally split. The daughter cells are genetically identical (unless a mutation occurs during replication). During binary fission, the DNA molecule divides and forms two DNA molecules. Each molecule moves towards the opposite side of the bacterium. At the same time, the cell membrane divides to form 2 daughter cells. In the process of cell division (in eukaryotes), first the division of nucleus takes place after which the division of cytoplasm (cytokinesis) takes place. After division, the new cells will grow and the process repeats itself. Although new mutations are a major source of variation in prokaryotic population's additional diversity arises from genetic recombination - the combining of DNA from two sources. Three mechanisms - transformation, transduction and conjugation - can bring together prokaryotic DNA from different cells (question 89). Recombination Recombination is the interruption and reconnection of DNA with the exchange of its segments. May be: 1. Heterologous New genes are introduced and exchanged between a pair of homologous DNA sequences. We distinguish here transposons, integrons. Transposons Moving within the genome and from the plasmid to the chromosome is called "jumping genes". By moving tr. certain genes can be started and stopped. They differ from the virus in that they lack a reproductive cycle, from the plasmid's inability to self-replicate and exist outside the chromosome. After incorporation - mutations , may carry stop codons , termination sequences, promoters. insertion sequence - the simplest type of transposon, it carries only the gene for trasposase and inverted repeats compound transposons - at least one gene in addition to IS, (genes for virulence factors , for resistance to ATB ) 2. Homologous Some bacteria change their properties by rearranging their own genes. We then distinguish between local inversion and gene conversion, eg in gonococci ( Neisseria gonorrhoeae ), when the antigenic composition changes, new serotypes are formed . There are a number of genes for antigens, only one is functional and the others are defective - multiplication and rearrangement of genes → a functional gene becomes defective and one of the defective ones becomes functional. Importance in Medicine Normal Flora - are microbes that inhabit the human body under normal circumstances, most commonly in the skin, eyes, mouth, upper respiratory, GI and urogenital tracts. The normal flora can is essential for humans - the bacteria serve as competition for invading pathogens over nutrients and place. In addition, some bacteria of the GI produce antimicrobial substances which keeps pathogenic microbes away. Bacteria of the gut provide important nutrients such as Vitamin K and aid in digestion and absorption of nutrients. If the bacteria are displaced from their normal sites in the body to abnormal sites, they may be pathogenic. For example, in a case that bacterium of the skin invades into the blood stream. Pathogenic microbes: Pathogenic bacteria are bacteria that can cause infection. Although most bacteria are harmless or often beneficial, several are pathogenic. It may be caused either by releasing of virulence factors from the bacteria or by the reaction of the immune system with the bacteria (for example - tuberculosis) Genetic engineering:Gene cloning - for gene we use several biological properties of bacteria. Restriction enzymes - used for cleavage of both strands of DNA to be replicated and a region in the plasmid. Plasmids - serves as vectors into which we insert desired DNA to be replicated. In addition, we make a use in the ability of plasmids to replicate independently from the chromosomal DNA as well as its antibiotic resistance property. Induced transformation - we insert the plasmid into E coli bacteria by induced transformation. PCR - we use DNA polymerase taken from bacteria that live in hot springs and thus their enzymes able to withstand high temperatures and not denatured.

what is the operon transcriptional regulation model? what is an inductive operon model? what is a repressive operon model? what is catabolite repression? (87)

Operon transcriptional regulation model The mechanism of transcriptional regulation in prokaryotes was described by F. Jacob and J. Monod in 1961. They found that the expression of a gene or group of contiguous structural genes of a metabolic pathway is controlled by two transcriptional regulatory elements: (i) a regulator gene the repressor, and (ii) the operator to which the repressor is linked. The unit of regulation of gene function, consisting of the operator, promoter and structural genes, was called the operon. They obtained their findings by studying lac operon mutations in E. coli. In addition to the promoter and operator, this operon comprises three structural genes that allow the bacterium to metabolize lactose: gene Z (encodes beta-galactosidase), gene Y (encodes beta-galactoside permease) and gene A (encodes beta-galactoside transacetylase). a) Inductive operon system The inductive operon system is typical for regulating the transcription of genes encoding catabolic reaction enzymes. The regulatory gene is transcribed constitutively (permanently). His product, the repressor, is tied to the operator of the operon. An operator is a segment of DNA that is part of the promoter of structural genes. Binding of a repressor to an operator prevents RNA polymerase from binding to the promoter and initiating transcription of structural genes. Under these conditions, the cell produces only about 1% of the maximum possible amount of protein (enzyme) encoded by the structural genes. When there is a substance in the environment that these enzymes can metabolize, this substance acts as an inducer. Inductors change the allosteric configuration of the repressor and thus make it impossible for it to connect to the operator. RNA polymerase can bind to the released promoter and initiate transcription of structural genes. In the case of the lac operon, the inducer is lactose. When lactose is present, it binds to the lac operon receptor and the bacteria produce enzymes that allow the use of lactose as an energy source. After depletion of lactose resources, lactose is also released from binding to the repressor. The free (active) repressor binds to the operon and transcription of the lac operon genes is repressed. b) Repressive operon system The repressive operon system is typical of genes encoding enzymes of anabolic reactions. The regulatory gene in this system produces an inactive repressor, i. that the repressor does not have the ability to bind to the operator. The repressor is activated by binding the repressor. A corepressor is usually a product of a metabolic chain, the synthesis of which is catalyzed by enzymes encoded by operon genes. The repressive operon model is the trp operon, which encodes enzymes for the synthesis of the amino acid tryptophan. The E. coli trp operon comprises 6 structural genes (trpL, trpE, trpD, trpC, trpB, trpA), the products of which catalyze metabolic steps from chorismic acid to tryptophan. If the bacterium has enough tryptophan for protein synthesis, transcription of the trp operon is blocked by binding of the repressor + compressor (tryptophan) complex to the operator. After depletion of tryptophan stocks in the cytoplasm, tryptophan bound to the repressor is released. Thus, the repressor is inactivated, does not bind to the operator, and RNA polymerase initiates transcription of the structural genes of the trp operon. Catabolite repression In a study of the lac operon, Jacob and Monod found that lactose did not induce transcription of the lac operon in the presence of glucose. Glucose prevented the induction of the production of other enzymes involved in the metabolism of other sugars. This phenomenon is called catabolite repression or the glucose effect. In the case of the trp operon, there is a second level of regulation of enzyme synthesis, which is independent of repression or derepression of the operon and which is related to the presence of a nucleotide sequence in the trpL region (determines the leader sequence of the polypeptide). The phenomenon was studied in detail and explained as attenuation, and the section of DNA in trpL that controls this phenomenon was called the attenuator.

ontogenesis of sex in mammals? process of sex determination in males? (94)

The dosage of the X-chromosome doesn't influence sex determination, but only the presence or absent of Y chromosome. Through analysis of human and mouse sex reversal syndromes (XX - males, XY - females) it was proved that a single gene - SRY (Sex determining region on chromosome Y) is responsible for initiation of male development. Actually and more accurately, the gene is termed T SPY (testis specific protein Y-linked), which is found in the SRY region. The SRY located on the short arm of chromosome Y, downstream of pseudo-autosomal region 1 (PAR1). In these sex reversal syndromes, which occur rarely, a crossing over event may occur in PAR1 - in that case, SRY gene is translocated to X chromosome and the result is 46 XX male. SRY is a transcription factor of HMG (high mobility group) family - a group of chromosomal proteins that are involved in the regulation of DNA dependent processes, such as transcription, replication, recombination and DNA repair mechanism. SRY expressed in the supporting cells of the indifferent gonad and triggers their differentiation into Sertoli cells. The T SPY gene product binds to promoters: The gene for cytochrome P450 aromatase, which converts testosterone to the female hormone estradiol. By this, it inhibits transcription of aromatase. The gene for MIS - Muller inhibitory substance, which is responsible for the differentiation of the testes and the regression of Mullerian duct derivatives - it activates its transcription. SOX9 is another gene found in the SRY region - it is a transcription factor that also belongs to the HMG family. Mutations in this gene cause Campomelic dysplasia - a syndrome in which XY genotype female-sex-reversal is combined with skeletal dysplasia (SOX9 is also necessary for cartilage development). Duplication of SOX9 gene causes XX male sex reversal. Decreasing SOX9 function lead to female development; increasing SOX9 boots male development. Sex determination in men and female: In males - SRY genes on Y chromosome is expressed in somatic cells and upregulates SOX9. SOX9 then determines the fate of somatic cells to become Sertoli cells. Sertoli cells preform many tasks in the male development: Secretory - it secrets AMH (anti-mullerian hormone) during early stages of fetal life; Androgen-binding protein (testosterone binding globulin( which increases testosterone concentration in the seminiferous tubules to stimulate spermatogenesis; Aromatase from Sertoli cells convert testosterone to estradiol to direct spermatogenesis. It induces differentiation of primordial germ cells (PGC) into spermatogonia - process accompanied with formation of testis cords from sertoli cells Sertoli cells responsible for Leydig cell differentiation - which in turn, Leydig cells produce androgens which govern testis descent and testosterone (stimulated by the pituitary hormone luteinizing hormone LH) - which is fully responsible for masculinization of external genitalia. Testosterone supports wolffian duct persistence and drives pubertal development. FGF9 - is a member of the fibroblast growth factor family (FGF) - and encodes for Glia-activating factor protein. It has a vital role in male development. Once activated by SOX9, it is responsible for forming a feedforward positive loop with SOX9 - increasing the levels of both genes. The absence of FGF9 causes an individual, even with X and Y chromosomes, to develop into a female, as it is needed to carry out important masculinizing developmental functions such as the multiplication of Sertoli cells and creation of the testis cords. INSL3 - Insulin-Like 3 is a protein encoded by the INSL3 gene - produced in gonadal tissues in males and females, and in male has a role in regulate growth and differentiation of gubernaculum testis - thus, mediating intra-abdominal testicular descent.

what is the genetic code? what are some major characteristics? what is the wobble hypothesis? (46)

The genetic code is the set of rules by which information encoded within genetic material (DNA or mRNA sequences) is translated into proteins by living cells. Translation is accomplished by the ribosome, which links amino acids in an order specified by mRNA, using transfer RNA (tRNA) molecules to carry amino acids and to read the mRNA three nucleotides at a time. The genetic code is highly similar among all organisms and can be expressed in a simple table with 64 entries. The code defines how sequences of nucleotide triplets, called codons, specify which amino acid will be added next during protein synthesis. With some exceptions, a three-nucleotide codon in a nucleic acid sequence specifies a single amino acid. In 1953 Watson and Crick solved the structure of DNA. They identified the base sequence as the carriers of genetic information, and later they confirmed the genetic code is a triplet nucleotides code. The genetic code can be used as a dictionary - matching the sequence of nucleotide bases to the sequence of amino acids forming a protein. 64 combinations of triplets are found (4 * 4 * 4), made of 3 nucleotides each. 61 of the codons are coding for amino acids - 20 different types of "codeable" amino acids (more amino acids exist that are not coded), whereas 3 codons - UAG UGA UAA - are stop (or termination) codons. When one of these codons is reached - the synthesis of the polypeptide stops. Specificity: the genetic code is specific, because a particular codon always codes for the same amino acid Universality: the genetic code is virtually universal; its specificity has been conserved from very early stages of evolution, with only slight differences in the manner in which the code is translated. Degeneracy: the genetic code is degenerate - redundant - although each codon corresponds to a single amino acid, a given amino acid may have more than one triplet coding for it. For example, Arginine (Arg) is specified by 6 different codons. Only Met and Trp have just one coding triplet. No overlapping, no comma - the genetic code is read from a fixed starting point as a continuous sequence of bases, taken three at a time without any punctuation between codon: AGCUGGCAU - is read as AGC UGG CAU. Reading Frame - A codon is defined by the initial nucleotide from which translation starts and sets the frame for a run of uninterrupted triplets, which is known as an "open reading frame" (ORF). For example, the string GGGAAACCC,1 if read from the first position, contains the codons GGG, AAA, and CCC; and, if read from the second position, it contains the codons GGA and AAC; if read starting from the third position, GAA and ACC. Every sequence can, thus, be read in its 5' → 3' direction in three reading frames, each of which will produce a different amino acid sequence (in the given example, Gly-Lys-Pro, Gly-Asn, or Glu-Thr, respectively). The actual frame from which a protein sequence is translated is defined by a start codon, usually the first AUG codon in the mRNA sequence. Isoaccepting tRNA's - different tRNAs that carry the same amino acid but have different anticodons -synonymous codons. Wobble allows this phenomena: Wobble hypothesis says that some non-standard pairing of bases could occur at the third position of a codon. When codon (mRNA) and anticodon (tRNA) join together, their first and second base strictly follows Watson and Creek rules; but their third base - may cause flexibility in pairing. The importance of this phenomena - it allows some tRNA pairing with more than 1 codon of the mRNA - from 30-50 types of tRNA - 61 sense codons can be matched.

process of eukaryotic DNA replication? what enzymes are invovled? how is it regulated? (43)

The process of eukaryotic DNA replication follows that of prokaryotic synthesis, with some differences. In eukaryotic cell, there are multiple origins of replication (versus single origin of replication in prokaryotes). Eukaryotic single-stranded DNA-binding proteins (in eukaryotes, SSBP is Replication Protein A - RPA) and ATP-dependent DNA helicases (MCM in eukaryotes) have been identified, whose functions are analogous to those of the prokaryotic enzymes. In contrast, RNA Primers, which in prokaryotes removed by the action of DNA Polymerase I 5🡪3 exonucleases, in eukaryotes - the primers removed by RNAse H, and Flap Endonuclease-1 - FEN1. Topoisomerases: as the two strands of the double helix are separated, positive supercoils in the region of DNA ahead of the replication fork as a result of over winding which interfere with further unwinding of the double helix. DNA Topoisomerase I - reversibly cleaves one strand of the double helix - do not require ATP - store the energy from the phosphodiester bond it cleaved - reusing the energy to reseal the strand. Each time a transient nick is created in one strand, the intact DNA strand is passed through the break before it is resealed - relieving supercoils. Topoisomerase II, from the other hand, cleaves both strands and use ATP, and causes a stretch of the DNA double helix to pass through the break. As a result, both negative and positive supercoils can be relieved . Eukaryotic cell cycle The events surrounding eukaryotic DNA replication and cell division in mitosis are coordinated to produce the cell cycle. The period preceding replication is G1 phase (gap 1). DNA replication occurs during the S (synthesis) phase. Following DNA synthesis, there is another period - G2, before M. Cells that have stopped dividing, such as mature T lymphocytes, found in G0 phase. They can be stimulated to reenter to G1 phase to resume division Origin recognition is done by ORC (origin recognition complex), following by binding of polymerase alpha to initiate primase action. Helicase activity is mediated by MCM protein, while SSBP - protein RPA (replication protein). So far: Primase activity achieved by Polymerase alpha, Helicase activity by MCM protein, and SSBP activity by Replication Protein A. Eukaryotic DNA Polymerases - they are designated by greek letters rather than by roman numerals: Polymerase alpha - multisubunit enzyme. One subunit has primase activity, which initiates strand synthesis on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. So in the eukaryotic case - the polymerase alpha accounts both for the Primer set (primase function) as well as the initiation of DNA synthesis. Polymerase epsilon and polymerase delta: once initiation of DNA synthesis starts, polymerase epsilon continues polymerase alpha on the leading strand, whereas polymerase delta elongates the Okazaki fragments of the lagging strand. Both have the 3 🡪 5 exonuclease activity to proofread the newly synthesized DNA. Polymerase betta and polymerase gamma - Polymerase betta is involved in the gap filling in DNA repair. Polymerase gamma is specific polymerase for mitochondrial DNA replication. Summary, steps involves: Identification of ORC and binding of proteins - Origin recognition complex. ATP-hydrolysis-driven unwinding of dsDNA to provide an ssDNA template (MCM - helicase activity) Stress relived by Topoisomerase I (non-ATP required, only one strand is cleaved), and II (ATP, both strands cleaved) Formation of the replication fork and synthesis of RNA Primer by Polymerase alpha Initiation of DNA synthesis (polymerase alpha) and elongation (polymerase epsilon in leading strand, polymerase delta in lagging strand) ligation of the newly synthesized DNA segments Regulation: All eukaryotic cells have gene products that govern the transition from one phase of the cell cycle to another. The cyclins are a family of proteins whose concentration increases and decreases at specific times during the cycle (thus - cyclins). They activate at the appropriate time different CDKs (cyclin-dependent kinases) that phosphorylate substrates essential for progression through the cell cycle. During G1 - cyclin D levels rise and allow progression beyond the restriction point: cyclin D activates CDK4 and CDK6. Cyclin D-CDK4 and CDK6 assemble an active serine-threonine protein kinase complex. One substrate for this kinase is the retinoblastoma protein (Rb) - cell-cycle regulator, because it binds to and inactivates transcription factor E2F necessary for the transcription of certain genes which needed for progression from G1 🡪 S. Phosphorylates Rb results in the release of E2F from Rb 🡪 Transcription occurs. This is the R-point: "restriction point" - after which, cell committed for division. The Late phase of G1, G1/S check-point, regulated by levels of cyclin E and CDK2. Cyclin A and CDK2 - initiation of DNA synthesis in S-phase. Cyclin B and CDK1 is rate-limiting for the G2/M transition. Excessive production of a cyclins, loss of CDK inhibitor, or activation of cyclin/CDK complex at an inappropriate time - might result in abnormal cell division. Bcl oncogene associated with B-cell lymphoma appears to be the cyclin D1 gene. Similarly, oncoproteins produced by DNA viruses target the Rb tumor-suppressor gene.

what is spermatogenesis? where and when does it occur? what are the outcomes? draw a schematic (27)

The process of spermatogenesis takes place in the testis; begins at puberty; there, at puberty, diploid (46 chromosomes) primordial germ cells (PGCs) divide in normal cellular mitotic division to produce still diploid cells called Spermatogonia. Each spermatogonium can undergo repeated rounds of mitosis, giving rise to numerous additional spermatogonia. Alternatively, a spermatogonium can initiate meiosis - and enter into prophase I. Spermatoginum that took the Meiosis pathway is now called Primary spermatocyte - the cell is still diploid, because the homologues chromosomes have not yet separated. Each spermatocyte completes regular meiosis I - in which reduction in the chromosome number occurred. One Primary spermatocyte produces two haploid Secondary spermatocytes that then undergo meiosis II - with each producing two haploid spermatids. Thus, each primary spermatocyte produces a total of four haploid spermatids, which mature in the seminiferous tubules and in the epididymis to develop into sperm. The process in which spermatids turn into mature sperm cell is called "Spermiogenesis". During Spermiogenesis - spermatids transformed into spermatozoa - acrosome is formed (head) - and contains enzymes which are needed in the penetration of the Zona Pellucida coat of the egg. In addition, the nucleus condense, and the spermatids form a structure contains head, neck, middle piece and tail; Most of the cytoplasm being shed. Golgi phase :in the Golgi complex, small PAS positive granules accumulate , merging into an acrosomal lysosome;the flagellum axonema begins to form;the chromosomes of the nucleus condense very tightly, the DNA is fixed in the crystalloid form by basic proteins - protamines (they replace histones ); all gene activity is suppressed. cap phase :the acrosome vesicle grows and caps the front edge of the nucleus. acrosomal phase :the final acrosome containing hydrolytic enzymes is formed (hyaluronidase - during fertilization it destroys the corona radiata, acid phosphatase, protease acrosin and others);the cell rotates to face the edge of the seminal vesicle channel with the anterior pole;the activity of the microtubule cuff stretches the core;the distal of the centrioles grows and forms the basal body of the flagellum (the second, proximal, after fertilization will form the dividing spindle in the egg);the mitochondria move to the proximal part of the flagellum, which they thicken to form the middle segment of the sperm; maturation phase :the remaining cytoplasm is discarded and phagocytosed by Sertoli cells;residual bodies remain from the bridges that connected the individual cells formed from one spermatogonia during development. Spermatogenesis is regulated by Luteinizing hormone (LH) produced by the pituitary gland; LH binds to receptors on Leydig cells (which are the interstitial cells found between seminiferous tubules) and stimulates the production of testosterone. Testosterone binds to Sertoli cells 🡪 promote spermatogenesis. FSH (follicular stimulating hormone) is also essential, because its binding to Sertoli cells stimulates testicular fluid production. In humans, the time required for spermatogonium to develop into a mature spermatozoa (sperm) is approximately 64 days and occur during all the male lifespan.

what is linkage? what are the requirements for linkage analysis? what are the types of markers that can be used? what is indirect DNA diagnostics? (16)

based on genetic distances that are measured in cM genetic distance of 1 cM is the distance b/t genes that show 1% recombination - that is in 1% of meioses the genes will not be co-inherited Mendel's third law- the principle of independent assortment - states that allele found on different gene pairs assort to gametes independently on one another. This is true for genes found on different chromosomes, but it is not always true for genes that are located close to each other on the same chromosome; two loci positioned close to each other on the same chromosome will tend to be inherited together and are said to be linked. Linkage analysis has proved to be extremely useful for mapping genes; it is based on studying the segregation of the disease with polymorphic markers from each chromosome - if a certain marker with known location found to segregate often with a disease - means, they are probably linked (=have low recombination frequency) 🡪 we can identify the location of the sequence account for the disease. Even though the whole human genome has been sequenced, the function of many genetic sequences remains unknown. For this reason, linkage analysis still plays an important role in studying the DNA, because it connects between the location and function of the genes. It is used mostly among families in which the character of interest is segregating. Genetic linkage analysis is one of the DNA indirect diagnosis method (direct analysis 🡪 essential to know the exact sequence of DNA segment and its loci on the chromosomes and by using probes we can discover if a person has the disease or not; indirect 🡪 linkage studies, we use known markers genes to find out whether a person has inherited particular gene region of interest). Genetic linkage analysis is used when we are not sure which gene is involved or if the disease is polygenic. requirements: The tested family should have the segregating desired trait as well as certain closely related markers that we'll be able to link the trait to at least one of them. The marker should show Mendelian inheritance and be highly polymorphic - reduces the chances for a person to be homozygous for the marker (to get same segments from both parents). If the person is homozygous for the marker, we won't be able to tell from which parent the offspring got the marker (=we can't link the marker to the disease). markers: Mendelian trait - a trait which must have a phenotype which is related for the genotype - such as the linkage between ABO blood group and Nail patella syndrome. Molecular markers - better options as markers - abundant in the genome and highly polymorphic (thus, highly specific). These markers may be SNP's, RFLP, and VNTRs (variable number tandem repeats). In order to localize the place of our interest, we should use pedigree, family tree, in which all members of family suspected to have genetic disease have to be examined. The affected family members can have some specific part of DNA in common - this part is usually transmitted throughout generations, whereas relatives which are healthy - won't have this DNA segment. In this case, we can attend that the part of DNA is connected with the involved gene. Segments of the analysis are called marker loci - usually DNA polymorphisms regions used as markers - it is typical for each person and can help us to recognize different genomes. If an offspring inherited specific segment which contained this polymorphism from one of his parents, we can deduce that linked genes - that are linked to the marker loci - inherited as well together with the marker.

translation in prokaryotes? enzymes? synthesis of aminoacyl-tRNA characteristics of ribosomes? steps? (88)

mRNA is translated from its 5'-end to its 3'-end (head to tail) - producing a protein synthesized from its amino N-terminal end to its carboxyl C-terminal end. Prokaryotic mRNA often have several coding regions (polycistronic), each coding region has its own initiation and termination codon. Process of translation is divided into 3 separate steps - initiation, elongation and termination - in general, eukaryotic and prokaryotic protein synthesis resembles in most aspects, with individual differences - one important difference is that translation and transcription are temporally linked in prokaryotes - translation starts before transcription is completed because the lack of a nuclear membrane in prokaryotes. Initiation - assembly of the components of the translation system - initiation complex - two ribosomal subunit, mRNA to be translated, aminoacyl-tRNA specified by the first codon in the message, GTP (energy for the process), and initiation factors - in prokaryotes only 3 are known, whereas in eukaryotes are many. The prokaryotic small subunit of ribosome (30S) recognizes the nucleotide sequence - AUG - that initiates translation by "Shine-Dalgarno" (SD) sequence - which is purine-rich sequence of nucleotide bases, located 6-10 bases upstream of the AUG (near the 5' end). There is no mRNA modification in prokaryotes (poly A, Cap, splicing) - only in tRNA and rRNA. In eukaryotic translation, there is no SD sequence in the mRNA, and the small ribosomal subunit binds close to the 5'-cap until it encounters the AUG. the initiating AUG is recognized by a special initiator tRNA which enters the P site on the small subunit. The initiator t-RNA is the only tRNA that goes directly to the P-site. The large ribosomal subunit (50S) then joins the complex, and a functional ribosome is formed with the charged initiating tRNA in the P-site (A-site is empty). the first amino acid of most bacteria is N-formylmethionine bound to a specific fMet tRNA . Usually, 1-3 N-terminal amino acids are cleaved post-translationally. N-formylmethionyl-tRNA fMet binds to the free 30S subunit of the ribosome mRNA is bound by the interaction of the 3 'end of a 16S rRNA with a Shine-Delgarno sequence near the 5' end (RBS - riobosome-binding site), usually 8 nucleotides from the AUG initiation codon fMet-tRNA interacts with anticodon with initiation codon AUG (sometimes GUG) The 50S subunit of the ribosome binds so that the fMet-tRNA is at the P site of the ribosome translation initiation requires initiation factors IF1, IF2 and IF3 and consumes energy in the form of GTP Elongation - addition of amino acids to the carboxyl end of the growing strand. The ribosome moves from the 5' to the 3' end of the mRNA that is being translated. Next stage is the delivery of the tRNA whose codon appears next on the mRNA template in the ribosomal A-site. The formation of the peptide bond is catalyzed by peptidyltransferase. After the peptide bond has been formed, what was attached to the tRNA at the P-site is now linked to the amino acid on the tRNA at the A-site. The ribosome advances 3 nucleotides toward the 3' end of the mRNA - translocation, movements of the uncharged tRNA from the P to the E site for release, and movement of the peptidyl tRNA from the A to the P site - requires GTP hydrolysis. Termination - when one of the 3 termination codons moves into the A-site: UAA, UAG, UGA, resulting in hydrolysis of the bond linking the peptide to the tRNA at the P site - release the new protein from the ribosome. Translation termination in a prokaryotic cell proceeds as follows: one of the three (usually) termination codons - UAA, UAG or UGA - gets to the A instead of the ribosome is recognized by one of three termination factors (RF-1, RF-2 or RF-3) the peptide is hydrolytically released by peptidyltransferase activity dissociation of ribosome subunits and translation proteins occurs. IF-3 remains bound to the 30S subunit, thus preventing reassociation with the 50S subunit t-RNA The tRNA molecule brings an amino acid residue to the ribosome. The following is typical for bacterial tRNA: about 60 types of tRNA (compared to 100-110 in a mammalian cell) length 73-93 nucleotides four - leaf clover secondary structure, L - shaped tertiary structure acceptor arm terminated by a CCA triplet dihydrouridine arm (D or DHU), pseudouridine arm (T or TΨC), variable arm anticodon arm Activation of tRNA by binding of an amino acid residue occurs via the enzyme aminoacyl-tRNA synthetase and consumes ATP: amino acid + ATP ↔ aminoacyl-AMP + pyrophosphate aminoacyl-AMP + tRNA ↔ aminoacyl-tRNA + AMP The reaction is thermodynamically driven by the decomposition of pyrophosphate Prokaryotic ribosome The bacterial ribosome, like the eukaryotic ribosome, consists of two subunits: - 30S subunit 16S rRNA (with 3 'end complementary to Shine-Delgarno sequence) 21 proteins - 50S subunit 5S rRNA, 23S rRNA (with peptidiyltransferase activity - catalyzes elongation of the peptide chain) 31 proteins

equation for selection against recessive homozygotes? (102)

measures the change in allele frequency as a factor of time in generations application can have paradoxical results ie if q =1 for a certain allele like CF, the entire population would have all had CF

structure and types of eukaryotic chromosomes? how are chromosomes classified? what is the centromeric index? descriptions of morphology? (30)

At the submicroscopic level, chromosomes consist of an extremely elaborate complex, made up of supercoils of DNA. The morphology of chromosome is best seen during cell division, when the chromosomes are maximally contracted and the constituent genes can no longer be transcribed (heterochromatin form). At this time, each chromosome can be seen to consist of two identical strands known as chromatids, or sister chromatids, which are the result of DNA replication having taken place during the S (synthesis) phase of the cell cycle. These sister chromatids can be seen to be joined at a primary constriction known as the centromere. Centromeres consist of several hundred kilobases of repetitive DNA and are responsible for the movement of chromosomes during cell division. Each centromere divides the chromosome into short and long arms, designated p (=petite) and q ('g' - grande). The tip of each chromosome arm is known as the telomere. Telomeres play a crucial role in sealing the ends of chromosomes and maintaining their structural integrity. Telomeres have been highly conserved throughout evolution and in humans they consist of many tandem repeats of a TTAGGG sequence. Gene mapping, describes the methods used to identify the locus of a gene and the distances between genes. The essence of all genome mapping is to place a collection of molecular markers onto their respective positions on the genome. Morphologically, chromosomes are classified according to the position of the centromere. If this is located centrally, the chromosome is metacentric, if terminal - it is acrocentric, and if the centromere is in a intermediate position, the chromosome is sub-metacentric. Telocentric - centromere located at the end of the chromosome, chromosome has only Q arm (not found in human). Acrocentric chromosomes sometimes have stalk-like appendages called satellites - that form the nucleolus of the resting interphase cell and contain multiple repeat copies of the genes for ribosomal RNA. A satellite chromosome or SAT chromosome has a chromosome segment that is separated from the main body of the chromosome by such a secondary constriction. holocentric - Centromere-forming units are scattered along the entire length of the chromosome (this type is not present in humans, it rarely occurs, for example, in Caenorhabditis elegans ). Individual chromosomes differ not only in the position of the centromere, but also in their overall length. Based on the three parameters of length, position of the centromere, and the presence or absence of satellites, cytogenetics were able to identify most individual chromosomes, or at least subdivide them into groups labeled A to G on the basis of overall morphology: A 1-3, B 4-5, C 6-12, D 13-15, E 16-18, F 19-20, G 21-22; X Y. In humans, the normal cell nucleus contains 46 chromosomes, made up of 22 pairs of autosomes and a single pair of sex chromosomes - XX for female, XY for male. One member of each of these pairs is derived from each parent. The Y chromosome is much smaller than the X and carries only a few genes of functional importance, most notably the testis-determining factor, known as SRY. Other genes on the Y chromosome are known to be important in maintaining spermatogenesis. In the female, each ovum carries an X chromosome, whereas in the male each sperm carries either an X or a Y chromosome. There is a roughly equal chance of either an X-bearing sperm or a Y-bearing sperm fertilizing an ovum. Somatic cells are said to have a diploid complement of 46 chromosomes, whereas gametes (ova and sperm) have a haploid complement of 23 chromosomes. Members of a pair of chromosomes are known as homologs. The centromeric index is a value used in cytogenetics to describe chromosomes . It is calculated according to the position of the centromere as the ratio of the length of the short arm p to the length of the whole chromosome p + q . Based on this value, we divide chromosomes into metacentric, submetacentric, acrocentric, etc. p / (p + q) = 0.5-0.39 - metacentric 0.38-0.26 - submetacentric 0.25-0.13 - subtelocentric 0.12-0 - telocentric

what is domain variability of antibodies? how it is regulated? (76)

Domain Variability the immune system is capable of making antibodies against virtually any antigen that might be encountered. Each person is capable of making about 1015 different antibody molecules. Antibodies are proteins, so the amino acid sequences must be encoded in the human genome. However, there are fewer than 105 genes in the genome, so how can this huge diversity of antibodies can be encoded? : The antibody genes are composed of segments. There are a number of copies of each type of segment, each differing slightly from the others. In the maturation of a lymphocyte, the segments are joined to create an immunoglobulin gene. There are multiple copies of each segment, so there are many possible combinations of the segments- a limited number of segments can therefore encode a huge diversity of antibodies. e.g. In the light chains, Kappa and Lambda chains are encoded by separate genes on different chromosomes - each gene is composed of three types of segments: V (variable - codes the variable region of the light chains), J (joining - short set of nucleotides that join the V and C segments together), C (constant - codes the constant region). Kappa gene - there are 30-35 different functional V gene segments, 5 different J genes, and a single C gene segment. Initially, an immature lymphocyte inherits all of the V gene segments and all of the J gene segments present in the germ line. In the maturation of the lymphocyte, somatic recombination within a single chromosome moves one of the V genes to a position next to one of the J gene segments. After somatic recombination has taken place, the combined V-J-C gene is transcribed and processed. The mature mRNA that results contains only sequences for a single V, J, and C segment; this mRNA is translated into a functional light chain. In this way, each mature human B cell produces a unique type of kappa light chain, and different B cells produce slightly different kappa chains, depending on the combinations. The gene that encodes the lambda light chain differs from the kappa gene in the number of copies of the different segments: 29-33 different functional V gene segments, 4-5 J and C gene segments. Somatic recombination takes place among the segments in the same way as in the kappa gene generating many possible combinations of lambda light chains. The gene that encodes the Ig heavy chain also has V J and C segments, as well as D segment (D-Diversity) - account for similar diversity. In addition to somatic recombination, other mechanisms add to antibody diversity: Each type of light chain can combine with each type of heavy chain The recombination process that joins V, J, D, and C gene segments is imprecise, and a few random nucleotides are frequently lost or gained at the junctions of the recombining segments - greatly enhances variation among antibodies. High rate of mutation - somatic hyper mutation is also occurs in Ig genes. T cell receptors are structurally similar to immunoglobulins and are located on the cell surface of the T-cells - they composed of one alfa and one beta chain held together by disulfide bonds - one end of each chain is embedded in the cell membrane and the other projects away from the cell and binds antigen. Like the genes that encode antibodies, the genes for the T-cell receptors chain consists of segments that undergo somatic recombination, generating an enormous diversity of antigen-binding site.

what are the ethical and legal aspects of medical genetics? fundamental principles? what are some examples? (124)

Ethics is the branch of knowledge that deals with moral principles, which in turn relate to principle of right, wrong, justice and standards of behavior. Traditionally, the reference points are based on a synthesis of the views of well-informed, respected, thinking members of society, and in this way, a code of practice evolves that is seen as reasonable and acceptable by the majority of population. In ethics, there is no absolute right or wrong, and individual views will vary widely. In complex scenarios, in which there may competing and conflicting claims to an ethical principle, practical decisions and actions often have to be based on balancing between many factors. Human genetics poses particular challenges in ethical consideration, because decisions in this field affects not only on an individual, but also on close relatives and the extended family. Just as patients need to balance risks when making a decision about a treatment option, clinician or counselor may need to balance the fundamental ethical principles one against the other. Fundamental Ethical principles: Autonomy - incorporating respect for the individual, his privacy, and the importance of confidential information together with informed consent . Beneficence- the principle of seeking to do good and therefore acting in the best interests of the patient. Non-maleficence - the principle of seeking, in overall, not to harm the patient - not to leave him in a worse condition than before the treatment Justice - incorporating fairness for the patient in the context of resources available, equity of access and opportunity to get proper treatment. A particular difficulty in medical genetics can be the principle of autonomy - given that we all share our genes with our biological relatives. Individual autonomy needs sometimes to be weighed against the principle of doing good, and doing no harm to close family members. Autonomy - It is the patient who should be empowered and in charge when it comes to decisions that have to be made - and he able to do so only if he gets proper, comprehensive, high quality information about all factors need to be considered. Important to stress that genetic consultation is voluntary - the patient's decision! It has the right to refuse getting information ("right to know, right not to know") Informed choice - the patient is entitlted to full information about all options available in a given situation, including the option and the consequence of not participating. No stress and pushing of the counselor regarding specific course of action. The information given to a patient should include details of the risks, limitations, implications and possible outcomes of each procedure. In the current climate, in which patients expect to get full information and the existence of 'doctor-patient' contract, some form of signed consent is being obtained for every action that exposes the patient - access to medical records, clinical photography, genetic testing and storage of DNA. The signed consent is called "positive reverse"; in a case patient refuses the treatment - "negative reverse" Legal aspect: there is no legal requirement to obtain signed consent for taking a blood test from which DNA is extracted and stored - DNA doesn't constitute human tissue in the same way as biopsy samples or cellular material, for which formal consent is required. However, consent is formally obtained where cellular material is used to obtain genetic information for another person. In clinical genetics, patients for clinical examination and genetic testing may be children or individuals which have some difficulties - who may lack capacity to grant informed consent. Person beyond the age of 16 has the right to self-decisions. Confidentiality - A patient has a right to complete confidentiality. There are many cases that patient, or a couple, would wish to keep private. Stigmatization and guilt may accompany the concept of hereditary illness. Traditionally, confidentiality may be "taken" only under extreme circumstances, such as when it is seems that that an individual's behavior could convey a high risk of harm to self or to others. This field may be encountered in many decisions, but the norm is according the principle that the patient must consent the release and sharing of its own information - with other people or even with other genetic and medical services. Universality - even though the traditional medical ethical thinking is to set the autonomy and the privacy of the individual as the most important value in medical genetics, growing appreciation of the ethical challenges posed by genetics has led to calls for a new pragmatism in bioethics. It is built on the concept that the human genome is common to all humankind, and can and should be considered a shared resource - because we have a shared identity at this level. The information gained from individual, family's or even population's genome may be beneficial far beyond the immediate and personal relevance - thus, it is need to be considered how best the genetic information is exchanged for the medical benefits. These ideas prompt the individual to consider his or her responsibility toward others, as well as to society, both in the present and in the future. Examples of ethical dilemmas and legal aspect in the genetic clinic: In Czech republic, from a legal perspective, the law sees the fetus as part of the mother's body, and thus, any decisions regarding diagnostic and preventive procedures during pregnancy is according the mother will. Abortion of women - may be her choice till the 12th of pregnancy. After the 12th week, abortion only in case the pregnancy threatened a woman's life \ there are proved severe difficulties for the fetus or in more severe cases that the fetus is not capable of life. If there are obvious genetic reasons for abortion (such as anencephaly, or major physical or mental handicap) - artificial abortion is possible till the 24th week of pregnancy. "Serious" condition is not defined in legislation, thus it can inevitably lead to controversy opinions. The most difficult problems in prenatal diagnosis are those involving autonomy and individual choice - particularly regarding to disease severity and who should make the decision that termination is justified. For example - parents whose first child, a boy, has autism, are expecting another baby. They have read that autism is more common in boys than girls, so they request sexing of the fetus with a view to terminating a male fetus but continuing if the sex is female. Overall, however, the risk of having another child with autism is only about %5 - such a request may be challenge for the clinician. There is wide consent that sex selection is not a legitimate reason to terminate pregnancy. Weird case - parents with inherited disease, such as achondroplasia (dwarfism) - that request to terminate pregnancy in case that there child is HEALTHY, means, doesn't have achondroplasia. Thus, the issue of autonomy and individual choice is being considered in this aspect as well.

what is selection? what are the types of selection? what is relative fitness? what is sexual selection? balancing selection? heterozygote advantage? frequency-dependent selection? (102)

Evolution by natural selection is a blend of chance and "sorting": chance in the creation of new genetic variations (as in mutation) and sorting as natural selection favors some alleles over others. Because of this favoring process, the outcome of natural selection is not random. Instead, natural selection consistently increases the frequencies of alleles that provide reproductive advantage, thus leading to adaptive evolution. The concept of natural selection is based on differential success in survival and reproduction: individuals in a population exhibit variations in their heritable traits, and those with traits that are better suited to their environment tend to produce more offspring than those with traits that are not as well suited. In genetic terms, selection results in alleles being passed to the next generation in proportions that differ from those in the present generation. For example - the fruit fly D. melanogaster has an allele that confers resistance to several insecticides, such as DDT. This allele has a frequency of 0% in laboratory strains of the fly established from flies collected in the wild in the 1930s, prior to DDT use. However, in strains established from flies collected after 1960 after 20+ years of DDT use, the allele frequency is 37%. The rise in frequency of this allele most likely occurred because DDT is a powerful poison that is a strong selective force in exposed fly populations. An allele that confers resistance to an insecticide will increase in frequency in a population exposed to that insecticide. By consistently favoring some alleles over others, natural selection can cause adaptive evolution - evolution that results in a better match between organisms and their environment. types Natural selection can alter the frequency distribution of heritable traits in three ways, depending on which phenotypes in a population are favored. Directional selection is a mode of natural selection in which an extreme phenotype is favored over other phenotypes, causing the allele frequency to shift over time in the direction of that phenotype. Under directional selection, the advantageous allele increases as a consequence of differences in survival and reproduction among different phenotypes. The increases are independent of the dominance of the allele, and even if the allele is recessive, it will eventually become fixed. i.e. Individuals that display a more extreme form of a trait have greater fitness than individuals with an average form of the trait. For example: an increase in the relative abundance of large seeds over small seeds led to an increase in beak depth in a population of Galapagos finches. Disruptive selection occurs when conditions favor individuals at both extremes of a phenotypic range over individuals with intermediate phenotypes. One example is a population of black-bellied seedcracker finches in Cameroon whose members display two distinctly different beak sizes. Small-billed birds feed mainly on soft seeds, whereas large-billed birds specialize in cracking hard seeds. It appears that birds with intermediate-sized bills are relatively inefficient at cracking both types of seeds and thus have lower relative fitness. Stabilizing Selection - acts against both extreme phenotypes and favor intermediate variants. This mode of selection reduces variation and tends to maintain the status quo for a particular phenotypic character. For example, the birth weights of most human babies lie in the range of 3-4kg. Babies who are either much smaller or much larger suffer higher rates of mortality. Regardless of the mode of selection, however, the basic mechanism remains the same - selection favors individuals whose heritable phenotypic traits provide higher reproductive success than do the traits of other individuals. Sexual Selection: It is a form of natural selection in which individuals with certain inherited characteristics are more likely than other individuals to obtain mates. Sexual selection can result in sexual dimorphism, a difference in secondary sexual characteristics between males and females of the same species. These distinctions include differences in size, color, ornamentation and behavior. In intrasexual selection, meaning selection within the same sex, individuals of one sex compete directly for mates of the opposite sex. In many species, intrasexual selection occurs among males. In intersexual selection, also called mate choice, individuals of one sex (usually the females) are choosy in selecting their mates from the other sex. In many cases, the female's choice depends on the showiness of the male's appearance or behavior. Male showiness may not seem adaptive in any other way and may in fact pose some risk. How do female preferences for certain male characteristics evolve in the first place? Female prefer male traits that are correlated with "good genes" as an indicative of a male's total genetic quality. If the trait preferred by females is indicative of a male's overall genetic quality, both the male trait and female preference for it should increase in frequency. Balancing Selection In diploid organisms, many unfavorable recessive alleles persist because they are hidden from selection when in heterozygous individuals. Selection itself may preserve variation at some loci, thus maintaining two or more forms in a population. Balancing selection refers to selective processes by which different alleles are kept in the gene pool of a population at frequencies above that of gene mutation. This usually happens when the heterozygotes for the alleles under consideration have a higher adaptive value than the homozygote. In this way genetic polymorphism is conserved. Heterozygote advantage: If individuals who are heterozygous (term of genotype, not phenotype) at a particular locus have greater fitness than do both kinds of homozygotes, they exhibit heterozygote advantage. In such a case, natural selection tends to maintain two or more alleles at that locus. Thus, whether heterozygote advantage represents stabilizing or directional selection depends on the relationship between the genotype and the phenotype. For example, if the phenotype of a heterozygote is intermediate to the phenotypes of both homozygotes, heterozygote advantage is a form of stabilizing selection (prefers intermediate phenotype). Frequency-dependent Selection: In frequency-dependent selection, the fitness of a phenotype depends on how common it is in the population. For example, the scale-eating fish of Lake Tanganyika in Africa. These fish attack other fish from behind. Some are "left-mouthed" and some are "right-mouthed". Simple Mendelian inheritance determines these phenotypes, with the right-mouthed allele being dominant to the left-mouthed allele. Because their mouth twists to the left, left-mouthed fish always attack their preys from the right. Similarly, right-mouthed fish always attack from the left. Prey species guard against attack from whatever phenotype of scale-eating fish is most common in the lake. Thus, from year to year, selection favors whichever mouth phenotypes is least common. As a result, the frequency of left and right mouthed fish oscillates over time, and balancing selection (due to frequency dependence) keeps the frequency of each phenotype close to 50%.

Mendel's experimental design? what are the three fundamental principles of hereditary that he observed? (1)

Examined single traits: allowed pea plants to self-fertilize for generations. Used crosses between varieties w/alternative forms: Applied numerical analysis to data for several generations. Law of uniformity (law of dominance) law of segregation law of independent assortment

what is gel electrophoresis? (54)

Gel electrophoresis - DNA fragments can be separated, and their sizes can be determined with use of gel electrophoresis. The fragments can be viewed by using a dye that is specific for nucleic acids or by labeling the fragments with a radioactive or chemical tag.

what is gene expression? how can it be regulated in eukaryotes? what are the levels at which it can be regulated? what are trans-acting and cis-acting factors? (51)

Gene expression refers to the multistep process that ultimately results in the production of a functional gene product, either ribonucleic acid (RNA) or protein. The first step in gene expression, is the use of deoxyribonucleic acid (DNA) for the synthesis of RNA (transcription) - is the primary site of regulation in both eu and prokaryotes. Regulations of transcription, the initial step in all gene expression, is controlled by regulatory sequences of DNA, and usually embedded in the noncoding regions of the genome. The interaction between these DNA segments and regulatory molecules, such as transcription factors, can engage or repress the transcriptional machinery, influencing the kinds and amount of products that are produced. Most of the specialized cells in a multicellular organism are capable of altering their patterns of gene expression in response to extracellular signals. If a liver cell is exposed to a glucocorticoid hormone, which released during periods of starvation or intense exercise, the liver produce glucose from amino acids and glycogen (gluconeogenesis). The set of proteins whose production is induced includes enzymes such as amino-transferase, which helps to catalyze amino acid into TCA cycle intermediates, and finally to glucose. Other cell types respond to glucocorticoids differently or not at all, demonstrate that different cell types often respond differently to the same extracellular signal. A cell can control the proteins it makes by: Controlling when and how often a given gene is transcribed - Transcriptional control Controlling the splicing and processing of RNA transcripts - RNA processing control Selecting which completed mRNAs are exported from the nucleus to the cytosol and determining where in the cytosol they are localized - RNA transport and localization control Selecting which mRNAs in the cytoplasm are translated by ribosomes - Translational control Selectively destabilizing certain mRNA molecules in the cytoplasm - mRNA degradation control Selectively activating, inactivating, degrading, or locating specific protein molecules after have been made - protein activity control. The most important point of control for most genes is the initiation of RNA transcription gene expression is regulated at several levels: at the chromosome level change of structure position effect = gene transfer from euchromatin to heterochromatin = change in function amplification = increase in the number of transcribed genes translocation = transfer of gene structures into the regulatory sphere of a strong promoter promoter insertion = increases gene expression also DNA rearrangements (deletions, insertions); influencing the cut regulation of transcription RNA polymerase - I in the nucleolus - transcribes the rRNA precursor (15 proteins) RNA polymerase - IIt ranscribes all pre- mRNAs (14 proteins) RNA polymerase - III transcribes all pre-tRNAs (17 proteins) regulation of cis-element transcription enhancers (amps) / silencers (attenuators) By creating a loop of DNA, the enhancer approaches the promoter to which specific activator proteins bind this binding prevents the RNA polymerase from being attached to the promoter only after a change in the conformation of the complex by the action of signaling molecules or binding of coactivators is the promoter released for the polymerase and transcription is started Transcription factors Zinc-fingers = proteins whose chains form short loops where the Zn 2+ atom is bound to 2 cysteines and 2 histamines; regulation of 5S rRNA transcription Leucine zippers = connect 2 α-helices by bonds between leucine moleculesHLH (helix - loop - helix)HTH (helix - turn - helix) Homeodomains regulation of transcription mediators = signaling molecules coordination activity:paracrine autocrine endocrine (hormones) regulatory gene cascade primarily regulated genes can subsequently regulate the expression of other genes through their own productthe whole control cascade can have a number of intermediate stagesat the end of the regulatory cascade, such factors (products) may arise which, in turn, suppress the activity of primarily regulated genes regulation by pre-mRNA modification after pre-mRNA transcription and 5´ cap binding during transcriptionpre-mRNA modification:by splitting the 3´ end and connecting the poly (A)intron splicing and exon junctionsOther possibilities of influencing alternative montage mRNA editing (change of pre-mRNA structure)cytoplasmic mRNA stability regulation at the translational level e.g., mRNA translation for ferritin production targeting genetic determination of protein targeting trans-acting factors Specific transcription factors are trans-acting DNA-binding proteins that function as transcriptional activators. They have at least two binding domains - the DNA-binding domain, and the transcription-activation domain. The DNA-binding domain contains specific structural parts, such as Zinc fingers, that facilitate the binding to consensus sequences in DNA. The transcription-activation domain recruits other proteins, such as the general transcription factors (TFIID, TFIIH, TFIIB, TFIIF) and coactivators. These facilitate formation of the transcription initiation complex (RNA Pol II and general transcription factors) at the promoter (TATA), and thus, activate transcription. Regulation is achieved by the formation of a multiprotein complex bound to DNA. *DNA-binding proteins can also inhibit transcription. Same response element can be found in different chromosome and fit to the same transcriptional activator (TF) which elicit coordinate transcription of different genes (e.g. thyroid hormone elicit many different metabolic changes in the cells that it acts in - gluconeogenesis, glycogenolysis, glycolysis, protein synthesis, synthesis of mitochondrial protein etc) Cis-acting regulatory elements The need to coordinately regulate a group of genes to cause a particular response is of key importance in multicellular organisms - a protein binds to a regulatory consensus element on each of the genes in the group, and coordinately affects the expression of those genes. Regulatory signals mediated by intracellular receptors - Includes receptors of steroid hormones (glucocorticoids, mineralocorticoids, androgens, vitamin A and D), thyroid hormones and more, all directly influence gene expression by functioning as specific transcription factors. These receptors contain a ligand-binding domain, DNA-binding domain and an activation domain. E.g. - steroid hormones such as cortisol (glucocorticoid) bind to soluble, intracellular receptors at the ligand-binding domain. Binding causes a conformational change in the receptor that activates it, causes it to enter the nucleus and binds via its zinc-finger part to nuclear DNA at a cis-acting regulatory element - which is called glucocorticoid-response element (GRE\HRE - hormone responsive element). The binding allows recruitment of co-activators to the activation domain of the receptor and results in increased expression of cortisol-responsive genes. Binding of the receptor-hormone complex to the GRE allows coordinate expression of a group of target genes, even when these genes are located on different chromosomes. The GRE can be located upstream or downstream of the genes it regulates and is able to function at great distances from those genes. In this example, the GRE function as a true enhancer. Regulatory signals can be mediated by cell-surface receptors, such as those for insulin and glucagon. Glucagon, for example, is a peptide hormone that binds to plasma membrane receptor on glucagon-responsive cells. This extracellular signal is then transduced to intracellular cAMP, which can affect protein expression and activity through a mediator protein - "cAMP response element-binding" or CREB protein, which is activated. Active CREB protein binds to a cis-acting regulatory element - cAMP response element (CRE), resulting in transcription of target genes with CREs in their promoters.

gene therapy in cancer? what are the types? what are the main methods? (117)

Gene therapy is the introduction of normal genes into cells in place of missing or defective ones, in order to correct genetic disorders. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve your body's ability to fight disease. If the patient has a disease that is caused by an error in a gene, gene therapy is a treatment that target to perform gene repair; it is believed that in the future this method will be possible not only to treat the diseases, but also to prevent them. Gene therapy as a treatment method is based on knowledge of how genes influence certain disease. Today it is obvious that each human disease has some connection with human genes which were obtained by inheritance from the parents. Slight differences in the genes of every human shape the human; unfortunately, some of these differences lead to the development of diseases, which are then transmitted from generation to generation. The advantage of gene therapy is to treat human disease in their "roots" - correct corrupted genes. Types of gene therapy: Somatic cell gene therapy - SCGT - changes/fixes genes in just one person - the targeted cells are the only ones affected and the changes are not passed on to that person's offspring. Germ line gene therapy - GGT - germ cells (sperm or eggs) are modified by the introduction of functional genes into their genomes. The changes that are made, adding or subtracting genes from the person's DNA, will be passed on to their offspring. This type of gene therapy raises a lot of ethical questions because it impacts the inheritance patterns of humans. Gene therapy may be either direct or indirect: Direct - correcting errors in DNA sequence which is responsible for the malignant transformation. E.g. removal of mutations in proto-oncogene or introduction of missing tumor-suppressor genes. Indirect - introduction of new genetic information into cells (tumor cells or other types) that leads to destruction of the tumor cells. e.g. introduction of DNA sequences that encode for proteins with different functions: stimulate anti-tumor immune response, altering tumor angiogenesis, activate inactive molecule such as antimetabolite to its cytotoxic activity in order to fight the tumor. The indirect method may be ethically problematic - the patient's must decide if he wants it. The safety of the procedure must be considered - favorable risk-to-benefit ratio. Gene transfer strategies Gene therapy can be accomplished "ex-vivo" or "in-vivo." Ex-vivo means the target cells are removed from the patient's body, the genes are fixed or replaced, and the cells are put back into the person. In-vivo means the genes are "injected", usually using a virus as a way to deliver the fixed or missing genes. There are 4 main methods used for gene therapy of cancer: Immunotherapy - its main task is to boost an immune response which will recognize and destroy the cancer cells. Currently gene therapy is being used to create recombinant cancer vaccines by the following way: The cancer cells are harvested from the patient and grown in vitro. These cells are then engineered to be more recognizable to the immune system by the addition of genes that produce either immune stimulating molecules (such as cytokines) or highly antigenic protein genes. These altered cells grown in vitro are killed, and the cellular contents are incorporated into a vaccine. Another method of immunotherapy is also by delivering immune-stimulatory genes (mainly cytokines) to the tumor in vivo. Once found in the cancer cells, these genes will produce proteins that unmask the cells from the immune system and encourage the development of anti-tumor antibodies. Oncolytic virus therapy - a new type of immunotherapy that uses genetically modified viruses to kill cancer cells. Virus is injected by doctor into the cancer cells, makes a copy of itself resulting in cell's bursting and dying. As the cells die, they release antigens which triggers the patient's immune system to target all the cancer cells in the body that have those same antigens. Gene transfer to the tumor cells - cancer growth can be arrested or reversed by treatment with gene transfer vectors that carry a single growth inhibitory or pro-apoptotic gene, or a gene that can recruit immune responses against the tumor. Many of these gene transfer vectors are modified viruses that retain the capability of the virus for efficient gene delivery. Loss of tumor-suppressor gene (e.g. P53) and over expression of oncogenes (e.g. Ras) are the main cause for cancer. It may be possible to correct an abnormality in mutated-P53 by inserting a copy of the wild-type gene; insertion of the wild type P53 gene into P53-deficient tumor cells has been shown to result in the death of tumor cells. The over expression of an oncogene such as Ras can be blocked at the genetic level by integration of an antisense gene whose transcript binds specifically to the oncogene RNA, disabling its capacity to produce protein and reduces the over-expression.

what is gene therapy? what are the principals? current possibilities? perspectives? (70)

Gene therapy is therapeutic delivery of nucleic acid into a patient's cells as a drug to treat disease - practically speaking - modifying human DNA by gene transfer. Gene therapy has risen due to the advances in molecular biology, leading to the identification of many important human disease genes and their protein products. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve your body's ability to fight disease. Gene therapy holds promise for treating a wide range of diseases, such as cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. The most common application by far will be the introduction of functional copies of the relevant gene into the appropriate target cells of a patient with a loss-of-function mutation Essential requirement of gene therapy for an inherited disorder: Identity of the affected gene, or at least of the biochemical basis of the disorder, must be known. Functional copy of the gene by cDNA clone of the gene or the gene itself must be available Knowledge of the pathophysiological mechanism of the disease must be sufficient to suggest that the gene transfer will correct the pathological process. Loss-of-function mutations require replacement with a functional gene; in disease due to dominant negative allele inactivation of the mutant gene or its product is necessary. Favorable risk-to-benefit ratio Appropriate regulatory components for the transferred gene - in thalassemia, for example, overexpression of the transferred gene would cause a new imbalance of globin chains, whereas low levels of expression would be ineffective. Appropriate target cell, which have a long half-life or good replicative potential, as well as much be accessible for introduction of the gene. Strong evidence of efficiency and safety Regulatory approval of Institutional Review Board The goal of the gene therapy is to improve a patient's health by correction of the mutant phenotype through delivery of normal gene to somatic cell. It must be focused in the somatic cell, without alter the germline of the patient because it would carry a substantial risk of introducing new mutation. Gene transfer strategies Gene therapy can be accomplished "ex-vivo" or "in-vivo." Ex-vivo means the target cells are removed from the patient's body, the genes are fixed or replaced, and the cells are put back into the person. In-vivo means the genes are "injected" somehow, usually using a virus as a way to deliver the fixed or missing genes. An appropriately engineered gene may be transferred into target cells by one of two general strategies: I. Ex vivo (outside the body) introduction of the gene into cells that have been cultured from the patient and then reintroduced after the gene transfer. II. The gene is injected directly in vivo into the tissue or extracellular fluid of interest (from which it is taken up by the target cells) usually by viral vector or plasmid - it is achieved by modifying the coat of a viral vector so that only the designated cells bind the viral particles. Ex Vivo - cells removed from the body 🡪 cultivation and incorporation with the desired gene 🡪 incorporated back into the original tissue The Target cell The ideal target cells are stem cells or progenitor cells with substantial replication potential. Introduction of the gene into stem cells can result in the expression of the transferred gene in a large population of daughter cells. Till now, bone marrow is the only target tissue used stem or progenitor cells. Genetically modified bone marrow stem cells have been used to cure immunodeficiencies, and they could be used for other diseases affecting blood cells. In addition, bone marrow could also be used for diseases that do not involve the blood system - such as PKU - the bone marrow cells will produce the enzymes which will meet the phenylalanine in the circulation. If the target cell cannot divide extensively in culture (and then be reimplanted), or if it doesn't have identifiable stem or progenitor cells, other approaches are needed. For example, use in hepatocytes - which can be briefly maintained in primary culture, transfected with a gene, and then returned to the patient or Endothelial cells - which are especially useful target for gene transfer because they line the walls of blood vessels and the protein product of a gene expressed in them can be released into the circulation to achieve a systemic effect. It takes place ex vivo (outside the whole organism). There are various techniques for introducing a gene into isolated cells: Physical methods allow direct insertion of nucleic acid into target cells (microinjection, microprojectiles or electroporation - introduction of a weak electric current into the cell suspension in the presence of the gene to be incorporated into the cell)efficiency <1% Chemical methods use for incorporation of genes into cells eg calcium phosphate, liposomes (improve passage through the cell membrane) Biological methods use viruses as DNA vectors , no. DNA viruses (papovaviruses, adenoviruses, herpes simplex virus) and retrovirusesrequired sequence transfer efficiency - almost 100%other vectors: plasmids - contain multiplied copies of the selected gene, are injected into the bloodstream or directly into the area of ​​tumor growthmonoclonal antibodiesin vitro proliferating tumor infiltrating lymphocytes (TIL) TILs after iv administration selectively infiltrate postoperative tumor residues from which they were isolated. Ex vivo, for example, the tumor necrosis factor (TNF) gene can be integrated into the TIL genome. The TNF gene (part of the TIL genome) is transcribed and the synthesized protein is secreted directly into the tumor tissue, which is destroyed by a necrotic process. Genetically controlled increase in immunobiological activity. Ex. retrovirus: First, it is genetically modified in vitro. The sequences encoding the viral proteins are removed, leaving only the expression control sequences (LTRs). This is followed by excision of the sequences encoding the production of the product selected for the respective gene therapy. These recombinant retroviruses have the ability to infect cells and incorporate exogenous genes into their genome, but they cannot replicate. An example of gene therapy of this type is the treatment of malignant glioma (a tumor with high mitotic activity) - it does not metastasize and is surrounded by nervous tissue that does not replicate. Ex. Herpes simplex virus (HSV): Its gene for the enzyme thymidine kinase has been integrated into the genome of the target cell and in it it converts an otherwise inactive dosage form (prodrug) into an active substance with a cytostatic effect. In Vivo techniques - doesn't require surgical treatment. Application of modified gene directly into cells of the body by using viral vectors. In order to replicate, viruses introduce their genetic material into the host cell, tricking the host's cellular machinery in order to use the viral proteins. Scientists exploit this by substituting a virus's genetic material with therapeutic DNA. A number of viruses have been used for human gene therapy, including retrovirus, adenovirus, lentivirus and adeno-associated virus. Adenoviruses - has ability to affect both replicating and non-replicating cells and to accommodate large amount of transferred genes. They are coding for proteins without integrating into the host cell genome. The immune system of the patient has a greater tendency to interfere with these viruses and to create reaction. Therefore, patients suffering from the symptoms of colds and runny nose. Retroviruses - their advantage is the complete suppression of viral DNA - the information that transmitted is only modified DNA. Their results are long lasting. The disadvantage is that it operates only on the newly formed daughter cells and has no effect on existing defective cells. Liposomes - fatty particles have the ability to carry the new, therapeutic genes to the target cells. Risks - unwanted immune system reactions (viruses may be seen as intruders), targeting the wrong cells (altered viruses may infect additional cells, not just the targeted cells, harm healthy cells), infection caused by the virus, possibility of causing a tumor (if the new genes get inserted in the wrong spot in the DNA). Non-viral methods present certain advantages over viral methods - such as large scale production and low host immune response. However, non-viral methods initially produced lower levels of gene expression, and thus lower therapeutic efficiency. Types of gene therapy Somatic gene therapy corrects the patient's genes without inheriting the corrected genes to future generations Germline therapy it would change genes already in the germ cells (sperm, egg) and the gene change would be transmissible to future generations. This type of therapy is not yet allowed. Gene therapy strategies Direct gene therapy error correction, ie. changes in the DNA sequence that is responsible for malignant transformation. E.g. removing a mutation in the proto-oncogene or introducing missing tumor suppressor genes not all cells can be repaired because the genotype is multifactorial in nature Indirect Gene Therapy introducing new genetic information into a cell (tumor or other type) that leads to the destruction of tumor cells. Ex. introduction of DNA sequences that encode, for example, the stimulation of an antitumor immune response or alter tumor angiogenesis and / or activate an inactive antimetabolite molecule to its cytotoxic activity strict ethical criteria, strict selection of patients, the safety of the procedure for both the patient and the nursing staff is considered, the effectiveness of the therapy Diseases that may be treated by gene therapy Genetic disorders - hemoglobinopathies, cystic fibrosis, hemophilia A and B - all parameters required for knowledge of the disease are available - their molecular mechanism, biochemical function and pathophysiology. Non genetic disorders - malignant melanoma, ovarian cancer, brain tumors, cardiovascular diseases. At present, gene therapy is still used rather rarely because they are still concerns about the safety in the procedures. It is used manipulation of human DNA - which can also give rise to ethical problems. In the past, there have been several cases where there was a serious disability in patients of gene therapy and several deaths was recorded. In summary, gene therapy used for replacing mutated genes, fixed mutated genes (by turning them off so that they no longer promote disease or by turning on healthy genes) and making diseased cells more evident to the immune system (in some cases, the immune system doesn't attack diseased cells because it doesn't recognize them as intruders) Gene therapy is now used more often in very serious diseases, which cannot be treated in another way and which usually end lethally. Introducing viral vectors may interfere with other DNA and alter, for example, tumor suppressor genes or proto-oncogenes.

what is a gene? what are some important components that are found near/within a gene? what is the structure and function of a gene? (47)

Gene: DNA sequence required to transcribe and code for an RNA molecule. A gene is the basic physical and functional unit of heredity. Genes, which are made up of DNA, act as instructions to make proteins. In humans, genes vary in size from a few hundred DNA bases to more than 2 million bases. The Human Genome Project has estimated that humans have between 20,000 and 25,000 genes. Every person has two copies of each gene, one inherited from each parent. Most genes are the same in all people, but a small number of genes (less than 1 percent of the total) are slightly different between people. Alleles are forms of the same gene with small differences in their sequence of DNA bases. These small differences contribute to each person's unique physical features. The total complement of genes in an organism or cell is known as its genome (The human genome contains about 3 × 10 9 nucleotides), which may be stored on one or more chromosomes. A chromosome consists of a single, very long DNA helix on which thousands of genes are encoded. The region of the chromosome at which a particular gene is located is called its locus. Each locus contains one allele of a gene; however, members of a population may have different alleles at the locus, each with a slightly different gene sequence. Introns and Exons: Exons - Areas of the gene the coding for RNA Intron - The non-coding sequences Both introns and exons are initially transcribed into RNA, and during post transcriptional modifications the introns are removed. Usually introns are longer than exons. Although they aren't coding for protein, they have many functions in gene expression regulation. Structural features of a typical human gene: The special structure of a gene allows it to form normal RNA molecules. Promoter - the molecular start signal for synthesis of RNA, located at the 5'-end of each gene. It is composed of several DNA elements specific for the promoter area. There are several different types of promoters, each have different regulatory properties. E.g. TATA box or CAT box. Enhancer - activating sequences - when interact with certain proteins it causes increased level of transcription. It can act with a distance - located independently either at the 5' or 3' ends of the transcription start site. Conversely, there are silencers. Locus control regions (LCR) - also located at 5' regions, found in various genes. It involved in timing and tissue specificity expression. Also involved with binding of proteins and transcription factors. Unique single-copy genes are genes coding for polypeptides that are involved in cellular functions. These include enzymes, hormones, receptors and structural and regulatory proteins. Multigene families: many genes have similar functions due to the fact that they are formed from gene duplication events during evolutionary changes. Gene families can be found physically close together or widely dispersed through the genome. Non-coding RNA genes: some genes do not encode for proteins, they encode for RNA. MiRNA (microRNA genes) - important group of non-coding RNA genes, takes part in controlling the expression or repression of other genes during development.

what are hereditary immunodeficienes? mode of inheritance? how can they be distinguished? how can they be diagnosed? what are some examples? genetic counseling options? therapy options? (83)

Genetic diseases arise as a result of mutations. Immunodeficiency is a condition where the influence of certain mutations causes to malfunction of the immune system, and this individual is prone to infectious diseases. Immunodeficiencies may also decrease cancer immune surveillance. Some people are born with defects in their immune system - Primary immunodeficiency. In secondary immunodeficiency, as occur in most cases, the condition is acquired. Primary immunodeficiency may be as a result of random mutation, but mostly it has hereditary causes. Currently, there are over 100 primary immunodeficiencies described. Advances in recent years in molecular genetics have helped locate the responsible gene responsible for the disorder. Most of these disorders show a recessive mode of inheritance, although dominant mode is known, but very rare. There are also some types of disorders with multifactorial inheritance mode. Relatively large amounts of the responsible genes located on the X-chromosome - thus, males are twice as likely to suffer from primary immunodeficiencies as females. The mutated DNA → mRNA sequences with substandard → dysfunctional (possibly none) → damaged protein function. According to impaired function, we distinguish between: Antibody deficits Cell deficiency Combined deficiency Disorders of the complement system Disorders of phagocytosis Disorders of cytokines, cytokines receptors And more.. In order to understand immunodeficiencies disorders, we need to take into account the complexity of the immune system: defect of one part of the system, may occur simultaneously with defect in other parts. E.g. in some combined immunodeficiency - we cannot find T-cells. B-cells, although forming in normal quantities, cannot be activated without the interaction with T-cells 🡪 their function impaired. Generally used as stimuli for testing for primary immunodeficiencies - frequent and repeated infections, children often fail to thrive and have shorter stature than their healthy peers. Another symptom is repeatedly complicated course of infectious diseases, which is relatively poorly respond to standard therapy. Diagnosis The basic tests performed when an immunodeficiency is suspected should include a full blood count and immunoglobulin levels. X-linked Agammaglobulinemia (XLA) - Bruton disease: Mutation in a specific tyrosine kinase enzyme, characterized by the failure of premature B-cells to differentiate into B-cells 🡪 causes absence of antibodies. Occur at frequency of 1/100,000 male infants (BTK gene is on chromosome X, thus, the disorder is mostly seen in males). XLA doesn't become apparent until the affected infant attains age of 6 months, when the trans-placental supply of maternal antibodies is depleted (the newborn is initially protected by the maternal antibodies). Patients with XLA are susceptible to certain viral and bacterial infections. Can be treated by intravenous Ig therapy. Common Variable immunodeficiency is a general term for disorders characterized by hypogammaglobulinemia. Males and females are affected equally; the prevalence of the disease is about 1/50000. The onset symptoms appear at the second or third decade of life and though most patient have normal numbers of mature B-cells, they have lack in plasma cells. Features of the disorder are impaired responses to infection, increased susceptibility to infections, developing autoimmune disorders and lymphoid tumors Mechanism: Some patients have mutation in B-cell receptors for certain growth factors, or in molecules involved in T and B cells interactions. The genetic basis of most cases isn't known. Isolated IgA deficiency - it is the most common of all the primary immunodeficiency diseases. Affects 1/700 people. IgA is the major Ig in mucosal secretions 🡪 involved in defending the airways and GI tracts. Most people with this condition doesn't have symptoms, however, some exhibit recurrent sino-pulmonary infections and diarrhea. In addition, they may exhibit significant association with autoimmune diseases. The pathogenesis seems to involve a block in the terminal differentiation of IgA secreting B-cells to plasma cells genetic counseling From the point of view of genetic counseling and prenatal diagnostics , the following facts are interesting: For many primary immunodeficiencies, we know the exact gene, its location and sequence. We can accurately identify the mutation and confirm the diagnosis using direct DNA diagnostic methods. Thanks to the known type of inheritance, we can estimate potential risks using a genealogical method based on family history . We can also use indirect DNA diagnostics ( RFLP ) to refine the estimate . In autosomal recessively inherited primary immunodeficiencies, an increased risk should be considered for related marriages and for marriages in population isolates. In X-linked primary immunodeficiencies, a different incidence should be expected in boys and girls. Determining the sex of the fetus can thus be of great importance in answering the question of whether the newborn will suffer from the relevant immunodeficiency. Cordocentesis is a very beneficial method for prenatal diagnosis of primary immunodeficiencies, because from the obtained umbilical cord blood we can not only isolate DNA for DNA diagnostics (for this purpose other invasive methods are usually chosen that can be used with less risk and earlier - AMC , CVS ), but we obtain and cellular elements of the fetus, which can be examined phenotypically . Even in the diagnosis of primary immunodeficiencies, the future lies in the routine use of DNA chips , which will make it possible to test (not only) a number of different types of immunodeficiencies at once. There is no real causal therapy for genetic diseases such as primary immunodeficiency . This would involve targeted repair of the mutated gene (primary DNA sequence). Advances in gene therapy give hope for the future; however, current gene therapy methods most often use retroviral carriers that insert the sequence into the genome more or less randomly. In X-linked SCID (severe combined immunodeficiency), gene therapy was performed as the first human disease. However, some patients treated in this way developed a subsequent leukemia, probably due to disruption of tumor suppressor genesretroviral carriers. Due to these complications, it is not yet possible to put this therapy into practice. Experimental gene therapy treatment for ADA (adenosine deaminase) deficiency has also met with some success . Thus, bone marrow stem cell transplantation remains the most common treatment for severe primary deficiencies . This method is particularly demanding to ensure a suitable donor with the greatest possible agreement in HLA antigens. Family members, especially of the same sex, are preferred as donors. Finding an unrelated donor is very difficult and, in addition, satisfactory agreement in minor HLA antigens cannot be expected. As regards immunoactive transplant tissue is to be expected with the risk of GVH reaction (= GHVR graft versus host reaction - graft versus host disease). Replacement therapy involves the intravenous administration of immunoglobulins ; there are also therapies based on defective enzyme substitution , as is the case with ADA deficiency. A suitable part of the therapy is the preventive administration of antibiotics , or antiviral drugs or antifungals. Depending on the type of immunodeficiency, some above-standard vaccinations can be considered . If the patient is threatened by autoimmune manifestations of the disease, immunosuppressive therapy also comes into play .

what are the major chromosomal aberrations found in cancer cells? what does genomic instability include? relevant examples? (114)

Genetic instability, which includes both numerical and structural chromosomal abnormalities, is a hallmark of cancer. Two prominent features of cancer cells are abnormal numbers of chromosomes (aneuploidy) and large-scale structural rearrangements of chromosomes. These chromosome aberrations are caused by genomic instabilities inherent to most cancers. Aneuploidy arises through chromosomal instability (CIN) by the persistent loss and gain of whole chromosomes. Chromosomal rearrangements occur through chromosome structure instability (CSI). Chromosome breaks associated with a chromosome rearrangement may occur within proto-oncogenes or tumor suppressor genes, disrupting their normal function and contributing into carcinogenesis. In addition, rearrangements of chromosomes may also bring together parts of different genes, creating a fusion protein (also known as chimeric protein) that stimulates some aspect of the cancer process; it can also translocate proto-oncogene to another location, when it gains new function and become oncogene - it is activated by different set of regulatory sequences. Abnormal numbers of chromosomes - CIN - chromosomal instability Aneuploidy, as said, is common in cancer cells and may contribute to cancer by altering the dosage of oncogenes and tumor-suppressor genes. CIN can arise from defects that affect different steps of chromosome segregation: Spindle checkpoint which should prevents aneuploidy by inhibiting the transition into anaphase until all the replicated chromosomes and the kinetochore attached properly to microtubules. Centrosome duplication. Mutations and mis-regulation of these processes are associated with various cancer types. Structural abnormalities - CSI - chromosome structure instability Point mutation and deletion - deletion of specific regions of chromosomes may result in the loss of particular tumor suppressor genes; usually, it requires mutation in both alleles in order for them to contribute to carcinogenesis (exceptions - in the case of Loss of heterozygosity + Haplo-insuffiency) Chromosomal translocation: Translocation events which relocate a proto-oncogene to a new chromosomal site that leads to higher expression Translocation events that lead to a fusion between a proto-oncogene and a second gene (chimeric genes) - form through the combination of portions of two or more coding sequences to produce new genes Gene amplification - production of multiple copies of the proto-oncogene, which increase the number of copies of proto-oncogene per cell up to several hundred times 🡪 become oncogene, leading to greater amounts of the corresponding onco-protein product 🡪 overexpression. Mutations in the anti-proliferative tumor-suppressor genes, which occur in the genome in 2 copies - thus mutation needed the intervention of both copies. Mutations in proto-oncogenes, from the other hand, is dominant characters - means that mutation of a single copy of a proto-oncogene turns it into oncogene and it is enough to produce symptoms. Examples: Chronic myeloid leukemia - reciprocal translocation between chromosome 9 and 22 occurs in about 95% of chronic myeloid leukemia - "Philadelphia chromosome" Retiboblastoma - deletion on chromosome 13 affects gene Rb1 - it is an embryonal tumor of the retina - occurs either sporadically (non-hereditary form - involve only one eye) or as familial-hereditary form which is inherited in an AD manner - usually bilateral - both eyes (or more than one site in one eye - multifocal). Burkitt's lymphoma Tumor-transformed B-lymphocytes are removed by immunological mechanisms with the crucial involvement of T cells . T-lymphocytes on their surface recognize virus -induced TSTA tumor-specific transplant antigens (TSTA) presented by MHC molecules . In the absence of T-cells or in the suppression of their activity, tumor growth develops rapidly. Most patients have a stable reciprocal translocation between chromosomes 8 and 14 - most commonly t (8; 14) (q24; q32). Malignancy occurring in Central Africa; an osteolytic jaw lesion is typical. Deletion of section of chromosome 11, in the region 11q15 . Wilms' tumor is a malignant tumor of the kidney that usually manifests in early childhood or even prenatally. Aniridia (lack of iris) and Wilms' tumor can manifest independently of each other. Many patients often have other malformations, mental retardation , genital malformations, and delayed physical development ( WAGR syndrome - a microdeletion syndrome ). In many patients with this association, a deletion of the 11q region is evident and one of the oncogenes, the so-called c-Ha-ras , is located at the site of the deletion .

what is hemoglobin? what are the genes for hemoglobin, where are they located? how does expression of the globin chains differ? how does the expression of globin chains change throughout the lifetime, what is responsible for this? what are the types? (62)

Hemoglobin is a metalloprotein that carries oxygen in the red blood cells of vertebrates. Without it, (cellular) respiration is impossible. The molecule consists of a protein component (globin) , which is represented by 4 polypeptide chains (two and two each are identical), and a prosthetic group - heme (= iron-containing pigment, which binds to oxygen and thus conditions the ability of the hemoglobin molecule to carry oxygen ). An adult has predominantly hemoglobin A (Adult), HbA in erythrocytes (98% of the total Hb in an adult). Hb A contains 2 alpha chains and 2 beta chains. The alpha globin chain consists of 141 amino acids, the beta chain of 146 amino acids. Hemoglobin genes There is more than one hemoglobin gene - the amino acids sequence of the globin proteins usually differ between species; these difference increase with evolutionary distance between the species (the most common hemoglobin sequences in humans and chimpanzees are nearly identical - differ by only one amino acid in both alpha and betta chains). Mutation in the genes for Hb proteins result in hemoglobin variants - many of these mutant gene forms functional hemoglobin that cause no disease, but some cause non-functional hemoglobin - cause a group of hereditary diseases termed hemoglobinopathies - such as sickle cell anemia and thalassemias (question 63). Variations in hemoglobin amino acid sequences, as with other proteins, may be adaptive. For example, hemoglobin has been found to adapt in different ways to high altitudes. Organisms living at high elevations experience lower partial pressures of oxygen compared to those at sea level. This presents a challenge to the organisms that inhabit such environments because hemoglobin, which normally binds oxygen at high partial pressures of oxygen, must be able to bind oxygen when it is present at a lower pressure. Different organisms have adapted to such a challenge. For example, mice that live in the mountains have four different genes (compared to lowland mice) that govern the oxygen-carrying capacity of their hemoglobin - the genetic difference enables highland mice to make more efficient use of their oxygen. Hemoglobin adaptation extends to humans as well. Studies have found that a small number of native Tibetan women have a genotype which codes for hemoglobin to be more highly saturated with oxygen. Natural selection seems to be the main force working on this gene because mortality rate of offspring is significantly lower for women with higher hemoglobin-oxygen affinity when compared to the mortality rate of offspring from women with low hemoglobin-oxygen affinity. Types in Humans - Hemoglobin variants are a part of the normal embryonic and fetal development. In embryo - Hemoglobin Gower 1 - (ζ2ε2), Hemoglobin Gower 2 (α2ε2), Hemoglobin Portland I (ζ2γ2) and Portland II (ζ2β2). In the fetus - Hemoglobin F - (α2γ2) - during the last seven months of development in the uterus, persists in the newborn until roughly 6 months old. It is able to bind oxygen with greater affinity than the adult form - giving the fetus better access to oxygen from the mother's bloodstream. After birth - Hemoglobin A (α2β2) - most common - 95%; Hemoglobin A2 (α2δ2) - 1.5%-3.5%; Hemoglobin F (in adults, HbF is restricted to a limited population of red cells called F-cells). Changes in the structure of hemoglobin in ontogenesis (the development of an individual feature from the earliest stage to maturity) are examples of gene regulation in ontogenesis. Changes in the expression of individual genes is called "Switching globin" - first synthesized Zeta and Epsilon chains (Gower 1), eventually after expression zeta and epsilon globin, Gower 2 and Portland are produced. Later, the zeta and epsilon genes are suppressed and the fetal period consists mainly of HbF. At birth, 70% of the RBCs contain HbF. The regulation of hemoglobin production in ontogenesis is associated with the localization of production of RBCs - begins in the yolk sac, than in the Liver and Spleen, and finally in the bone marrow: Hemoglobin synthesized in a complex series of steps - the heme part is synthesized in the mitochondria and the cytosol of immature RBCs, while the globin protein parts are synthesized by ribosomes in the cytosol. Production of hemoglobin continues in the cell throughout its early development from the proerythroblast to the reticulocyte in the bone marrow. At this point, the RBCs nucleus is lost; even then, residual rRNA allows further synthesis of hemoglobin until the reticulocyte loses its RNA soon after entering the vasculature. Genes for globin chains Cluster of genes related to the alpha gene is located on the 16th chromosome - Alpha globin locus contains of 4 genes - alpha 1 (HBA1), alpha 2 (HBA2), and two pseudogenes (non-functional copies of the alpha 1 and 2 genes - sequences are similar to globin genes but produce no identifiable message or protein product). Groups of genes related to beta gene are located on the 11th chromosome. Knowledge of the structure of groups of genes for hemoglobin chain explains the different clinical manifestations of mutations in the genes for alpha and beta chain. Mutation in the beta gene effects on heterozygotes - 50% of the chains (because there is only one beta gene); mutation of the alpha gene affecting only 25% of the hemoglobin molecules (because in chromosome 16 there are two alpha gene copies). Translations studies have shown that alpha and betta chains are synthesized in roughly equal proportions. However, the beta globin mRNA is slightly more efficient in protein synthesis than the alpha globin mRNA - this difference is compensated in RBC by a relative excess of alpha-globin mRNA. The most important level of regulation of expression of globin genes appears to occur at the level of transcription - except of the promotor sequences of the various globin genes - there are sequences 6-20kB upstream (5') to the globin genes. This region is called "Locus Activation Region" - LAR or LCR (locus control region): When the LCR region binds protein NF-E1, the DNA forms loop - which its size determines the activation loci for which gene will be transcribed. The beta-globin locus is composed of five genes located on chromosome 11, and except of the beta globin gene, the delta, gamma-A, gamma-G and epsilon globin genes are also located in the same loci. Expression of all of these genes is controlled by single locus control region (LCR) - and the genes are differentially expressed throughout development. The arrangement of the genes directly reflects the temporal differentiation of their expression during development, with the early-embryonic stage version of the gene located closest to the LCR.

what is a heteroduplex analysis? (66)

Heteroduplex analysis - heteroduplex is a double-stranded molecule of nucleic acid originated through the genetic recombination of single complementary strands derived from different sources - such as from different homologous chromosomes or even from different organisms. In this method, control DNA is denatured, and allowed to anneal with denatured sample (tested) of DNA. If the sequence of the reference DNA differs from the sample, both original homoduplexes (similar strands) and heteroduplexes (mutated strands) are formed - consisting of one strand of control and one strand of sample DNA. The heteroduplex will have a different shape due to single difference match (except of the point mutation, the rest of the nucleotides are exactly the same and will anneal to each other). Then, by applying to electrophoresis of DGGE (denaturing gradient gel electrophoresis) - the fastest migrating band is the homoduplex band, whereas heteroduplexes with mismatches migrate slowly. Because they contain a segment which is lacking H-bond (the mismatched pair) - they separate earlier and stop first in the gel.

What is immunotolerance? what is self-tolerance? what are the two primary mechanisms that provide the immune system with immunotolerance? what is induced tolerance? (81)

Immunological tolerance is the failure to mount an immune response to an antigen. It differs from immunodeficiency by being antigen specific - whereas immunodeficiency (non-specific intolerance) is generally failure of the immune system to mount response, tolerance is only for a specific antigen - thus called specific tolerance. Immunotolerance can be either natural self-tolerance or induced tolerance. Self-tolerance - (a good thing!) - Refers to a lack of immune responsiveness to one's own tissue antigens - non responsive to self-antigens. During production of B and T-cells receptors, there is an element of random gene rearrangement - which may result in B or T-cells receptors against "self" MHC. One of the major functions of the thymus in T-cells maturation is the "self-tolerance test" - a T-cell that attacks "self" antigens, is immediately destroyed. There are two primary mechanisms that provides the immune system with self-tolerance: Central and Peripheral tolerance Central tolerance: self-destruction of lymphocytes during their maturation in the central lymphoid organs - thymus and bone marrow. Maturation of T-cells in the thymus: Many "self" antigens are present by thymic APC's in association with MHC II. Any immature T-cell that shows too strong affinity to such self-antigen goes through apoptosis - Negative selection. Positive selection also occurs in the thymus, where clones that have a very low or no affinity at all to MHC molecules (thus - aren't functionally efficient) - also destroyed. In addition, transcription factors that induce the expression of peripheral tissue antigens were discovered in the thymus cells - making the thymus an "immunologic mirror" of the whole body. Maturation of B-cells in the bone marrow: same processes as T-cells in the thymus appear regarding B-cells maturation in the bone marrow - these B-cells that react against "self" MHC's going through apoptosis. "Receptor editing", specifically to B-cells in bone marrow - some self-reactive B-cells may undergo a second round of rearrangement of their antigen receptor genes, thus express new receptors that are no longer self-reactive. Peripheral tolerance: Many self-antigens may not be present in the thymus, so T-cells bearing receptors for such autoantigens can escape to the periphery, the same relevance regarding B-cells. Thus, there are several mechanisms in the peripheral tissues that cover \ backup the central tolerance - ensures that the escaped lymphocytes won't induce autoimmune response. Ignorance - Potentially self-reactive T-cells are not activated at immunoprivileged sites - where antigens are expressed in non-surveillanced areas. This can occur in the testes (blood-testis barrier due to tight junction between adjacent sertoli cells - prevents autoimmune attacks against spermatogenic cells which first appear long after the central self-tolerance is established). Anatomical barriers such as blood-testis barrier or Blood-brain barrier separate the lymphocytes from the antigen. Some antigens are at too low concentration to cause an immune response. Split tolerance - as many pathways of immunity are interdependent, they do not all need to be tolerised - for example, tolerised T-cells won't activate autoreactive B-cells; without the help from T-helper CD4 cells, the B-cells won't be activated. Suppression - auto reactive T-cells are inhibited by T-reg (CD25) cells which secret immunosuppressive cytokines. experimentally, removal of Treg cells leads to autoimmune reactions (e.g. AIDS) Anergy - absence of the normal immune response to a particular antigen: Activation of T-cells requires two signals: Recognition of antigen in association with self MHC molecules on APC's (first signal). Costimulation: Co-stimulatory signals provided by the APC's - it is the second signal from an APC to a T-cell, which rescues an activated T-cell from anergy, allowing it to produce the cytokines necessary for production of additional T-cells. If the second costimulatory signals are not delivered, the T cell becomes anergic and cannot respond to the antigen. B-cells can also become anergic, as said, in the case they encounter antigen in the absence of T-helper cells. Induced Tolerance- "non-self" material can trigger safeguarding mechanisms against the immune response. It is useful to cause induce tolerance in the case of transplantation and in order to prevent allergies. By administration of antibodies against some molecules on the surface of T-helpers (CD4) or against APC's, we can induce a state of tolerance. Repeated low doses of antigens can induce toleration. "Antigen suicide" - antigens coupled to toxic drugs radioisotopes, bind to specific B-cells which result in the death of both the B-cells and the antigens, without exposing other cells to danger. In the experiment , tolerance can also be induced to antigens of allogeneic origin - after inoculation of allogeneic cells or tissue transplantation into a newborn organism that does not respond because it does not have a mature immune system. Tolerance can also be induced in an adult host whose immune system has been suppressed - by radiation, drugs, lymphocyte antibodies, etc. Tolerance can be induced in adult animals without attenuation of the immune system - depending on the dose, route of administration and nature of the antigen - low and high doses of antigen usually induce tolerance, medium doses immunity; very high doses of antigen can cause depletion of the immune system ( clonal exhausts ) - it is caused by stimulation of all cells capable of responding, no memory cells are formed - the organism does not respond to repeated supply of antigen.

disturbances of ontogenesis of sex determination? (94)

In humans, the sex is genetically determined, as evident through studies of Turner and Klinefelter syndrome. Turner Syndrome - cytogenetic abnormality with karyotype 45, X - develop as women - have all Mullerian duct derivatives (uterus, oviducts) and do not have Wolffian duct derivatives. Klinefelter syndrome - 47, XXY - is the set of symptoms that result from two or more X chromosomes in males - the individual is male because he has Y chromosome - and one of the paired X chromosomes become inactivated. The primary feature is sterility. From these two syndromes, it can be concluded that Y is the male determining chromosome - whenever present, male developmental progress is initiated. When the chromosome is absent - female developmental pathways are initiated; Gonadal dysgenesis Turner syndrome - karyotype 45, X. True hermaphroditism The basis of ovaries and testes , chimera 46, XX / 46, XY (fusion of two zygotes). Male pseudohermaphroditism Testes and female or ambiguous genitalia , testicular feminization - insensitivity to testosterone , no receptors are expressed for it, karyotype 46, XY, incomplete insensitivity to testosterone, 5α-reductase gene mutation , mosaic 45, X / 46, XY. Female pseudohermafroditizmus ovaries, masculinization of the external genitalia overproduction of testosterone in the adrenal glands 21-hydroxylase block ( adrenogenital syndrome , AR , gene located in the HLA class III region )

what is cell fractioning? (54)

In order to perform nucleic acid analysis, we need to obtain DNA fragments. Cell fractionation is the process of producing pure fractions of cell components in order to study them (e.g. purify proteins or DNA molecules). The process involves two basic steps: disruption of the tissue and lysis of the cells, followed by centrifugation, in which the highly dense cellular components will sink down, whereas the supernatant will be in the higher part of the tube. The supernatant will be separated and go through another round of centrifugation which will again yield supernatant and remnants of denser cell's fraction. The first step in cell fractionation is tissue disruption and cell lysis. The goal is to disaggregate the cells and break them open with minimum damage to the cellular fraction of interest. Three basic methods of breaking up the tissues and cells are: I. Homogenization, II. Sonication, III. Osmotic lysis. The method of choose depend on the tissue, cell type and particular cell fraction of interest. Homogenization involves the use of a mechanical homogenizer, like a blender, to break the tissue apart and lyse the cells. When using gentle mechanical procedures (called homogenization) the plasma membranes of cells can be ruptured so that the cell contents are released, either by high-frequency sound, or by using a mild detergent to make holes in the plasma membrane such as Proteinase K with SDS buffer (sodium dodecyl sulfate). We can also force cells through a small hole using high pressure. The resulting thick soup - homogenate (extract) contains large and small molecules from the cytosol, such as enzymes, ribosomes and metabolites, as well as all of the membrane-enclosed organelles - include the nucleus. When carefully conducted, homogenization leaves most of the membrane-enclosed organelles intact. Sonication involves the use of ultrasound to disrupt the cell, often used when prokaryotic cells are to be lysed. Osmotic lysis is often the method of choice when dealing with cells that are vulnerable to osmotic stress - such as RBCs, which are very sensitive to the tonicity of the surround fluid - if we introduce the RBCs into hypotonic fluid, there is a net inward osmosis resulting in the continuous uncontrolled swelling of the cells until the cells burst - lyse (hemolyze). Examination of the RNA or DNA from a particular gene requires that we be able to distinguish the specific DNA segments or RNA molecules corresponding to that gene from among all the many other segments present in a sample of cells or tissue; the problem is to find the specific DNA fragment in which we are interested in, from within a complex mixture of genomic DNA containing millions DNA fragments generated by restriction enzyme. With RNA samples, the problem is to detect and measure the amount and the quality of a particular mRNA transcript in an RNA sample from a tissue in which the desired mRNA account for only 1/1000 of the total RNA transcripts (the rest are non-coding RNA such as rRNA, tRNA) The solution to the problem of detecting one rare sequence among many involves use of gel electrophoresis to separate the molecules by size, then carrying out nucleic acid hybridization with a probe to identify the molecule of interest.

what are the principles of therapy of heritable diseases? (71)

In order to treat heritable diseases, it is essential to understand the genetic disease at a molecular level. The goal in genetic disease treatment is to eliminate or improve the effect of the disorder. Conventional approach to treatment of genetic disease Before using new treatments of gene therapy, it is often better to consider conventional approaches to the treatment of genetic disease: in PKU, for example, dietary restriction is very useful. In congenital adrenal hyperplasia, hormone replacement may be used. In other disorders, supplementation with a vitamin or co-enzyme can increase the activity of the defective enzyme. If a genetic disorder is found to be the result of a deficiency (or abnormality) in a specific enzyme or protein - treatment could involve replacement of the deficient or defective enzyme or protein - Enzyme Replacement Therapy. For most of the inborn errors of metabolism (enzyme deficiency) - recombinant DNA techniques may be used to biosynthesize the missing or defective gene product. In some genetic disorders, drug therapy is the best solution - statins can help to lower cholesterol levels in hypercholesterolemia (inhibiting endogenous cholesterol biosynthesis) Tissue transplantation - replacement of diseased tissue - an example is renal transplantation in adult polycystic kidney disease or lung transplantation in patients with cystic fibrosis. Islet transplantation for treating type I diabetes mellitus, prepared from donated pancreases and injected into the liver of the recipient. Recombinant DNA technology allows the biosynthesis of gene products for the treatment of certain inherited diseases - e.g. Insulin. Microorganisms can be used to synthesize insulin from human insulin gene. This is inserted into recombinant DNA vector such as a plasmid and cloned in a microorganism such as Escherichia Coli. In this way, large quantities of insulin can be made. The synthetically produced genes cannot contain the introns found in the genes, because E. coli do not possess a means for splicing of the mRNA after transcription. Gene Therapy (question 70) - in general, it is the injection of material to human somatic cell in order to eliminate the mutation causing the disorder. The gene may be transferred through viruses or by non-viral vector, and may be injected into the genome in vivo or in vitro by culturing cells with the desired gene, and then inject them back into the patient body. Gene therapy may work on the DNA itself using its mechanism for mutation repair, or it may affect the translation of the mRNA of the gene product. RNA Modification - targets mRNA, either by suppressing mRNA levels or by correcting/adding function to the mRNA. It is divided to Antisense Oligonucleotides and RNA interference. Anti sense Oligonucleotides therapy used to modulate the expression of genes associated with malignancies and other genetic disorders. The principle is sequence-specific binding of an antisense oligonucleotide to a target mRNA that results in inhibition of gene expression. RNA Interference - siRNA (small-interfering RNA) is a small RNA molecule that causes interference by cleavage the target mRNA. It works through the targeted degradation of the mRNAs. For example, bevasiranib, a siRNA therapy that designed to silence the genes that produce vascular endothelial growth factor - believed to be responsible for the vision loss in patients with the "wet" form of age-related macular degeneration. Targeted Gene Correction - repair genes in situ through the cellular DNA repair machinery. Stem Cells therapy - The ability of an embryonic stem cell to differentiate into any type of cell means that the potential application of ESC therapy are vast - using them as vehicles for genes that mediate phenotype correction through gene-transfer technology. The strategy starts with removing cells (e.g. fibroblasts) from a patient with a monogenic disorder (single-gene disorders) and then transferring the normal gene using a vector (or perhaps, by correcting the mutation in vitro). The nucleus from a corrected cell is then transferred to an enucleated egg obtained from an unrelated donor by somatic cell nuclear transfer. The egg, which now containing the genetically corrected genome of the patient, is activated 🡪 develop into blastocyst in vitro 🡪 corrected stem cells are derived from the inner cell mass. The stem cells are then directed to differentiate into a specific cell type and transferred to the patient, thereby - correcting the disorder. Germline gene therapy intervention in the gamete , zygote or embryonic cells at a very early stage of development genetic change is found in all cells of the newly formed organism The influence of bb, from which gametes are formed , is therefore transmissible to offspring we cannot estimate the results in future generations of ethical barriers before performing germline gene therapy Somatic cell gene therapy performing a genetic change in somatic cells or tissues tissues are selected according to the type of disease manipulations in cells can be performed ex vivo, in vivo Ex vivo = cells are harvested into a suitable environment and returned after therapy In vivo = suitable for bb, which is impossible to cultivate or return to the body the vector carrying the gene could be inserted directly into the tissue the success of these experiments is very low most suitable bb for gene therapy - long life , proliferate, can be easily obtained Bone marrow stem cells are an example - but they are poorly insulated Modification of somatic cells Several approaches: introducing a functional copy of the gene into bb, the mutant gene remains unchanged repairing the mutant gene or placing a working copy of the gene in place of the mutant gene targeted inhibition of gene expression targeted destruction of specific cells (significant in tumors) destruction of specific cells of the immune system The goal is long-term expression of the introduced gene. Thus, the foreign gene must integrate into the chromosome of the host cell, and bb must have the ability to further divide. The foreign gene is therefore subsequently transferred to daughter cells - there is a different integration of the gene , with subsequent cycles is therefore situated in other places. Gene therapy eg transformation by cloned genesdisease: adenosine deaminase deficiency ( severe combined immunodeficiency ) [1] Enzyme induction eg barbituratesdisease: congenital non-hemolytic jaundice (see differential diagnosis of jaundice ) Enzyme replacement eg tissue transplantationdisease: mucopolysaccharidosiseg enzyme substitutiondisease: trypsin deficiency Protein substitution eg antihemophilic globulindisease: hemophilia Vitamin substitution eg vitamin Ddisease: vitamin D resistant rickets Product substitution ex cortisonedisease: adrenogenital syndromeeg thyroxinedisease: congenital hypothyroidism Substrate restriction in the diet AMK - Phenylalaninedisease: phenylketonuria (see Disorders of aromatic and branched chain amino acid metabolism )eg sugars - galactosedisease: galactosemiaeg fats - cholesteroldisease: hypercholesterolemia (see Hereditary disorders of fat metabolism ) Drugs that reduce the excess product of defective metabolism eg cholestyraminedisease: hypercholesterolemia (obsolete in the era of statins)eg penicillaminedisease: M. Wilson Replacement of the institution eg kidney transplantationdisease: polycystic kidney disease Removal of organ eg colectomydisease: familial colon polyposis

what is inbreeding? what is cosanguinity? consanguineous marriages and their risks? how does inbreeding affect HW? how to measure cosanguinity risks? (103)

Inbreeding is a situation in which individuals from a small population tend to choose their mates from within the same population. It may be from cultural, geographical or religious reasons. In this situation - parents may still have a common ancestry within the past few generations, especially if the parents originally are from common geographical or ethnical areas. Inbreeding increases the chances of mating between two heterozygous carriers of an autosomal recessive disorder. An inbreed individual can thus receive two copies of a gene that was carried by a common ancestor - these 2 copies are 'identical by descent' - meaning they came from both parents, from a common origin (which is different from 'identical by nature' - the non-inbred homozygous). increases the chances of mating b/t two heterozygous carriers of an autosomal disorder can receive two copies of a gene that was carried by a common ancestor - copies are identical by descent Consanguinity - Consanguinity ("blood relation", from the Latin consanguinitas) is the property of being from the same kinship as another person. In that aspect, consanguinity is the quality of being descended from the same ancestor as another person. Autosomal recessive disorders occur in individuals who have two copies of an allele for a particular recessive genetic mutation. Except in certain rare circumstances, such as new mutations or in a case of uniparental disomy (UPD - occurs when a person receives two copies of a chromosome or of part of a chromosome from one parent, and no copy form the other parent), both parents of an individual with such a disorder will be carriers of the gene. These carriers do not display any signs of the mutation and may be unaware that they carry the mutated gene. Since relatives share a higher proportion of their genes than do unrelated people, it is more likely that related parents will both be carriers of the same recessive allele, and therefore their children are at a higher risk of inheriting an autosomal recessive genetic disorder. The extent to which the risk increases depends on the degree of genetic relationship between the parents; the risk is greater when the parents are close relatives and lower for relationships between more distant relatives, such as second cousins, though still greater than for the general population. The measurement of consanguinity risks: measured by the coefficient of inbreeding - F - the probability that a homozygote is identical by descent - have received both alleles at a loci from the same ancestral source. F= (1/2)^n+1 N - number of generations in genealogy. The coefficient of breeding can range from 0-1, when: 0 indicates that mating in large population is random 1 indicates that all alleles are identical by descent r = (1/2)^n *proportion of heterozygotes decreases by 2fpq Dahlberg relationship = the dependence of the relative proportion of AR homozygotes born from a cousin's marriage to the gene frequency and frequency of these marriages in the population.

What is a DNA microarray? what are the applications? (54)

Microarray technology A DNA microchip is usually a glass plate to which thousands of different DNA sequences are attached in a certain order. These molecules serve as probes for hybridization to test samples. Use of microchip technology: Gene expression analysis - comparison of expression in cells and tissues in different situations (tumor cells) Genotype analysis - determining whether an individual is homozygous or heterozygous for a given polymorphism Genetic testing - identification of the mutation carrier, diagnosis of hereditary disease

mutations in a population's perspective what is the significance? how can mutations be expressed? what is the frequency of mutations? how does frequency differ from rate? how does this apply to HW? on allelic frequencies? how does this relate to selection? (105)

Mutation, in population genetics aspect, is the random and permanent inherited change of genetic material. Mutation is the ultimate source of genetic variation in the form of new alleles - It enriches the population of new alleles. In addition, mutation may influence the direction of evolution when there is mutation bias, i.e. different probabilities for different mutations to occur. For example, recurrent mutation that tends to be in the opposite direction to selection can lead to mutation-selection balance. The mutation selection balance is equilibrium in the number of deleterious alleles in a population that occurs when the rate at which deleterious alleles are created by mutation equals the rate at which deleterious alleles are eliminated by selection. The mutation can be either on the chromosomal level (large-scale mutations) or on the genes level - point mutation and can be expressed in many ways: Tolerated - effects are minimal, may form natural selection: Neutral mutation - do not change the reproductive ability of carrier Favorable mutation - increases reproduction ability and improve the carrier function Disadvantageous - loss or damage of gene function. Forbidden - lethal mutation, incapable for reproduction. Each person carries 12 genes with harmful recessive mutation - about 3-5 of them are lethal in homozygous form. The frequency of mutation in humans in 10^-5 to 10^-6. Mutation frequency and mutation rate: Mutation frequency and mutation rates are highly correlated to each other: Mutant frequency is defined as the proportion of mutant cells in a population. It should be distinguished from mutation rate, which relates to the rate at which mutation events arise, and is generally expressed as events per cell division. Mutation and Hardy-Weinberg equilibrium: In a certain population, mutation is the means by which new alleles are created and join the gene pool, but it is also very random, thus mutation will have a very subtle effect on allele frequencies. Mutation rates are of the order 10−5 to 10−6, and the change in allele frequency will be, at most, the same order. Recurrent mutation will maintain alleles in the population, even if there is strong selection against them. The effect of mutation on allelic frequencies: Mutation can affect the rate of one genetic variant increases on the expense of another genetic variant. The mutation changes are affected by: Mutation rate - how many new cases in defined number of births - M (allele A 🡪 allele a) The frequency of allele A in the population - P The equation to calculate the mutation change in the population is by multiple the ratios of new cases to number of births (mutation rate - M), by the frequency of allele A (non-mutant) - P: ∆P= -u x P Backward mutation can also occur, affected by: Backward mutation rate (how many a 🡪 A appear) - V Allelic frequency of a (recessive trait) - q ∆q=-q x v Both equations together: ∆q= u x P-Q x v Equilibrium of allelic frequencies: In equilibrium, the increase in q due forward mutation is equal to the increase in p due to backward mutation - thus, there is no net change between allelic frequencies. Equilibrium of mutation and selection: see image

What is multifactorial inheritance? how can the risk of involvement be estimated? what are some examples of disease with a multifactorial etiology? What do the terms liability and the threshold model refer to? what is familial aggregation? (8)

Multifactorial inheritance means that many factors are involved in causing a birth defect. The factors are usually both genetic and environmental, where a combination of genes from both parents, in addition to unknown environmental factors, produce the trait or condition. In a family where the selected multifactorial hereditary diseases have been documented, the estimate risk of further occurrence is calculated according Edward's formula: risk of involvement r = (√f) n Where f is equal to the relative frequency of the disease in the population and n is equal to the number of first-degree relatives with the disease Among the diseases with a multifactorial etiology generally include: coronary heart disease ; bronchial asthma ; selected cases of congenital malformations - neural tube defects rare defects and diseases (population frequency <1%) congenital malformations (VVV) as clefts in the face (lip, palate) cardiac VVV nerve tube clefts incorrect development of the hip joint esophageal narrowing defects and diseases with medium frequency (<5%) a significant proportion of severe mental illnesses such as schizophrenia (personality cleft) bipolar psychosis (manic depression) weak-mindedness (oligophrenia) diseases with a high population frequency high blood pressure (hypertension) type II diabetes mellitus obesity gastrointestinal ulcer disease immune disorders - allergies (eg asthma, atopy) liability - the heritability of the disease, likelihood that two identical twins that are separated at birth will grow up to develop the same disease - collectively describes all of the genetic and environmental factors that contribute to the development of a multifactorial disease - liability for a group of people can be estimated based on the number of affected individuals within that group At the point an individual accumulates a certain liability they will be affected by the disorder, the level of liability at which this occurs is referred to as the threshold level. The liability required to exceed the threshold level is the same in all individuals however individuals with affected relatives (especially first degree relatives ) will have a higher chance of exceeding the threshold level and being affected due to inherited genetic factors and possibly shared environmental factors too. As a general rule the later in life a multifactorial disorder develops the more it is dependent on environmental factors (=the lower the heritability) Family aggregation, also known as familial aggregation, is the clustering of certain traits, behaviours, or disorders within a given family. Family aggregation may arise because of genetic or environmental similarities. When 2 related individuals in a family have the same disease, they are said to be concordant (inheriting the same genetic characteristic). Members of the same families are usually exposed to the same environment - which may increase the probability for a disease to appear. When gene are important contributors to a disease the frequency of disease concordance increases as the degree of relativeness increases - the most extreme example is monozygotic (identical) twins.

what are methods used for the detection of specific mutations? (66)

Mutation-specific RFLP analysis - the mutation is part of restriction site of specific restriction enzyme. An allele which has the mutation (or polymorphism) - restriction site won't be recognized and allele which doesn't have the polymorphism/mutation in the recognition site will be recognized. When viewing the DNA segment after Southern blotting and suitable probe - allele with the mutation won't be cut (single heavy band appear in the electrophoresis) whereas allele with no polymorphism will be cut - 2 shorter bands with different lengths. ASO - Allele specific oligonucleotide - is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a southern blot assay. It is common tool used in genetic testing and molecular biology research. Typically, 15-21 nucleotide bases in length, designed in a way that makes it specific for only one version (allele) of the DNA being tested. These probes can be designed to detect a difference of as little as 1 base in the target's genetic sequence, a basic ability in the assay of SNPs. Example - detection Sickle Cell anemia which is caused by point mutation in the gene encode for beta-hemoglobin - The altered sequence substitutes a valine amino acid into glutamate. To test for the presence of the mutation in a DNA sample, an ASO probe would be synthesized to be complementary to the altered sequence, and as a control, another ASO would be synthesized for the normal sequence. After examination of the strand in which full-strong annealing occur (without the single mismatch) we can dedicate what is the exact sequence and whether there is mutation or not (the other strand will form weaker bonding which can be washed). Diagnosis of trinucleotide repeat expansion - also known as triplet repeat expansion (disorder) - are a set of genetic disorders caused by trinucleotide repeat expansion, a kind of mutation where trinucleotide (triplets) repeats in certain genes exceed the normal, stable threshold, which differs per gene. Amplification by PCR of gene region containing the repeated CAG sequence 🡪 apply in polyacrylamide gel electrophoresis 🡪 number of repeats associated with development of disease can be assumed. For example, Huntington's disease occurs when there are more than 35 CAG repeats on the gene coding for the protein HTT. In healthy people there is less then 35 CAG repeats (triples).

what is Northern blotting (54)

Northern blotting is a standard approach for determining the size and abundance of the mRNA from a specific gene in a sample of RNA. RNA cannot be cleaved by the restriction enzymes used for DNA analysis; however, different RNA transcripts are naturally of different lengths, depending on the size and number of exons within a transcribed gene. Thus, total cellular RNA obtained from a particular cell type can be separated according to size by gel electrophoresis. After the electrophoresis, the RNA is transferred to a filter which is incubated with a denatured, labeled probe that hybridizes to one or more specific RNA transcripts. After exposure of the washed filter to x-ray film, one or more bands may be apparent, revealing the position and abundance of the specific transcript of interest.

what is ontogenesis? how is it controlled? what are morphogens? how does fertilization occur? what are the stages of development? what mechanisms are involved? what are the development gene families? (91)

Ontogenesis is the origination and development of an organism, usually from the time of fertilization of the egg to the organism's mature form. It is the developmental history of an organism within its own lifetime (in contrast to phylogeny - evolutionary history of a species). Key point: all cells in the body have the same set of genes, but differ in their expression. Morphogene - signaling molecule that acts directly on cells to produce specific cellular response, depending on its local concentration. It is produced by cells in particular region of an embryo. It occurs during early development in order to perform a concentration gradient which drive the process of differentiation of unspecified stem cells into differential cell types ultimately forming all the tissues and organs of the body. Cells, thus, undergo specification to different fates. Morphogene, thus, controls differentiation and predetermination of cells, and their effect depends on the concentration of their product - when the effect occurs only in locations where the concentration reaches a threshold level. A fetus is recognizably human after about 12 weeks of pregnancy - the first trimester. Normal development requires an optimum maternal environment but genetic integrity is fundamental - which leads to the field of developmental genetics. Brief embryological reminder: Fertilization leads to the formation of a zygote - start of pre-embryonic stage. During the next stage -cleavage - mitotic cell divisions transform the zygote into a hollow ball of cells - blastula. The blastula forms the blastocyst, which consists of an inner cell mass (embryoblast - future embryo) and the outer cell mass (trophoblast - future placenta). Next, gastrulation takes place - the embryonic stage starts. During the embryonic stage, the craniocaudal, dorsovetral and proximodistal axes are established. During gastrulation, the primitive streak formed at the caudal end of the embryo. The germinal layers of the trilaminar disc give rise to ectodermal, mesodermal and endodermal structures. The neural tube is formed and neural crest cells migrate to form sensory ganglia, sympathetic nervous system, pigment cells and bone and cartilage in parts of the face. The final stage - fetal stage, is characterized by rapid growth and development as the fetus (no longer referred to as embryo) matures into a viable human infant. On average, the development takes approximately 38 weeks. Generally, human developmental has been divided into: Embryonic stage - during which all the major organ system are established Post embryonic stage - in humans begins with the fetal period, 9 weeks after fertilization. The time at which development stops is controversial: according to some, it ends after the second decade of life, while others say that even aging can be regarded as part of development. The pattern of formation of the organism and its morphogenesis depends on the molecular level on several cell behaviors, which monitored and controlled by genes: Cellular proliferation - hyperplasia Growth in the size of cell - hypertrophy Differentiation of stem cells into mature cells Cellular migration, communication and signaling Cell death - apoptosis Developmental Gene Families Genetic studies have identified several genes and gene families that play important roles in early developmental processes. Many developmental genes produce transcription factors, which control RNA transcription from the DNA template by binding to specific regulatory DNA sequences to form complexes that initiate transcription by RNA polymerase. Transcription factors can switch genes on and off by activating or repressing gene expression. Important transcription factors control many other genes in coordinated cascades and feedback loops involving the regulation of fundamental embryological processes such as induction (extracellular signals give rise to a change from one cell fate to another), segmentation, migration, differentiation and apoptosis. It is believed that these processes are mediated by growth factors, cell receptors and chemicals - all known as morphogens. Maternal-effect genes - the phenotype of an organism is determined not only by the environment it experiences and its genotype, but also by the environment and genotype of its mother. It occurs when an organism shows the phenotype expected from the genotype of the mother, irrespective of its own genotype, often due to the mother supplying mRNA or proteins to the egg. The Segmentation genes - play a role in the early stages of development. It is family genes whose function is to specify tissue pattern The segmentation genes are set of genes that control the differentiation of the embryo into individual segments, affecting the number and organization of the segments. Mutations in these genes usually disrupt whole sets of segments. The segmentation genes include - gap genes, HOX genes, Pair-rule genes (PAX), genes-polarity segments. Gap genes - involved in the development of the segmented embryos - affect the basic differentiation of the embryo. They are defined by the effect of a mutation in that gene - which causes the loss of contiguous body segments. Each gap gene is necessary for the development of section of the organism. E.g. Knirps, giant, hunchback. Pair-rule genes - segmentation gene which are expressed as a result of differing concentrations of gap gene proteins, which encode transcription factors controlling pair-rule gene expression. They are defined by mutation in that gene, which causes the loss of the normal developmental pattern in alternating segments: It was found that each gene is expressed in alternate parasegments - embryo divided to 15 parasegments. Fushi tarazu, even-skipped Segment-polarity genes affect affect the organization of segments. Mutations in these genes cause part of each segment to be deleted and replaced by mirror image of part or all of an adjacent segment. The gap genes, pair-rule genes, and segment-polarity genes act sequentially, affecting progressively smaller regions of the embryo. First, the products of the egg-polarity genes activate or repress the gap genes, which divide the embryo into broad regions. The gap genes, in turn, regulate the pair-rule genes, which affect the development of pairs of segments. Finally, the pair-rule genes, influence the segment-polarity genes, which guide the development of individual segments. When the major axes have been established - segmentation genes determine the number, orientation and basic organization of the body segments. Tissue-specific genes After the segmentation genes have established the number and orientation of the segments, homeotic genes become active and determine the identity of individual segments. Eye normally arise only on the head segment, whereas legs develop only on the thoracic segments. The products of homeotic genes activate other geens that encode these segment-specific characteristics. Mutations in the homeotic genes cause body parts to appear in the wrong segments. Homeotic genes create addresses for the cells of particular segments, telling the cells where they are within the regions defined by the segmentation genes. Mutation 🡪 wrong address. Homeotic genes are expressed and activated by specific concentrations of the proteins produced by the segmentation genes. For example, the homeotic gene Ubx is activated when the concentration of Hunchback protein (a product of gap gene) is within certain values. These concentrations exist only in the middle region of the embryo - so Ubx is expressed only in these segments. The homeotic genes encode regulatory proteins that bind to DNA - homeobox region, which is similar in all homeotic genes and encodes amino acids that serve as a DNA-binding domain. These homeobox are also present in segmentation genes and other genes that play a role in spatial development. HOX genes - homeobox containing genes - is the mammalian version of the homeobox genes. HOX genes have been found in all animals studied so far. They are group of related genes that control the body plan of an embryo along the cranio-caudal (i.e. head to tail) axis. After the embryonic segments have formed, the HOX proteins determine the type of segment structures (e.g. legs, arms, vertebrae) that will form on a given segment. The protein product of each HOX gene is a transcription factor - each gene contains a well-conserved DNA sequence known as the homeobox. The essence of multicellularity is the ability to express only certain portions of the genome in particular cells at particular times (done by transcription factors that turn on and off specific genes as required). It involves a cascade of hundreds of genes.

what is PCR? what are the steps? purpose? (54)

PCR is an alternative to molecular cloning for generating unlimited amounts of a sequence of interest. It can selectively amplify a single molecule of DNA several billion fold in a few hours. PCR is an enzymatic amplification of a fragment of DNA (the target) located between two primers which are designed so that one is complementary to one strand of a DNA molecule on one side of the target sequence and the other primer is complementary to the other strand of the DNA molecule on the opposite side of the target sequence - they "flank" the target sequence. DNA polymerase enzyme is then used to synthesize two new strands of DNA with the sequence located between the primers as the template; repeated cycle of: Heat denaturation Annealing - Hybridization of the primers Elongation - enzymatic DNA synthesis The repetitive cycle results in the exponential amplification (2, 4, 8 , 16, 32... copies) of the target DNA sequence. Thus, PCR amplification can generate sufficient quantities of specific genes from DNA samples for the analysis of mutations. Particular portions of a gene (usually the exons) are rapidly amplified with use of primers known to be specific to the gene. PCR is an extremely sensitive technique that is faster, less expensive, more sensitive and less demanding of patients samples than any other method for nucleic acid analysis; it eliminates the need to prepare large amounts of DNA or RNA from tissue samples and becoming a standard method for analysis of samples for research, clinical diagnosis and for forensic and law enforcement laboratories. PCR is also quantitative method - the number of cycles required to reach a randomly threshold is a measure of how much template was initially present at the start of the PCR: the fewer cycles - the more template must have been present at the beginning - technique called "real-time PCR".

evolution of homo sapiens? what is paleoanthropology? what are the traits that differentiate us from other primates? what is our family tree? what are examples of evolution? what are the two main models of evolution? (122)

Paleoanthropology- the study of human evolution based mostly on fossil study such as teeth due to their high mineralization compare to bone they stay longer. One of the groups of Primates is Anthropoids (other group - Prosimians) - within the anthropoids, Apes and Human are called Hominoids, has even larger brain then other primates. Humans and apes are very close in molecular level, e.g. amino acid sequence of Chimpanzee for Hemoglobin is almost identical for that of a human (evidence for evolutionary similarity). Hominine Evolution - evolutionary changes from early hominines to modern humans evidenced by difference between apes and humans - curvature of the spine provides better balance for standing on both feet for human; apes do not have curvature. Humans have flatter face and better teeth arrangement. They also have larger brain, much more complex, divided into lobes and hemispheres. Whereas apes designed to climb on trees (with the exception of gorillas), humans do not, however, we have well-functioning arms and legs. Humans are classified as the highest order of primates and share common traits that differentiate us from others: 1. Flexible hands and fit with 5 digits containing opposable thumb to grab things 2. Front facing eyes- 3D vision important in judging distance and depth 3. Relatively large brain due to increase sensory input; hemispheres, lobes. 4. Strong social organism 5. Human pelvic is shorter and boarder, allow better attachment of muscles used for upright walking and enough space for giving birth to a baby with big brain. Ramapithecus Widespread in Africa, Europe and Asia at the Miocene and Pliocene transition. At an upright position, 100-110 cm tall, he moved mostly quadrupedally (four at a time, ankle toes bent at the fist). A flat brain with a volume of about 350 cm³. He collected seeds and other plant food in the gangs. Historical and now obsolete idea of ​​the development of the genus Homo, E. Haeckel, 1877 Australopithecus Widespread in Africa at the transition of the Pliocene and Pleistocene, there were several species, but mutual phylogeny is unclear, adaptive radiation of species of the genus Australopithecus occurred 3 million years ago in Africa. 115-125 cm tall, weighing about 25-35 kg, was able to walk upright, walking bipedally. The head had a more arched cranium with a volume of about 490 cm³ and the cerebral cortex was richly grooved. He was an omnivore with a predominant meat diet. genus Homo _ It probably originated about 2 million years ago in Africa. In terms of species identification, in the case of fossils, the biological definition of a species , assuming the possibility of successful crossing to form viable and fertile hybrids only within a given species, is not applicable. However, the possibility of comparing the genomes of fossil representatives brings interesting findings in this direction . Homo erectus A figure adapted to walking, based on that proportion for leaving Africa and migrating to other parts of the world (Indonesia, China). They could make tools, they communicated. They had a flat face, a spherical skull, a supraorbital wall, the skulls have a typical postorbital constriction (can be seen in this picture ), the capacity of the skull was about 1100 cm³. He lived and hunted in steppe landscapes with sparse forests, lived in caves, made pointed fist wedges, axes and scrapers from stones, knew fire. Homo habilis _ _ He lived in Central and East Africa in the ancient Pleistocene. In a number of somatic traits, he was more advanced than Australopithecus (average cranial capacity about 750 cm³, had a typical human teeth and an upright figure). The arch of the feet , the toes ended with nails, also corresponded to bipedal walking . Homo floresiensis Skeletal remains and other minor finds in Indonesia indicate the possibility of a separate species with a relatively small cerebellum. He was small in height (height 1 m, weight 25 kg, "hobbit" from Flores, where he lived 18,000 years ago). However, there is much controversy surrounding his recognition [2] . Homo neanderthalensis The robust human species, adapted to cold weather, evolutionarily follows the findings of European fossils Homo heidelbergensis. It was documented in the fossil record 220 to 35,000 years ago, so he was a contemporary of H. sapiens, with whom he crossed. Skull with superciliary arches, brain capacity 1400-1450 cm³, lived and hunted in hordes, knew and carefully maintained the fire, made stone tools from flint chips, and worked relatively hard on wooden tools. He created a primitive communal society, vocal expressions gradually developed into simple speech - the same variant of the FOXP2 gene was found in genetically studied fossils as in today's humans. FOXP2 is a gene that affects the ability to learn a language, located on chromosome 7. If this gene is mutated, patients are unable to articulate speech, there is a lack of development of the appropriate nerve centers. The same variant of this gene has been found in both Neanderthals and Dennis as in modern humans. He probably had a system of cults and rituals, burying his dead in the center of the caves. The first personal ornaments are documented. HERC2 gene Molecular genetic research on Homo neanderthalensis fossils has led to the discovery of gene variants that are co-responsible for blue-eyed, fair skin and redness in humans today. The same SNP (single nucleotide polymorphisms ) was found in the HERC2 gene in the studied fossils, which causes blue-bone in today's humans. The HERC 2 gene product does not itself participate in pigmentation, but the SNP in its sequence affects the transcription of the OCA2 gene (mutations in this gene cause oculocutaneous type 2 albinism ), its function is crucial for the character of iris pigmentation. Homo denisoviensis A human species named after Dennisov after the site of the fossil find (Denis Cave in the Altai). Described only on the basis of molecular analysis of the fossil genome (finger joint and stool). In the fossil record 48 to 30,000 years ago, he was a contemporary of both Neanderthals and modern humans. Research shows that they have contributed 4-6% of their genome to the gene pool of today's Melanesans . [3] This is therefore one of the evidences of the crossing of different developmental branches of the genus Homo . Homo sapiens The first representatives of this species appeared about 200,000 years ago in East Africa, their physical constitution (tall, slender figure) was closest to today's Maasai. The average brain volume of the found fossils was 1500 cm³, they hunted large game. In Europe and Siberia, after their settlement, the first dwellings (earthworms) are documented. They were the first to dress (skin), to communicate using articulated speech. Crossing with the Neanderthals, who inhabited the cold regions of the north before the arrival of modern man, may have contributed to the adaptation to the cold climate (see below). Fine art was created 40,000 years ago, which is considered to be the first revolutionary act in the history of mankind to enable communication through representative material symbols.

127. prenatal screening of inborn errors of development? 128. Prenatal diagnostics of heritable diseases, possibilities of prevention 129. Prenatal diagnostics of chromosomal aberrations, possibilities of prevention 130. Prenatal diagnostics of inborn errors of development, possibilities of prevention what are the goals of prenatal diagnositics? what are the indications? what are the methods used for diagnostics (invasive and non-invasive)? possibilities to prevent heritable, inborn diseases?

Prenatal diagnosis or prenatal screening are aspects of prenatal care (Fetal medicine - unborn person) that focus on detecting anatomic and physiologic problems with the zygote, embryo or fetus as early as possible, either before gestation even starts. Prenatal diagnosis includes inter-disciplinary approach, in which clinical genetics, obstetrics and gynecology, as well as clinical biochemistry and imaging methods cooperate together. In prenatal diagnosis there is a usage of medical tests to detect problems such as neural tube defects, chromosome abnormalities and gene mutations that would lead to genetic disorders and birth defects - such as spina bifida, cleft palate, Tay-Sachs, Sickle cell anemia and more. Congenital chromosomal aberrations are diseases caused by changing the number of chromosomes (genomic abnormalities) or changing the structure of the chromosome (chromosome abnormalities). Congenital means that they are present in all cells of the body and can be transferred between generations. They are also called as gametic. Estimates of incidence is 5-8 cases per 1000 births. typical chromosomal aberration: Numerical aberrations of autosomes: Down syndrome (21), Edwards syndrome (18), Patau syndrome (13). Numerical aberrations of gonosomes: Turner syndrome (45,X), Klinefelter syndrome (47, XXY), syndrome 47, XXX or 47 XYY. Tasks of prenatal diagnosis Main goal - perform a systematic usage of genetic screening for high-risk pregnancies and prenatal testing of fetuses to reduce the number of affected newborns. Inform parents about the diagnosis of the fetus and its prognosis and allow them to prepare psychologically, socially, financially and medically for a baby with a certain healthy problem or disability. Specific measures for the further course of pregnancy, labor management (such as optional caesarean section) or for follow-up care (e.g. decision to perform the childbirth in a special center which provide surgical options or with specific child-care specialists) Initiate prenatal fetal therapy (e.g. intra-umbilical transfusion in anemic fetus) In case of unfavorable diagnosis, optionally artificial terminate of pregnancy for genetic and development reason (until the 24th week of pregnancy in the CR, or in extremely unfavorable diagnosis like incompatible with life, e.g. Anencephaly, allows to terminate a pregnancy at any time). Indications for prenatal screening - particularly irregular examinations: Family history of genetic condition or chromosome abnormality - incidence of congenital defects and hereditary diseases in the family or personal history. Such as presence of a known translocation, inversion or insertion in one parent A chromosomal anomaly in an existing child of couple. Molecular test for single gene disorder Increased maternal (35+) or paternal (40+) age Abnormal maternal serum screening results or ultrasound findings Strong family history of cancer Following an abnormal result from non-invasive screening (such as US) examples for methods of prenatal screening and diagnosis Generally, the methods can be classified as non-invasive methods or invasive methods - with different level of invasiveness. Non-invasive methods: External examination of woman's uterus from outside of the body, Fetal heartbeat - listening to the fetal heartbeat Examination of biochemical markers - Triple test (also known as triple screen) is an investigation of maternal serum levels performed during the second trimester (after 16th week of gestation) to classify a patient as either high-risk or low-risk for chromosomal abnormalities and neural tube defects. The triple screen measures three parameters: Levels of AFP - alpha-fetoprotein - encoded by AFP gene. It is major plasma protein produced by the yolk sac and the liver during the fetal development and it is the most abundant plasma protein found in the fetus. It is measured through the analysis of maternal blood or amniotic fluid. High levels of alpha-fetoprotein may indicate neural tube defects, yolk sac tumors or other tumors hCG - human chorionic gonadotropin - which is a hormone produced by the placenta after implantation. Some cancerous tumors produce this hormone (thus, elevated level may indicate tumors). Unconjugated Estriol - a natural steroidal estrogen that synthesized in large amount during pregnancy and 90-95% of the amounts, during pregnancy, should be conjugated (in the form of estriol glucuronide and estriol sulfate. Low level of AFP, UE together with high levels of hCG may indicate increased risk for down-syndrome. Low levels of all the three - trisomy 18 (Edward's syndrome) High level of AFP - may cause to uncovered skin - neural tube defects (e.g. spina bifida) Unconjugated Estriol levels reflects the overall risk of pregnancy Ultrasounds examinations - sounds waves are used to create real-time visual images of the developing fetus in the uterus. It can provide a variety of information about the health of the mother, the timing and progress of the pregnancy, and the health and development of the fetus. The embryo should be seen approximately 5-6 weeks after gestation. US examination occurs in the 6th, 13th, 20th and 32nd week of pregnancy and it is standard care for pregnant women. Signs possible - retardation of development, little or much amniotic fluid, disproportions of development. In the first trimester, a standard US examination typically includes: gestational sac size, location, number, identification of the embryo and yolk sac, measurement fetal length, fetal number (amniotic sacs, chorionic sac numbers), fetal heart activity, assessment of fetal anatomy and more. In the 13-14 weeks of pregnancy - US Nuchal Translucency is carried to evaluate the thickness of the nuchal area between the skin and the cement that covers the cervical spine (when too thick - an increased risk of chromosomal aberrations). Week 30 - screening in order to measure the size and position of the fetus. Nuchal Translucency Screening Increased nuchal translucency in the fetus is associated with increased risk of chromosomal abnormality and other diseases. Sonographic measurement of the thickness of the nuchal fold between 11th and 13th weeks of pregnancy, together with maternal age and biochemical markers allows an individualized risk of aneuploidies such as trisomy 21, 13, and 18 to be calculated. Invasive methods: The main purpose of invasive testing is to obtain a tissue sample of the fetus for testing karyotype or for molecular genetic analysis in order to exclude chromosomal abnormalities or genetic disorders. Chromosome analysis requires cellular material, thus an appropriate intervention is needed. These methods are risky and pricey, thus offered only on the basis of specific indications from previous standard tests. Amniocentesis - involves taking a sample of amniotic fluid with a needle through the abdominal wall under US control, usually performed during the 16-18th week of gestation. It allows examination of fetus cells that will be floating in the amniotic fluid and can be separated and tested. The fetal DNA is examined for genetic abnormalities, most commonly is to determine whether a baby has certain genetic disorders or chromosomal abnormalities, such as down syndrome. *amniotic fluid reminder - the protective fluid found in amniotic sac (the inner membrane - amnion, encloses the amniotic cavity where the fetus develop, the outer membrane - chorion - is part of the placenta and connected to the umbilical cord). CVS - Chorionic villus sampling - usually taken at 10-12th weeks of gestation (earlier then AMC) determine chromosomal or genetic disorders in the fetus by sampling the chorionic villus and testing it with FISH or PCR. Possible reasons for having a CVS include abnormal first trimester screen results, increased nuchal translucency, family history of a chromosomal abnormality or other genetic disorders, advanced maternal age (associated with increased risk of Down syndrome). Umbilical cord blood sampling - PUBS - Cordocentesis - examines blood from the fetal umbilical cord to detect fetal abnormalities. PUBS provides a means of rapid chromosome analysis and is useful when information cannot be obtained through CVS, AMC or US (or if the test's results were inconclusive). This test carries a significant risk of complication and is typically reserved for pregnancies determined to be at high risk for genetic defect. Fetoscopy - an endoscopic procedure during pregnancy to allow access to the fetus, the amniotic cavity, the umbilical cord and the fetal side of the placenta. A small incision is made in the abdomen, and an endoscope is inserted through the abdominal wall and uterus into the amniotic cavity.

how are proteins translocated to the nucleus? (50)

Protein Targeting into nucleus: The nuclear envelope consists of outer and inner membranes and has inter membranous space between them. The outer membrane is continuous with ER and has ribosomes on it. Proteins for the nucleus are synthesized on free ribosomes in the cytosol and imported into nucleus through 3000-4000 nuclear pores known as nuclear pore complexes which are special gates. All the proteins found in the nucleus are synthesized in the cytoplasm - histones, ribosomal proteins, DNA and RNA polymerases, Transcription factors. The proteins that are imported into nucleus are in fully folded state and do not require any chaperones. Proteins imported into nucleus have targeting signal sequences on them which are called nuclear localization signals (NLS). Each one has 4-8 amino acids and they are internal sequences and not terminal. NLS is not cleaved from the protein. Due to this feature proteins can re-enter the nucleus whenever the nuclear envelope is lost during cell division.

what is the process of translation of membrane and excretory proteins? what is protein sorting and targeting? what are targeting signals and what do they do? difference b/t post-translational and co-translational targeting? (50)

Protein targeting or protein sorting is the biological mechanism by which proteins are transported to the appropriate destinations in the cell or outside of it. Proteins can be targeted to the inner space of an organelle, different intracellular membranes, plasma membrane, or to exterior of the cell via secretion. This delivery process is carried out based on information contained in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases. Proteins destined for cytoplasm and those to be incorporated into mitochondria and nuclei are synthesized on free ribosomes in the cytoplasm. Proteins destined for cellular membranes, lysosomes and extracellular transport, use a special distribution system. The main structures in this system are the rough endoplasmic reticulum (RER) and Golgi complex. Proteins to be directed to their destinations via Golgi complex are synthesized by ribosomes associated with endoplasmic reticulum. Targeting signals: Protein sorting requires proper address labels which are in the form of peptide signal sequences. A signal sequence that directs the protein to its target is present in the form of 13-35 amino acids in the newly synthesized protein itself. It is the first to be synthesized and is mostly present at the amino N-terminal, sometimes at the carboxyl C- terminal. It is known as signal sequence or leader sequence. *N & C Terminals - N terminal refers to the start of a protein or polypeptide terminated by an amino acid with a free amine group. C Terminal - also known as carboxy-terminal - is the end of an amino acid chain terminated by a free carboxyl group. Proteins to be transported carried inside a membrane bound vesicles. These proteins are called cargo proteins. An embedded or integrated protein is carried in the membrane of the vesicle, while secretory protein is carried within the lumen of the vesicle. The vesicle buds off from the donor surface and fuses with the target surface, releasing its contents into the target organelle and the membrane protein is incorporated into the membrane of the target organelle. The process is repeated during the passage of protein from ER to Golgi to lysosomes and from Golgi to plasma membrane. transmembrane proteins: Functions of receptors, transmembrane channels, surface antigens. They are synthesized in GER, released and transported to membranes. Their incorporation and anchoring allows for anchoring topogenic sequences, forms an α-helix, and anchors the protein in the phospholipid bilayer. Transmembrane proteins thus have 2 signal sequences - leader and anchor the leader sequence becomes part of the GER membrane as it passes and fixes the nascent protein in the membrane;the location of the sequences on the polypeptide determines the length of the extra- and intracellular portions of the protein. Post-translational targeting - proteins that destined to the nucleus, mitochondria and peroxisomes. Co-translational targeting - from the free floating ribosomes into the ER through translocon 🡪 golgi 🡪 lysosomes, plasma membrane or proteins to be secreted.

what are proto-oncogenes? general characteristics? -( mutation effect, number of mutant alleles, role in a cell, examples??) what are oncogenes? what are the types? what (111)

Proto-oncogene is simply a normal gene found in the organism that could become an oncogene due to mutations or increased expression; i.e. Oncogenes are the altered forms of normal genes - proto-oncogenes. - **proteins that act pro-mitotically - inhibit apoptosis - stimulates cell proliferaiton - gain of function mutations, 1 allele - GFs and their receptors, adapters, transducers, kianses, TFs Proto-oncogenes code for prunctions of Proto-oncogenes: Cancers have characteristics that indicate loss of the normal function of proto-oncogene that then referred to oncogene. They have a role control of cellular proliferation and differentiation, in the process of signal transduction: complex multistep pathway from the cell membrane 🡪 cytoplasm 🡪 nucleus, involve variety of types of proto-oncogene product involved in positive and negative feedback loops necessary for accurate cell proliferation and differentiation. (Signal transduction - question 23) Which type of mutations? Mutations in proto-oncogene that transform it into oncogene may be due to: Point mutation Chromosomal translocation: translocation events which relocate a proto-oncogene to a new chromosomal site that leads to higher expression; translocation events that lead to a fusion between a proto-oncogene and a second gene (chimeric genes) e.g. Philadelphia chromosome Gene amplification - production of multiple copies of the times, leading to greater amounts of the corresponding onco-protein product. Chimeric genes - form through the combination of portions of two or more coding sequences to produce new genes Mutations in the anti-proliferative tumor-suppressor genes, which always occur in the genome in 2 copies - thus mutation needed the intervention of both copies (two-hit hypothesis). Mutations in proto-oncogenes, from the other hand, is dominant characters - means that mutation of a single copy of a proto-oncogene turns it into oncogene and it is enough to produce symptoms. Types of oncogenes - growth factors, receptors, cell-cycle factors, apoptosis factors signal molecules (ligands) receptors FGFR - fibroblast growth factor EGFR - epidermal growth facctor ErbB-1; HER1 HER2/neu subfamilies of closely related receptors tyrosine kinases adapters Sos, Grb transducers Ras kinase cascades MAPK cascade transcription factors promitotic stimulation - tyrosine kinase receptors Growth factors - also known as Mitogens - the transition of a cell from G0 to the start of the cell cycle is governed by growth factors. Growth factors stimulate cells to grow by binding to GFR (growth-factors receptors). The best known oncogene that acts as a growth factor called SIS. Mutation in gene coding for growth factors may lead to non-appropriate start of cell cycle. Growth factors can be, for example, VEGF (vascular endothelial growth factor), NGF (nerve GF), PDGF (platelet-derived), FGF (fibroblast GF). Many oncogenes encode proteins that form growth factor receptors with tyrosine kinase activity - mutations, rearrangement and amplification of the oncogenes encode for these receptors may result in ligand-independent activation 🡪 signal transduction that occur without signal arrival 🡪 improper gene expression. Mutation in each component of the pathway - from tyrosine-kinase receptor activation till the formation of cyclins. E.g. growth factors pathway through MAPK pathway (GRB2 🡪 SOS 🡪 Ras 🡪 Raf 🡪 MEK 1/2 🡪 ETK 🡪 Fos, Jun, myc) - each one of them may act as oncogene if produced in high amount or non-responsive for inhibition (Ras inactivation by GTPase - in many cancer can be mutated and not-responsive). Cell-cycle factors - cancer cells can increase in number by increased growth and division, or accumulate through decrease apoptosis. The progress through the cell cycle is regulated at the check-points, through CDK factors; abnormalities in regulation of inhibitory factors lead to activation of the CDK resulting in cellular transformation with uncontrolled cell division. E.g. Overproduction of cyclins or CDKs, over-production of E2F, overproduction of MDM2 (accounts for inhibition of P53). Telomerase as an oncogene - mutation in the gene encoding for telomerase; Telomerase is an enzyme found in stem cells and responsible for preventing the natural process of telomere shortening in dividing cells - eventually results in their inability to replicate. Most cancer cells has mutation that activate the telomerase genes 🡪 enabling them unlimited number of replications and proliferations.oteins that help to regulate cell growth and differentiation that upon acquiring an activating mutation it becomes a tumor-inducing agent - an oncogene. Currently 40 types of oncogenes have been identified. Currently there are 40 kinds of known proto-oncogenes; 16 of which have been demonstrated a direct correlation with tumor proliferation; examples: K-Ras - tumors of esophagus, colon, pancreas Cyclin E - liver tumors Her-2 - breast tumors When a virus infects a cell, a proto-oncogene may become incorporated into the viral genome through recombination. Within the viral genome, the proto-oncogene may mutate to an oncogene that, when inserted back into a cell, causes rapid cell division and cancer. Because the proto-oncogenes are more likely to undergo mutation or recombination within a virus, viral infection is often associated with cancer. Philadelphia chromosome: The Philadelphia chromosome or Philadelphia translocation is a specific genetic abnormality in chromosome 22 of leukemia cancer cells (particularly CML - chronic myeloid leukemia). This chromosome is defective and unusually short because of reciprocal translocation of genetic material between chromosome 9 and chromosome 22, and contains fusion (chimeric) gene called BCR-ABL1. ABL1 gene on chromosome 9 juxtaposed onto the BCR gene on chromosome 22, coding for hybrid protein (chimeric gene): a tyrosine kinase signaling protein that is always on, causing the cell to divide uncontrollably.

how can gene expression at mRNA level be regulated (51)

Regulation by processing of mRNA - mRNA modifications before it is exported from the nucleus to the cytoplasm - capping at the 5' end, polyadenylation tail at the 3' end and splicing - variations of these events can affect gene expression. Splice-site choice - differential co-transcriptional processing, particularly the use of alternative splice sites, may change the proteins to be synthesized. Over 60% of the approximately 25,000 genes in the human genome undergo differential splicing. The use of alternative polyadenylation and transcription start sites is also seen in many genes - this explains how 25,000 genes can give rise to different much larger amount of proteins. mRNA editing - even after mRNA has been processed, it may undergo additional post-transcriptional modification in which a base in the mRNA is altered, in a process known as RNA editing. mRNA stability - how long an mRNA remains in the cytosol before it is degraded influences how much protein product can be produced from it. E.g. Iron metabolism - transferrin is a plasma protein that transports iron. Transferrin binds to cell-surface receptors that get internalized and provide iron to erythroblasts. The mRNA for the Transferrin receptors has several cis-acting iron-responsive elements (IREs) at its 3'-end. When the iron concentration in the cell is low, the iron regulatory proteins (IRPs) bind to the iron responsive elements and stabilize the mRNA for these transferrin receptors, allowing prolonged synthesis of these receptors. RNA Interference (RNAi) is another mechanism which controls gene expression - it causes gene silencing through decreased expression of mRNA, either by repression of translation or by increased degradation. RNAi is mediated by short noncoding RNAs called microRNAs (miRNA) or by siRNA (dsRNA) - mediated by interaction with protein from Argonaute family. Translation of mRNA - regulation of gene expression can also occur at the level of translation. One mechanism by which translation is regulated is through phosphorylation of the translation initiation factor - eIF-2. Phosphorylation of eIF-2 inhibits its function and so inhibits translation at the initiation step. Phosphorylation is catalyzed by kinases that are activated in response to environmental conditions, such as amino acid starvation, heme deficiency in erythroblasts etc.

how can gene expression be regulated at the DNA level? (51)

Regulation through modification to DNA Gene expression is also influenced by the availability of DNA to the transcriptional apparatus, the amount of DNA, and the arrangement of DNA. Access to DNA - DNA is found complexed with histone and nonhistone proteins to form chromatin. Transcriptionally active, light stained, decond ensed chromatin (Euchromatin) differs from the more condensed, inactive form (heterochromatin). Active chromatin contains histone proteins that have been covalently modified at their amino terminal ends by an addition of acetyl residue (post-translation modification) by Histone Acyltransferase - such modifications decrease the positive charge of these basic proteins, thereby decreasing the strength of their association with negatively charged DNA. This relaxes the nucleosome, allowing transcription factors access to specific regions on the DNA. Amount of DNA - a change up or down in the number of copies of a gene can affect the amount of gene product produced. Arrangement of DNA - A process which allows the generation of different type of proteins from a single gene - providing diversity. E.g. the process by which antibodies (immunoglobulins) are produced by B lymphocytes involves permanent rearrangements of the DNA in these cells. Ig's consist of two light and two heavy chains, with each chain containing regions of variable and constant amino acid sequence. The variability is the result of somatic recombination of segments within both the light and heavy chain genes, providing diversity needed for the recognition of an enormous number of antigens. Mobile DNA elements - Transposons are mobile segments of DNA that move in an essentially random manner from one site to another on the same or a different chromosome. Movement is mediated by Transposase, an enzyme encoded by the transposon itself. Movement can be direct, in which transposase cuts out and then inserts the transposon at a new site (CUT AND PASTE) or replicative - in which the transposon is copied and the copy inserted elsewhere while the original remains in place (COPY AND PASTE). Replicative transposition frequently involves an RNA intermediate - in which cases of RNA intermediate needed, the transposon is called retrotransposon. Transposition has contributed to structural variation in the genome, but also has the potential to alter gene expression and even to cause disease. Although the vast majority of retrotransposons in the human genome have lost the ability to move, some are still active

postnatal screening of heritable diseases? postnatal prevention and therapy of heritable and inborn diseases? what is screening? what is the criteria? what are some examples? what are the required tests done in CZ? (126) (132)

Screening = systematic targeted search of certain diseases, before the clinical manifestation of them, in the effort to prevent its possible effects. Newborn screening is a public health program of screening in infants shortly after birth for a list of conditions that are treatable, but not clinically evident in the newborn period. This type of screening is referred to as "all-over" screening (in contrast to "selective" - which only high-risk population is screened). Some of the conditions included in newborn screening programs are only detectable after irreversible damage has been done, in some cases sudden death is the first manifestation of a disease. Most newborn screening tests are done by measuring metabolites and enzyme activity in whole blood samples collected on specialized filter paper ("screening card"), however many countries are starting to screen infants for hearing loss using automated auditory brainstem response and congenital heart defects using pulse oximetry (noninvasive method for monitoring a person's oxygen saturation (SO2). Infants who screen positive undergo further testing to determine if they are truly affected with a disease or if the test result was a false positive. Follow-up testing is typically coordinated between geneticists and the infant's pediatrician or primary care physician. Criteria for screening: The disease is diagnosable, and in case of delayed treatment - the baby may be affected severely Disorder has sufficient incidence in the population There is an effective treatment There is screening test with high sensitivity and sufficient specificity Test is reasonable cheap (favorable cost/benefit ratio) Newborn screening programs have been introduced on a widespread basis for phenylketonuria, galactosemia , congenital hypothyroidism, hemoglobinopathies, cystic fibrosis and more. In all of these disorders early treatment can dramatically prevent the development of learning disability. In each country there are different rules regarding which diseases are screened (but, commonly, all screen for phenylketonuria, galactosemia and hypothyroidism). All neonates born in the Czech Republic are subjected to neonatal laboratory screening (NLS) of congenital or inherited diseases listed below by the method of taking a so-called dry drop of blood on a neonatal screening card between 48 and 72 hours of age. The goal of neonatal screening is rapid diagnosis and early treatment of newborns with these diseases. hyperphenylalaninemia and phenylketonuria ; method: tandem mass spectrometry; congenital hypothyroidism ; method: determination of thyroid stimulating hormone (TSH) by fluoroimmunoassay (FIA); congenital adrenal hyperplasia ; method: determination of 17alpha-OH-progesterone by immunoassay methods; cystic fibrosis ; method: determination of immunoreactive trypsinogen (IRT) level by immunoassay method; selected inherited metabolic disorders : method: tandem mass spectrometry. Phenylketonuria - PKU is an inborn error of metabolism that results in decreased metabolism of the amino acid phenylalanine. Mal-metabolism of phenylalanine 🡪 hyperphenylalaninaemia 🡪 damages the CNS and affects the mental development. Incidence - 1/10000. Routine biochemical screening of newborn infants for phenylketonuria was recommended after it had been shown that a low-phenylalanine diet could prevent the severe learning disabilities that previously had been a hallmark of this condition. Most affected children are persuaded to adhere to the low-phenylalanine diet until early adult life. One method of testing is checking phenylalanine levels after the child received milk-diet which contains high enough phenylalanine. Level of phenylalanine above 20mg/dl considered as PKU, reduced level of tyrosine (because PKU persons cannot synthesize tyrosine from phenylalanine). The most common screening test is called "Guthrie test", and it is carried out on a small sample of blood obtained on the filter paper disc. The filter paper is put in bacterial medium and competitive growth inhibitor - evaluation of the bacterial growth (which grown in the presence of phenylalanine in the blood spot). Woman with phenylketonuria should adhere to strict low-phenylalanine diet before and during pregnancy because high phenylalanine levels are toxic to the developing brain. Galactosemia - a rare genetic metabolic disorder that affects an individual's ability to metabolize sugar galactose properly. Classic galactosemia affects 1/50,000 newborn infants and usually presents with vomiting and severe metabolic collapsing within the first 2-3 weeks of life. Newborn screening is based on a modification of the Guthrie test with subsequent confirmation by specific enzyme assay. The early introduction of appropriate dietary restriction can prevent the development of serious complications such as liver failure, learning disability and cataracts. Congenital Hypothyroidism -It is inadequate thyroid hormone production n newborn infants due to anatomic defect in the gland, iodine deficiency or inborn error of thyroid metabolism. The test is based on examining of either thyroxine or thyroid-stimulating hormone. It is relatively common - 1/4000 incidence - thus, suitable for screening. The treatment with lifelong thyroxine replacement is extremely effective in preventing the developmental problem associated. Cystic Fibrosis - it is a genetic disorder (AR) which caused by the presence of mutations in both copies of the gene for cystic fibrosis transmembrane conductance regulator (CFTR protein), chloride transporter - which is involved in production of sweat, digestive fluids and mucus. When CFTR is not functional, secretions which are usually thin - becomes thick. Newborn screening is based on the detection of a raised blood level of immunoreactive trypsin, which is a consequence of blockage of pancreatic ducts in the uterus (symptoms of CF). The rationale for screening is that early treatment with physiotherapy and antibiotics improve the long-term prognosis. Sickle cell disease and Thalassemia - Sickle cell disease and thalassemia are genetic disorders caused by errors in the genes for hemoglobin (in sickle cell - fragile Hemoglobin S is formed because of point mutation, in thalassemia, the hemoglobin polypeptide chains are not produced fully). Newborn screening based on hemoglobin electrophoresis. Early prevention may reduce morbidity and mortality. In the case of sickle cell disease - treatment involves the use of oral penicillin, in order to reduce the risk of infection resulting from immune deficiency, because even in western countries with good medical facilities, a significant proportion of affected babies die as a result of infection in early childhood. Congenital adrenal hyperplasia - any of several AR diseases resulting from mutations of genes for enzymes mediating the biochemical steps of production adrenal gland hormones from cholesterol (mineralocorticoids, glucocorticoids and sex hormones). Other conditions for screening - inborn errors of metabolism of amino acids and fatty acids. Adult general population screening is mainly focused on diseases of the circulatory system and certain types of cancer.

what is Southern blotting? (54)

Southern Blotting technique allows to find and examine a number of DNA fragments of interest from a collection of millions restriction enzyme fragments. It is the standard method for examining particular fragments of DNA cleaved by restriction enzymes. DNA is isolated usually from lymphocytes (samples can be taken from other sources, like skin fibroblasts, amniotic fluid or chorionic villus cells for prenatal diagnosis or biopsy samples from liver, kidney placenta etc.). The DNA fragments are put into gel electrophoresis and are separated on the basis of size (small fragments move through an electric field more rapidly and farther away than do larger ones). The separated DNA is stained with a fluorescent DNA dye. The smear of double-stranded DNA fragments is denatured with a strong base to separate the two complementary DNA strands; the now single-stranded DNA molecules are then transferred from the gel to a piece of filter paper by blotting To identify the one or more fragments of interest among the millions of fragments on the filter, a single-stranded labeled probe is incubated with the filter under conditions that favor formation of pairing of complementary double-stranded DNA molecules. Then, the unbound molecules are washed away. The filter (with its bound radioactive probe) is exposed to x-ray film to reveal the position of the fragments to which the probe hybridized. The pattern of fragments containing sequences complementary to the probe generated with each restriction enzyme is revealed.

what is teratogenesis? when is the most critical period? what are some types of deformities? what are teratogens? how are they classified? what about maternal diseases? what are some specific effects that need to be taken into account regarding teratogenesis? (98)

Teratogenesis is a process leading to the formation of a congenital anomaly; the production of deformity in the developing embryo, called also teratogeny. Teratogens generally refer to external factors that are able to give rise to congenital malformations. Like mutagens, teratogens can be divided into 3 main groups - biological, chemical or physical nature. (Teratogen - a substance which can cause physical defects in a developing embryo whereas mutagen is a material that induces genetics changes - mutations - in the DNA) first two weeks are completely cell from teratogens exposition during the 1st trimester has the most severe consequences process leading to the formation of congenital anomaly long and complex process depends on species gestation age dose of teratogen genotype of fetus and mother Congenital anomaly, also known as birth defects and congenital malformations, is a term used to describe functional, structural, behavioral and metabolic disorders present at birth. In 40-50% of the defected persons the cause is unknown. Genetic factors (such as chromosome abnormalities and mutant genes) account approximately to 28% Environmental factors - 7-10% Combination of the factors - 20-25% There are several types of deformities (question 100): Malformations - during formation of structures - may result in complete or partial absence of a structure Disruptions - morphological alterations of already formed structures which caused by destructive processes such as defects produced by amniotic bands Deformations - result from mechanical forces that mold a part of the fetus over a prolonged period; clubfeet, for example, are caused by prolonged compression in the amniotic cavity. Syndrome - a group of anomalies occurring together and have a specific determined common cause Association - non-random appearance of 2 or more anomalies that occur together more frequently than by chance alone (associated to each other; linked anomalies). Teratogens divide into 3 groups: *not all teratogens are mutagens!! Biological nature - infectious agents such as viruses (rubella, herpes, HIV, influenza etc.), bacteria (syphilis), parasite (toxoplasmosis, caused by toxoplasma gondii). The highest risk to the fetus is a primary infection of the mother and fetus during early pregnancy. infectious congenital rubella (Gregg syndrome) microcephaly PDA (patent ductus arteriosus) cataracts TORCH toxoplasma other (HIV Varicella, syphilis and Parvovirus B19) rubivirus cytomegalovirus herpesvirus Chemical nature - such as number of substances used in industry and agriculture such as organic solvents, heavy metals. Important groups are drugs and medicines - such as drugs that cause cytostatic (cancer treatment), antibiotics, anticonvulsants, warfarin, ACE inhibitors. Antibiotics, such as tetracyclines, can cross the placental barrier and deposits on the sites of active calcification - bones and teeth, may result in diminished growth of long bones, discoloration of the teeth and more. Alcohol - ethanol, whose using during pregnancy may cause fetal alcohol spectrum disorder, which its extreme is FAS - fetal alcohol syndrome - involves defects of the head (microcephaly), eyes (shortened palpebral fissures), mouth (orofacial clefts), face (short nose, thin upper lip), joint defects and more. Smoking - nicotine, a chemical present in smoke, is a vasoconstrictor and this role on uterine blood vessels can lead to diminished supply of nutrients and oxygen to the fetus - this in turn can cause chronic fetal hypoxia. Anticoagulants - warfarin can cross the placental barrier (in contrast to heparin), it is inhibitor of Vitamin K reductase; the critical period of warfarin is 6-12 weeks during which it can cause hypoplasia (underdevelopment) of the nasal cartilages and CNS defects. Physical nature - ionizing radiation such as X-ray and gamma-radiation - they can cause death of embryonic cells, chromosome abnormalities, mental deficiency and deficient physical growth. The severity is related to absorbed dose, dose rate and developmental stage. Large doses of ionizing radiation can cause anencephaly, spina bifida and microcephaly, depending on the stage of the fetal development. High temperatures (hyperthermia) can cause CNS damage. Mechanical Teratogen, a physical teratogen, may cause deformations. One of the functions of the amniotic fluid is to act as a cushion and a support medium for the embryo - reduction in amounts of amniotic fluid in some pathological cases induced deformations, such as deformations of the limbs because of limited mobility of the embryo's, which is necessary for limbs and musculature development. Maternal diseases: diabetic mothers have 40% increased chance of teratogenic effects - neonatal deaths, abnormally large infants, congenital malformations. Evidence suggests that altered glucose levels play a role (rather than insulin levels). Thus, strict control of maternal glucose levels beginning before conception and throughout gestation reduces the occurrence of malformation. Mother with PKU - at risk for having infants with intellectual disability The factor which causes birth defects and the effects of certain teratogen on causing abnormality depends on: Susceptibility of the embryo genome to a certain teratogen; as well as the maternal genome, which is also important with respect to drug metabolism (mothers with PKU may bring offspring with mental retardation as a result of increased phenylalanine concentration) Susceptibility to certain teratogen varies with the developmental stage at the time of exposure - the most sensitive period is the 3rd-8th weeks - the period of embryogenesis The birth defect depends on the dose and duration of exposure to the teratogen Each teratogen act in a specific mechanism on developing cells and tissues - for example, inhibition of a specific biochemical or molecular process Four manifestations possible - death, malformation, growth retardation and functional defect. Specifics effect The effect of teratogens is complex and the simplification mutagen = teratogen is definitely not valid. Several specificities need to be taken into account in the action of teratogens: Factor doses The dose of the teratogenic agent is often critical. Low doses of teratogen may not cause a birth defect at all, they may cause a milder disability, or even another type of defect. The time factor The sensitivity to the effect of individual teratogens is not the same throughout pregnancy. In general, the effect of teratogens during the first trimester of pregnancy has the worst prognosis , but the effect of teratogens in the second and third trimesters also has an adverse effect. Within individual teratogens, the time factor is applied as a "critical period" during which the fetus is sensitive to a particular teratogen, or when an organ / system develops - the development of which is adversely affected by the effect of the teratogen. Applying the same dose of the same teratogen at different stages of pregnancy can have significantly different effects. The "All or Nothing" rule is the reaction of the early stages of the embryo (during the period of embryogenesis ) to the action of teratogens. Congenital malformations do not occur during this period - the embryo can either repair all the damage ad integrum - or it will disappear. In the following period - organogenesis - the action of teratogens already causes developmental defects. Factor of genetic and species Sensitivity to the action of individual teratogens is also affected by the genetic makeup of a particular individual. Although within one species this variability may not be significant - interspecies variability may be significant. This is particularly important in relation to testing the teratogenic effects of drugs and chemicals in laboratory animals, as the same dose of the same teratogens can be a major teratogen in humans but not in the species used laboratory animal (resistance of mice to the teratogenic effects of thalidomide was one of the reasons for the outbreak of thalidomide affair ).

structure of prokaryotic chromosomes? what is their importance? (85)

The genome of a prokaryote (prokaryote = before nucleus) is structurally different from a eukaryotic genome and in most cases has considerably less DNA. Prokaryotes generally have circular chromosomes, whereas eukaryotes have linear chromosomes. In addition, in prokaryotes, the chromosome is associated with many fewer proteins than are the chromosomes of eukaryotes; prokaryotes also lack nucleus - their chromosome is located in the nucleoid, a region of cytoplasm that is not enclosed by a membrane. Chromosome consists of double-stranded circular DNA. Usually the size of the chromosome ~200kb. Bacterial mRNA is generally polycistronic - means that it can encode more than one polypeptide separately within the same RNA molecule (eukaryotes mRNA is monocistronic - one gene per one mRNA molecule) Plasmid - In addition to its single chromosome, a typical prokaryotic cell may also have much smaller rings of independently replicating DNA molecules called plasmids - mostly carrying only few genes - usually non-essential genes. Plasmids ranged in size between 1.5-20kb. Organization: The genome of prokaryotes is often significantly larger than the cell itself. How is it possible that the genetic information does fit into the cell? Eukaryotes solve this problem by wrapping DNA around the histones. However, prokaryotes do not contain histones (with a few exceptions). To compress their DNA, Prokaryotes using fiber rolled into small rolls - supercoiling. The fibers are twisted so tightly that the final consequences loops overlap to form one big ball. Distinguishes two types of collapse - positive (DNA turns are in the same direction as the helix) or negative (DNA is coiled in the opposite direction than the helix). Most bacteria during normal growth are negatively coiled. Prokaryotes reproduce asexually most often and are haploid (there is always only one copy of the gene). Prokaryotes often contain several plasmids (extrachromosomal stored DNA molecule - linear or circular). Unlike chromosomal DNA, plasmid DNA is typically smaller and encodes genes that are not necessary for survival. Often, however, give to the cell some advantage (eg resistance to antibiotics). Replication of plasmids is independent of chromosomal replication. Prokaryotes need to cram all their genes within one chromosome, so it doesn´t remain too much space for the non-coding sequences. While in eukaryotes, the share of non-coding parts of DNA is about 98%, in prokaryotes it is only 12% Methylation of bacterial DNA: The DNA of most bacteria contains methylated adenine or cytosine nucleotides. The methylation is carried out by a methyltransferase enzyme and it serves the bacteria in cases of injection by bacteriophages (a virus that infects and replicates within a bacterium). Bacteriophages insert their DNA into the bacterial DNA, and thanks to the methylation, restriction enzymes can recognize which DNA belongs to the bacteria and which is a foreign DNA segment. After recognition, the enzyme cleaves the DNA at a restriction site and getting rid of the foreign DNA. GC Islands - the bacterial chromosome contains regions known as pathogenic islands. The differ from the rest of the chromosome in their G+C content, and are usually surrounded by repeated sequence genes that encode for tRNA's. Transponsons - The bacterial DNA consist of transponsons - which are DNA segments that able to jump from one location in the DNA to another. Importance of Bacterial chromosome: Disadvantageous for humans - the ability of the bacteria to pass or acquire genes from other cells (even dead cells) provide the bacteria with survival advantage, which may be very disadvantageous for human. For instance, a non-pathogenic bacterium may acquire pathogenic islands from other bacteria of the same species, and it will become pathogenic as well. In this manner, bacteria that are found normally in our body and aren't harmful, may turn to be harmful by the presence of another pathogenic bacteria that invaded our body and transform its genes. In addition, pathogenic bacteria may acquire survival properties such as antibiotic property, or may acquire a more hazardous properties that will cause more damage to their host. Advantageous for humans - in genetic engineering - there is a wide usage of plasmids as vectors, and in bacteria as a host-cell that proliferate and produce many copies of wanted genes. It is useful for research, diagnostic and therapies (production of insulin).

what are the steps of eukaryotic translation? what is the wobble hypothesis? how is translation regulated? (45)

The Wobble Hypothesis explains why multiple codons can code for a single amino acid (61 codons, 20 amino acids). One tRNA molecule (with one amino acid attached) can recognize and bind to more than one codon, due to the less-precise base pairs that can arise between the 3rd base of the codon (mRNA) and the base at the 1st position on the anticodon (tRNA). The process of protein synthesis translates the 3-letter alphabet of nucleotide sequences on mRNA into the 20-letter alphabet of amino acids that constitute proteins. In eukaryotes, each mRNA has 1 coding region for 1 protein - monocistronic RNA. Initiation - involves the assembly of the components of the translation system before peptide bond formation occurs. These components include the two ribosomal subunits, the mRNA to be translated, the aminoacyl-tRNA specified by the first codon in the message (Start codon), GTP (Which provides energy for the process), and initiation factors that facilitate the assembly of this initiation complex - eIF's (IF - initiation factors), bound to the small ribosomal subunits. Once the pre-mRNA finished its maturation process and become mRNA, it gets out from the nucleus through the nuclear pores. The small ribosomal sub unit binds to the 5'-end of the mRNA by recognizing the 5 capping head, and slides on it - till it reaches to the starting codon (AUG). The scanning process requires ATP. The Kozak consensus sequence plays a major role in the initiation of the translation process. Then, attachment of the large sub-unit occurs, forming a functional ribosome with charged initiation tRNA at its P site, and empty A site. Elongation - Elongation depends on eukaryotic elongation factors eEF. At the end of the initiation step, the mRNA is positioned so that the next codon can be translated during the elongation stage of protein synthesis. The initiator tRNA occupies the P site in the ribosome, and the A site is ready to receive an aminoacyl-tRNA. Translocation - after the formation of the peptide bond (between the amino acids in the P site and A site) by an enzyme peptidyl transferase - the amino acid chain that was attached to the P site is moved and now linked to the amino acid at the A site. The ribosome advances 3 nucleotides towards the 3' end - causes the empty tRNA to move to the E site (exit site), and the polypeptide chain to the P site. The A-site is empty, ready for the next tRNA molecule - next triplet codon. Termination - termination occurs when one of the three termination codons move into the A site (UAA, UGA, UAG). The recognition of stop codon is done by releasing factor - eRF - recognizes the termination codon, binds to it, and causes the hydrolysis of the bond linking the peptide chain to the P-site tRNA. Recycling of the synthesis factors - after termination, the ribosome subunits, tRNA, rRNA and protein factors can be recycled. Polysomes - a complex of 1 mRNA and more than 1 ribosome attached to it, translated simultaneously. Iron metabolism - transferrin is a plasma protein that transports iron. Transferrin binds to cell-surface receptors TfR that get internalized and provide iron to erythroblasts. The mRNA for the Transferrin receptors has several cis-acting iron-responsive elements (IREs) at its 3'-end (non-coding region of the mRNA, i.e. - beyond the termination codons UAA UAG UGA). When the iron concentration in the cell is low, the iron regulatory proteins (IRPs) bind to the iron responsive elements and stabilize the mRNA for these transferrin receptors, allowing prolonged synthesis of these receptors. RNA Interference (RNAi) is another mechanism which controls gene expression - it causes gene silencing through decreased expression of mRNA, either by repression of translation or by increased degradation. RNAi is mediated by short noncoding RNAs called microRNAs (miRNA - ssRNA) - which mainly act on the 3-UTR regulatory elements, and either cleaved the Poly A tail (decrease stability) or just make the translation activity less efficient. Another RNAi is siRNA (dsRNA) - mediated by interaction with protein from Argonaute family - binds complementary mRNA and induce its degradation.

what is RNA? what are the types? what is the structure and their functions? (42)

The genetic master plan of an organism is contained in the sequence of deoxyribonucleotides in its DNA. However, it is through the ribonucleic acid - RNA - the "working copies" of the DNA, that the master plan is expressed. The copying process, during which a DNA strand serves as a template for the synthesis of RNA, is called transcription. Transcription produces one of the types of RNA - Messenger RNA (mRNA) - that are translated into sequences of amino acids (which are polypeptide chains - compose proteins). Transcription of DNA also produces other types of RNAs: ribosomal RNAs (rRNA), transfer RNA (tRNA) and additional small RNA molecules that perform specialized structural, catalytic, and regulatory functions are aren't translated - non-coding RNAs (ncRNAs); only about 2% of the genome codes for proteins. The final product of gene expression, therefore, can be RNA or protein, depending upon the transcripted gene. There are three major types of RNA that participate in the process of protein synthesis. Each is transcripted by different polymerase. They differ from each other in size, function and special structural modifications: rRNA tRNA mRNA Like DNA, these types of RNA are unbranched polymeric molecules, composed of nucleoside monophsphates joined together by 3' 🡪 5' phosphodiester bonds. However, they differ from DNA in several ways: They are considerably smaller the DNA They contain ribose instead of deoxyribose They contain different pyrimidine - uracil instead of thymine. They exist as single strands rRNA - Ribosomal RNA rRNAs are found in association with several proteins as components of the ribosomes - the complex structures that serve as the sites for protein synthesis. rRNA make up about 80% of the total RNA in the cell. Ribosomes contain two major rRNA and 50 or more associated proteins. The ribosomal RNAs form two subunits - large and small subunits. The large rRNA subunit serves as catalyzer of the translation process - RNA with catalytic activity is termed ribozyme. rRNA is formed in the nucleolus and combines with appropriate proteins in the nucleolus to form the ribosomal subunits (40S and 60S). tRNA - Transfer RNA tRNAs are the smaller of the three major types of RNA molecules. There is at least one specific type of tRNA molecule for each of the 20 amino acids commonly found in proteins. Together, tRNAs make up about 15% of the total RNA in the cell. Each tRNA serves as an "adaptor" molecule that carries its specific amino acid, covalently attached to its 3'-end, to the site of protein synthesis. There it recognizes the genetic code sequence on an mRNA, which specifies the addition of its amino acid to the growing peptide chain. The common features of tRNA molecules allow them to bind to the binding sites on ribosomes (TC arm - ribosome binding site) and to mRNA. The Anticodon arm base sequence of tRNA molecules varies which causes some variable features in its structure. The variable features give each type of tRNA a distinctive 3D shape. This allows the correct amino acid to be attached to the 3' terminal by an enzyme called tRNA-aminoacyl synthetase. There are 20 different tRNA activating enzymes - one for each of the 20 different amino acids. Energy from ATP molecule is needed for the attachment of amino acids. A high-energy ester bond is created between the amino acid and the tRNA. D-loop ;according to the dihydrouracil content. Anticodon loop ;contains a trio of bases complementary to the codon of a given AMK;allows the inclusion of the AMK-tRNA complex in the correct place in proteosynthesis. V-loop ;variable, differs in both size and ranked bases between tRNA molecules for different AMKs. Dog loop (ψ) ;according to the pseudouridine content. mRNA - Messenger RNA mRNA, also known as coding RNA, comprises only about 5% of the RNA in the cell, yet is by far the most heterogeneous type of RNA in size and base sequence. It carries genetic information from DNA for use in protein synthesis. This involves transfer of mRNA out of the nucleus and into the cytosol. In addition to the protein-coding regions that can be translated, mRNA contains untranslated regions (UTR) at its 5'- and 3' ends, long sequence of adenine nucleotides (poly A tail) on the 3'-end of the RNA chain, plus a "cap" on the 5'-end consisting of a molecule of 7-methylguanosine MicroRNA (miRNA) - functions in RNA-silencing and post-transcriptional regulation of gene expression- they function via base-pairing with complementary sequence within mRNA molecules. As a result, these mRNA molecules are silenced, by either: Their cleavage into pieces destabilization through shortening of the poly A tail in the 3' end Induce less efficient translation of the mRNA. They are similar to siRNA - small-interference RNA. But in the case of siRNA, they are double-stranded rather than single stranded. They interfere with mRNA expression by elicit their degradation. Small nucleolar RNA (snoRNA) - guides modification of RNA molecules, especially rRNA. After transcription of rRNA, they are nascent (similar to premature mRNA before splicing: pre-rRNA - and undergo processing steps to generate the mature rRNA: nucleoside modifications such as methylation, which is guided by snoRNA. Small Nuclear RNA (snRNA) - accounts for the splicing process of pre-mRNA.

what is the immune response? what are the types of immunity? what are the difference b/t innate and adaptive immunity? what is antigen recognition? what is cell-cooperation? (77)

The immune system contains a number of different components and uses several mechanisms to provide protection against pathogens, but most immune responses can be grouped into two major classes: humoral and cellular immunity. Also it is convenient to think of these classes as separate systems, they interact and influence each other significantly. Humoral immunity centers on the production of antibodies activated B-cells - plasma cells - antibodies circulate in the blood and other body fluids, binding to specific antigens and marking them for destruction by phagocytic cells. Antibodies also activate as set of proteins called complement that help to lyse cells and attract macrophages. Cellular immunity is conferred by T-cells. After a pathogen such as a virus has infected a host cell, some viral antigens appear on the cell membrane. Proteins, called T-cell receptors, on the surfaces of T-cells, bind to these antigens and mark the infected cell for destruction. T-cell receptors must simultaneously bind a foreign antigen and self-antigen (MHC). The immune system recognize an almost unlimited number of foreign antigens, even though each mature B-cell produces antibodies against a single antigen, and each T cell is capable of attaching to only one type of foreign antigen. If each lymphocyte is specific for only one type of antigen, how does an immune response develop? - Clonal selection - when a foreign protein enters the body, only a few lymphocytes will be specific for this particular antigen, but when binding occurs, that lymphocyte is stimulated to divide and proliferates, producing a large population of identical cells (clone) each of which is specific for that particular antigen. This initial proliferation is known as a primary immune response. Subsequently, most of the lymphocytes in the clone die, but a few continue to circulate as memory cells. When these memory cells encounter the same antigen, they quickly give rise to another clone of cells - Secondary immune response. This secondary immune response is the basis for vaccination - which stimulates a primary immune response to an antigen and results in memory cells that can produce secondary immune response. Three sets of proteins are required for immune response: antibodies, T-cell receptors, and the MHC antigens. The immune system in all its forms is our defense mechanism against microorganisms, insects and other pathogens. The immune defense mechanisms can be divided into innate and adaptive immune system: Innate immunity - consists of non-specific defense mechanisms that act immediately following infection. This is the primary line of defense against the invading pathogens. It is not specific, means that it attacks all pathogens with equal likelihood. Physical barriers - there are several anatomical structures that act as physical barriers to pathogens which prevent pathogens from entering and create an inhospitable environment in which pathogens cannot grow: Skin - our skin consists of several layers that create a first line of defense against invading pathogens. The skin also contains glands that secrete fatty acids which cover the skin - creates an environment in which most bacteria cannot grow. Mucus and Cilia - found in the passage ways (i.e respiratory pathway) - goblet cells found along our air passageways produce a sticky layer of mucous that traps pathogens; the cilia can then be used to move the pathogens to the outside or to our stomach. Acidity of stomach - the parietal cells in the stomach release HCl, creates very acidic environment that kills off most of the pathogens that enter the stomach. Tears and saliva - lysozyme found in tears and saliva helps breakdown cell walls of bacterial cells. Inflammation - once the anatomical physical barriers are breached and the pathogen enters our tissue, the innate immunity then initiates the process of inflammation - blood flow to the area increases, brings WBCs - neutrophils, eosinophils, basophils and macrophages (increased phagocytosis). Although the inflammatory response begins as a local response, sometimes the entire body may become involved. Fever helps the body fight infection by increase phagocytosis. Basophils - release histamine, which causes the dilation of blood vessels leading to the infected area (🡪 Redness). In addition, the capillaries in the infected area become more permeable, which causes edema (swelling) and allows the passage of antibodies into the tissues. Neutrophils - Engulf bacterial cells and other harmful agents and kill them. Eosinophils - specialized to fight parasites. Macrophages - phagocytosis Mast cells - situated within the tissue and release histamine and cytokines that promote inflammation. NK cells - Kill infected cells and cancer cells in non-specific response. They release cytokines, as well as perforins - enzymes that destroy target cells by produce pores in their membranes, finally leads to their apoptosis. Cytokines Large, diverse group of peptides and proteins that serve as important signaling molecules and perform regulatory functions. They help regulate the intensity and duration of immune responses. E.g. when infected by viruses, certain cells respond by secreting cytokines called interferons which inhibit viral replication and activate NK cells that have anti-viral actions. Interleukins are another group of cytokines secreted mainly by macrophages and lymphocytes and regulate interactions between cells. The complement system Phagocytes secrete cytokines that activate the complement system, which complements the action of other defense responses. It consists of more than 20 proteins present in plasma and other body fluids. Complement activation involves a cascade of reactions; each component acts on the next in the series. Generally, complement proteins are active against many antigens, and their actions are non-specific: they lyse viruses and bacteria, they coat pathogens so the macrophages and neutrophils phagocytose them, they attract WBCs to the site of infection (chemotaxis) and more. Adaptive (acquired) immune response - specific immune response Unlike the innate immune system, the adaptive is highly specific to particular pathogen and provides long-lasting protection (e.g. someone who recovers from measles is now protected against measles for his life time). It divides into humoral and cellular responses. The acquired immunity relies on the capacity of immune cells to distinguish between the body's own cells and unwanted invaders. Whereas NK lymphocytes cells are important in non-specific immunity, T and B cells function in specific immunity: T-cells are responsible for cell-mediated immunity (cellular response), whereas B -cells are responsible for antibody-mediated immunity (humoral response). Each B-cell, while mature in the bone marrow, genetically programmed to encode a receptor that can bind with a specific type of antigen. When a B cell comes into contact with an antigen that binds to its receptors - the B cell becomes activated; the process requires the participation of T-helper cell. The activated B cell divides rapidly to form clone of identical cells, some differentiate into plasma cell (antibody secretion - binds to the antigen that originally activated the B cells) whereas others become long-living memory B cell. T cells mature in the thymus - negative selection - T cells that react to self-antigens undergo apoptosis, positive selection - only T-cells that are functioning against foreign antigens become mature). T cells are distinguished by the TCR - T cell receptor, which recognizes specific antigens. Two main populations - CD8 (cytotoxic) and CD4 (T helper). T-regs are sub-population of T-helper cells and help regulate immune responses by suppressing the function of certain T cells. Antigen-presenting cells activate T-helper cells Macrophages, dendritic and B-cells function as APCs that display foreign antigens as well as their own surface proteins. When APC ingest pathogen, lysosomal enzymes inside the APC degrade most of the bacterial antigens, and then the APC displays fragments of the foreign antigens on its cell surface in association with a type of self-molecules - MHC II; the complex of MHC II antigens combine with foreign antigen from bacteria presented to T-helper cells. Once activated, T-helper cells secrete cytokines Interleukin 2 - a growth factor that stimulates the activated cells to proliferate, giving rise to a clone of T-helper cells (clonal expansion). T-helper cells in the site of infection produce cytokines which attract macrophages to the site and promote the destruction of intracellular pathogens. Others function mainly in promoting antibody-mediated immunity. Regarding T-cytotoxic cells, it has more than 50,000 identical TCRs that bind to one specific type of antigen. It recognizes antigens presented to them as part of a foreign antigen-class I MHC glycoprotein complex. In order to be activated, it requires at least two signals in addition to the presented antigen - a co-stimulatory signal and an interleukin cytokine signal. Once activated, it gives rise to a clone of many effector Tc cells. Effector Tc cells when combines with antigen on the surface of the target cell, destroys the cell by secrets perforins that perforate the plasma membrane of the target cell (like NK) 🡪 apoptosis. antibody-mediated immunity - cell-cooperation B-cells are responsible for antibody-mediated immunity. Antibody molecules serve as cell-surface receptors that combine with antigens - only a B cell with a receptor (antibody) complementary in shape to a particular antigen can bind that antigen. The antigen enters the B cell by receptor-mediated endocytosis, and then degraded into peptide fragments; The B cell displays peptide fragments together with MHC protein class II on its surface. The activation of B cells involves APCs and T-helper cells. When an APC (may be dendritic, macrophages or the same B-cell) displaying a foreign antigen-MHC II complex contacts a T-helper cell with complementary T-cell receptors (reminder - T-cell receptors, like Ig's is super-diverse and during its formation, somatic recombination of gene occurs) - the T-helper is activated 🡪 binds with the foreign antigen-MHC complex on the B cell and releases interleukins which activate the B cells. Once activated, a B cell divides by mitosis, giving rise to a clone of identical cells which mature into plasma cells that secrete antibodies (some activated B cells do not differentiate into plasma cells, rather, become memory B cells). These antibodies are specific to the antigen that activated the original B cell (it is important to remember that the specificity of the clone is determined before the B cell encounters the antigen). The antibody secreted by the B cell is soluble form of the same B-cell receptor of the activated B cell. Sequence of events: Pathogens invades body 🡪 APC phagocytoses pathogen 🡪 foreign antigen-MHC complex displayed on APC surface (MHC II) 🡪 T-helper cell binds with foreign antigen-MHC II complex 🡪 activated T-helper cell interacts with a B-cell that displays the same antigen 🡪 B cell activated 🡪 clone of B cells produced 🡪 B cells differentiate, becoming plasma cells 🡪 Plasma cells secrete antibodies 🡪 antibodies form complexes with pathogen 🡪 pathogen is destroyed. Tc activation Tc develops from precursors upon contact with antigen, in such a way that the antigen is absorbed by the APC (antigen presenting cell) upon entry into the organism and, after processing, antigen fragments are exposed on the surface of the APC , some together with class I molecules, others with molecules Class II HHK. Complexes of antigen and class I molecules are recognized by Tc lymphocyte receptors with the help of CD 8 molecule , antigens with molecules II. class are recognized by T H cell lymphocytes using the CD4 molecule , antigen binding to the TCR is an important signal for T cell activation, additional signals are provided by adhesive molecules, similar to B lymphocytes , T H and Tc begin to divide, to which cytokines also contribute : IL-1 produced by APC cells, IL-2 produced by T H lymphocytes, mature Tc lymphocytes recognize infected cells through their receptor, which express the viral antigen and the class I molecule on their surface, Tc binds to the target cell and, through hydrolytic enzymes and proteins (perforin) contained in the vesicles, kills the cell, the cytotoxic lymphocyte survives this dramatic event and can kill other target cell

what is chromosomal determination of sex? what genes are involved? when does sex differentiation occur? what is the process? what is X-inactivation? why is it necessary? (93)

The sex of an individual is determined by the X and Y chromosomes. The presence of an intact Y chromosome leads to maleness regardless of the number of X chromosomes present; absent of Y chromosome 🡪 female development. Although the sex chromosomes are present from conception, differentiation into a phenotypic male or female doesn't commence until approximately 6 weeks. Up to this point, both the Mullerian and Wolffian duct systems are present, and the embryonic gonads, are still undifferentiated. From 6 weeks onwards, the embryo will develop into female unless testis-determining factor initiates a sequence of events that prompt the undifferentiated gonads to develop into testes. SRY - Testis-determining factor SRY gene is located in short arm of the Y chromosome - sex-determining region. It consists of a single exon that encodes a protein of 204 amino acids. Expression of SRY triggers off a series of events that involves other genes, leading to the medulla of the undifferentiated gonad developing into a testis - in which the Leydig cells begin to produce testosterone. This leads to stimulation of the Wolffian ducts, which form the male internal genitalia, and also to masculinization of the external genitalia. SRY gene also upregulates SF1 gene - which acts through another transcription factor (SOX9) to induce differentiation of Sertoli and Leydig cells. The Sertoli cells in the testes produce a hormone known as MIF - Mullerian inhibitory factor (also known as MIS - substance), which causes the mullerian duct system to regress. In the absence of normal SRY expression, the cortex of the undifferentiated gonad develops into an ovary. The mullerian duct forms the internal genitalia. The external genitalia fail to fuse and grow as in the male, and instead - evolve into normal female external genitalia - "the default pathway". Even though it is referred to as the default pathway, there are specific genes that induce ovarian development - for example, DAX1, is a member of the nuclear hormone receptor family and located on the short arm of the X chromosome, it downregulates SF1 activity - preventing differentiation of Sertoli and Leydig cells. In the absence of MIS production by Sertoli cells, the paramesonephric ducts are stimulated by estrogens to form the uterine tubes, uterus, cervix and upper vagina. Estrogens also act on the external genitalia at the indifferent stage to form the labia majora, labia minora, clitoris and the lower vagina. Without the stimulating effects of testosterone, the Wolffian ducts regresses. Normally, sexual differentiation is complete by 12-14 weeks gestation, although the testes do not migrate into the scrotum until late pregnancy.

what are transplantations? what are the types? what are the types of reactions? how can the host respond to the grafts? how can survival of the graft be improved? what are histocompatibility systems? (79)

Transplantation is the transfer of body part or tissue from one organism to another, or to other locations within the same organism. The major barrier to transplantation of organs from one individual to another is the immunologic rejection of the transplanted tissue by immune cells and antibody reactions that destroy the graft. The rejection of allograft (a tissue from a donor of the same species but not genetically identical) occurs as response to MHC molecules. Types of transplants: Auto-transplant - transferring tissue on the same body, doesn't induce any reaction - 'self' MHC. Iso-transplant - transferring tissue between organisms with the same genetic basis - e.g. identical twins Allo-transplant -between two same species organisms, most frequent. Risk of rejection 🡪 immune response. Xeno-transplant - from one species to another, very rapid rejection response happens. Most often the response is IgM or cell-mediated response. Although B-cells and antibodies contribute to the rejection reaction, T-cells have the major role. Their reaction is dependent on antigen presentation via MHC through T-helper cell and lymphokines (subset of cytokines produced by the T-cells). The result is production of interleukin 2 (cytokine signaling molecule) which activate the CD8 (T Cytotoxic) cells. By testing on mice proved the T-cells role in rejection - surgical removal of the thymus together with radiotherapy to remove the rest of immunocompetent T cells in the body - there was no rejection to the graft. From the other hand, injection of T-cells of these mice leads to rapid and aggressive response. The response is mostly directed against vascular endothelial cells or parenchymal cells of the organ. Types of reactions: Hyperacute - rare, particularly in patients who already have antibodies in their blood (produced by blood transfusion, pregnancy or previous graft rejection) - there is a vascular endothelial cell destruction, leading to clot formation and blockage of blood supply to the graft. Acute rejection - days to weeks till rejection occurs. May be cellular rejection or humoral rejection. Cellular - T cytotoxic cells direct recognition, Humoral rejection - APC's MHC II recognition 🡪 T-helper activation 🡪 cytokines 🡪 B cells to plasma cells 🡪 antibodies. Chronic rejection - dependent on genetic differences between donor and recipient, slow process, can take months to years. Often occurs in transplant kidney, even ten years after the surgery. Graft reaction against host - GvHR (graft vs Host reaction) - if the graft contains T-cells, they recognize the recipient cells as foreigners. Graft begins to reject the recipient, and the recipient, which is usually immunosuppressed - unable to attack these aggressive foreign T-cells. Graft is highly dangerous for the recipient responses Newborn - not fully developed immune system Adult with discarded immune system (as a result of radiation, immunodeficiency pathology, etc) There are two mechanisms by which the host immune system recognizes and respond to MHC molecules of the graft: Direct recognition - host T-cells directly recognize the foreign MHC molecules expressed on the graft cells. They do not recognize them as self - thus CD8 T-cells (cytotoxic) attack them and kill the graft cell. Since Dendritic cells in the graft express high level of MHC - they are believed to have a major role in the direct recognition of the T-cells as non-self tissue. Indirect recognition - similar to the presence of foreign antigen. APCs (such as macrophages, dendritic cells or B-cells) recognize the foreign MHC molecules, pick a portion of these and migrate with them to the lymph nodes. There, they present the antigen by MHC II molecules (reminder - MHC II molecules found only on APC's membranes). CD4 T-helper cells react with these molecules, activated, proliferate and secrete cytokines which causes B-cells to differentiate and secrete antibodies that recognize the host cell's antigen and attack them. Methods for improving graft survival Matching HLA of donor and host (recipient) - HLA is highly polymorphic, virtually infinite number of phenotypes resulting from different alleles combinations at these loci. HLA loci are haplotypes (inherit as a "full-set" from one parent), and therefore, has little chance of going through recombination process. For this reason, the best possibility match will be in the family of the recipient. H-Y antigen: Tissue graft from males cannot be donated to females of the same strain. These incompatibilities were found to be due to histocompatibility antigen known as H-Y. Males has both antigen for X and Y, though females do not have antigen H-Y. Thus - females can donate to males, but females will form antibodies against males graft and thus reject the transplantation. Immunodepression of the recipient is necessary in all organ transplantation, except in the case if identical twins. Due to immunodepression, sometimes HLA matching is not even attempted in urgent cases (e.g. - liver, heart, lung transplantations). Though immunodepression enables flexibility in the transplantation rules, it has disadvantages by making the host body vulnerable to infective agents. The major histocompatibility complex (MHC) serves to present alloantigens to T cells. If T-cells recognize antigens as foreign to the body, the graft rejects. In humans, we are talking about HLA = Human Leukocyte Antigen. In mice, the H-2 system. Everyone inherits the HLA gene from their parents. It is encoded on chromosome 6 . The resulting type is a combination of maternal and paternal information. Individual cells of the human body express MHC molecules to varying degrees. Cells that normally have little activity are stimulated after exposure to interferon gamma (IFNg) or tumor necrosis factor(TNF). The MHC molecule consists of two alpha chains, two immunoglobulin-like domains, and a beta moiety that connects them. It is the alpha helices that are on the outside of the membrane that play a major role in T-cell activation.

how are proteins translocated to the ER? (50)

Transport of Proteins into ER: A short N-terminus signal sequence at the beginning of the growing protein chain determines whether a ribosome synthesizing the proteins binds to ER or not. The protein synthesis always begins on free ribosomes. As the signal sequence emerges out of the ribosome, the large ribosomal sub-unit binds to ER membrane. This is decided by the type of signal sequence. This is the first sorting as the ribosome binds to ER, forming rough ER. Translocation takes place into the ER while growing chain is still bound to the ribosome. This is called co-translational translocation. The process is facilitated by the signal sequence recognition mechanism - consists of a signal recognition particle (SRP) present in the cytosol. SRP binds to the signal sequence of the nascent protein as soon as it emerges out of ribosome and directs it towards the ER membrane. The binding of SRP stops further synthesis of protein chain when it is about 70 amino acids long. This prevents it from folding. The SRP-ribosome complex binds to an internal membrane protein receptor in the ER wall. At this point GTP hydrolysis hydrolyses frees SRP which is ready for the next round of directing next nascent protein of ER. Now lengthening of polypeptide restarts which enters ER lumen. Ribosome is aligned to a channel, called translocon, in the wall of ER. It allows the elongating chain to enter through the translocon into the ER lumen. As the growing polypeptide chain emerges into the ER lumen, the signal sequence is cleaved by a peptide called signal peptidase. Once inside the lumen of ER, the protein undergoes folding and several modifications for which the ER lumen contains a number of enzymes. The most common processing is glycosylation which involves addition of carbohydrates to the protein chain in Asparagine residue (N-glycosylation post-translation modification - formation of glycoprotein). The Golgi complex has a role in protein transportation: it acts as a switching center (post-office) for proteins to various destinations. Both ER and Golgi apparatus are flattened cisternae. Transport of proteins from one compartment (donor) to the next one (target) is carried out in transport vesicles. The vesicles contain cargo proteins in their lumen and integral membrane proteins in their membranes. The vesicles bud off from ER and fuse with the cis-compartment or receiving compartment of Golgi. In this process cargo proteins are delivered into the lumen of Golgi and membrane proteins become part of the membrane of the target vesicles. The proteins which glycosylated (O-glycosylation), folded, modified and sorted in ER, keeping the modification process in the Golgi cisternae. Starting from the cis-compartment to medial compartment and lastly to trans-Golgi network proteins are exported to the end target. In trans-golgi network proteins are further sorted to be delivered to lysosomes, for secretion outside the cell and to plasma membrane according to signals present in the nascent proteins.

what is DNA? what is the structure? (41)

The nucleus contains the hereditary material in the form of chromosomes, which is composed of a single DNA double helix. DNA is a chain of nucleotides (nucleoside = without the phosphate group), which are individual molecules that forming the nucleic acid. Each nucleotide is composed of a nitrogenous base, and a backbone composed of sugar molecule and a phosphate molecule. The nitrogenous bases fall into two types: Purines - Adenine, Guanine (purines - two heterocycle rings - one imidazole - two nitrogens, 3 carbons, and one pyrimidine - two nitrogens, 4 carbons) Pyrimidines - Cytosine, Thymine, Uracil (one ring of pyrimidine) Primary structure - linear sequence of nucleotides that are linked together by phosphodiester bonds Secondary structure - interactions between bases in the double strand - two strands of DNA are held together by hydrogen bonds (in RNA - secondary structure consists of only single polynucleotide that may sometime folds between complementarity regions and - may form regions of double stranded) Because only the base differs in each of the four types of subunits, each polynucleotide chain in DNA is analogous to a necklace (the backbone) strung with four types of beads (the four bases A, C, G, and T). There are two different types of nucleic acid, ribonucleic acid (RNA) which contains the five carbon sugar ribose, and deoxyribonucleic acid (DNA) - in which the hydroxyl (OH) group at the 2nd position of the ribose sugar is replaced by a hydrogen (an oxygen molecule is lost - hence the name "deoxy") DNA and RNA both contain the purine bases adenine and guanine, and the pyrimidine cytosine, but Thymine occurs only in DNA and Uracil is found only in RNA. According to Watson and Crick, DNA molecule is composed of two chains of nucleotides arranged in a double helix. The backbone of each chain is formed by phosphodiester bonds between the 3' and 5' carbons of adjacent sugars, the two chains being held together by hydrogen bonds between the nitrogenous bases, which point in toward the center of the helix. Each DNA chain has a polarity determined by the orientation of the sugar-phosphate backbone - the sugar-phosphate backbone forms the structural framework of nucleic acids, including DNA and RNA. This backbone is composed of alternating sugar and phosphate groups, and defines directionality of the molecule. The nucleotides are linked together covalently by phosphodiester bonds through the 3′-hydroxyl (-OH) group of one sugar and the 5′-phosphate (P) of the next. Thus, each polynucleotide strand has a chemical polarity; that is, its two ends are chemically different. The 3′ end carries an unlinked -OH group attached to the 3′ position on the sugar ring; the 5′ end carries a free phosphate group attached to the 5′ position on the sugar ring. The members of each base pair can fit together within the double helix only if the two strands of the helix are antiparallel—that is, only if the polarity of one strand is oriented opposite to that of the other strand. A consequence of these base-pairing requirements is that each strand of a DNA molecule contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand. A purine in one chain always pairs with a pyrimidine in the other chain, with specific pairing of the base pairs: guanine in one chain always pairs with cytosine in the other chain (G - C, triple H-bonds), and adenine always pairs with thymine (A - T, double H-bonds), so that this base pairing forms complementary strands. The hydrophilic part of the deoxyribose is the phosphate backbone of each chain, faces toward the outside whereas the hydrophobic bases are stacked inside to the center (their hydrophobic interactions donate to the stability of the base pairs) The spatial relationship between the two strands in the helix creates a major and minor groove. These grooves provide access for binding of regulatory proteins.

what happens to phenotype expression of non-allelic interaction with inhibition? what are the phenotypic ratios? (4)

aka dominant-recessive interaction a gene by itself has no phenotypic effect, but inhibits the expression of another non-allelic gene in rice, purple leaf colour is due to allele P, and p causing green colour. Another non-allelic dominant gene I inhibits the expression of P but is ineffective in recessive form (ii). Thus the factor I has no visible effect of its own but inhibits the color expression of P. Therefore, a different phenotype arises only with the opposite combination of genes, recessive in the first gene and dominant expression in the second gene. Phenotypic fission ratios: F2 generation: 13: 3 Bc - F1 x P ( aabb ): 3: 1 Inhibitory factor with partial dominance: Sometimes an inhibitory gene shows incomplete dominance thus allowing the expression of other gene partially.

what is pharmacogenetics? what is the aim? what are the ways to assess the effect of genes on drugs? how does the human genome influence the effects of drugs? what are examples of how variations are revealed by the effects of drug? how can drug metabolism be assessed? (134)

pharmacogenetics is the discipline that comes from both pharmacology and genetics. It is the study of inherited genetic differences in drug metabolic pathways, which can affect individual responses to drugs, both in terms of positive therapeutic effect as well as negative adverse effects. Pharmacogenetics describes the influence of genes on the efficiency and side effects of drugs. The aim of pharmagenomics is the development of individualized treatment procedures and the identification of the most suitable type and dosage of drugs for a particular patient. Pharmacogenomics, which is different term, describes the interaction between drugs and the genome (multiple genes). Pharmacogenetics is important because negative drug reactions are a major cause of morbidity and mortality. It is also likely to be of increasing importance in the future, particularly as a result of the development of new drugs from information that has become available from the human genome project. The ways in which many drugs are metabolized vary from person to person and can be genetically determined. Association - A relatively suitable method is to use association studies. These deal with the association between phenotype and genotype . E.g. a case-control method that compares the frequency of polymorphisms between a side effect group and a control group (which did not occur) after administration of the same drug. The disadvantages of this method are, above all, the need for the surveyed groups to be as similar as possible, apart from the observed trait (representation of men and women, ethnic group, age, etc.). SNP - Of all polymorphisms in humans, about 90% [1] . of which they form the SNP . These most often cluster into haplotypes . Chromosomes are composed of short segments that have undergone a minimal number in evolution [Crossing-over, its mechanism and significance | of recombinant changes]]. For this reason, haplotype mapping of the human genome has begun ( HapMap project https://www.ncbi.nlm.nih.gov/variation/news/NCBI_retiring_HapMap/ ). If all haplotypes can be mapped, it will be possible to identify those at risk for a particular drug . Today, SNP mapping can be performed using SNP chips . Amplichip - A tool that allows tens of thousands of genes to be tested simultaneously in a single sample . Short sections of single-stranded DNA with a known nucleotide sequence are placed on a 1.5 x 1.5 cm chip. Based on complementarity, fluorescently labeled DNA from the analyzed sample binds to them. The results are evaluated by computer. The disadvantage of this method is its high price. The human genome influences the effects of drugs: Pharmacokinetics - describes the metabolism of drugs - their uptake, their conversion to active metabolites, and their detoxification or breakdown Pharmacodynamics refers to the interaction between drugs and their molecule targets (e.g. drugs and its receptor) Palliative drugs that do not act directly on the cause of a disease, but rather on its symptoms (e.g. drugs that do not influence the cause of pain but merely the perception of pain in the brain). Genetic variations revealed by the effects of drugs - examples: Isoniazid - one of the drugs used in the treatment of tuberculosis (infectious disease caused by mycobacterium tuberculosis, generally affects the lungs - chronic cough with blood-containing sputum, fever etc.) - it is rapidly absorbed from the gut, resulting in an initial high blood level that is slowly reduced as the drug is inactivated and excreted. The metabolism of isoniazid allows two functionally (due to genetics factors) different groups to be distinguished: rapid (blood levels of the drugs fall rapidly) and slow (...). Family studies shown that slow in-activators of isoniazid are homozygous for an AR allele of the liver enzyme N-acetyltransferase (isoniazid metabolism), with lower activity levels. Slow in-activators have a significantly greater risk of developing side effects, whereas rapid in-activators have an increased risk of liver damage from isoniazid. Succinylcholine sensitivity - a drug which produces muscular relaxation, used for anesthesia for intubation (placement of tube into the trachea to maintain open airway) - by causing short-term skeletal and respiratory muscles (but...cause short-lived apnea). However, about one patient in every 2000 has a period of apnea that can last 1 hour or more after the use of succinylcholine (instead of only 2-3 minutes; during this time the anesthetist maintain respiration by artificial means). It was found that the apnea in such instances could be corrected by transfusion of blood or plasma from a normal person. In patients which are highly sensitive to succinylcholine, the plasma pseudo-cholinesterase (which is plasma enzyme responsible for destroy the drug) in their blood destroys the drug at a markedly slower rate than normal (or even entirely deficient). Succinylcholine sensitivity is inherited as an autosomal recessive trait due to mutation. Glucose 6-phosphate Dehydrogenase Variants - Hemolytic anemia occurs after administration of anti-malarial drugs Primaquine. During the 2nd World War, this phenomenon was observed mainly among African Americans soldiers. The basis was the deficiency of glucose-6-phosphate dehydrogenase - X-linked Recessive inherited disease. Coumarin Metabolism - Coumarin anticoagulant drugs, such as warfarin, are used in the treatment of a number of different disorders to prevent the blood from clotting. Warafarin is metabolized by the cytochrome P450 enzyme, encoded by a gene which has other two variants. The variants result in decreased metabolism. Consequently, these patients require a lower warfarin dose to maintain their target value, and may be at increased risk of bleeding. The development of the Pharmacogenetics field that occurred as a result of increased understanding of the influence of genes on the efficiency and side effects of drugs has led to the increasing development of personalized, individualized, medicine - where the treatment for a particular disease is dependent on the individual's genotype. Pharmacogenomics is defined as the study of the interaction of an individual's genetic makeup and response to a drug. The distinction between pharmacogenetics and pharmacogenomics is that pharmacogenetics describes the study of variability in drug responses attributed to individual genes, whereas pharmacogenomics describes the study of the entire genome related to drug response. The expectation is that inherited variation at the DNA level results in functional variation in the gene products that play an essential role in determining the variability in responses, both therapeutic and adverse (שליליים), to a drug. It is estimated that around 15% of hospital patients will be affected by an adverse drug reaction. The objective of adverse-event pharmacogenetics is to identify a genetic profile that characterizes patients who are more likely to suffer such an adverse event. From the other hand, there is no doubt that the cost-effectiveness of drugs is improved if they are prescribed only to those patients likely to respond to them. Several drugs developed for the treatment of various cancers have different efficiency depending on the molecular biology of the tumor. Genetic profiling is a step toward personalized medicine - this information can be used to select the appropriate treatment at the correct dosage and to avoid adverse drug reactions. Drug metabolism applications - absorption - tissue distribution - exclusion either the liver breaks down or it is transferred to the target cells, in the target cells, the drug has a therapeutic effect , followed by degradation and excretion The rate of metabolism is affected by enzymes - genetic equipment. As a sign, we monitor the level of the drug in the blood after administration of the standard dose. The distribution of concentration values ​​is: continuous unimodal (Gaussian) curve polygenic inheritance discontinuous bi or trimodal curve monogenic inheritance We also monitor the decline in levels over time - there are individual differences in response to drugs, including side effects. The dose is calculated per body area.

what is selection? what does it depend on? what are the effects of certain alleles? (102)

process by which organisms that are better adapted tend to survive and breed whilst those less adapted tend not to results when fitness (probability that an organism will pass on its genes) depends on genotype certain alleles can increase or decrease fitness effects can be dominant, recessive, co-dominant or additive also will depend on the genetic background (other genes) and environment

What is a cloning vector? how does this work with amplification? (54)

the original plasmid, the DNA molecule that can carry foreign DNA into a host cell and replicate it there thanks to the discovery of enzymes - restriction endonucleases - it was possible to obtain fragments of different lengths , which can be further analyzed. Propagation itself is based on the formation of recombinant DNA molecules, which are created by combining different sections of DNA molecules derived from taxonomically different species. Another important DNA molecule necessary for DNA replication is the so-called vector into which a DNA fragment is inserted. The recombinant vector is then introduced into a host organism, such as a bacterium. Restriction endonucleases Restriction endonucleases are enzymes isolated from bacteria that cleave double-stranded DNA in specific sequences. Enzymes protect bacteria from the ingress of foreign DNA. Endonucleases cleave DNA to form overhangs called cohesive or sticky ends and DNA fragments that are of defined length. These are mostly palindromes . Restriction endonucleases can cleave recombinant DNA molecules in the presence of a DNA ligase enzyme and complementarity between sticky ends, e.g., an EcoRI enzyme isolated from E. coli cleaves the GAATTC sequence. Restrictions are needed to obtain sufficient DNA. Vectors Plasmids - occur in bacteria Lambda (λ) phage - belongs to bacteriophages Cosmids - A combination of plasmid DNA and lambda phage Artificial yeast chromosomes - possibility to clone very long fragments (up to 2Mpb) Bacterial artificial chromosomes

what are some commonly used methods of nucleic acid analysis? what are the two basic approaches? what are the applications? (54)

two basic methods: DNA replication: in living organisms or in vitro by chemical process (PCR) molecular hybridization Application: gene structure analysis, mapping, function analysis of genetically determined diseases prenatal diagnosis detection of mutant allele carriers diagnosis and pathogenesis of the disease biosynthesis - insulin , growth hormone ... treatment of genetic diseases - gene therapy basic discovery for development Selective propagation of specific DNA fragments from genomic DNA or cDNA. Amplification can be performed in vivo or in vitro using DNA polymerase cell fractioning gel electrophoresis southern blot northing blot PCR DNA sequencing

evolution of genes and genomes? mechanisms? (120)

A gene arises from a gene. The genes are similar. Duplications and gradual gene diversifications have resulted in gene families and superfamilies. Genes can arise by the following mechanisms: exon rearrangement gene duplication retrotransposition gene fusion and cleavage Genome evolution is the process by which a genome changes in structure (sequence) or size over time. Genome evolution is a constantly changing and evolving field due to the steadily growing number of sequenced genomes. Human genome has size of approximately 3.2Gb, and total of 20,000 genes. Each gene averages about 27,000 base pairs. Eukaryotic genomes evolve over time through many mechanisms including sexual reproduction which introduces much greater genetic diversity to the offspring than the prokaryotic process of replication in which the offspring are theoretically genetic clones of the parental cell. Other mechanisms for genome evolution are gene duplication, retro-transportation, shuffling exons and genes fusion. Mechanism of genome evolution: Gene duplication - Gene duplication is the process by which a region of DNA coding for a gene is duplicated. It may be duplication of an entire gene, a portion or even a cluster of genes. This can occur as the result of an error in recombination or through a retrotransposition event. A transposable element (transposon) is a DNA sequence that can change its position (or copy itself and transfer to another place) within a genome, by "copy and paste" mechanism sometimes creating or reversing mutations and altering the cell's genome size. Transposition often results in duplication of the transposon. Retrotransposition (transposons via RNA intermediates) are genetic elements that can amplify themselves in a genome, and are one of the two subclasses of transposon, where the other is DNA transposon, which does not involve an RNA intermediate. Around 42% of the human genome is made up of retrotransposons, while DNA transposons account for about 2-3%. The retrotransposons' replicative mode of transposition by means of an RNA intermediate rapidly increases the copy numbers of elements and thereby can increase genome size. A retrotransposons copied themselves to RNA and then back to DNA they may integrate back to the genome. The second step of forming DNA may be carried out by a reverse transcriptase, which the retrotransposon encodes. Because of accumulated mutations, most retrotransposons are no longer able to retrotranspose. Duplicate genes are often immune to the selective pressure under which genes normally exist. This can result in a large number of mutations accumulating in the duplicate gene code. This may render the gene non-functional or in some cases confer some benefit to the organism. Mutation - Spontaneous mutations often occur which can cause various changes in the genome. Mutations can either change the identity of one or more nucleotides, or result in the addition or deletion of one or more nucleotide bases. Such changes can lead to a frameshift mutation, causing the entire code to be read in a different order from the original, often resulting in a protein becoming non-functional. Exon Shuffling - a molecular mechanism accounts for the evolution of new genes. May also refer to "regrouping exons" - It is a process through which two or more exons from different genes can be brought together ectopically (Ectopic recombination is recombination in which crossing over occurs at non-homologous, rather than along homologous, loci), or the same exon can be duplicated, to create a new exon-intron structure. Exon shuffling may occur due "transposon mediated exon shuffling" or crossover during sexual recombination of parental genomes. Another mechanism for exon shuffling is alternative splicing - differential splicing, which is a regulated process during gene expression that results in a single gene coding for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. Fusion genes - another mechanism of genome and gene evolution is formation of fusion genes, which is a hybrid gene formed from two previously separate genes. It can occur as a result of translocation, deletion, or chromosomal inversion. Gene fusion plays a key role in the evolution of gene architecture. Duplication, sequence divergence, and recombination are the major contributors at work in gene evolution. These events can probably produce new genes from already existing parts. When gene fusion happens in non-coding sequence region, it can lead to the mis-regulation of the expression of a gene now under the control of the regulatory sequence of another gene. If it happens in coding sequences, gene fusion cause the assembly of a new gene, then it allows the appearance of new functions.

how is gene expression regulated in prokaryotes? what are the general mechanisms? what are operon systems? how do they function? (87)

Gene expression is regulated in prokaryotic cells: transcriptional regulation ; mRNA stability ; translation regulation ; post-translational modification of polypeptides . Role of operators and the Lac Operon Operons contain an operator- a segment of DNA that regulates the activity of the structural genes of the operon. If the operator isn't bound by a repressor molecule, RNA polymerase passes over the operator and reaches the protein-coding genes region, but if a repressor molecular is bound to the operator, the polymerase is blocked. As long as the repressor is bound to the operator, no proteins are made. However, an inducer molecule (such as allolactose) binds to the repressor, causing the repressor and prevents it from blocking the transcription. Transcription in prokaryotes is fundamentally different from that in eukaryotic regulation . The basic difference is that in prokaryotes , one regulatory region may be common to multiple genes , the transcription of which is then affected together. In eukaryotes, there is always one promoter region per gene and vice versa. We refer to such bound genes as operons . The site where the RNA polymerase anneals is called the promoter by default , and the site where the regulatory proteins anneal are referred to as the operator . The entire operon is then expressed as a single mRNA molecule . We distinguish operons in two types: catabolic and anabolic . An example of a catabolic operon is the Lac operon - in the presence of lactose , which has an inductive effect on transcription, several genes responsible for lactose catabolism are transcribed at the same time. In contrast, the typical anabolic operon is the Trp operon - in the presence of tryptophan , the transcription of genes responsible for the anabolism of this amino acid is inhibited. Transcription factors We generally divide regulation into positive and negative . A number of proteinaceous transcription factors are involved in regulation, which must recognize a particular region on the DNA molecule - at sites in the deep groove of the DNA. Their DNA binding domains contain various conserved motifs (evolutionarily sparing sequences of proteins that are able to bind to DNA), with transcription factors for the operon system mostly containing β-sheets . Other canned motifs are the following: I. HTH (helix - turn - helix) and HLH (helix - loop - helix) = 2 α-helices connected by a short chain AMK → form a "turn"; II. HTH homeodomains - involved in ontogenesis; III. Steroid receptors ; IV. Zinc - fingers = 1 α-helix and 1 β-ridge - held in a constant position by a Zn atom; V. Leucine zippers = connect 2 α-helices by bonds between leucine molecules; VI. β- sheets - operon system. Posttranslational modifications = removal of the first methionine from the N-terminus of the polypeptide, removal of the signal peptide from the N-terminus Cascade control genes expressed in a certain order early transcription genes later late transcription genes genetically programmed cascade early transcription gene promoters have signal sequences to which the host cell's sigma-factor RNA polymerase binds initiates their transcription Another regulation Among the early transcription genes is the gene for viral RNA polymerase, which specifically binds to the promoters of viral late transcription genes Among the early transcription genes is a gene for a protein that replaces the host RNA polymerase σ-factor and ensures the specificity of bacterial binding to the promoters of late transcription genes TRANSLATION REGULATION In prokaryotes, multigenic mRNA is transcribed to encode multiple proteins (enzymes), usually one metabolic chain. Detailed studies have shown that although these genes are expressed simultaneously as part of a single operon, different amounts of individual proteins (enzymes) are formed during translation. This is made possible by the following mechanisms: a) Uneven translational initiation efficiency of different operon genes, different rates of ribosome movement in mRNA intergene regions, and mRNA hairpin formation that affect the rate at which ribosomes move across mRNA and different rates of mRNA degradation. b) Post-translational regulatory mechanisms In addition to the described regulation of transcription and translation, regulations on the level of enzyme activity are described in prokaryotes. Sufficient end product of a particular metabolic pathway can inhibit the activity of the first enzyme of that pathway. This mechanism is called feedback inhibition or enzyme inhibition by end products. Enzymes capable of this reaction have, in addition to the substrate binding site, a binding site (s) for the final product of the metabolic chain. Upon binding of the final product, the allosteric configuration of the enzyme molecule changes, thereby reducing its affinity for the substrate. These proteins are called allosteric proteins.

what are indirect diagnostics of hereditary diseases? what are some advantages, disadvantages? (67)

Indirect diagnostics is made without knowing the mutation or even the gene that is responsible for the mutation. It is possible to perform if the chromosomal position of the mutant gene is roughly known - "Linkage studies". Linkage studies - Sometimes, the error in the gene responsible for causing a disease has not been identified. In these cases, we use "markers gene" to find out whether a person has inherited the crucial region of the genetic code that is passing through the family with the disease. Markers are DNA sequences located close to the area of interest (as said, we roughly know the location of the potential mutant gene) - the area is so closed to a gene, so usually they are inherited together - they are said to be "linked genes". In this way, if someone has the set of linked markers, he or she will also have the disease-causing gene. The family member with the disorder in question is studied first as in direct DNA analysis. The pattern of the person's markers is compared to other relatives. If there is an exact match, then those relatives are also likely to have the faulty gene. The accuracy of linkage studies depends on how close the markers are to the faulty gene. In some cases, a reliable marker is not available and the test, therefore, cannot give any useful information to the healthy family members. In many cases, several family members are needed to establish the most accurate set of markers to determine who is at risk for the disease in the family. In indirect methods, the main instrument is a pedigree. By sampling the proband, his parents and his siblings we can assemble a pedigree. The next is the use of Southern blotting, in order to divide the DNA according to restriction endonucleasis and the DNA fragments should be lined according to their size. We can now know the gene fragments pattern of each one of the examined individuals. In the indirect methods, we used two types of probes - intragenetic and extragenetic. The probe is a segment of the DNA or RNA which binds specifically to some part of the DNA strand. The intragenetic probe located right on the spot of the examined gene, whereas the extragenetic probe lies just close to it - referred to as "linked gene" region. The Extragenetic probe is complementary to the segment of DNA which is our "flanking" sequence - i.e. so close to the gene of interest that probably if it exists in both the parent and the child - thus the target gene is also present (inherited together). After the DNA is synthesized and the probes bind, we should identify which segment of DNA was inherited from which parent, putting the results of electrophoresis into the scheme of the family tree. If we are able to determine an origin of inherited DNA segments - the family is called to be informative. *Disadvantages - complete family is necessary, diagnostics can be unsuccessful due to lack of information in selected DNA markers (uninformative), recombination can cause misdiagnosis - if recombination occurred, the linked gene are not relevance - we have to consider the risk of recombination. The indirect methods arise from genetic polymorphism - genetic polymorphism are used as DNA markers for mutant genes identification (they are inherited - thus, really specific for parent 🡪 child connection, and could use as useful marker that is exist in both father and son - means that the sequence as well as the linked gene of interest inherited together). The kinds of Polymorphisms that take into considerations: Tandem repeat polymorphisms - minisatellites, microsatellites SNPs - common variations in the human genome - when occur within the recognition sequence of a restriction enzyme will cause restriction length polymorphism (RFLP) - fragments produce by that restriction enzyme will be of different lengthes. Structural polymorphism which is inherited to offspring - insertions, deletions, insertions. Trinucleotide repeats Steps for indirect method: Distinguish of the two chromosomes of the parent who may have transmitted the disease Find whether the marker allele and the disease allele are linked (segregate together or not) Discover which parent passed allele to the proband Summary - linked markers are used in family studies to discover whether or not the child (patient) inherited the high-risk chromosome from a heterozygous parent. The test is of a family and gives information about the segregation of a chromosomal segment in the family.

What are mutator genes? examples? what is genome stability? (113)

Most advanced tumors contain cells that exhibit a dramatic variety of chromosome anomalies, including extra chromosomes, missing chromosomes and chromosome rearrangements. Some cancer researchers believe that cancer is initiated when genetic changes occur that cause the genome to become unstable. Thus, genome instability refers to a high frequency of mutations within the genome. Although humans surrounded by environmental exogenous agents that may be mutagens (carcinogens), cancers are relatively rare outcomes of these encounters. This results from the ability of normal cells to repair DNA damage and maintain the genome stability. DNA repair mechanism maintains the integrity and stability of the genome. This ability is provided by Caretakers tumor-suppressor genes which are capable of detecting mutations and repair them. In case the damage is too severe, they promote apoptosis. Mutations in the tumor-suppressor genes are usually inherited, and they greatly increase the risk for development of cancers; usually, they are recessive factors - means that in order to carcinogenesis to occur both alleles should be mutated. In some cases they may act like dominant (Haplo-insufficiency) due to the dosage effect - even though the heterozygote produces product, it is not in sufficient amount 🡪 may cause cancer. Increased frequency and the accumulation of mutations in the cell is one of the causes of malignant transformation. Damage in the DDR - DNA damage response - pathways promotes critical steps in the development of cancer. Mutator gene is a gene that increases the mutation frequency of other genes - DNA repair genes typically have a mutator phenotype which means that if a DNA repair mechanism become mutated and exhibit its mutator phenotype - instead of repairing damages it will increase the mutation frequency 100-1000 times 🡪 increase the mutated oncogenes or mutated tumor-suppressor genes 🡪 promote even more rapid cancerous progress. Hereditary non-polyposis Colon cancer: also known as Lynch syndrome - Autosomal dominant condition Mutation in MMR - mismatch repair genes - this encoded proteins that recognize and repair erroneous insertion of bases that can arise during DNA replication as well as repairing some forms of DNA damage. In case of mutator phenotype of these genes it results in MSI - Microsatellite instability. Cells with abnormally functioning MMR (mismatch repair) are unable to correct errors that occur during DNA replication and consequently accumulate errors 🡪 creation of novel microsatellite fragments. Microsatellite instability may result in colon cancer, gastric cancer, ovarian cancer and more. MMR genes another group of mutator genes. The encoded proteins correct the base misalignment during DNA replication (but not complementary). The manifestation of mutations of these genes is instability at the nucleotide level, instability of microsatellite loci (MIN) - microsatellite instability (incorrect base pairing causes changes in the length of microsatellite sequences - their lengthening or shortening). Instability of microsatellite sequence lengths leads to replication errors. Mutations are recessive. Microsatellite sequences are distributed throughout the genome and are inherited in length. They are repetitive sequences of dinucleotides or trinucleotides, there are 50,000-100,000 (CA) n repeats in the human genome. Germline mutations, especially hMSH2 (human MutS homolog 2), hMLH1, hPMSI and hPMS2 genes are the basis of hereditary non-polyposis colorectal cancer (HNPCC) - inherited autosomal dominantly , familial occurrence is considered to affect 3 or more family members with a relationship of 0.5 disease before the age of 50. Familial occurrence of HNPCC accompanied only by the finding of cancer of the colon or rectum (so-called Lynch syndrome I ). In addition, about 30% of HNPCC patients develop carcinomas in other organs (endometrium, pancreas, stomach, urinary tract). This is the so-called Lynch syndrome II . The HMSH2 (chrom. 2p15-p22), hMLH1 (chrom. 3p21.3), hPMSI (2q31-33) and hPMS2 (chrom. 7p22) genes are responsible for correcting base mismatches (MMRs). Their mutations predispose to Lynch syndrome.

processes of reproduction in prokaryotes? what is horizontal gene transfer? what is conjugation? what is required? what is transformation? applications? what is transduction? applications? (89)

Prokaryotes do not reproduce sexually and their genetic variation can result from a combination of rapid reproduction and mutation. E-coli reproduces by binary fission in a human intestine, the amount of new E.coli cells arise per day is 2 X 1010, and even though the mutation rate is small, 2,000 bacteria have mutations per day, thus can increase genetic diversity quickly due to short generation times and large population of E-coli; Although new mutations are a major source of variation in prokaryotic populations, additional diversity arises from genetic recombination - the combining of DNA from two sources. In Eukaryotes, meiosis and fertilization combine DNA from two individuals, but meiosis and fertilization don't occur in prokaryotes. Instead, 3 other mechanisms - transformation, transduction and conjugation can bring together prokaryotic DNA from different cells. This movement of gene from one organism to another is called Horizontal gene transfer. Transformation In transformation, the genotype (and possibly the phenotype) of a prokaryotic cell is altered by the uptake of foreign DNA from its surroundings. Transformation occurs when bacteria, which have specialized cell-surface proteins, recognize DNA from closely related species and transport it into the cell. Once inside the cell, the foreign DNA can be incorporated into the genome by homologous DNA exchange. E.g. - harmless strain of Streptococcus pneumoniae can be transformed into pneumonia-causing cells, if the cells are exposed to DNA from a pathogenic strain. When a nonpathogenic cell takes up a piece of DNA carrying the allele for pathogenicity, he replaces its own allele with the foreign allele and produces a recombinant: its chromosome contains DNA derived from two different cells. Transduction Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector - for example, viral transfer of DNA from one bacterium to another. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA (as occurs in conjugation). A virus that carries prokaryotic DNA may not be able to replicate because it lacks some or all of its own genetic material, but the virus can attach to another prokaryotic cell (recipient) and inject prokaryotic DNA acquired from the first cell (donor). If some of this DNA is then incorporated into the recipient cell's chromosome by crossing over - a recombinant cell is formed. Phages infects a bacterial cell (the donor) that carries A+ and B+ alleles on its chromosome (brown) The phage DNA is replicated, and the cell makes many copies of the proteins encoded by its genes. Certain phage proteins halt the synthesis of proteins encoded by the host cell's DNA, and the host cell's DNA may be fragmented. Fragment of bacterial DNA carrying the A+ allele happens to be packaged in a phage capsid. The phage carrying the A+ allele from the donor cell infects a recipient cell with alleles A_ and B_ ; crossing over at two sites allows donor DNA to be incorporated into recipient DNA, and the genotype of the resulting recombinant cell (A+B-) differs from the genotypes of both the donor and the recipient. Medical applications - resistance to anti-biotic drugs inserted into bacterium, and correcting genetic diseases by direct modification of genetic errors. Conjugation and Plasmids DNA is transferred between two prokaryotic cells (usually same species) that are temporarily joined. The DNA is transferred in one-way - one cell donates the DNA, and the other receives it. First, a Sex Pilus, a flexible tube of protein subunits extends from the donor cell and attaches to a recipient cell. The pilus pulls the two cells together, and formation of Mating Bridge occurs. The ability to form pili and donate DNA during conjugation results from the presence of a particular piece of DNA called the F factor (F for fertility). The F factor, in the case of E-coli, consists of about 25 genes. These F factor genes can exist either as a plasmid or as a segment of DNA within the bacterial chromosome. In a case the F-factor is a plasmid - it is called F plasmid. Cells that contain the F plasmid - F+ - function as DNA donor during conjugation and the cells lacking the F factor - F- - function as DNA recipients. The F+ condition is transferable, and cell with such factor can converts F- to F+ if a copy of the entire F plasmid is transferred. In a case the F-factor found in the chromosome - the cell is called an Hfr cell (high frequency of recombination). Hfr cell functions as a donor during conjugation. When chromosomal DNA from an Hfr cell enters into a recipient and their DNA is combined - the recipient cell becomes a recombinant bacterium with a new genetic variant. R plasmids and antibiotic resistance - sometimes, mutation in a chromosomal gene of a pathogen can confer resistance - it may make it less likely to transport antibiotic into its cell. Mutation in a different gene may alter the intracellular target protein for an antibiotic molecule, reducing its inhibitory effect. In other cases, bacteria have "resistance genes" which code for enzymes that destroy or hinder the effectiveness of certain antibiotics. Such resistance genes are often carried by plasmids known as R plasmids (R - resistance). Resistant strains of pathogens are becoming more common, making the treatment of certain bacterial infections more difficult. The problem is compounded by the fact that many R plasmids, like F plasmids, have genes that encode pili and enable DNA transfer from one bacterial cell to another by conjugation - spreading the resistance property.

what are environmental mutagens? how do these differ from teratogens? how are they classified? (99)

Teratogen - a substance which can cause physical defects in a developing embryo whereas mutagen is a material that induces genetics changes - mutations - in the DNA - see also question 57. With all written above about teratogen relevance for this question, some more information regarding to mutagens (question 57): The same 3 groups that affected teratogenesis is relevance to mutagenesis - physical, chemical and biological mutagens. Physical Mutagens Ionizing radiation - such as X-rays, has a high energy and passes through the tissue. When passing through the tissue it may cause collisions with atoms and release their electrons (excitement), forming free radicals and ions, which can react with other molecules, cellular structures including DNA. Absorbed radiation dose is measured in units of Gray (Joul per kg). Mutagenic effect depends on dose, exposure time, phase of the cell cycle and the quality of reparation mechanisms. The ionizing radiation may cause chromosomal breaking and alternations as well as simply gene mutation. Electromagnetic radiation of short wavelength and higher energy than the visible radiation (e.g. - Ultraviolet). UV radiation, even though has less energy than X-rays, also capable of increasing electron energy and cause excitation of atoms. It is absorbed by many organic molecules, especially DNA molecules - thus, it is powerful mutagen, especially for unicellular organisms. In multicellular organisms UV damages only the superficial cells - in humans it can cause skin cancers. Nowadays, risk on UV radiation increases with decreasing the content of ozone in the atmosphere. Purines, when affected - being hydrated, Pyrimidines - formation of pyrimidine dimers (dimers of thymine), which cause mutations in two ways: Changes the structure of the DNA double helix, thus prevents the progression of DNA polymerase and interrupt the replication of DNA; Repair mechanism doesn't function well - it may result in incorrect classification of bases Chemical Mutagens Induced mutation may be caused by chemical factors: Deamination - base changing as a result of removal of amine group. Base Analogs - they are chemical compounds that are sufficiently similar to the normal nitrogen bases of DNA; they are occasionally incorporated into DNA during replication in place of normal bases. Many of these analogs have pairing properties unlike those of the normal bases, and thus they can produce mutations by causing incorrect nucleotides to be inserted during replication. Alkylating agents are a class of chemotherapy drugs that bind to DNA and prevent proper DNA replication. They have chemical groups that can form permanent covalent bonds with nucleophilic substances in the DNA. These agents may lead to crosslinking between two or more molecules by a covalent bond - either in the same strand (intrastrand crosslink) or in the opposite strands of the DNA (interstrand crosslink). DNA replication is blocked by crosslinks, which causes replication arrest and cell death if the crosslink is not repaired. Hydroxylation agents - may change cytosine to hydroxylaminocytosin which pairs with adenine - changes C—G to A—T pairing. Biological mutagens: Viruses - Virus DNA may be inserted into the genome and disrupts the genetics function Bacteria - some bacteria can cause inflammation, causing DNA damage and reducing efficiency of DNA repair system. Transposons - elements that able to move from one site of the genome to another; two types - LINE and SINE (long and short interspersed nuclear element). Their movements in the genome may have a mutagenic effect.

genetics and clinical importance of the Rh blood group system? (73)

The Rh blood group system is one of 35 current human blood group systems. It is the second most important blood group system after ABO groups, consists of highly polymorphic - 50 defined blood group antigens found on RBCs, among which the five antigens D, C, c, E and e are the most important. The commonly used terms - Rh factor, Rh positive and Rh negative refer to the D antigen only. Rhesus blood type named after the rhesus monkey, since they were initially used in the research to make the anti-serum for typing blood samples. Inheritance Rh phenotypes are identified by identifying the presence or absence of the Rh surface antigens. If both of a child's parents are Rh negative, the child will definitely be Rh negative (17% of European population). Otherwise the child will be Rh+. The D antigen is inherited as one gene - RHD - on the short arm of the 1st chromosome. If the gene codes for RhD on RBCs is present - the individual is Rh+. There is no d antigen - lowercase "d" indicates the absence of the D antigen (the gene is usually deleted). D antigens generally induces a strong antibody response in an individual that lacks the D antigen (recessive homozygote dd - Rh negative). [Epitopes - antigenic determinant - the part of an antigen that is recognized by the immune system; a specific piece of the antigen to which an antibody binds] The Epitopes for the next 4 most common Rh antigens (C, c, E, e) are expressed on RhCE protein, encoded by RHCE gene, found also on chromosome 1. RHD and RHCE are closely linked structural genes (very strong gene linkage) - combination of alleles of both genes is transmitted from generation to generation in a block - haplotypes. Antigens C, c, E, e are not as strong and important as the D antigen, but may provoke formation of antibodies. Expression of these antigens is controlled by one gene and is the result of alternative splicing of the primary transcript RHCE gene. There is codominance between alleles C/c and E/e. Rh Factor An individual either has, or doesn't have, the Rh factor on the surface of their RBCs. This term refers only to the most immunogenic D antigen of the Rh blood group system: Rh+ does have the D antigen, Rh- doesn't. In contrast to the ABO blood group, immunization against Rh can generally only occur through blood transfusion or placental exposure during pregnancy in woman (doesn't occur naturally). Erythroblastosis fetalis - Hemolytic disease of the newborn It is a disease of the fetus and newborn child characterized by agglutination and phagocytosis of the fetus's RBCs, most often occurs when the mother is Rh negative and the father is Rh positive; the baby inherited the Rh-positive antigen from the father, and the mother develops anti-Rh agglutinins from exposure to the fetus's Rh antigen. In turn, the mother's agglutinins diffuse through the placenta into the fetus and cause RBC agglutination 🡪 the agglutinated RBCs subsequently hemolzyed and release hemoglobin into the blood 🡪 the fetus macrophages convert the hemoglobin into bilirubin 🡪 baby have jaundice - skin become yellow; the antibodies can also attack and damage other cells of the body. The newborn is usually anemic at birth, and the anti-Rh agglutinins from the mother usually circulate in the infant's blood for another 1-2 months after birth, destroying more and more RBCs. The hematopoietic tissues of the infant attempt to replace the hemolyzed RBCs - liver and spleen become greatly enlarged, produce RBCs in rapid production rate many in early forms - including many nucleated blastic forms - immature RBC's contains nucleus. As said, first exposure to Rh-positive antigen do not elicit serious reaction (mother doesn't develop sufficient anti-Rh agglutinins to cause harm); however, about 3% of second Rh-positive babies exhibit some signs of erythroblastosis fetalis; 10% of 3rd babies and the incidence rises with subsequent pregnancies. The severe anemia is a major cause of death of these fetus, but many children who survived the anemia exhibit permanent mental impairment or damage to motor areas of the brain because of precipitation of bilirubin in the neuronal cells, causing destruction of many - Kernicterus process. Rh Immune response: When RBCs containing Rh factor (D) are injected into a person whose blood doesn't contain the Rh factor (into a Rh negative person) - anti-Rh agglutinins develop slowly, reaching maximum concentration of agglutinins about 2-4 months later, and the extent of immune response vary between people. If a Rh-negative person has never before been exposed to Rh-positive blood, transfusion of Rh+ blood into this person likely cause no immediate reaction, but anti-Rh antibodies can develop in sufficient quantities during the next 2-4 weeks to cause agglutination of the transfused cells which will then hemolyzed by the macrophage system. Thus, a delayed transfusion reaction occurs, usually mild. Upon subsequent transfusion of Rh-positive blood into the same person, the transfusion reaction is greatly enhanced and can be immediate and severe.

What is the Law of Independent Assortment and how does this "law" relate to meiosis? (1)

each pair of alleles segregates independently of the other pairs of alleles during gamete formation occurs during anaphase I only applies to genes on different chromosomes (not homologous) or genes that are far apart on the same chromosome

what are the repair mechanisms for double-stranded breaks? (58,59)

1. Nonhomologous end-joining- the broken ends are directly ligated without the need for a homologous template. The process takes place in 3 stages: End binding NHEJ is initiated by recognition and binding of the Ku protein to the broken DNA ends. Ku is a heterodimer of Ku70 and Ku80 that forms the DNA-binding component by forming a ring that encircles the DNA. By forming a bridge between the broken ends, Ku acts to structurally support and align the DNA ends, protect them from degradation and prevent binding to unbroken DNA. Ku effectively aligns the DNA, while still allowing access of polymerases, nucleases and ligases to the broken DNA ends to promote end joining. Once Ku is in place, it recruits the catalytic subunit of DNA-PKcs, which is a serine/threonine kinase. DNA PKcs phosphorylate DNA ligase IV (and XRCC4), which affect their interaction with Ku and other proteins. End-processing: NHEJ requires two DNA blunt ends in order to join them together. In some cases, terminal processing of the DNA ends is required before ligation, due to one strand overhang (בולט מעל). Single-stranded overhang can be trimmed off via nuclease activity occurs by Artemis protein which arrives and bind to the complex formed and phosphorylated by DNA-PKcs. Ligation The DNA ligase IV complex, consisting of the catalytic subunit DNA ligase IV and its cofactor XRCC4, performs the ligation step of repair. Homologue recombination repair- After a double-strand break occurs, BRCA1, BRCA2 and ATM become activated. ATM is a kinase protein that becomes activated in response to DNA damage and phosphorylates BRCA1. BRCA1, BRCA2 and Rad51 cause resection - exonucleases perform resection of the DNA on either side of the break occurs. RPA (Replication protein A) - which perform the function of SSBP of prokaryotes - has high affinity for ssDNA, binds the single strand and holds it in place. Rad51, a recombinase protein binds to the strand with RPA, and begin searching for DNA sequences similar to that of the single strand DNA but in the homology chromosome. After finding such a sequence, the single-stranded strand invades into the similar/identical homology DNA - strand invasion. If it occurs during mitosis, the sister chromatid (which is identical to the damaged DNA) provides the template for repair. In meiosis, the recipient DNA may by similar but not necssarily identical. The displacement loop - D loop - is formed during strand invasion, caused due to displacement of one strand from the homology chromosome. DNA polymerase extends the end of the invading strand, using the homology as template. The other non invading strand, also synthesizes DNA using the displacement strand as a template. Whether recombination results in chromosomal crossover is determined by how the double Holliday junction is cut: chromosomal crossover will occur if one Holliday junction is cut on the crossing strand (along the horizontal purple arrow) and the other Holliday junction is cut on the non-crossing strand (along the vertical orange arrowheads). From the other hand, if the two Holliday junctions are cut on the crossing strands along the horizontal purple arrows at both Holliday junctions - chromosomes without crossover will be produced. microhomology-mediated end joining

what are the methods to analyze linkage? (16)

3-point experiment - Three-point experiment: We have locus A, B and C. We know the position of loci A and C. We want to find B. If B is between A and C, then recombination between A and B occurs with probability x, recombination between B and C is probability y. The probability of double recombination (ie between A and B and between B and C) is significantly lower and is equal to the product of the probabilities x and y. The correct order of loci A, B and C is therefore determined using double recombinants, which must be the least. two-point linkage analysis In case that location of the trait is unknown, at least one marker for each chromosome is required (the more markers 🡪 more accurate results). The location of the linked marker, which segregates with the trait more often than would be expected by chance only, will indicate us that the trait and the marker found near each other on the same chromosome with high probability. If we had more than 1 marker that segregates together with the trait more often - we can be sure regarding our decision which chromosome contains the trait, and where the trait is localized on the specific chromosome - by using recombination frequencies of the trait and the markers. LOD score - Logarithm of the odds a statistical procedure used to estimate whether two loci are likely to lie near each other on a chromosome - and are therefore likely to be inherited together as a linkage group. We calculate the ratio of the probability for having a linkage with the probability that chance occurred (no linkage - there is independent assortment between the two genes); the log of the ratio is the LOD score. LOD score of 3 or higher considered as high probability for linkage. LOD score of -2 or less is taken as proof that the loci are not linked. LOD score = log (probability of birth sequence with a given linkage value /probability of a birth sequence with no linkage) Autozygosity Mapping - autozygosity occur when individuals are homozygous at a particular locus by descent from a common ancestor (referred to inbred families - consanguineous marriage). There is high probability that children that are homozygous for a disease also have the same markers that are linked to the disease; Thus, a search can be made for shared areas of homozygosity in affected relatives using highly polymorphic markers such as microsatellites. In pedigree with a relatively large number of affected individuals, some of them will also share the linked disease-marker. The marker location is known, thus we can localize the disease loci as well. Linkage disequilibrium (used in association studies) - defined as the association of two alleles at linked loci more frequently than would be expected by chance, and is also referred to as allelic association. (LD means simply a nonrandom association of alleles at two or more loci, and detecting LD does not ensure either linkage or a lack of equilibrium.) The concept and the term relate to the study of diseases in populations rather than families, because in families the association between specific alleles and the disease in question holds only within an individual family - in a separate affected family a different marker may show association with the disease (because the markers are polymorphic). The significance of linkage disequilibrium is that even though the markers are usually highly polymorphic - still, unrelated people have both the diseases and the markers (which generally, if the genes weren't associated - it occurs with very low probability). By locating these markers which are in disequilibrium and are linked to the diseases, we can also find the mutated DNA sequence responsible for the disease. Linkage disequilibrium analysis is different than the genetic analysis - in genetic analysis we are looking for certain highly polymorphic markers that will appear with the disease - but only within certain family that we examine - if we'll examine a different family, we will have to use different markers; linkage disequilibrium means that there is a disequilibrium with the presence of the highly polymorphic marker in a population of people that aren't connected to each other. In addition, LDs provide information on the occurrence of new mutations or genetic drift. The question of the number of markers needed to describe LD on the genome to identify genes associated with a given disease is very unclear. If we had a sufficiently detailed description of the structure of the human genome (by sequencing), we could reduce the number of markers needed to mark LD on the genome and focus only on gene-rich sites.

structure and function of prokaryotic cells? what is a prokaryote? how can they be distinguished? DNA, RNA structure? extra-cellular structures? what are achaea and cyanobacteria? (84)

A prokaryote is a unicellular organism that lacks a membrane-bound nucleus (karyon), mitochondria, or any other membrane-bound organelle - that's what differentiate them from eurokaryote, which contains nuclei and organelles bound in membranes. biologists now estimate that each human being carries nearly 20 times more bacterial, or prokaryotic, cells in his or her body than human, or eukaryotic, cells. Prokaryotes can be divided into two domains, Archaea and bacteria. Bacteria are much smaller than eukaryotic cell - average size only 1-5 µm (much smaller than the average 10-100µm of many eukaryotic cells) Also the shape of prokaryotic cells varies, most prokaryotic cells fall into one of the three following categories, each category gives the cell its own advantages: Rod shaped - large surface area, better metabolic exchange with the environment Spherical - less prone to dry Spiral- usually motile In the prokaryotes, all the intracellular water-soluble components (proteins, DNA and metabolites) are located together in the cytoplasm enclosed by the cell membrane, rather than in separate cellular compartments. Structure of prokaryotes: A key feature of nearly all prokaryotic cells is the cell wall - located outside the cell membrane - and maintains cell shape, protects the cell and prevents it from bursting in a hypotonic environment. Salt can be used to preserve foods because it causes food-spoiling prokaryotes to lose water, preventing them from rapidly multiplying. Most bacterial cell walls contain peptidoglycan - a polymer composed of modified sugars cross-linked by short polypeptides. It encloses the entire bacterium. Gram-positive bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids. In contrast, Gram-negative bacteria have a relatively thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. The plasma membrane Prokaryotic cells can have multiple plasma membranes. Prokaryotes known as "gram-negative bacteria," for example, often have two plasma membranes with a space between them known as the periplasm. As in all cells, the plasma membrane in prokaryotic cells is responsible for controlling what gets into and out of the cell. A series of proteins stuck in the membrane also aid prokaryotic cells in communicating with the surrounding environment. Among other things, this communication can include sending and receiving chemical signals from other bacteria and interacting with the cells of eukaryotic organisms during the process of infection. Whereas eukaryotes possess mitochondria responsible for production of energy and processing of nutrients, in prokaryotes the proteins found on the plasma membrane contain enzymes associated with these functions. Many of the plasma membrane's lipids contain molecules called hopanoids that are derived from the same precursors as the human's cholesterol, and also assist in maintaining membrane stability. Cytoplasm The cytoplasm in prokaryotic cells is a gel-like, yet fluid, substance in which all of the other cellular components are suspended. It is very similar to the eukaryotic cytoplasm, except that it does not contain organelles. Recently, biologists have discovered that prokaryotic cells have a complex and functional cytoskeleton similar to that seen in eukaryotic cells. The cytoskeleton helps prokaryotic cells divide and help the cell maintain its plump, round shape. Ribosomes Prokaryotic ribosomes are smaller (70S compared to the human 80S - The 70S ribosome is made up of a 50S and 30S subunits) and have a slightly different shape and composition than those found in eukaryotic cells. Bacterial ribosomes, for instance, have about half of the amount of ribosomal RNA (rRNA). Despite these differences, the function of the prokaryotic ribosome is virtually identical to the eukaryotic version. Just like in eukaryotic cells, prokaryotic ribosomes build proteins by translating messages sent from DNA. Genetic material (DNA and RNA) All prokaryotic cells contain large quantities of genetic material in the form of DNA and RNA. Because prokaryotic cells, by definition, do not have a nucleus, the single large circular double-stranded of DNA containing most of the genes needed for cell growth, survival, and reproduction is found in the cytoplasm - in a region which is called nucleoid - usually a singular, circular chromosome. The DNA of prokaryotes is called "genophore" - commonly referred to prokaryote chromosome. As in eukaryotic cells, the prokaryotic chromosome is intimately associated with special proteins involved in maintaining the chromosomal structure and regulating gene expression. In addition to a single large piece of chromosomal DNA, many prokaryotic cells also contain small pieces of DNA called plasmids. These circular rings of DNA are replicated independently of the chromosome and can be transferred from one prokaryotic cell to another through pili, which are small projections of the cell membrane that can form physical channels with the pili of adjacent cells. The transfer of plasmids between one cell and another is often referred to as "bacterial sex." The genes for antibiotic resistance, or the gradual ineffectiveness of antibiotics in populations, are often carried on plasmids. If these plasmids get transferred from resistant cells to nonresistant cells, bacterial infection in populations can become much harder to control. Inclusion bodies: granular structures found in the cytoplasm and act as food reserves. Extra Cellular structures - mainly involved either with locomotion of the cell or its attachment to surfaces. Flagella - Thin-hair structure, used for locomotion in many bacteria. Each flagellum is hollow but rigid cylindrical filament, made of the flagellin protein. It attaches to a basal body, which secures it to the cell wall and plasma membrane, and provides the flagellum with energy required for its movements. Pili - the resemble a short flagella, but they do not penetrate to the plasma membrane and do not associated with motility. Their function is to anchor the bacterium to surfaces. Glycocalyx - Either as a loosely bound slim layer, protect the bacteria against dryness, and important to the bacteria attachment to other organisms tissue, or, may be thick capsule - offer protection against phagocytic cells of the immune system. Archea - constitute a domain and kingdom of single-celled microorganisms. These microbes are prokaryotes, meaning that they have no cell nucleus or any other membrane-bound organelles in their cells. Archaea and bacteria are generally similar in size and shape. Despite this morphological similarity to bacteria, archaea possess genes and several metabolic pathways that are more closely related to those of eukaryotes, notably the enzymes involved in transcription and translation. Archaea were initially viewed as extremophiles living in harsh environments, such as hot springs and salt lakes, but they have since been found in a broad range of habitats, including soils, oceans and the human colon, oral cavity, and skin. Cyanobacteria Gram-negative phylum of bacteria (phylum = below kingdom, above class). The only prokaryotes with plantlike, oxygen-generating photosynthesis (obtain energy from photosynthesis). The name "cyanobacteria" comes from the color of the bacteria (blue). Like other prokaryotes, cyanobacteria have no membrane-sheathed organelles. Photosynthesis is performed in distinctive folds in the outer membrane of the cell. Cyanobacteria lives in a wide variety of habitats such as moist soils and in water.

what is a virus? what are their structures? origin? how do they replicate? what are their cycles? how are they classified? relevance in medicine? (90)

A virus is a small infectious agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. While not inside an infected cell, viruses exist in the form of independent particles - viral particles, also known as virions. They consist of three parts: The genetic material made from either DNA or RNA. The genome of the virus may be dsDNA, ssDNA or RNA. A protein coat - called Capsid - which surrounds and protects the genetic material. The capsid is responsible for attaching the virus to the surface of the host cell and for the nucleic acid's protection. It is made by subunits - capsomers - which are proteins that encoded by the virus genetic material. - capsids with cubic (icosahedral) symmetry form a regular icosahedral. It is a body with 12 vertices and 20 walls of equilateral triangles. Capsids with spiral (helicoidal) symmetry are difficult to distinguish from a nucleoid and therefore we speak of a nucleocapsid. It consists of a nucleic acid helix to which the capsomeres are closely aligned. The virus family is determined by electron microscopy according to the diameter and pitch of the thread. Capsid of cubic symmetry in adenoviruses Viruses with complex symmetry, we classify flagellar bacteriophages here, where the head is formed according to cubic symmetry, while flagellar according to binary. Poxviruses, the largest animal viruses, have a nucleoid shaped like a double-sided disk. Lenticular lateral bodies are placed in its concavities and as a whole it is wrapped in a wrapper. In some cases - envelope of lipids that surrounds the protein coat. The envelope is typically derived from portions of the host cell membranes, but includes some viral glycoproteins which are encoded by the virus. They may help viruses to avoid a host immune system. The glycoproteins on the surface of the envelope serve to identify and bind to receptor sites on the host's membrane. The lipid viral envelope then fuses with the host's membrane, allowing the capsid and viral genome to enter and infect the host. • The average virion is about 20-300nm. Viral replication cycles: Attachement, Penetration and uncoating, Replication and assembly, Release. Viral populations do not grow through cell division, because they are acellular. Instead, they use the machinery and metabolism of a host cell to produce multiple copies of themselves, and they assemble in the cell. In short - after attachment to the target, penetration and uncoating, the genetic material of the virus injected into the host. It can either be RNA virus, which will reverse transcript to form cDNA 🡪 incorporation, or can be DNA virus which incorporate into the genome. Anyway, the replication cycle based on the formation of viral particles by the host, which will eventually ends in release of the viral particles and spread to other hosts. In bacteriophages specifically, the replication cycle can be either lytic or lysogenic cycle; in the lytic cycle - entrance to cell following directly by viral components production till lysis and release, while in lysogenic - incorporation into the genome following in dormant period till entrance to lytic cycle. The life cycle of viruses differs greatly between species but there are six basic stages in the life cycle of viruses: Attachment (adsorption) is a specific binding between viral capsid proteins and specific receptors on the host cellular surface. This specificity determines the host range of a virus. E.g. HIV virus (which is a RNA virus) able to penetrate only T-helper cells by binding to their specific CD4 receptors. Penetration: Virions enter the host cell through receptor-mediated endocytosis or membrane fusion. This is often called viral entry. Uncoating is a process in which the viral capsid is removed: This may be by degradation by viral enzymes or host enzymes or by simple dissociation; the end-result is the releasing of the viral genomic nucleic acid. Replication of viruses involves primarily multiplication of the genome. Replication involves synthesis of viral messenger RNA (mRNA) from "early" genes, viral protein synthesis, possible assembly of viral proteins, then viral genome replication mediated by early or regulatory protein expression. Assembly -self-assembly of the virus particles by forming new protein capsomers to construct the capsid and replicate the viral DNA/RNA. release - Viruses can be released from the host cell by lysis (The Lytic cycle), a process that kills the cell by bursting its membrane and cell wall if present: This is a feature of many bacterial and some animal viruses. Some viruses undergo a lysogenic cycle where the viral genome is incorporated by genetic recombination into a specific place in the host's chromosome. Whenever the host divides, the viral genome is also replicated. The viral genome is mostly silent within the host. However, at some point, the provirus or prophage may give rise to active virus, which may lyse the host cells. Enveloped viruses (e.g., HIV) typically are released from the host cell by budding. During this process the virus acquires its envelope, which is a modified piece of the host's plasma or other, internal membrane. Prior to budding, the virus may put its own receptor onto the surface of the cell in preparation for the virus to bud through, forming an envelope with the viral receptors already on it. Though budding does not immediately destroy the host cell, this process will slowly use up the cell membrane and eventually lead to the cell's demise. RNA Viruses vs DNA virus A DNA virus is a virus that has DNA as its genetic material and replicates using a DNA-dependent DNA polymerase. The nucleic acid is usually double-stranded DNA (dsDNA) but may also be single-stranded DNA (ssDNA). RNA viruses - RNA is the genetic material of some medically important human viruses including HIV, common colds and more. RNA Viruses capable of integrating into the genomes of their hosts - also called "retroviruses". Because the retroviral genome is RNA, whereas that of the host is DNA, a retrovirus produces reverse transcriptase, an enzyme that synthesizes cDNA from either a RNA template. RNA 🡪 cDNA 🡪 copies to make dsDNA 🡪 recombine into the host DNA. The DNA copy of the viral genome then integrates into the host chromosome. A viral genome incorporated into the host chromosome is called a provirus, and it is replicated by host enzymes when the host chromosome is duplicated. The provirus undergoes transcription to produce numerous copies of the original viral RNA genome. This RNA encodes viral proteins and serves as genomic RNA for new viral particles. As these viruses escape the cell, they collect patches of the cell membrane to use as their envelopes. Another classification of RNA viruses is plus-strand and minus-strand RNA. These RNA viruses that directly insert their RNA into the cytoplasm of the host and not incorporate to the host DNA, rather, synthesize their protein products directly from their RNA can be plus-strand - means that the RNA is directly used to form protein, or minus-strand - which the RNA is first need to transcribed into mRNA before translation can take place. Importance of viruses in medicine Biological studies - viruses have been used in molecular and cellular biology studies. These viruses provide the advantage of being simple systems that can be used to manipulate and investigate the functions of cells. They have been used extensively in genetics research and understanding of the genes and DNA replication, transcription, RNA formation, translation, protein formation and basics of immunology. Viruses are being used as vectors or carriers that take the required material for treatment of a disease to various target cells. Viruses in bacteriophage therapy - highly specific viruses can target, infect and destroy pathogenic bacteria. Bacteriophages are believed to be the most numerous types of viruses accounting for the majority of the viruses present on Earth. Viruses in cancer prevention and control - the key element of gene therapy are the introduction of functioning genes into the cells of a human patient. This new gene shows desired functions and corrects defective or non-operational genes within those cells. The most common target has been cancers - "adenoviruses" are most commonly used as vectors, and can be engineered both to enhance specificity and to minimize unwanted effects. Vaccines - Vaccines against polio, measles, chicken pox etc. use live and weakened viruses causing the disease or dead virus particles. These, when introduced into a healthy individual, help the immune system to recognize and mount an immunity against the virus. The body remembers the organism and attacks it in case of a later infection thus preventing the disease. Diseases caused by DNA viruses: smallpox, herpes, chickenpox origin There are three main theories that try to explain the origin of viruses: Most viruses originated and developed in parallel with primitive cells. Probably the first RNA (a structure capable of replicating itself) developed in two lines: viral and cellular. If true, RNA viruses would be older than cellular life forms. The most complex viruses, poxviruses , are thought to have arisen by regressive development from single cells or from cellular organelles ( mitochondria , chloroplasts). Other viruses probably originated from cellular material that acquired the ability to exist in part independently. The independence of the RNA molecule, which encodes RNA polymerase and to which the protein coat gene was added, may have been the beginning of RNA virus formation. The emergence of DNA viruses was probably based on the independence of transposons or from a primitive cell in which the DNA had not yet been organized into chromosomes. Should mutation of genes leading to a protein capable assemble into icosahedral clipboard may arise a virus whose genome has been further enriched other genes.

what is codominance? (1)

AB blood type, MN blood group Heterozygote with showing both. variation on dominance relationships between alleles; in this variation, the two alleles each affect the phenotype in separate, distinguishable way. For example, the human MN blood group is determined by codominant alleles for two specific molecules located on the surface of RBCs, the M and N molecules. Individuals homozygous for the M allele (MM) have RBCs with only M molecules; individuals homozygous for the N allele (NN - N molecules; but both M and N molecules are present on the RBCs of individuals heterozygous for the M and N alleles (MN). It is not intermediate between the M and N phenotypes - which distinguishes codominance from incomplete dominance. Both M and N phenotypes are exhibited by heterozygotes, since both molecules are present.

what are antibodies? what are epitopes? genetics of immunoglobulins - types, functions? what are the classes and how are they so diverse? what are the segments of the light chain? why are B-cell receptors and T-cell receptors important? how are they strucutured? (78)

Antibodies bind to antigen's epitopes. Antigen is a molecule that is recognized by cells of the immune system and may elicit immune responds. The cell of the immune system binds only to a small part of the antigens, the epitopes. The response of the organism may be either cellular (cell's attacking the antigen by phagocytosis) or humoral (antibodies bind to antigen, label it and destroy it). Antibodies (immunoglobulins) are glycoproteins which composed of two identical light and two identical heavy chains, bound by disulfide bonds and non-covalent forces. The light chains can either appear as 2 lambda light chains or 2 kappa light chains. The isolated carboxyl terminal portion of the heavy chains molecule is called the Fc region, which is recognized by receptors present on several cell types, leads to binding of the antibody to the cells surfaces. Near the amino terminal part of the light and heavy chains, the first 110 amino acids are variable among different antibody molecules, thus the region is called the variable region, gives the antibodies their diversity. Antigen binding site of the antibody consist of one light chain and one heavy chain. Antibodies can appear as either free antibodies or membrane bound antibodies: Free antibodies - circulating in plasma or locate in tissues or epithelial secretions. They are secreted by plasma cells (which differentiated from B-lymphocytes). Their receptors recognize and bind to a specific epitopes. Membrane bound antibodies - are integral membrane proteins on the surface of lymphocytes (IgD IgM on B cell, TCR on T-cell, IgG on macrophages, neutrophils and eosinophils, IgE on mast cells and basophils). Type of antibodies - IgG - most abundant, can cross the placental barrier. Main immunoglobulin in the secondary immune response (IgM in primary response 🡪 class switching 🡪 IgG). IgA - main immunoglobulin found in secretions, tetramer. IgM - together with IgD is the major immunoglobulin found on B lymphocytes. Have both membrane bound and free circulating form. It is formed in the primary immune response to antigen, and following formation of memory B-cells, they going through class-switching, which changes the constant region of IgM and IgD to IgG/IgA/IgE - but, with same antigen specificity (variable region stays the same) - in a process called class switching. IgM composed of multimer antibodies - 5 units, joined together by "J-peptide" joining. IgE - secreted from plasma cells. Its Fc region has great affinity for receptors present in basophils and mast cells, causing allergic reaction. Action of immunoglobulins: They can agglutinate cells and to precipitate soluble antigens, thus neutralizing their effect on the body. Stimulate phagocytosis of microorganisms and other particles when they cover them, in a process which is called Opsonization. It occurs due to the presence of receptors for Fc region of IgG on phagocytic cells - macrophages, eosinophils and neutrophils. Antibodies activates the complement system, which is group of ~20 plasma proteins produced mainly in the liver (most important is C3). Antibodies have complement-binding site on their constant region, which following binding to pathogen - elicit the complement binding and activation ("classical pathway of the complement system" - C3 inactivated become activated 🡪 phagocytosis/MAC - membrane attack complex). The complement system stimulates phagocytosis by phagocytic cells, induce lysis of microorganisms by acting on their cell membrane. Generation of antibody diversity The diversity of the antibodies is achieved by several mechanisms: Somatic recombination of the antibodies gene segments - each antibody is composed of segments. Each segment has number of copies which are slightly differ from each other. Different combination of these copies leads to formation of diversity in antibodies, each one of them acts on specific antigen (in the germ cell - all the genes are present, but when the cell matured - i.e. become somatic cell - recombination occurred) The Light chains of antibody composed of 3 types of segments: V segments - encode the variable region, has 30-35 copies J segments - encode a short set of nucleotide that join (junctional segment) the V and C segments. There are 5 different J genes. C segments - encode for the constant region of the chain, has one gene, the depends on the type of response (class switching), found on chromosome 14; the first transcription is of IgM or IgD, following class switching - IgG. Initially, immature lymphocyte inherits all of the possible segments present in the germ line. During its maturation, a somatic recombination within a single chromosome occurs, and one of the V genes move next to one of the J genes, while others segments are deleted. The V-J-C gene is transcribed, forming mRNA which is translated into a light chain. Heavy chains are also arranged in V, J, and C segments, though they also contain D segments. The D segments are joined to the J segments and then V segment joins the D-J complex - gives even higher variability. Each type of light chain can combine with each type of heavy chain, leads to diversity of antibodies. The recombination process that joins V, J, D and C gene segments is imprecise, and a few random nucleotides are frequently lost or gained at the junctions of the recombining segments - greatly enhance the junctional diversity. Somatic hypermutation - high rate of mutation is a characteristic of immunoglobulin genes and contribute to its diversity. Recognition of foreign antigen is possible by the existence of two types of molecules acting as receptors: B-Cell receptors (BCR) T-Cell receptors (TCR) B-cell receptor (BCR) is a transmembrane receptor protein located on the outer surface of B-cells. The receptor's binding moiety is composed of a membrane-bound antibody that, like all antibodies, has a unique and randomly determined antigen-binding site (VDJ recombination). When a B-cell is activated by encounter with an antigen that binds to its receptor, the cell proliferates and differentiates to generate a plasma cells and memory B-cells population (requires activation by T-helper II cell - which occurs following secretion of cytokines IL-4 IL-5 IL-6). The ability of the immune system to recognize antigens depends on receptor-forming molecules on B and T cells; both cell populations can recognize a huge number of antigens. The cellular and molecular processes leading to this diversity are as follows for both types of receptors: First of all, these are changes that have accumulated during evolution and are passed down from generation to generation when the original gene was "multiplied" by a process of repeated duplication . At the same time, mutations took place that differentiated these genes, so they are not exact copies of the original gene (so the so-called gene family was created). When these different genes remain stored in one chromosomal region, they form a gene complex- it contains many genes or segments that encode variable regions and only one or a few segments encode a constant region. Within the complex, other changes take place in somatic cells (somatic diversification), which, however, are not passed on to the offspring (these are various rearrangements of complex genes in individual cells, which result in a definitive DNA segment encoding a specific receptor). Genetic B receptors Ig B cell receptors Immunoglobulin chains are encoded by three gene complexes: IgH - for heavy chain (chromosome 14), IgK and IgL for light chains κ [kappa] (chromosome 2) and λ [lambda] (chromosome 22). The BCR has two crucial functions upon interaction with the antigen: One function is signal transduction, involving changes in receptor shape. The second function is to mediate internalization of the antigen (get it into the cells), and present the peptides to helper T-cells, elicits immune response. B cell receptors are composed of two parts: Membrane-bound immunoglobulin molecule of one isotype: IgD, IgM (most commonly found on B-cells is IgM). Signal transduction moiety - a heterodimer called Ig-alpha and Ig-beta (CD79), bound together by disulfide bridges. Each member of the dimer spans the plasma membrane and has a cytoplasmic tail, bearing immunoreceptor tyrosine-based activation motifs (ITAM) - which are important for signal transduction in immune cells. The tyrosine residues within these motifs become phosphorylated following interaction of the receptor molecules with their ligands (tyrosine-kinase receptor), and form docking sites for other proteins involved in the signaling pathways of the cell. Like B-cells, each mature T-cell has genetically determined specificity for one type of antigen that is mediated through the cell's receptors. T-cell receptors are structurally similar to immunoglobulins and are located on the cell surface. Most of the receptors are composed of one alpha and one beta polypeptide chains held together by disulfide bonds. One end is embedded in the cell membrane, the other projects away from the cell and binds antigens. Each T-cell receptor possesses a constant region and a variable region. The variable regions of the two chains provide the antigen-binding site. T receptor is encoded by three gene complexes: TCR-alpha stored on chromosome 14, TCR-beta + TCR-gamma on chromosome 7. The genes that encode the alpha and beta chains of the T-cell receptor are organized like those that encode the heavy and light chains of immunoglobulins: each gene is made up of segments that undergo somatic recombination before the gene is transcribed. For example, the human gene for the alpha chain initially consists of 44 to 46 V (variable) gene segments, 50 J (junctional\connecting) gene segments, and a single C (constant) gene segment. The order of the field is V J C. The organization of the gene for the beta chain is similar, except that it also contains D segments. Alpha and beta chains combine randomly. During development of T-lymphocytes, the segments will be built as follows: One of the V-alpha (variable segment) genes is connected to one of the J-alpha, and there is a deletion of DNA, which is deposited between the selected genes. Finally, mRNA introns excised, and completed "treatment" of the mRNA is done. TCR heterodimers are associated with additional polypeptides, with which they form a TCR-complex = a group of 3-5 polypeptides that are required for TCR expression on the surface of the T cell and for signal transmission into the cell. Heterodimer composed of 2 polypeptide chains : 95% - α and β chains (TCR2), 5% - γ and δ chains (TCR1). TCR2 TCR2 chains α and β are transmembrane polypeptides, composed of an external part that is anchored in the plasma membrane by a transmembrane part and a short cytoplasmic region.

genetic control of antibody production? what are antibodies? how are they produced? structure? types? (76)

Antibody, also known as immunoglobulin (Ig) is a large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to identify and neutralize pathogens such as bacteria and viruses. The antibody recognizes a unique molecule of the harmful agent (an antigen). Each tip of the Y of an antibody contains a "paratope" (lock - also known as Fab region - antigen-binding fragment) that is specific for one particular epitope (key) on an antigen, allowing these two structures to bind together with precision. By this binding mechanism, an antibody tags the pathogen or infected cell for attack by other parts of the immune system, or can neutralize its target directly. The ability of an antibody to communicate with the other components of the immune system is mediated via its Fc region (the base of the Y) which contains a conserved glycosylation site involved in these interactions - The Fc fragment determines the secondary biological functions of antibody molecules (except of its antigen-binding region) - by binding complement and Fc receptors on a number of different cell types involved in the immune response. The production of antibodies is the main function of humoral immune system. Antibodies are secreted by B-cells of the adaptive immune system - activated B-cells, which are called Plasma cells. Antibodies can occur in two physical forms - a soluble form that is secreted from the cell to be free in the blood plasma, and a membrane-bound form, that is attached to the surface of a B-cell and is referred to as the B-cell receptor (BCR). The BCR facilitates the activation of the B-cells and their differentiation into plasma cells or memory B cells (which will survive in the body and remember that same antigen, so the B-cells can respond faster upon future exposure). In most cases, interaction of B-cell with a T-helper cell is necessary to produce full activation of the B-cell and therefore, antibody generation following antigen binding. Structure: Antibodies made of basic structural units - each with two large heavy chains and two small light chains - total of 4 polypeptide chains, the two chains are held together in a Y-shape by disulfide bonds and non-covalent interactions. The light chains of an immunoglobulin come in two basic types - kappa and lambda chains. An Ig molecule can have 2 kappa or 2 lambda chains, but it cannot have one of each type. Both the light and the heavy chains have a variable region at one end, and a constant region at the other end; the variable regions of different Ig molecules vary in amino acid sequence, whereas the constant regions of different Ig are similar in sequence. The variable regions of both light and heavy chains make up the antigen-binding regions and specify the type of antigen that the antibody can bind. There are five basic classes of immunoglobulins, each is defined by the type of heavy chain found in the immunoglobulin. The different classes of antibodies have different functions. IgG - 75-80%, produced in large amount during immune responses. The only Ig that crosses the placental barrier and transported to the fetus circulatory system - protects the newborn. IgA - found in secretion, such as nasal, bronchial, vaginal, intestinal, tears. IgA resistant to enzymes, thus found in secretory areas where it provides protection against the proliferation of microorganisms. IgD - functions mainly as an antigen receptor on B cells that haven't been exposed to antigens. It has been shown to activate basophils and mast cells. IgM - 10%, together with IgD found on the surface of B lymphocytes. IgM bound to the membrane of a B lymphocyte functions as its specific receptor for antigens, and this interaction results in proliferation of B lymphocytes into anti-body-secreting plasma cells, secreting IgM when bound to antigen. IgE - its Fc region has affinity for receptors present on mast cells and basophils surfaces. It attaches to these cells after being secreted by plasma cells. After encounter the antigen that elicited the production of this specific IgE, the antigen-antibody complex triggers secretion of biologically active substances such as histamine, heparin etc, elicit allergic reaction. It has been estimated that humans generate about 10 billion different antibodies, each capable of binding a distinct epitope of an antigen. Although a huge repertoire of different antibodies is generated in a single individual - the number of genes available to make these proteins is limited by the size of the human genome.

causes of tumors? genes responsible? what is carcinogenesis? what are the stages? what are carcinogens? what are the types? (110)

Carcinogenesis is the formation of a cancer, in which normal cells are transformed into cancer cells; the process is characterized by changes at the cellular, genetic and epigenetic levels and abnormal cell division. Cell division, under normal circumstances, is balanced between proliferation and programmed cell death by regulation of both processes to ensure the integrity of tissues and organs. According to the somatic mutation theory, mutations in DNA that lead to cancer disrupt these orderly processes by disrupting the programming regulating the processes which results in uncontrolled cell division and the evolution of those cells by natural selection in the body. More than one mutation is necessary for carcinogenesis - for example, 15 "driver mutations" (initiators) and 60 "passenger" mutations are found in colon cancers. Mutations in those certain types of genes that play vital roles in cell division, apoptosis, and mutations in DNA repair genes will cause a cell to lose control of its cell proliferation. In order for a normal cell to transform into a cancer cell, genetic changes can occur at many levels: Gain or loss of entire chromosomes - Aneuploidy; may cause due to errors in mitosis. Mutation affecting a single DNA nucleotide Silencing or activating a microRNA that controls expression of genes Epigenetic changes that alter whether genes are expressed or not. Carcinogenesis is divided into 3 phases: Initiation - initial genetic event - critical mutation gene - the neoplastic cells has selective advantage on normal cells, allows it for excessive growth, promote invasiveness and the ability to form metastases. Graduation - promotions stage that takes years in which the affected cells are stimulated and proliferate. Progression - Further gradual accumulation of genetic changes - uncontrolled growth, alteration of critical points in cell cycle, deregulation of DNA - transcription factors. The tumor initially remains in the place of its origin, but the activation of other factors will begin to spread into the surroundings and perform metastasis. Over a period of time, many tumors become more aggressive, acquire greater malignant potential - tumor progression, and become less responsive to therapy. There are two broad categories of genes that are affected by these changes: Oncogenes - a gene that has the potential to cause cancer; Proto-oncogene is a normal gene that may become an oncogene due to mutations or increased expression. Proto-oncogenes are usually genes that encode for transcription factors, growth regulating proteins or proteins involved in cell survival and cell to cell interaction; Oncogene, thus, is a mutated proto-oncogene which results in abnormal stimulation of cell division and proliferation. Mutation transforming proto-oncogene into an oncogene can results in gain of function mutation in the proto-oncogene, mutation in the regulatory elements or simply increase in amount of the proto-oncogene. Oncogenes considered dominant because even if they appear in a single allele they can lead to cellular transformation. Tumor suppressor genes - inhibit cell division, survival or other properties of cancer cells; prevent uncontrolled growth. They are often disabled by cancer-promoting genetic changes. They can be divided into two general groups: Gatekeepers - control cell growth and prevent tumor by regulating the transitions of cells through checkpoints in the cell cycle; promote apoptosis if needed. Mutations in gatekeepers genes that lead to loss of function lead to uncontrolled cell accumulation; e.g. mutation in retinoblastoma protein. Caretakers - protect the integrity of the genome by encode proteins responsible for detecting and repairing mutations and proteins that involve in normal dis-junction of chromosomes during mitosis. Loss of function mutation 🡪 malfunction repair mechanism, mutations accumulate in proto-oncogene and gatekeeper genes results in promoting cancer. TP53 - the guardian of the genome - is an example for tumor suppressor gene. Mutations in miRNA - which have function as negative regulator of genes; they inhibit gene expression post-transcriptionally by repressing translation or by preforming mRNA cleavage. Mutations in miRNA can lead to formation of neoplastic cells due to: I. Increasing expression of oncogenes - because repressing translation level of oncogenes is low II. Decreasing expression of tumor suppressor genes - if mutations in miRNA cause it to be overexpressed, it over represses translation of tumor suppressor genes. The majority of cancers are non-hereditary, called - "sporadic cancers". About 30% of sporadic cancer do have some hereditary component that is currently undefined, while the majority - 70% of sporadic cancers, have no hereditary component. malignant cell transformation - In tumor growth, the regulation of the cell cycle transition from G1-phase to S-phase is most often impaired . There are several mechanisms of regulation and they are very complex. Three complexes of cyclin-dependent kinases (CDKs) with different types of cyclins: cyclin D + CDK4 cyclin D + CDK6 cyclin E + CDK2 Endogenous agent and Carcinogens: As said, DNA damage and deficient DNA repair - considered to be the primary cause of cancer. DNA damage occurs naturally in the body due to Endogenous agents as well as due to exogenous agents. Endogenous agents may be due to aging (older people are more likely to develop cancer), endogenous hormones in excess concentrations such as testosterone and estrogen which promote the growth of tissues in specific target organs such as prostate and breast, excess bile acids due to high fat diet, Race - blacks face a 10% higher risk than whites, Japanese, Chinese and Filipinos with 25% lower than the risks faced by whites.. Carcinogens- Exogenous agents -- any substance or radiation that is an agent directly involved in causing cancer, with the ability to damage the genome or to the disruption of cellular metabolic processes. Chemical - any chemical that may cause a change to DNA and perform mutations (http://www.psr.org/environment-and-health/confronting-toxics/examples-of-environmental-carcinogens.html). Physical - UV light from solar radiation causes DNA damage that is important in melanoma; X-rays, gamma rays and cosmic rays are called ionizing radiations and may excite electrons from the atoms they encounter - changing stable molecules into free radicals and reactive ions. When these type of radiations attack the DNA - it may alter the structure of bases and break bonds, may result in dsDNA breaks. Biological - such as viruses that have an oncogenic potential by producing viral genes that interact with growth regulating proteins encoded by proto-oncogenes and tumor suppressor genes. Bacteria can cause inflammations, during which oxidative species are produced, causing DNA damage and reduce the efficiency of DNA repair mechanism. A deficiency in DNA repair would cause more DNA damages to accumulate, and increase the risk for cancer. Individuals with an inherited impairment in the germ line that causes mutation in any of the 34 DNA repair genes are at increased risk of cancer.

what is apoptosis? what are the pathways? what are the clinical outcomes of its dysregulation? genetic control of apoptosis? significance in development? (95), (96)

Cells that are infected, damaged or have reached the end of their functional life span often undergo "programmed cell death" - Apoptosis ("Falling off"), occurs in multicellular organisms. 50-70 billion cells die each day due to apoptosis in the average human adult. In contrast to necrosis, in which traumatic cell dies as a result from acute cellular injury, apoptosis is highly regulated and controlled process that confers advantages to the organism. Defective apoptotic processes have been implicated in a wide variety of diseases - excessive apoptosis causes atrophy, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer. During this process, cellular agents chop up the DNA and fragment (nuclear fragmentation - chromosomal DNA fragmentation) the organelles and other cytoplasmic components. The cell shrinks and becomes lobed - "blebbing". The cell's parts are packaged up in vesicles that are engulfed and digested by phagocytic cells (macrophages), leaving no trace. Apoptosis, by this mean, protects neighboring cells from damage that they would otherwise suffer if a dying cell merely leaked out all its content, including its many digestive enzymes. Importance of Apoptosis - shapes the tissues and organs in development (e.g. fingers separation, connection between brain cells in children, sexual development), prevents excess of proliferating cells, prevent from infected cells to harm others. Signals that trigger apoptosis can come from either outside or inside the cell. Outside the cell, extrinsic pathway - signaling molecules released from other cells can initiate a signal transduction pathway that activates the genes and proteins responsible for carrying out cell death. Within a cell whose DNA has been damaged - intrinsic pathway, the cell kills itself because it senses cell stress - a series of protein-protein interactions can pass along a signal that similarly triggers cell death. Both pathways induce cell death by activating caspases - which are proteases (protein degrading enzymes) Mechanisms - intrinsic and extrinsic pathways Once apoptosis has begun, it inevitably leads to the death of the cell. The two best-understood activation mechanisms are the intrinsic pathway (also known as the mitochondrial pathway) and the extrinsic pathway. Both pathway ends in activation of Caspase 3. (Intrinsic - Caspase Nine; Extrinsic - Caspase Eight) The intrinsic pathway - mitochondrial path way The mitochondria is essential to the cell's life - without them, cell ceases to respire aerobically and quickly dies. Pro-Apoptotic proteins (e.g. bax) that target mitochondria affect them in different ways - they may cause mitochondrial swelling through the formation of membrane pores, or they may increase the permeability of the mitochondrial membrane and cause apoptotic effectors to leak out. Mitochondrial pro-apoptotic proteins known as SMACs - Second Mitochondria-derived Activator of Caspases - released into the cell's cytosol following the increase in permeability of the mitochondria membranes. SMACs bind to proteins that inhibit apoptosis (IAPs). These inhibitors of apoptosis proteins are group of proteins that mainly act on the intrinsic pathway and block apoptosis by blocking caspases. When SMACs bind to these IAPs, it blocks them from functioning and prevents them from arresting the process 🡪 Caspases can be activated. Cell stress 🡪 activation of pro-apoptotic proteins (e.g. by p53) 🡪 Mitochondrial membrane more permeable 🡪 SMACs proteins released to cytosol 🡪 binds apoptosis inhibitor proteins 🡪 prevent them from blocking caspases activation. Caspases are endoproteases that hydrolyze peptide bonds; they are tightly controlled by their production as inactive zymogens that gain catalytic activity following signaling events promoting their aggregation into dimer complexes. There are two types of caspases - initiator caspases and effector caspases. The activation of initiator requires binding of activator protein; Effector caspases are then activated by this active initiator through proteolytic cleavage 🡪 active effector caspases then proteolytically degrade a host of intracellular proteins to carry out apoptosis (initiator activated by proteins 🡪 activated initiator causes activation of effector caspases). Activation of Caspases: Cytochrome C is also released from mitochondria due to formation of a channel (holes) - created by protein Bax and/or Bak from the BCL-2 proteins family - the Mitochondrial Apoptosis-induced Channel - MAC - in the outer mitochondrial membrane. Once Cytochrome C is released, it binds with APAF1 - Apoptotic Protease Activating Factor. Cytochrome C-APAF1 complex then bind to pro-caspase-9 (initiator caspase) to create a protein complex known as an Apoptosome. The Apoptosome function is to cleave the pro-caspase to its active form of caspase 9 - which in turn, activates the effector caspase 3. Cytochrome C released from MAC 🡪 binds to APAF1 🡪 binds to procaspase 9 🡪 produce Apoptosome 🡪 pro-caspase-9 to activated caspase-9 (initiator caspase) 🡪 activate effector caspase 3 🡪 apoptosis. Extrinsic pathway - TNF-path, Fas path The two extrinsic pathway theories are TNF-induced (tumor necrosis factor) model and the Fas-Fas ligand-mediated model - both involving receptors of the TNF receptor (TNFR) family coupled to extrinsic signals. In these two models, there is clustering of receptors that bind a ligand. Upon binding, cytoplasmic adapter proteins are recruited 🡪 Exhibit corresponding death domains that bind with the receptors. TNF Path - TNF-alpha is a cytokine produced mainly by activated macrophages, and is the major extrinsic mediator of apoptosis. Most cells in the human body have receptors (TNFR) of TNF-alpha. The binding of TNF-alpha to TNFR initiate the pathway that leads to caspase activation. It occurs due to activation of intermediate membrane proteins, called TRADD - TNF Receptor-Associated Death-Domain 🡪 activation of FADD - Fas-Associated Death Domain protein. FADD then associates with procaspase-8 via dimerization of the death effector domain. At this point, a death-inducing signaling complex -DISC - is formed, resulting in the auto-catalytic activation of procaspase-8.Once caspase-8 is activated - it cleaves caspase 3 - the execution phase of apoptosis is triggered - catalytic activation of peptide linkages. Moreover, caspase 8 activates BID that becomes tBID, which is pro-apoptotic protein that stimulates the intrinsic pathway. Fas path - the Fas receptor (first apoptosis signal, transmembrane protein, part of the TNF family) binds the Fas ligand 🡪 formation of DISC (death inducing signaling complex) - which contains the FADD (first apoptosis death-domain), caspase-8 and caspase-10, which then activates procaspase 3 🡪 caspase, as well as bid 🡪 tBID.. Pro-apoptotic factors: Bak, Bax, tBID, PUMA, p53 Anti-apoptotic factors: Bcl-2, Bcl-Xl; AIF (binds with released SMAC an inactivated). Genes and proteins associated with apoptosis TP53 Gene - encode to produce the protein p53 - regulates cell division in the G1 checkpoint. The p53 is a transcription factor that activates the expression of genes for factors that inhibit cell proliferation. One of these is the protein p21 - which stops the development of the cell division cycle in order to perform DNA repair. When DNA damage is irreparable, p53 participates in the induction of apoptosis. P53 activates Bax that perform holes in mitochondrial membrane (and inhibits bcl-2). p53 mutations are present in 50% of tumors (e.g. Li-Fraumeni syndrome) Bcl-2 and Bax-Bak genes - are the genes that encode for products that regulate apoptosis. They form homodimers (dimer of Bax) or heterodimers (dimer of Bcl-2-bax). Bcl-2 suppresses apoptosis while Bax induces. Depending on whether the predominant amount of Bax homodimers (induces) or Bax-bcl-2 heterodimers (inhibits) - apoptosis occurs or doesn't occurs. P35 protein - coded by certain viruses and inhibits apoptosis - virus can live and propagate in the host cell. Protein P35 is cleaved by caspases and thereby causes them to be occupied - preventing them from active induction of apoptosis of the cell. P35 may be referred to as survival signal. Survival factors (signals) - such as hormones, cytokines, growth factor - are necessary for cells life. If they are not present in enough concentration, it leads to apoptosis. These include - PDGF, FGF, HGF, IGF. Their action is to activate PI3K pathway which ends with activation of ATK - ATK is a kinase that perform many functions, one of them is to inhibit Bax from performing apoptosis. Apoptosis and development -apoptosis is a physiological process that leads to programmed cell death without the development of inflammatory reactions. Examples for importance of apoptosis in development: Limbs - apoptosis that leads to the development of individual fingers (fault to occur - syndactyly - AD) Muller or Wolffian ducts - apoptosis of one of them, depends on which sex is evolving. Kidney - apoptosis takes part in the morphogenesis of tubules and glomeruli and keep occur throughout life. PNS - peripheral nerves are usually in excess during development, only those who are connected with muscles and have relevance function - survive, the rest - going through apoptosis. Development of the heart - apoptosis occurs in certain stages in the development (septum). Dis-regulation of apoptosis may be either inability to perform apoptosis or may lead to hyperactive apoptosis. Inability to perform apoptosis - can result due to damage in one of the pathways or due to overexpression of IAP proteins that inhibit apoptosis. Mutation in proteins that induce apoptosis - the genes coding for these proteins may be referred to as part of the tumor suppressor genes, and thus, it is required that both alleles which produce apoptotic proteins will be damaged - Damage in production of P53 - the "guardian of the genome" - responsible to arrest the cell cycle temporarily or permanently and induce apoptosis of cells with irreversibly DNA damage by regulating Bax and Bak. Bax and Bak are part of the intrinsic pathways, and their damage will cause inability of the cell to perform apoptosis. Moreover, damage in the genes coding for the death receptors, or for the ligands that attach to these receptors - will prevent apoptosis. Overexpression of proteins that inhibit apoptosis - IAPs - due to mutation of an oncogene; can be done either by translocation of the oncogene into a region where it either has no regulatory sequence or by a regulatory sequence of another protein which is highly expressed. It can also be done by amplification of genes. Clinical outcomes of in-ability to perform apoptosis - Tumors - apoptosis prevent excess of cells proliferation; no apoptosis - accumulation of cells (stomach, skin) - may be malignant tumor. Developmental defects - apoptosis has a great role in shaping the tissues and the organs in the body (hand plate) Immune diseases - Immune-deficiencies - no apoptosis to pathogens which occur due to release of apoptosis-stimulate substances by WBCs Autoimmune diseases - during maturation of T and B cells, those cells that show too high affinity for self-cells will go through apoptosis (central tolerance mechanism) - no apoptosis 🡪in tolerant B and T-cells may attack the body and cause autoimmune disease. From the other hand, hyperactive apoptosis may occur - due to overexpression of pro-apoptotic genes or loss of function of the anti-apoptotic genes - it may cause developmental problems, immunodeficiency, tissue damage and neuro-degenerative diseases. Immunodeficiency - HIV virus affects T helper lymphocytes and induces their apoptosis by increasing the mitochondrial pathway, by increasing the Fas-mediated apoptosis and by deactivation of anti-apoptotic proteins Bcl-2. Neuro-degenerative diseases - Huntington disease is an AD disorder in which specific cell death occurs in the brain by mutation in gene coding for the protein Huntingtin - Htt. Htt acts as an anti-apoptotic agent preventing apoptosis. A mutation in Htt damages its function as an anti-apoptotic agent and also causes activation of caspases. Process of apoptosis: Cell shrinkage and rounding because of cytoskeleton breakdown by caspases Cytoplasm appears dense; organelles are tightly packed Pyknosis - chromatin undergoes condensation into compact patches against the nuclear envelope Karyorrhexis - nuclear envelope becomes discontinuous and the DNA inside is fragmented Cell membrane shows irregular buds - blebs (Blebbing) The cell break apart into several vesicles called apoptotic bodies - which are then phagocytosed

what are congenital anomalies? how can they be classified? (2 sets) what are some examples? what are the causes? how can birth defects be screened? pre-natal (non-invasive vs. invasive) and post-natal how can they be prevented? (primary vs. secondary) (100)

Congenital anomalies are deviations from normal prenatal development. They arise from abnormal ontogeny and caused by genetic factors, environmental factors or both groups of factors. Clinical severity of birth defects is different - from insignificant at all, may be just cosmetic variations, to a lethal defect that causes the death of its wearer even in-utero or shortly after birth. Birth defects vary widely in cause and symptoms - any substance that causes birth defects is known as teratogen. The genetic or environmental factors may include errors of morphogenesis, infection, epigenetic modifications on parental germline, or a chromosomal abnormality. The outcome of the disorder will depend on complex interactions between the prenatal deficit and postnatal environment. Animal studies indicate that the mother's (and likely the father's) diet, vitamin intake and glucose levels prior to ovulation and conception have long-term effects on fetal growth and adolescent and adult disease. Animal studies have shown that paternal exposures prior to conception and during pregnancy result in increased risk of certain birth defects and cancers. Birth defects can be divided into 4 groups: Malformation - caused by abnormal development of organ or tissue due to intrinsic abnormality in one or more genetic program operating in development. They may result in complete or partial absence of a structure or in alterations of its normal configuration. Most malformations occur at the 3rd to 8th week of gestation. Example - limb malformations such as Meromelia (partial absence of the limb), Amelia (complete absence of extremities) and micromelia (abnormally short segments of the extremities). The drug Thalidomide which serves as sleeping pill has teratogenic effect that can cause these malformation. Disruption - morphological alternations result from destruction of irreplaceable normal fetal tissue - pathological processes that disrupt the development of organ or tissue, which the development was originally normal. It may result from vascular insufficiency, trauma or teratogens. Example - "Amnion Disruption" - partial amputation of fetal limb associated with strands of the amniotic tissue. Deformation - caused by extrinsic factors such as mechanical forces that mold a part of the fetus over a prolonged period - damages previously healthy organ or tissue. They are especially common during the second trimester and may result from constraint of the fetus due to twin or triplet gestations or prolonged leakage of amniotic fluid. Dysplasia - disorder at the organ level that is due to problems with tissue development. examples NTD anencephaly encephalocele spina bifida maligna occulta abdominal wall effects omphalocele gastrochisis Examples of primarily structural congenital disorders - a limb anomaly is called dysmelia. These include all forms of limbs anomalies, such as - polymelia, polydactyly, polysyndactyly Primarily metabolic disease - also referred to as an inborn error of metabolism, most of these are single gene defects - usually heritable. Many affect the structure of body parts but some simply affect the function. Other well defined genetic conditions may affect the production of hormones, receptors, structural proteins and ion channels. According to the complexity and frequency of birth defects, they can be divided further into 4 groups: Isolated defects - the defects are not associated with other defects or anomalies (e.g. isolated polydactyly) Sequence - the multiple defects that arise as a result of the pathological cascade of events caused by the primary pathological interference. - multiple anomalies that result from pathological cascade caused by a primary insult Potter's sequence more or less fatal anbnormality of the kidneys initial cause is that fetal kidneys are not functioning fetal urine becomes an important part of the amniotic fluid not enough amniotic fluid lungs require hydrostatic pressure of amniotic fluid Association - certain types of birth defects tend to develop along with other typical defects - "linked defects" - selected congenital anomalies that tend to develop all together VATER association - vertebral anomalies anal atresia cardiac anomalies trachoesophageal fistual renal/urinary anomalies limb defect Syndrome - a complex phenotypic traits (anomalies) which are typical of defined clinical diagnosis - example - down syndrome Causes of birth defects Causes of congenital defects may be different. Generally, it can be said that an abnormal prenatal development and formation of congenital defects may contribute genetic factors, environmental factors and the combination of the two. The exact ause of many types of birth defects remains unknown. Genetic causes Chromosomal aberrations - structural or numerical abnormalities in karyotype, may manifest themselves as a syndromes (down syndrome 21, Edwards 18, patau 13, turner 45,X) Monogenic birth defects - caused by mutations in a single gene. These include some major congenital malformations of the skeleton such as achondroplasia. Particular advantage in this case is the possibility of target molecular genetic diagnostics to verify diagnosis. Multifactorial congenital defects represent a very large group that etiologically stands at the interface between defects on the genetic defects and environmental factors. External Factors - Teratogens External factors which are able to give rise to congenital malformations or risk of such defects significantly increase generally known as teratogens. It can be divided into 3 groups: Teratogens of biological nature - infectious agents such as viruses, bacteria. Teratogens of chemical nature - chemicals used in industry and agriculture, as well as drugs and medicines. Antibiotics, anticonvulsants, warfarin, ACE inhibitors etc. Cytostatic (chemotherapy). Teratogens of physical nature - different types of ionizing radiation (X-ray, gamma etc.) as well as high temperature and mechanical teratogens. Prenatal diagnosis - involves a set of procedures and methods used in the diagnosis of unborn human. Developmental defects are probably the most important group of diagnoses that can be diagnosed prenatally. The used methods are divided into two groups: Non-invasive diagnosis - Used primarily in screening programs; safe both for mother and fetus. Biochemical examination of specific markers in maternal blood, ultrasound scans. Invasive prenatal diagnosis - used when there is an increased risk of certain birth defects - relatively riskier methods. Amniocentesis, Chorionic villous sampling and cordocentesis, fetoscopy (which is barely used nowadays). post-natal - examination of the newborn - delivery unit (palate, eyes, heart, spine, arms, congenital cataract, urination, defecation) - screening of hip joint dysplasia - regular checks at the GP prevention primary: - optimal time for reproduction (age) - exclusion of possible teratogens - good nutrition including vitamins (supplementation with folic acid (0.4 mg) to prevent NTD - good treatment of diseases, careful medication - genetic counseling secondary: prenatal diagnostics of severe anomalies termination of pregnancy for genetic reasons up until the 24th week * secondary prevention is why the number of severe anomalies in births are so low

genetic determination of body plan in development? what is the process? what is gastrulation? how do morphogens play a role? what are some other important gene families? (92)

Critical function of the developing organism is to specify the spatial relationship of structures within the embryo. In early development, the organism must determine the relative orientation of a number of body segments and organs: Head to tail (cranial-caudal) Dorso-ventral axis Anterior-posterior axis The body plan is a process that directs the cells differentiation into their proper fates forming the right structures in the right locations. All the somatic cell contains all of the genetic information, but each cell shows different properties after being differentiated - occurs due to changes in the cell's gene expression - process which maintained throughout life and passed into daughter cells - "cell memory", which achieved through epigenetics (modification of the phenotype without changing the genotype) - chromatin remodeling, master regulator, miRNA(micro)/ siRNA(small-interfering) - microRNA functions in RNA silencing and post-transcription regulation of gene expression, siRNA operates within the RNA interference pathway (cause to inhibition of expression). Formation of the body plan: First stage in formation begins with gastrulation - 3 germ layers formation begins by the formation of the primitive streak at the midsaggital plane of the embryonic disc which elongates in caudo-cranial directions, where at the cranial end the primitive streak is expanded to form the primitive node. The primitive streak becomes deeper, forming the primitive groove through which epiblast cells fall, forming the 3 germ layers. Thus, the primitive streak contributes to the body plan in three ways: It determines all 3 body axes - dorsoventral, craniocaudal, mid-lateral. It forms the 3 germ layers It forms the two main signaling centers in the embryo - the primitive node and the endoderm. Genetic determination of the body plan is not fully understood in human; since it is immoral to do expirements on human embryos, animal models such as drosophila revealed several genes and gene families that play important roles in early developmental processes. Many developmental genes produce certain molecules that causes induction, segmentation, migration, differentiation and programmed cell death. These molecules can be - transcription factors (such as growth factors), cell receptors and morphogens. Morphogens are extremely important during early development. They affect cells differentiation according to its concentration - generally, morphogen is a protein whose concentration gradient affects the developmental fate of the surrounding region. Egg-polarity genes play a crucial role in establishing the two main axes of development in fruit flies. There are two sets of egg-polarity genes: one that determines the anterior-posterior axis, and the other determines the dorsal-ventral axis. These genes work by setting up concentration gradients of morphogens within the developing embryo. The egg-polarity genes transcribed into mRNAs in the course of egg formation in the maternal parent, and after fertilization, the mRNAs are translated into proteins that distributed in the cytoplasm. The translated proteins are examples of genetic-maternal effect (genes that originate in the maternal parent and influence the phenotype of the offspring), distributed asymmetrically in the cytoplasm - giving the egg polarity (direction) due to different concentration in different regions. Important example for this mechanism is the morphogens that determine the dorsal-ventral axis, which defines the back and belly. One type of morphogen encode to a protein which is called "dorsal". Dorsal protein remains in the cytoplasm in one part of the embryo, and in the other side is taken up into cells nuclei and degraded there (do not function as transcription factors in these areas). Thus - forming a smooth gradient of dorsal protein concentration: The area of cytoplasm rich with dorsal protein will be the dorsal side of the embryo, whereas the other side, where the nuclei degraded the protein, will be the belly. Inside the nucleus, dorsal protein acts as a transcription factor, binding to regulatory sites on the DNA and activating or repressing the expression of other genes - cause to different structure to be formed in different areas (dorsal structures in the dorsal areas, ventral structures in the ventral areas). Development is a complex process - consisting of numerous events that much take place in highly specific sequence. After the dorsal-ventral axis and the anterior-posterior axis are established by maternal genes, these genes encode mRNAs and proteins that are localized to specific regions within the egg and cause specific genes to be expressed in different regions on the embryo (HOX genes). The proteins of these genes then stimulate other genes, which in turn stimulate others in a cascade of control. In human, there are four clusters of HOX genes, each type - A - D, found on a different chromosome, and arranged in groups from 1-13. These genes transcribe for transcription factors that regulate other gene expression. The expression of HOX genes occurs from 3' to 5' and corresponds to cranial and caudal body parts - in the cranial segments, the HOX genes which will be expressed are closer to the 3' end of the DNA olecule, while in the caudal regions, genes closer to 5' end of the DNA molecule will be expressed. For example - in the cranial region, HOXA1, HOXB1, HOXC1, HOXD1 will be expressed (while HOXA13 etc. will be expressed in the caudal regions). Another important gene families - SHH genes, PAX genes, SOX genes, and TGF betta-family genes: SHH genes - are part of the Hedehog family - a class of morphogens which are responsible for the determination of the positions of different parts of the body. They play a key role in regulating vertebrate organogenesis, such as in the growth of digits on limbs and organization of the brain PAX genes - paired box genes are a family of genes coding for tissue specific transcription factors. Their key role is to specify and maintain progenitor cells through use of molecular mechanisms such as gene activation or inhibition. SOX genes - code for proteins that bind to DNA through a HMG domain (high mobility group). This domain interacts with the minor groove in the double helix - induces a bend in the DNA. The bend allows other regulatory factors to bind with the promoter regions of genes that encode for important structural proteins. TGf Betta-family - FGF (fibroblast growth factor) - family of growth factors with members involved in angiogenesis, mesoderm induction, antero-posterior patterning, limb development, neural induction and neural development and more. BMP - bone morphogenic protein - has ability to induce formation of bone and cartilage, regulation of embryo polarity, neurulation and development of neural plate. WNT - structurally related genes encode secreted signaling proteins.

what are population polymorphisms? how do these differ from mutations? what are the types of polymorphisms? stable vs. transient polymorphism? (104)

DNA sequences of the exact same region (loci) on a chromosome are remarkably similar among chromosomes from different individuals. A randomly chosen segment of about 1000 bp (base pairs) contains on average only one base pair that varies between the two homologous chromosomes. A gene is said to be polymorphic if more than one allele occupies that gene's locus within a population. For example, in dogs the E locus, which controls coat pattern, can have any of five different alleles, known as E, Em, Eg, Eh, and e. A polymorphic variant of a gene may lead to the abnormal expression or to the production of an abnormal form of the gene; this may cause or be associated with disease. Where monomorphism means having only one form and dimorphism means there are only two forms, the term polymorphism is very specific term in genetics and biology, relating to the multiple forms of a gene that can exist. The term does not extend to character traits with continuous variation such as height (even though this may be a heritable aspect). Instead, polymorphism refers to forms that are discontinuous (have discrete variation), bimodal (having or involving two modes), or polymodal (multiple modes). For example, earlobes are either attached or they are not, it is an either/or situation and not like height, which is not a set number or another. Polymorphism was originally used to describe visible forms of genes, but the term is now used to include hidden modes such as blood types, which require a blood test to decipher. In addition, the term is sometimes used incorrectly to describe visibly different geographical races or variants, but polymorphism refers to the fact that the multiple forms of a single gene must occupy the same habitat at the same time (which excludes geographical, race or seasonal morphs) - same population. Genetic polymorphism refers to the occurrence of two or more genetically determined phenotypes in a certain population (in proportions that the rarest of the characteristics cannot be maintained just by recurrent mutation). Polymorphism promotes diversity and persists over many generations because no single form has an overall advantage or disadvantage over the others in terms of natural selection. A population where the gene frequency of the most common allele is less than or equal to 0.99 (99%) is polymorphic for a given trait . However, this stated value is not an objective limit, but was only set by agreement . It is best to determine the degree of polymorphism using heterozygosity , which is defined by: H=1-∑ x^2, where m = number of alleles of the monitored gene and x i = gene frequency of the i-th allele (CHW applies: x 1 + x 2 ... + x m = 1) or verbally as a representation of individuals in a population who are heterozygous for a particular locus Polymorphism vs Mutation: Mutations by themselves do not classify as polymorphisms. A polymorphism is a DNA sequence variation that is common in the population. A mutation, on the other hand, is any change in a DNA sequence away from normal (implying that there is a normal allele running through the population and that the mutation changes this normal allele to a rare and abnormal variant). In polymorphisms, there are two or more equally acceptable alternatives and to be classified as a polymorphism, the least common allele must have a frequency of 1% or more in the population. If the frequency is lower than this, the allele is regarded as a mutation. There are different types of polymorphisms, and the classification is according to how the sequence varies between different alleles: SNP's - single nucleotide polymorphism - variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population (e.g. > 1%). For example, at a specific base position in the human genome, the base C may appear in most individuals, but in a minority of individuals, the position is occupied by base A. There is a SNP at this specific base position, and the two possible nucleotide variations - C or A - are said to be alleles for this base position. SNPs underlie differences in our susceptibility to disease; a wide range of human diseases, e.g. sickle-cell anemia, β-thalassemia and cystic fibrosis result from SNPs. The severity of illness and the way our body responds to treatments are also manifestations of genetic variations. Haplotype - a haplotype is a group of genes within an organism that was inherited together from a single parent - it an describe a pair of genes inherited together from one parent on one chromosome, or it can describe all of the genes on a chromosome that were inherited together from a single parent because of genetic linkage. In the SNP's terms - the term haplotype can also refer to the inheritance of a cluster of single nucleotide polymorphisms (SNPs). SNP's as markers - by examining haplotypes, scientists can identify patterns of genetic variation that are associated with health and disease states. For instance, if a haplotypes is associated with a certain disease, then scientists can examine stretches of DNA near the SNP cluster to try to identify the gene or genes responsible for causing the disease. Insertion-deletion polymorphism - "Indel" - An insertion/deletion polymorphism, commonly abbreviated "indel," is a type of genetic variation in which a specific nucleotide sequence is present (insertion) or absent (deletion) - insertion or deletion of about 2-100 nucleotides. Indels can be either simple or multiallelic; Simple - only two alleles affected, multiallelic - variable number of segments of DNA is affected, repeated one by one at specific location in the chromosome. Multiallelic indels are further classified to microsatellite variation (STRP's) and minisatellite (VNTR's): Microsatellite variation (STR's): Short tandem repeats - STRs are short sequences of DNA, normally of length 2-5 base pairs, that are repeated numerous times in a head-tail manner, i.e. the 16 bp sequence of "gatagatagatagata" would represent 4 head-tail copies of the tetramer "gata". The polymorphisms in STRs are due to the different number of copies of the repeat element that can occur in a population of individuals. Minisatellite (VNTRs): Variable number of tandem repeats, similarly to STR's, VNTR's is a location in a genome where a short nucleotide sequence is organized as a tandem repeat (one after another). These can be found on many chromosomes, and often show variations in length between individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. Individual repeats can be removed from (or added to) the VNTR via recombination or replication errors, leading to alleles with different numbers of repeats. Usage of mini-satellites in DNA fingerprinting - the pattern and length of the VNTR's is highly different between individuals, only identical twins show an indistinguishable pattern. Due to these facts, detection of a number of minisatellite polymorphisms was one of the first methods of DNA matching in forensic medicine (investigation of crime). Copy number of polymorphism - abbreviation CNV: Consists of variation in the number of copies of larger segments of the genome - 200+ base pairs. It is a phenomenon in which sections of the genome are repeated and the number of repeats in the genome varies between individuals in the human population. Restriction fragment length polymorphism - RFLP - polymorphisms in the patterns of fragments produced when DNA molecules are cut with the same restriction enzymes (samples are taken from 2 individuals and compared). The different patterns are inherited. RFLP provides a large number of genetic markers that can be used for mapping. Stable polymorphism gene frequencies do not change; eg population in CHW equilibrium , or polymorphism maintained by the frequency of heterozygotes , or mutations and back mutations. Transient polymorphism In a population where, due to selection , one allele is gradually replaced by another, as is the case, for example, with selection against homozygotes .

what is epigenetics? what are the relevant modifications? what are some associated diseases? what is genomic imprinting? what is the significance? what is x-inactivation? (52)

Epigenetics is the study of heritable changes in gene expression that does not involve changes to the underlying DNA sequence, i.e. a change in phenotype without a change in genotype — which in turn affects how cells read the genes. Epigenetic change is a regular and natural occurrence but can also be influenced by several factors including age, the environment/lifestyle, and disease state. Epigenetic modifications can manifest as commonly as the manner in which cells terminally differentiate to end up as skin cells, liver cells, brain cells, etc (during development; i.e. all cells in the body have the same genetic material, but each cell according to the cell type and tissue that it is found in, is guided to express certain genes, while others are silenced - it is achieved by epigenetics mechanisms). Beyond developmental aspect, epigenetic change can have more damaging effects that can result in diseases like cancer. The term also refers to the changes themselves: functionally relevant changes to the genome that do not involve a change in the nucleotide sequence. At least three systems including DNA methylation, histone modification (methylation + acetylation) and non-coding RNA (ncRNA)-associated gene silencing (microRNA, small-interference RNA) are currently considered to initiate and sustain epigenetic change. Each of which alters how genes are expressed without altering the underlying DNA sequence. DNA methylation: DNA methylation is a process by which methyl groups are added to DNA and modifies the function of the DNA. When located in a gene promoter, DNA methylation typically acts to repress gene transcription. DNA methylation plays an important role for epigenetic gene regulation in development and disease. Methyl group is added to cytosine or adenine groups. The methylation occurs by DNA methyltransferase, which uses SAM (S-adenosylmethionine) as a donor for methyl group. The methyl group lies in the major groove and acts as a signal for enzyme Histone deacetylases - which removes acetyl group from histones, and thus, the methyl group indirectly repress transcription. Cytosine that are methylated are cytosine that found in CG islands. If the CG islands found in the promoter and the Cytosine is methylated - the genes are turned off. Genes whose products are necessary in all tissues have unmethylated promoter CG islands. DNA methylation is extremely important in diseases - particularly in cancer. The DNA at promoters of tumor suppressor genes, those genes encode for proteins that can slow down cell growth or cause cell death, is often abnormally methylated in cancer. Since those genes are inactive - cells can proliferate. Similarly, the DNA at promoters of oncogenes, those that can increase cell growth and survival, is frequently unmethylated in cancer, causing an increase in those genes activity. DNA Methylation is related also to genome silencing, not only of individual genes, but also through genetic imprinting and X-chromosome inactivation. Imprinting refers to a situation whereby only one of the two alleles of a gene is expressed, whereas the other one is silenced, in part by DNA methylation. For a certain gene, the maternal allele may be normally silenced, whereas the parental allele may be normally expressed. X-chromosome inactivation refers to the fact that of the two X-chromosomes in a female cell, normally only one is active while the other is silenced and is seen in heterochromatin form - known as the Barr body. Histone modification: Histones undergo post-translational modifications that alter their interaction with DNA and nuclear proteins. The H3 and H4 histones have long tails protruding from the nucleosome, which can be covalently modified at several spaces. Modifications of the tail include methylation, acetylation, phosphorylation and ubiquitination. The core of the histones H2A and H2B can also be modified. Combinations of modifications are thought to constitute a code - "histone code", and affect gene regulation. Histone Acetylation is the addition of acetyl groups to histone tails by Histone Acyltransferase - results in increase activation of the related genes. When Acetyl is introduced to histone tails, it removes the positive charge of the histone tail, reducing the affinity to the negative charged phosphate groups of DNA. When the interaction between histone tails and phosphate groups of DNA is reduced - the chromatin opens - Euchromatin form - allows for transcription to occur by increase access of transcription factors to DNA through the structural changes in the nucleosome. Thus, the process of histone acetylation is tightly involved in the regulation of many cellular process including chromatin dynamics and transcription, gene silencing, cell cycle progression, apoptosis, differentiation, DNA replication and DNA repair. Histone methylation - takes place on arginine and lysine amino acids by histone methyltransferases. It can either induce or repress gene expression. Phosphorylation - catalyzed by histone kinases. Phosphorylation occurs in serine/threonine/tyrosine resides - addition of a negatively charged phosphate group lead to major changes in protein structure (role of phosphorylation in controlling protein function). Ubiquitination of histones can take place. Beyond regulation of gene expression, marking sites of DNA damage is an important function for histone modification. Some transcription factors have domains that recognize the specific histone modification and may be affected accordingly. One example of an epigenetic change in eukaryotic biology is the process of cellular differentiation. During morphogenesis, totipotent stem cells become the various pluripotent cell lines of the embryo, which in turn become fully differentiated cells. In other words, as a single fertilized egg cell - the zygote - continues to divide, the resulting daughter cells change into all the different cell types in an organism, including neurons, muscle cells, epithelium, endothelium of blood vessels, etc., by activating some genes while inhibiting the expression of others. Cancer was the first human disease to be linked to epigenetics. Studies used primary human tumor tissues, found that genes of colorectal cancer cells were substantially hypomethylated compared with normal tissues. DNA hypomethylation can activate oncogenes and initiate chromosome instability, whereas DNA hypermethylation initiates silencing of tumor suppressor genes. Mental Retardation Disorders. Epigenetic changes are also linked to several disorders that result in intellectual disabilities. For example, the imprint disorders Prader-Willi syndrome and Angelman syndrome, display an abnormal phenotype as a result of the absence of the paternal or maternal copy of a gene, respectively. In these imprint disorders, there is a genetic deletion in chromosome 15 in a majority of patients. The same gene on the corresponding chromosome cannot compensate for the deletion because it has been turned off by methylation, an epigenetic modification. Genetic deletions inherited from the father result in Prader-Willi syndrome, and those inherited from the mother, Angelman syndrome (important to study these syndromes as an example for genomic imprinting). Genomic imprinting is the epigenetic phenomenon. For most genes, we inherit two working copies - one from mom and one from dad. But with imprinted genes, we inherit only one working copy. Depending on the gene, either the copy from mom or the copy from dad is epigenetically silenced. Silencing usually happens through the addition of methyl groups to adenine/cytosine DNA residues of the silenced gene during egg or sperm formation. under normal circumstances, paternal and maternal copies of a particular gene have the same potential for their expression in any human cell. Genomic imprinting is a process that fundamentally changes this potential, because as a result, the activity of a particular gene is limited to one chromosome, depending on which parent it was inherited from . The epigenetic tags on imprinted genes usually stay put for the life of the organism. Regardless of whether they came from mom or dad, certain genes are always silenced in the egg, and others are always silenced in the sperm. In diploid organisms (like humans), the somatic cells possess two copies of the genome, one inherited from the father and one from the mother. Each autosomal gene is therefore represented by two copies, or alleles, with one copy inherited from each parent at fertilization. For the vast majority of autosomal genes, expression occurs from both alleles simultaneously. In mammals, however, a small proportion (<1%) of genes are imprinted, meaning that gene expression occurs from only one allele. The expressed allele is dependent upon its parental origin. For example, the gene encoding insulin-like growth factor 2 (IGF2) is only expressed from the allele inherited from the father; this is called maternal imprinting (the maternally derived allele is imprinted, i.e., silenced). Another example of epigenetic is X-inactivation. It is a process by which one of the copies of the X chromosome present in females is inactivated (imprinted). The inactive X chromosome is silenced by its being packaged in such a way that it has a transcriptionally inactive heterochromatin structure. Barr body - the condensed chromatin (Heterochromatin) form which the inactivated X-chromosome is found at. The condensation is controlled by X-inactivation specific transcript gene - XIST - that is located on the X-chromosome inactivated center (XCI) - on the inactivated chromosome.

process of sex determination in females? (94)

Female sex differentiation is initiated in the primodial gonad when SRY gene is not expressed. Primoridal germ cells become oocytes precursors, proliferate and recruit follicular cells - undergo first meiotic division. After the first meiotic division, they arrest in diplotene stage of meiosis I until ovulation. In late fetal life, all oocytes, still primary oocytes, have halted at this stage of development called the dictyate. After menarche, these cells then continue to develop - only few every menstrual cycle. Feminine external genital develops and wolffian duct disappears in the absence of testosterone. In the absence of AMH - mullerian duct differentiate into uterus and oviducts.

what is a species? what are reproductive barriers? what are the types? what is speciation? how does it occur? (119)

In biology, a species is the basic unit of biological classification. Species are groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups. A genetic species is a set of individuals or populations with sufficiently similarity of DNA. Species are distinctly different kinds of organisms. Birds of one species are, under most circumstances, incapable of interbreeding with individuals of other species. Indeed, the "biological species concept" centers on this inability to successfully hybridize, and is what most biologists mean by "distinctly different." That concept works very well when two different kinds of birds live in the same area. Population whose members interbred in nature to produce fertile offspring and do not interbred with members of different species (said to be "reproductively isolated"). Another method for define species is the "morphological species concept" - which define different species as such with visible structural difference (in plants, for example, different number of flowers). Species hypothesized to have the same ancestors that placed in one genus, based on similarities in their physical abilities and when available - in DNA sequence. Reproductive barriers keep a given species genetically distinct from other species. Reproductive isolation prevents interbreeding between two different species, preserve genetic integrity of each species. Evolution of internal (i.e., genetically-based) barriers to gene flow is necessary for speciation to be complete. If internal barriers to gene flow do not evolve, individuals from the two parts of the population will freely interbreed if they come back into contact. Whatever genetic differences may have evolved will disappear as their genes mix back together. Speciation requires that the two incipient species be unable to produce viable offspring together or that they avoid mating with members of the other group. Prezygotic isolation: Temporal or habitat isolation - Any of the factors that prevent potentially fertile individuals from meeting will reproductively isolate the members of distinct species. The types of barriers that can cause this isolation include: different habitats, physical barriers, and a difference in the time of sexual maturity. When factors change, especially physical barriers, often, species will branch off. Behavioral isolation - The different mating rituals of animal species creates extremely powerful reproductive barriers, termed sexual or behavior isolation, that isolate apparently similar species in the majority of the groups of the animal kingdom ("e.g. mating dance). Mechanical isolation - Mating pairs may not be able to couple successfully if their genitals are not compatible ("lock and key") Gametic isolation - the gametes of the fertile individuals should be "in the right place" (uterus, for example), in the "right time" (during or adjacent to ovulation) - barriers such as thick mucosa may cause gametic isolation and prevent fertilization. Post-zygotic isolation: Zygote mortality and non-viability of hybrids - egg or ovule is fertilized but the zygote does not develop, or it develops and the resulting individual has a reduced viability. Hybrid sterility - A hybrid has normal viability but is deficient in terms of reproduction or is sterile. Speciation: evolution of new species - formation of two species from a single species - occurs when a population becomes reproductively isolated from other populations of the species. The gene pools of the two separated populations begin to diverge in genetic composition. When a population is sufficiently different from its ancestral species that there is no genetic exchange between them 🡪 speciation occurs. Allopatric speciation - or geographic speciation is speciation that occurs when biological populations of the same species become isolated from each other to an extent that prevents or interferes with genetic interchange. This can be the result of population dispersal leading to emigration, or by geographical changes such as mountain formation, island formation, or large scale human activities. The separated populations then undergo genotypic or phenotypic divergence as: (a) they become subjected to different selective pressures, (b) they independently undergo genetic drift, and (c) different mutations arise in the gene pools of the populations. Geological processes - Geological processes can fragment a population through such events as emergence of mountain ranges, canyon formation, glacial processes, the formation or destruction of land bridges etc. On a global scale, plate tectonics is a major geological factor that can lead to the separation of populations to result in the distribution of species. Population dispersal is used to describe migratory events, either in the form of range expansion (natural movement away from parents) or jump dispersal (crossing of barriers), which may lead to genetic isolation. If the smaller population fragment becomes genetically isolated from the parental group, it may be subjected to its own unique mutations, selection forces, and genetic drift effects; thus, it will follow its own evolutionary pathway. Peripatric speciation - sub-form of Allopatric speciation. New species are formed in isolated peripheral populations - similar to allopatric in that populations are isolated and prevented from exchanging genes, however, peripatric proposes that one of the populations is much smaller than the other (founder effect, bottleneck). Genetic drift is often proposed to play significant role in this evolution. Parapatric speciation - parapatry is the relationship between organisms whose ranges do not significantly overlap but are immediately adjacent to each other; they do not occur together except in a narrow contact zone. This minimal contact zone may be the result of unequal dispersal or distribution, incomplete geographical barriers. A parapatric population distribution may result in non-random mating and unequal gene flow, which can then produce an increase in the dimorphism between populations. In parapatric speciation, there is an intrinsic barrier of nonrandom mating and distinct selection pressures that create unequal gene flow. Sympatric speciation is the process through which new species evolve from a single ancestral species while inhabiting the same geographic region. Reproductive isolation mechanisms evolve at the start of the speciation process. It results in polymorphism in which each of the characteristic has advantage over the other.

What is the major histocompatibility complex (MHC)? what are the classes? significance for transplant rejection? major disease associated with HLA loci? (80)

MHC is a genetic system that is primarily responsible for distinguishing self from non self. In humans, the MHC is called HLA - human leucocyte antigen. MHC is large complex genes, which determine the surface antigens located in the plasma membrane of cells. In short: main physiological function of MHC proteins is to present antigens or fragments to cells of the immune system (particularly T-cells); using these MHC molecules immune cells corporate. When tissues are transferred from individual to individual, the transplanted tissues may be rejected by the recipient. This graft rejecting is due to an immune response that occurs when antigens on the surface of grafted tissue are detected and attacked by T-cells in the host organism. The antigens that elicit graft rejection are referred to as histocompatibility antigens, and they are encoded by a cluster of genes called the Major Histocompatibility complex - MHC genes. T cells are activated only when the T-cell receptor simultaneously both a foreign antigen and the host cell's own histocompatibility antigen. Through their T-cell receptors, T cells bind to both the HC protein and the foreign antigen and secrete substances that ether destroy the antigen-containing cell or activate other B and T cells. We know 5 HLA complexes: HLA - A, HLA - B, HLA - C, HLA - D, HLA - DR (D-region related - in relation to area D). Each of them has a number of alleles (today at least 20 alleles are known for HLA - A, 40 alleles for HLA - B, 8 and more for the other three). A set of HLA genes on one chromosome forms a haplotype , so an individual has two haplotypes (from each parent) and 5 determinants in each. The order of areas in the MHC is different for different animal species. HLA genes are so tightly bound that they act as a unit. The probability that 2 siblings will have the same haplotypes is 1/4. A large number of alleles at each of the five loci allows for HLA variability. If the HLA system is highly polymorphic, it can be used as the sole genetic marker in population research or in determining paternity. MHCs are cluster of genes found on chromosome 6, and their product is an integral membrane protein found on the surface of all body's cells. They serve as indicators for T-cells to distinguish between "self" and "non-self" peptides. The human MHC is also called the HLA (human leukocyte antigen) complex. MHC molecules can be classified into two types: Class I - found on the surface of every nucleated cell. Each MHC molecule on the cell surface displays a molecular fraction of a protein - called an epitope - which gives the body's immunity the ability to recognize it as "self" cell - thus do not generate any immune reaction. In case of viral infection or tumor cells, different peptides are joined to the proteins generated from the MHC genes, and thus they aren't recognized as "self" anymore - CD8+ T-cells (cytotoxic) able to identify them and elicit immune response. The body's T-Cells learn to recognize self from non-self during their maturation in the thymus - in a "selection mechanism" of the thymus: they are exposed to self-MHC molecules that are normally found in the body, and in case they trigger an immune response against them - they destroyed; thus, prevent auto-immune responses. MHC II molecules are a class of MHC normally found only on antigen-presenting-cells such as dendritic, mononuclear phagocytes (macrophages), some endothelial cells and B-lymphocytes. The antigens presented by class II peptides are derived from extracellular proteins (not cytosolic as in MHC I). Loading on MHC class II molecule occurs by phagocytosis; extracellular proteins are endocytosed, digested in lysosomes, and the resulting epitope peptide fragments are loaded onto MHC class II molecules prior to their migration to the cell surface. Because class II MHC is loaded with extracellular proteins, it is mainly concerned with presentation of extracellular pathogens (e.g. bacteria that might be infecting a wound or the blood). Class II molecules interact mainly with immune cells, like T-helper (CD4) which then triggers an appropriate immune response. Class III molecules have physiologic roles unlike classes I and II, but are encoded between them in the short arm of human chromosome 6. Class III molecules include several secreted proteins with immune functions: components of the complement system, cytokines and more.. Binding of class I and class II MHC molecules to antigens: The sequence of events by which antigens made in a cell are processed, bound to class I MHC proteins, and displayed at the cell surface: (e.g. in a virus-infected cell\tumor cell) Proteins in the cell are continuously digested by proteasomes and antigenic fragments are transferred to the RER where they associate with class I MHC proteins synthesized there The class I MHC-antigen complex is transferred to the Golgi region Golgi vesicles transport the complex to the cell membrane presenting the antigen at the outer surface Formation of complex between class II MHC proteins and antigens internalized by the cell: Synthesis of class II MHC molecules in the RER of APC's Transfer of the molecules to the Golgi region and formation of Golgi vesicles. The Golgi vesicles fuse with a lysosome containing antigens processed after endocytosis and digestion of microorganisms and debris by lysosomal enzymes. Antigens form complexes with class II MHC molecules The MHC II-antigen complex are exposed at cell surface Contrary to B cells, which recognize soluble antigens or antigens present on cell surfaces, T lymphocytes recognize only small peptides displayed by MHC molecules. T cells from an individual recognize as 'self' the MHC II-antigen complex only if the MHC molecule belongs to the same individual ("self-MHC") due to "central tolerance" of T maturation in the thymus. Because different individuals express different MHC, cell or organ transplantation between distinct individuals induces an intense immune reaction. Transplant rejection In a transplant procedure, as of an organ or stem cells, MHC molecules act themselves as antigens and can provoke immune response in the recipient, thus causing transplant rejection. Each human cell expresses six MHC class I alleles (one HLA-A, -B, and -C allele from each parent) and six to eight MHC class II alleles. The MHC variation in the human population is high, at least 350 alleles for HLA-A genes (and polymorphism for the other types as well). Any two individuals who are not identical twins will express differing MHC molecules. All MHC molecules can mediate transplant rejection (but some of them have less affect). Ankylosing spondylitis In patients with some diseases, some alleles of the HLA loci occur more frequently than in groups of healthy patients. The association of these diseases with certain HLA alleles is evident. Many of these diseases affect the joints, endocrine glands and skin . One of the strongest associations is M. Bechtěrev (ankylosing spondylitis) - it occurs together with the HLA-B27 allele, it affects and gradually immobilizes the joints of the spine and limbs, especially the hip joint .

what are the aims of medical genetics? what are the basic principles? what cases are typically dealt with? (123)

Medical genetics is the branch of medicine that involves the diagnosis and management of hereditary disorders. Medical genetics differs from human genetics in that human genetics is a field of scientific research that may or may not apply to medicine, while medical genetics refers to the application of genetics to medical care. For example, research on the causes and inheritance of genetic disorders would be considered within both human genetics and medical genetics, while the diagnosis, management, and counselling people with genetic disorders would be considered part of medical genetics. In contrast, the study of typically non-medical phenotypes such as the genetics of eye color would be considered part of human genetics, but not necessarily relevant to medical genetics (except in situations such as albinism). Accurate diagnosis using cytogenetic and molecular genetic testing, determining the risk of recurrence of the pathology and possible preventive measures - are the main tasks of clinical genetics. Medical genetics encompasses many different areas, including clinical practice of physicians, genetic counselors, and nutritionists, clinical diagnostic laboratory activities, and research into the causes and inheritance of genetic disorders. Examples of conditions that fall within the scope of medical genetics include birth defects and dysmorphology, mental retardation, autism, and mitochondrial disorders, skeletal dysplasia, connective tissue disorders, cancer genetics, teratogens, and prenatal diagnosis. Medical genetics is increasingly becoming relevant to many common diseases. It is important to note that clinical genetics is strictly non-directive field. All decisions, such as decision on abortion in the case of serious developmental defects in the fetus, are completely voluntary and geneticist task is solely provide enough information for the patient's free choice. Medical genetics based on principles: Prevention - despite there are some advances in gene therapy, prevention is still the most important task of medical genetics. Prevention focusing in identifying genetic risk of defects or diseases, as well as to identify the external factors that may have an impact on conditions that lead to the potential defect. Planned parenthood is good method taking place in order to prevent birth defects - not only total prevention applies, but also planned administration of folic acid, for example, in order to prevent congenital neural tube defects. Diagnosis - the main task is to detect congenital defects or genetic diseases and accurately classify them. Prenatal diagnosis deals with the examination of the fetus, while postnatal diagnosis applies to already born individuals. There are various tests - biochemical, cytogenetic, molecular genetics, imaging - on the basis of which it is possible to diagnose a disease or defect. Treatment of defects and diseases - treatment at the molecular level (DNA or RNA) is still in the development stage. Thus, in the medical genetics field, it is still consider much more common to deal with prevention. In certain diseases, e.g. metabolic disorders - the treatment doesn't necessary need to be surgical or medical - and proper special diet will help to relief the symptoms. Registartion and monitoring - development countries tend to monitor and register the incidence of diseases, increase the awareness of the population and the success of prenatal diagnosis. Spectrum of cases: Infants and children with suspected congenital malformations Children or adolescents with psychiatric findings Children and adolescents with growth failure, impaired sexual development People with family history of frequent occurrence of cancer at a younger age (which is suspected to family history of genetic cancer syndrome) Persons or couples suffering from sterility (long unsuccessful attempt to conception) or repeated abortions; Couples that coming to preconception consolation - may be due to birth defects or genetical diseases in family history. Pregnant women, for which screening tests (biochemical, ultrasound) drew attention to the increased risk of congenital malformations, or in case there is increase due to age (generally over 35 years). Clinical genetics has to cooperate with almost all medical fields - especially cytogenetic and molecular genetic laboratories, gynecology and obstetrics, oncology, plastic surgery, psychiatry and neurology etc.

what is nutrigenetics? how could DNA influence metabolism? what assumptions is it based on? what are some relevant nutritional tools? (134)

Nutrigenetics Nutrigenetics aims to identify how genetic variation affects response to nutrients. This knowledge can be applied to optimise health, and prevent or treat diseases. The ultimate aim of nutrigenetics is to offer people personalized nutrition based on their genetic makeup. Due to naturally occurring mutations, humans differ in their DNA (polymorphism). The most common type of DNA polymorphisms are SNPs. SNPs may influence the way individuals absorb, transport, store or metabolize nutrients. This may determine requirements for different nutrients and this assumption forms the basis for nutrigenetic sciences. Obesity - A major goal for nutrigenetic researchers is to identify genes that make certain individuals more susceptible to obesity and obesity-related diseases. The thrifty gene hypothesis is an example of a nutrigenetic factor in obesity. The thrifty gene theoretically causes bearers to store high-calorie foods as body fat, a most likely as an evolved protection against starvation during famines. In the long run, nutrigenetics should allow nutritionists and physicians to individualize health and diet recommendations. Consequently, preventive medicine, diagnostics and therapies could be optimized. underlying assumptions Substances contained in food (micro- and macronutrients) act directly or indirectly on the human genome and thus change its structure or gene expression. Under certain circumstances, diet can be a significant risk factor for many diseases in some individuals. Some of the target genes in food are likely to play a role in the onset, incidence, course and severity of some chronic diseases. The extent to which diet affects the balance between health and disease may depend on an individual's specific genetic makeup. Nutritional intervention based on knowledge of both a particular nutritional status and needs and genotype (individualized nutrition) can be used to prevent, alleviate or treat chronic diseases. One of the important tools of nutritional genomics is the profiling of gene transcriptome expression , eg using cRNA or cDNA chips, analogous procedures have been developed to monitor expression at the protein level ( proteome - especially two-dimensional electrophoresis and various forms of mass spectrophotometry) and metabolites ( metabolome ). The specific expression profiles of genes, proteins and metabolites in response to a given dietary component or nutritional regime form so-called "dietary signatures", which are further examined at the level of specific cells, tissues and whole organisms to understand the effect of nutrition on health and disease balance. A classic example of a nutrigenetic interaction is persistent lactose tolerance in adulthood. In young mammals, including humans, functional lactase is an essential enzyme for the breakdown of lactose present in milk into monosaccharides - glucose and galactose. Lactase expression in small intestinal enterocytes is tightly controlled during development - it is attenuated during the fetal period, increases around birth and decreases again after weaning. Most adults do not tolerate lactose naturally - they consume large amounts of milk (which contains 4-8% lactose) with abdominal pain, flatulence or diarrhea, because undigested lactose causes osmotic transport of water to the lumen of the small intestine and is fermented by bacteria in the intestinal microflora. Acidified dairy products are a cultural adaptation to lactose intolerance, which, due to their significantly lower lactose content and sometimes the presence of lactase-secreting bacteria (Lactobacillus acidophilus), do not cause digestive problems in intolerant individuals. On the other hand, there are a number of adults, especially in the European population, who do not show signs of lactose intolerance and we are talking about so-called lactase persistence .

what are leukocytes? general characteristics? what are the types? functions of each? specific vs. innate immunity? what is immunophenotyping? (74)

Our bodies are exposed continually to bacteria, viruses, fungi and parasites, all of which occur normally and found in many places in the body. Many of these infectious agents are capable of causing serious abnormal physiological functions; we are also exposed to other highly infectious bacteria and viruses besides those that are normally present, and these agents can cause acute diseases. Our bodies have a special system for combating the different infectious and toxic agents - the system composed of blood leukocytes (WBCs). The leukocytes are the mobile units of the body's protective system. They are formed partially in the bone marrow (granulocytes and monocytes, as well as few lymphocytes). After formation, they are transported in the blood specifically to different parts of the body where they are needed - providing a rapid and potent defense against infectious agents. Immunocompetent cells mediate the immune response. There are two groups: Cells of the innate immunity - Macrophages and granulocytes Cells of specific (adaptive) immunity - T and B lymphocytes General characteristics of WBC's: Types: there are six types of WBC's: Granulocytes - Neutrophils, Eosinophils, Basophils Agranulocytes - Monocytes, Lymphocytes and occasionally, Plasma cells that secrete antibodies. In addition, there are large numbers of Platelets, which are fragments of Megakaryocyte - which is type of cell similar to the WBCs found and found in the bone marrow. The granulocytes and the monocytes protect the body against invading organisms by ingesting them (phagocytosis) or by releasing antimicrobial or inflammatory substances that have multiple effects that aid in destroying the offending organism. The lymphocytes and plasma cells (activated B-lymphocytes) function mainly in connection with the immune system. The platelets activate the blood clotting mechanism. Functions - Overview: Granulocytes Neutrophils - Play roles in the destruction of bacteria and the release of chemicals that kill or inhibit the growth of bacteria. Eosinophils - Function in the destruction of allergens and inflammatory chemicals, and release enzymes that disable parasites. Basophils - Secrete histamine which increases tissue blood flow via dilating the blood vessels, and also secrete heparin which is an anticoagulant. Agranulocytes Lymphocytes: Lymphocytes function in destroying cancer cells, cells infected by viruses, and foreign invading cells. In addition, they present antigens to activate other cells of the immune system. They also coordinate the actions of other immune cells, secrete antibodies and serve in immune memory. Monocytes: function in differentiating into macrophages, which are large phagocytic cells, and digest pathogens, dead neutrophils, and the debris of dead cells. Like lymphocytes, they also present antigens to activate other immune cells. The primary cells that participate in the immune response are lymphocytes, plasma cells, mast cells, neutrophils, eosinophils, and cells of the mononuclear phagocyte system. Specific adaptive immunity cells Lymphocytes - are classified as B, T and NK (Natural killer) cells. B and T cells differ based on their life history, surface receptors and behavior during an immune response. B lymphocytes originate, mature and become functional in the bone marrow, T lymphocytes precursors are in the bone marrow and going through proliferation and differentiation in the thymus. Each B lymphocyte that leaves the bone marrow or each T lymphocyte that leaves the thymus has just one type of surface receptor that recognizes one specific epitope. Soon after a lymphocyte is first exposed to the epitope it recognizes, a stimulus to cell proliferation occurs, leading to an amplification of that particular lymphocyte population. B-Lymphocytes - The surface receptors able to recognize antigens are IgM antibodies. The encounters with the epitope leads to several cycles of cell proliferation of these B-cells, followed by differentiation of most of these cells into plasma cells- secrete antibodies against the same epitope. Some other cells will turn into B memory cells which are able to react very rapidly to a second exposure to the same epitope. In most cases, activation of B cell requires the assistance of T-Helper cell (CD4). T-Lymphocytes - 65-75% of blood lymphocytes. To recognize epitopes, all T cells have on their surfaces a molecule called T-Cell receptor (TCR). In contrast to B-lymphocytes which recognize "free" soluble antigens or antigens present on cell surfaces, T lymphocytes recognize only epitopes that form complexes with MHC proteins on the cell surface of other cells. There are 3 subpopulations of T-cells: T-Helper cells - CD4 - produce cytokines that promote differentiation of B cells into plasma cells, activate macrophages to become phagocytic, active T-cytotoxic cells, induce inflammatory reaction. Cytotoxic T cells - CD8 - act directly against foreign cells or virus-infected cells by secret perforins into holes in the membrane of the target cell Regulatory T cells - play crucial roles in allowing immune tolerance - maintaining unresponsiveness to self-antigens and suppressing excessive immune responses. Natural Killer cells - comprise 10-15% of the lymphocytes of circulating blood, they attack virus-infected cells, transplanted cells and cancer cells without previous stimulation - part of the innate immune response. Major Histocompatibility Complex (MHC) & Antigen Presentation One individual expresses one set of MHC I and one set of MHC II proteins (APC's only), these proteins are unique to that person and they are integral membrane proteins present on the cell surfaces. All nucleated cells have I proteins, but class II proteins exist only on small group of cells called antigen-presenting cells (APCs). Antigen-Presenting Cells (APC's) - found in many tissues, and includes dendritic cells, macrophages and B lymphocytes. APC's recognize the foreign cells by the absence of HLA-I (MHC I) and phagocytose it. The main feature of APC's cells is the presence of MHC II on their surfaces, which interact with CD4+ (T-Helper) cells. CD8 (Cytotoxic) interacts with MHC I molecules, which can be presented by any nucleated cell. APCs, being recognized by helper lymphocytes, are essential for triggering and development of a complex immune response. Cells of Innate immunity - Monocytes, Neutrophils, Eosinophils, Basophils, Mast cells The main function of the innate immunity cells is to initiate a non-specific immune response. The destruction of antigens involved mostly phagocytosis and activation of specific immune responses. They migrate from blood to the tissue on the basis of chemotaxis, which participates in the defense response. Complement is a non cellular component of immunity - it is a group of blood serum proteins, which bind to the complex of antigen-antibody, strengthens the destruction of cellular antigens phagocytes, or may itself have destructive effects. Neutrophil - defense against bacteria - are the most abundant type of granulocytes and the most abundant (60-70%) type of white blood cells in most mammals. They form an essential part of the innate immune system. They are formed from stem cells in the bone marrow. They are short-lived and highly motile, or mobile, as they can enter parts of tissue where other cells/molecules wouldn't be able to enter otherwise - a process which is called diapedesis (can squeeze through the pores of the blood capillaries). Neutrophils may be subdivided into segmented neutrophils and banded neutrophils (or bands). The main function of Neutrophils is phagocytosis and kill bacteria. They contain bactericidal agents that kill most bacteria even when the lysosomal enzymes fail to digest them; it is important because some bacteria have protective coats or other factors that prevent their destruction by digestive enzymes. The eosinophils normally constitute about 2% of all the blood leukocytes. They are weak phagocytes, and exhibit chemotaxis. They produced in large numbers in people with parasitic infections (causes eosinophilia), and they migrate into tissues diseases by parasites. Eosinophils attach themselves to the parasites by way of special surface molecules and release substances that kill many of the parasites. Eosinophils have a special tendency to collect in tissues in which allergic reactions occur; This action is caused by the fact that many mast cells and basophils participate in allergic reaction. The mast cells and basophils release an eosinophil chemotactic factor that causes eosinophils to migrate toward the inflamed allergic tissue. The eosinophils are believed to detoxify some of the inflammation inducing substances released by the mast cells and basophils and probably also phagocytize and destroy allergen-antibody complexes, thus preventing excess spread of the local inflammatory process. Basophils and Mast cells - The basophils in the circulating blood are similar to the large tissue mast cells which located immediately outside many of the body's capillaries. Both cells liberate Heparin into the blood - a substance that can prevent blood coagulation. In addition, they release histamine, bradykinin and serotonin. They play an important role in some types of allergic reactions because the type of antibody that causes allergic reactions, IgG (immunoglobulin E) has a special tendency to become attached to mast cells and basophils. Then, when the specific antigen for the specific IgE antibody subsequently reacts with the antibody, the resulting attachment of antigen to antibody causes the mast cell or basophil to rupture and release large quantities of histamine, bradykinin, serotonin, heparin slow-reacting substance of anaphylaxis and a number of lysosomal enzymes. These substances cause local vascular and tissue reactions that cause many of the allergic manifestations Immunophenotyping Immunophenotyping is a test used to identify cells on the basis of the types of markers or antigens present on the cell's surface, nucleus, or cytoplasm. This technique helps identify the lineage of cells using antibodies that detect markers or antigens on the cells, hence the "immuno-" prefix. An example is the detection of tumor marker, such as in the diagnosis of leukemia. It involves the labelling of white blood cells with antibodies directed against surface proteins on their membrane. By choosing appropriate antibodies, the differentiation of leukemic cells can be accurately determined. The labelled cells are processed in a flow cytometer, a laser-based instrument capable of analyzing thousands of cells per second. Method: A solution of antibodies (serum) is poured over sample containing examined cell If the sample contains the antigens for which the antibodies are specific - the antibodies will bind the cells The cells are washed to prevent non-specific binding; the specific antibodies remain bound The cells with the bound antibodies exposed - declares cells presence The exposure of the antibodies can be done by several ways: Immunofluorescence Flow cytometry - if the speciemen is cells suspended in lkquid, the cells can be run through a flow cytometer - this will expose cells in a stream of liquid, one at a time, to a beam of laser light. If they bear the bound antibodies, they will fluoresce and the cytometer will record the signal. Immunohistochemistry - identify cells by the antibodies that have attached to them.

what is a polymorphism? what are the types? How do these differ from mutations? what can they be used for? (53)

Polymorphism is the existence of two or more variants ( alleles , sequence variants, proteins , etc.) that are represented in a population with a frequency ≥ 0.01 [1] . We usually consider them to be non-pathogenic sequence variants. The polymorphism itself or a combination thereof may contribute to the disease . Polymorphic moieties are found primarily in non-coding DNA sequences because they are not subject to selection . The most common form of polymorphism is base change. Nucleic acids are carriers of hereditary information; their most important ability is replication. Nucleic acids, such as DNA or RNA, composed of nitrogenous base, a five carbon sugar (ribose in RNA or deoxyribose in DNA), and phosphate group. Nucleoside is nucleotide without the addition phosphate group. The phosphate group connected to the 5' carbon by ester linkage. The nitrogen base connected to the first carbon with N-glycosidic bond. The differences in the nucleotide sequence between individuals can be single base pair changes (SNP), deletions, insertions, or even changes in the number of copies of a given DNA sequence. SNPs (single nucleotide polymorphisms) are the most common type of DNA polymorphism in humans. An example of an SNP would be if a cytosine (C) nucleotide is present at a particular locus in one person's DNA but a thymine (T) nucleotide occurs at the same locus in another person's DNA. Generally, if an inheritable mutation is observed in a population at frequency higher than 1%, it is referred to as DNA polymorphism. Such variations are observed mainly in non-coding DNA sequences because mutations in these sequences may not have any immediate effect or impact on the individual's reproductive ability - thus, it may transfer to its offspring. As a result, these mutations keep on accumulating generation after generation, and form one of the bases of variability of human genomes. Mutations by themselves do not classify as polymorphisms. A polymorphism is a DNA sequence variation that is common in the population. A mutation, on the other hand, is any change in a DNA sequence away from normal (implying that there is a normal allele running through the population and that the mutation changes this normal allele to a rare and abnormal variant). In polymorphisms, there are two or more equally acceptable alternatives and to be classified as a polymorphism, the least common allele must have a frequency of 1% or more in the population (means that the most common allele must to have frequency of less than 99%). If the frequency is lower than this, the allele is regarded as a mutation. types: SNP's - single nucleotide polymorphism - variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population (e.g. > 1%). For example, at a specific base position in the human genome, the base C may appear in most individuals, but in a minority of individuals, the position is occupied by base A. There is a SNP at this specific base position, and the two possible nucleotide variations - C or A - are said to be alleles for this base position. SNPs underlie differences in our susceptibility to disease; a wide range of human diseases, e.g. sickle-cell anemia, β-thalassemia and cystic fibrosis result from only one (!) different nucleotide - SNPs. The severity of illness and the way our body responds to treatments are also manifestations of genetic variations. Insertion-deletion polymorphism - "Indel" - An insertion/deletion polymorphism, commonly abbreviated "indel," is a type of genetic variation in which a specific nucleotide sequence is present (insertion) or absent (deletion) - insertion or deletion of about 2-100 nucleotides. tandem repeats: Tandem repeats are made of successive identical repeat units. They vary in length of repeat unit (e.g. dinucleotide repeat - ACACACACAC) as well as number of the repeat units. The largest repeats, which tend to be composed from large repeat units are called satellites. Satellite DNA - 10-15% of the repetitive DNA sequences in human genome; range between 100 to 500 base pairs for each repeat unit. They consist of large series of tandemly repeated non-coding DNA sequences, and are clustered around the centromere of the chromosome. These sequences can be separated from the whole genes with density-gradient centrifugation - thus named "satellites". Mini satellite DNA - are shorter tandem repeats, in the range of 10-100 base pairs for each repeat unit. Found in the sub-telomeric regions of the chromosomes. They are often highly polymorphic as to the number of repeat units in a repeat (the length of the region) and used as genetic markers - VNTR - variable number of tandem repeats - useful for DNA Fingerprint technique (E.g., each repeat unit composed of 50 bases, and the polymorphism is regarding how many times it is repeated). Certain mini satellites are hypothesized to have regulatory functions. Usage of mini-satellites in DNA fingerprinting - the pattern and length of the VNTR's is highly different between individuals - i.e. highly polymorphic. Only identical twins show an indistinguishable pattern. Due to these facts, detection of a number of minisatellite polymorphisms was one of the first methods of DNA matching in forensic. Obtaining of DNA sample from a crime area and matching the minisatellite of the sample with possible suspected criminals or with database. Microsatellites - most common form of repetitive sequences. Also known as Short tandem repeats. STRs are short sequences of DNA, normally of length 2-5 base pairs, that are repeated numerous times in a head-tail manner, i.e. the 16 bp sequence of "gatagatagatagata" would represent 4 head-tail copies of the tetramer "gata". The polymorphisms in STRs are due to the different number of copies of the repeat element that can occur in a population of individuals. They represent 0.5% of the genome and are highly polymorphic thus very often used as genetic markers. Trinucleotide Expansion: Unstable transmission of some repetitive sequences, called expansion. E.g. expansion of CAG trinucleotide in a coding sequence of the gene for Huntington disease leads to a production of an abnormal protein Huntingtin, and causes clinical manifestation of Huntington disease. Huntington disease is a progressive neurodegenerative disorder with late and variable age at onset causing to dementia and progressive disorders of movement. It is AD trait and caused by CAG expansion in the HD gene - 99% of normal genes contain less than 30 CAG triplets, but mutated genes in patients with HD contain always 40 or more of the CAG repeats. Interspersed repeats - transposable elements: Interspersed repeats are repeated DNA sequences located at dispersed regions in a genome. They are also known as mobile elements/transposable elements. Transposable elements are segments of DNA that can move from one location in the genome to a target sequence in another. After many generations, such sequence (the repeated unit) could spread over various regions. Depending on their length, moderately repetitive sequences are classified as LINEs and SINEs (moderate in the aspect that each bulk is relatively short - 6000/7000 in LINE or less than 500 in SINE; in contrast, the tandem repeats that occur in the same bulk are longer - even though each repeat is much shorter). Both types appear to be retrotransposons - arrive through an RNA intermediate by the action of reverse transcriptase that transcribed an RNA template into DNA (regular transposons - may be either cut and paste or copy and paste, while retrotransposon are always copy and paste). LINEs stands for Long Interspersed Nuclear Elements - compose approximately 21% of the human genome. 6000-7000 base pairs long, interspersed in many copies in different regions through the genome. SINEs stands for short interspersed nuclear elements - are typically less than 500bp long and have no protein coding potential. The main SINE family in humans if formed by Alu elements; the amount of Alu elements in the human genome account for about 11% of its mass. Microsatellite repeat sequences, which consist of 1-5 base pairs (ACACACACAC) and repeated up to 5 times, can be both dispersed (interspersed) as well as grouped in tandem repeats. These microsatellite sequences most commonly are found as dinucleotide repeats of AC on one strand, and TG on the opposite strand. The AC repeat sequences occur at 50,000-100,000 locations in the genome. At any locus, the number of these repeats may vary on the two chromosomes (e.g. chromosome 1 that arrive from dad can have 30 repeats, while its homologue that arrive from the mother has only 25) - thus, providing heterozygosity of the number of copies of a particular microsatellite number in an individual. This is a heritable trait, thus, such repeats are useful in constructing genetic linkage maps: most genes are associated with one or more microsatellite markers, so the relative position of genes on chromosomes can be assessed, as can the association of a gene with a disease. Large number of family members can be screened for a certain microsatellite polymorphism. Association of a specific polymorphism with a gene in affected family members, and the lack of this association in unaffected members - may be the first clue about the genetic basis of a disease. Forensic medicine usage minisatellite VNTRs Parental test Detection of certain disease - e.g. Huntington in trinucleotide expansion (microsatellite) Genetic marker - used for association analysis of diseases Genetic marker - used for constructing genetic maps

what is a population? HW conditions? what is genetic drift? what can cause it? what is sampling error? (107)

Population is a group of individuals of the same species that live in the same area and interbreed, producing fertile offspring. Members of a population typically breed with one another and thus on average they are more closely related to each other than to members of other populations. According to Hardy-Weinberg equilibrium theory, in a population that is not evolving, allele and genotype frequencies will remain constant from generation to generation, provided that only Mendelian segregation and recombination of alleles are at work. Such population is said to be in Hardy-Weinberg equilibrium. Conditions for Hardy-Weinberg equilibrium: The Hardy-Weinberg approach describes a hypothetical population that is not evolving. But in real populations, the allele and genotype frequencies often do change over time. Such changes can occur when at least one of the following five conditions of Hardly-Weinberg equilibrium is not met: No mutations - The gene pool is modified if mutations alter alleles or if entire genes are deleted or duplicated Random mating - If individuals tend to mate within a subset of the population, such as their near neighbors or close relatives, random mixing of gametes doesn't occur and genotype frequencies change. No natural selection - differences in the survival and reproductive success of individuals carrying different genotypes can alter allele frequencies. Extremely large population size - the smaller the population, the more likely it is that allele frequencies will fluctuate by chance from one generation to the next (genetic drift) No gene flow - by moving alleles into or out of populations, gene flow can alter allele frequencies. Such hypothetical population will produce gametes carrying genes that perfectly represent the parental gene pool. However, no real population is infinity large, and in fact, the genes carried are just sample of the parental gene pool. Genetic drift Genetic drift is a variation in the relative frequency of different genotypes in a small population, owing to the chance disappearance of particular genes as individuals die or do not reproduce A change in allele frequencies caused by random events Sampling error - the deviation from the expected ratio is the sampling error and that's what the genetic drift is based on: If you flip a coin 1000 times, a result of 700 heads and 300 tails might make you suspicious about that coin. But if you flip a coin only 10 times, an outcome of 7 heads and 3 tails would not be surprising. The smaller the number of coin flips, the more likely it is that chance alone will cause a deviation from the predicted result: chance events can also cause allele frequencies to fluctuate unpredictably from one generation to the next, especially in small populations - a process called genetic drift. The model shows how genetic drift might affect a small population of the wild flowers - drift leads to the loss of an allele from the gene pool, but it is matter of chance that the Cw allele is lost and not the Cr allele. Such unpredictable changes in allele frequencies can be caused by chance events associated with survival and reproduction. Perhaps a large animal stepped on the three Cw Cw individuals in generation 2, killing them and increasing the chance that only the Cr allele would be passed to the next generation. Allele frequencies can also be affected by chance events that occur during fertilization. For example, suppose two individuals of genotype CrCw had a small number of offspring. By chance alone, every egg and sperm pair that generated offspring could happen to have carried the Cr allele and not the Cw allele. The founder effect - when a few individuals become isolated from a larger population, this smaller group may establish a new population whose gene pool differs from the source population. The founder effect probably accounts for the relatively high frequency of certain inherited disorders among isolated human populations. For example, in 1814, 15 British colonists founded a settlement on Tristan De Cunha, a group of small islands in the Atlantic Ocean midway between Africa and South America. Apparently, one of the colonists carried a recessive allele for retinitis pigmentosa, a progressive form of blindness that afflicts homozygous individuals. Of the founding colonists - 240 descendants on the island and 4 had retinitis pigmentosa - frequency of the allele is ten times higher on Tristan da Cunha than in the populations from which the founders came. The Bottleneck effect - A severe drop in population size, as a result, for example, from a fire or flood, can cause the bottle neck effect - the population has passed through a "bottle-neck" that reduces its size. By chance alone, certain alleles may be overrepresented among the survivors, others may be underrepresented, and some may be absent altogether. Even if a population that has passed through a bottleneck ultimately recovers in size, it may have low levels of genetic variation for a long period of time, and by chance could have an increase in the frequency of harmful alleles.

genetic aspects of populations? what is a population? what is a gene pool? what is the Hardy-Weinberg equilibrium? what are the conditions? what are the applications? (101)

Population is a group of individuals of the same species that live in the same area and interbreed, producing fertile offspring. Members of a population typically breed with one another and thus on average they are more closely related to each other than to members of other populations. We can characterize a population's genetic makeup by describing its gene pool, which consists of all copies of every type of allele at every locus, in all members of the population. If only one allele exists for a particular locus in a population, that allele is said to be fixed in the gene pool and all individuals are homozygous for that allele. But if there are two or more alleles for a particular locus in a population, individuals also may be either homozygous or heterozygous. When studying a locus with two alleles, the convention is to use p to represent the frequency of one allele and q to represent the frequency of the other allele. Thus, p, the frequency of the Cr allele in the gene pool of this population, is p = 0.8 (80%). Because there are only two alleles for this gene, the frequency of the Cw allele, represented by q, must be Cw = q = 1-p = 0.2 (20%). For loci that have more than two alleles, the sum of all allele frequencies must still equal 1 (100%). The Hardy-Weinberg equation In a population that is not evolving, allele and genotype frequencies will remain constant from generation to generation, provided that only Mendelian segregation and recombination of alleles are at work. Such population is said to be in Hardy-Weinberg equilibrium. To determine whether a population is in Hardy-Weinberg equilibrium, it is helpful to think about genetic crosses in a new way. Previously, we used Punnett squares to determine the genotypes of offspring in a genetic cross; Here, instead of considering the possible allele combinations from one cross, we'll consider the combination of alleles in all of the crosses in a population. Imagine that all the alleles for a given locus from all individuals in a population are placed in a large bin - gene pool for that locus. "Reproduction" occurs by selecting alleles at random from the bin. By using the numbers from the wildflower population: In that population of 500 flowers, the frequency of the allele for red flowers Cr is p = 0.8, and Cw = 0.2. In other words, the bin holding all 1000 copies of the flower-color gene in the population would contain 800 Cr alleles and 200 Cw alleles. Thus, the probability that an egg or sperm contains a Cr or Cw allele is equal to the frequency of these alleles in the bin. Using the rule of multiplication , we can now calculate the frequencies of the three possible genotypes; must be add up to 1 (100%): The probability that two Cr alleles will come together is p X p = 0.8 X 0.8 = 0.64; Thus - about 64% of the plants in the next generation will have genotype Cr-Cr. The frequency of Cw-Cw individuals is expected to be about q X q = 0.2 X 0.2 = 0.04 - 4%. The frequency of heterozygote Cr-Cw can arise in two different ways. If the sperm provides the Cr allele and the egg provides the Cw allele, the resulting heterozygotes will be p X q = 0.8 X 0.2 = 0.16 (16%); if the sperm provides the Cw allele and the egg the Cr allele, the heterozygous offspring will make up q X p = 0.2 X 0.8 = 0.16 (16%). Thus, the frequency of heterozygotes is the sum of these possibilities: pq + pq = 2pq = 0.16 + 0.16 = 0.32, 32%. The equation for Hardy-Weinberg equilibrium states that at a locus with two alleles, the three genotypes will appear in the following proportions: Conditions for Hardy-Weinberg equilibrium: The Hardy-Weinberg approach describes a hypothetical population that is not evolving. But in real populations, the allele and genotype frequencies often do change over time. Such changes can occur when at least one of the following five conditions of Hardy-Weinberg equilibrium is not met: No mutations - The gene pool is modified if mutations alter alleles or if entire genes are deleted or duplicated Random mating - If individuals tend to mate within a subset of the population, such as their near neighbors or close relatives, random mixing of gametes doesn't occur and genotype frequencies change. No natural selection - differences in the survival and reproductive success of individuals carrying different genotypes can alter allele frequencies. Extremely large population size - the smaller the population, the more likely it is that allele frequencies will fluctuate by chance from one generation to the next (genetic drift) No gene flow - by moving alleles into or out of populations, gene flow can alter allele frequencies. Departure from these conditions usually results in evolutionary change, which is common in natural populations. But it is also common for natural populations to be in Hardy-Weinberg equilibrium for specific genes. This can occur if selection alters allele frequencies at some loci but not others. Applying the Hardy-Weinberg equation: It is often used as an initial test of whether evolution is occurring in population. It has also medical applications, such as estimating the percentage of a population carrying the allele for an inherited disease. For example, consider phenylketonuria (PKU) - a metabolic disorder that results from homozygosity for a recessive allele and occurs in about 1/10,000 babies in the US. Left untreated, PKU results in mental disability and other problems. To apply the Hardy-Weinberg equation, we must assume the following assumptions: No new PKU mutations are being introduced into the population (condition 1) People neither choose their mates on the basis of whether or not they carry this gene, nor generally mate with close relatives (condition 2) Ignore any effect of differential survival and reproductive success among PKU genotypes (condition 3) There are no effects of genetic drift or of gene flow from other population into the US (condition 5). Frequency of individuals in the population born with PKU will correspond to q2 (frequency of homozygotes recessive). Because the allele is recessive, we must estimate the number of heterozygotes rather than counting them directly as we did with the pink flowers. Since we know there is 1/10000 babies with PKU, the frequency (q) of the recessive allele for PKU is q= 0.0001 = 0.01, and the frequency of the dominant allele is 0.99 (1-0.01). The frequency of carriers, heterozygous people who do not have PKU but may pass the PKU allele to offspring is 2pq: 2pq = 2 X 0.99 X 0.001 = 0.0198 - approximately 2% of the US population.

genetics in presymptomatic diagnostics what is screening? what is the criteria for screening? screening for breast, cervical, and colorectal cancers? prevention of cancer? (116)

Pre-symptomatic is a type of predictive testing of an asymptomatic or unaffected individual who is at risk of a specific genetic disorder. In cancer, usually we use cancer screening test. It means checking your body for cancer before you have symptoms. Cancer screening may involve blood tests, urine tests or medical imaging. "Universal screening" (population screening) involves screening everyone, usually within a specific age group; "Selective screening" identifies people who are known to be at higher risk of developing cancer, such as people with a family history of cancer. The main concern regarding screening is that it can lead to false positive results and subsequent invasive procedures; it is also can lead to false negative results, where an existing cancer is missed. Thus screening tests must be effective, safe and with acceptably low rates of false positive and false negative results. If signs of cancer are detected, more definitive and invasive tests are performed to reach a diagnosis. Screening for cancer can lead to cancer prevention and earlier diagnosis which may results in higher rates of successful treatment and extended life. In diagnostic of pre-symptomatic prone-to-cancer patients we'll follow their family history. Cancer, although usually sporadic, has genetic factor. Diagnosis of hereditary predisposition to cancer is done by using hybridization by probes or sequencing of the relevance genes the exist in the family. Chronic myeloid lymphoma occurs as a result of reciprocal translocation between chromosome 9 and 22 - the disease is often suspected on the basis of a complete blood count, which shows increase granulocytes of all types. In order to perform diagnosis of the disease, we can use FISH (detect chromosomal translocation). Criteria for use of screening: Disease must be frequent in the population/in the family Disease must have a relatively high morbidity There is an effective treatment in the early stages Detecting features available, and the test is relatively inexpensive The test method should be sensitive and specific with low false-negative or false-positive results Breast cancer Breast cancer is a cancer that develops from breast tissue. It is the most common cancer for women. The investigation and detection of breast cancer is the responsibility of gynecologists, who should do an annual preventive examination by palpation. Symptoms found? 🡪 Testing. Mammogram reveals 95% of all cancer, complemented examination in Ultrasound. There is general agreement that breast screening reduces mortality from the disease. Recommendations to attend to mammography screening vary across countries (in Israel, women over 50 - recommended to attend mammography once a two years; if there is family history/BRCA genes 🡪 over age of 40); mammography proved to reduce 30% mortality. Mammography is an X-ray examination; the effect of irradiation during our lives adds up, so frequent testing may be more harmful than beneficial. Risk factors for breast cancer: Obesity Lack of physical exercise Drinking alcohol Hormone replacement therapy during menopause Ionizing radiation Early age at first menstruation Family history - about 5-10% of cases are due to genes inherited from a person's parents, including BRCA1 and BRCA2. Cervical cancer Treatment of early cancer of the cervix is relatively easy, thus screening is absolutely crucial and beneficial. Cervical screening is the process of detecting and removing abnormal tissue or cells in the cervix before cervical cancer develops; screening followed by a biopsy. The initial stage of cell changes doesn't have any symptoms, so it may be detectable only through screening as part of routine gynecological examination once a year. An important role in the development of the disease is Human papilloma virus infection - HPV - which appears to be involved in the development of more than 90% of cases. Other risk factors include smoking, weak immune system, birth control pills, starting sex at a young age and having many sexual partners. Cervical screening is done mainly by the conventional cytology - called Pap smear, in which the physician collecting the cells smears from the cervix which is sent to a laboratory for evaluation. Colorectal cancer The basis are stool occult blood tests and primary screening colonoscopy : occult bleeding tests (TOKS) are recommended for people over the age of 50 once a year - they are available from general practitioners people over the age of 55 undergo TOKS every two years or colonoscopy every ten years Colorectal cancer is one of the first three most common cancers in the Czech Republic. Their treatment has significantly better results in early detection - ideally for people who do not yet experience difficulties. Nationwide screening was launched in 2009. Symptoms of colorectal cancer specific : weight loss, diarrhea and constipation - change in stool regularity, frequent bowel movements, abdominal pain , convulsions, non -specific : fatigue, nausea , increasing abdominal volume, fever or subfebrile Cancer prevention Defined as active measures to decrease the risk of cancer; the vast majority of cancer cases are due to environmental risk factors, and many, but not all, of these factors are controllable lifestyle choices. Thus, cancer is considered a largely preventable disease. Greater than 30% of cancer deaths could be prevented by avoiding risk factors including: tobacco, overweight, insufficient diet, physical inactivity, alcohol, sexual transmitted infections and air pollution. Not all environmental causes are controllable, such as naturally occurring background radiation, and other cases of cacner are caused through hereditary genetic disorders, and thus it is not possible to prevent all cases of cancer. Vaccination - vaccines have been developed that prevent infection by some carcinogenic viruses. Human Papillomavirus vaccine decreases the risk of developing cervical cancer; hepatitis B vaccine prevents infection with hepatitis B virus and thus decreases the risk of liver cancer. Dietary - obesity and alcohol consumption - increased risk, diet low in fruits and vegetables and high in red meat 🡪 increased risk. Consumption of coffee is associated with a reduced risk of liver cancer.

what are proteins? what are some of their major functions? typical structure? what is the relevance of genetic polymorphisms? (61)

Proteins are large macromolecules consisting of one or more long chains of amino acid residues bonded together by peptide bonds (polypeptides). Peptide bond connects by single covalent bond the amino group of one amino acid with the carboxyl group of the second amino acid. The start of the polypeptide chain is called the N-terminus (free amino group - found in the 5' end of the nucleotide sequence), and the other end is the C-terminus (free carboxyl group on the 3' end of the nucleotide sequence) They perform a vast array of functions within organisms, including catalyzing metabolic reactions (enzymes), DNA replication, responding to stimuli and transporting molecules from one location to another (transporters). Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code which specifies 20 standard amino acids. Shortly after or during synthesis, the residues in a protein are often modified by post-translational modification, which alters the physical and chemical properties, folding, stability, activity and ultimately - alters the function of the proteins. Sometimes proteins have non-peptide group attached, which can be called prosthetic groups or cofactors. Once formed, proteins only exist for a certain period of time and are then degraded and recycled by the cell's machinery through the process of protein turnover. Main functions: Enzymes - catalyze biochemical reactions and vital for metabolism Structural and mechanical factors - cytoskeleton (scaffold, maintains cell shape, actin and myosin in muscle Cell signaling (ability of cells to perceive and respond to their environment is the basis of development and homeostasis); (receptors) Immune response (antibodies) Cell adhesion (the process in which cells interact and attach to a surface or to another cells or the ECM components, mediated by interactions between cell-adhesion proteins of the cell surface) Cell cycle Maintains plasma oncotic pressure - fluid volume in different body compartments Source for energy - glucogenic and ketogenic amino acids Components of ECM - collagen, proteoglycans, glycoproteins Structure - based on the arrangement of amino acids in the chain and is very important for the protein structure: Primary structure - defined by the amino acid sequence in the chain, bonded by peptide bonds Secondary structure - The polypeptide backbone of the primary protein doesn't assume a random 3D structure but instead, forms regular arrangements of amino acids that are located near each other in the linear sequence - the arrangement, which are the secondary structure, may be alpha-helix, betta-sheet and betta-turn, bonded together by H-bonds between amino groups and carboxyl groups. Alpha-helix - twisted string; betta-pleated sheet - two parallel or antiparallel arranged chains. Tertiary structure - refers to the spatial arrangement of amino acids that are found far away from one another along the polypeptide chain and bonded together by disulfide bridges, hydrophobic interactions, H-bonds, electrostatic interaction as well as van der walls interaction. Quaternary structure - some proteins may consist of 2 or more polypeptide chains that may be structurally identical or totally unrelated. The polypeptide chains held together by intermolecular non-covalent interactions. The sub-units may work together (cooperatively) as in hemoglobin, or independently of each other. Denaturation of proteins involves the disruption and possible destruction of both the secondary and tertiary structures. Since denaturation reactions are not strong enough to break the peptide bonds, the primary structure (sequence of amino acids) remains the same after a denaturation process. Denaturation disrupts the normal alpha-helix and beta sheets in a protein and uncoils it into a random shape. Denaturation occurs because the bonding interactions responsible for the secondary structure and tertiary structure are disrupted. In tertiary structure there are four types of bonding interactions between "side chains" including: hydrogen bonding, ionic interactions, disulfide bonds, and non-polar hydrophobic interactions which may be disrupted. Therefore, a variety of reagents and conditions can cause denaturation (e.g. Heat - disrupt H-bonds and non-polar hydrophobic interactions by increasing the kinetic energy and causing the molecules to vibrate so rapidly and violently that the bonds are disrupted). The most common observation in the denaturation process is the precipitation or coagulation of the protein. Genetic polymorphism of Proteins Polymorphism is a term for a condition where there are at least 2 genetic variants (alleles) in the population, where the frequency in the population of each allele is at least 1% (if it is less or equal to 1% - it is referred to as random occurrence - mutation). Polymorphisms have their bases in the DNA structure. Thus, protein polymorphism occurs due different DNA bases found in the population in the same loci. It may be either change in repetitive sequences (many copies of nucleotides, which vary in the population in their length and number) or spot polymorphism (point mutation, also called SNP's - single nucleotide polymorphism - which some diseases occurred to single nucleotide change).

genetic information of the mitochondria...how it differs from nuclear, mode of inheritance, gene functions, etc what are mitochondrial diseases and what are the types? what is the main mode of inheritance for mt diseases? (65)

Small but important subsets of genes encoded in the human genome reside in the cytoplasm of mitochondria. Mitochondria genes are inherited almost exclusively from the oocyte (maternal pattern of inheritance aka extranuclear). Human cells can have hundred-thousands of mitochondrion in their cytoplasm, each containing a number of copies of small circular dsDNA which is referred to as mtDNA - Mitochondrial DNA, 1% of the total DNA in cell. Mitochondrial DNA molecule is only 16,000 base pairs and encodes only 37 genes, which include two types of rRNA's, 22 tRNA's and 13 protein subunits for enzymes. The products of these genes function in mitochondria, although the majority of the proteins within the mitochondria are products of nuclear genes. Each mitochondrion contains 2-10 copies of mtDNA genes thus each cell contains thousands of copies of mtDNA (in contrast to diploid cell nuclear DNA which contains two copies of each gene). According to the endosymbiotic theory - nuclear and mitochondrial DNA are thought to be separate by evolutionary origin - with mtDNA derived from circular genomes of bacteria that were engulfed by early ancestors of today's eukaryotic cells. As said, mtDNA is inherited from the mother. Mechanisms for this include simple dilution - an egg contains on average 200,000 mtDNA molecules, whereas a healthy human sperm was reported to contain on average only 5 molecules. In addition, degradation of sperm mtDNA in the male genital tract and in the fertilized egg leads to degradation of sperm mtDNA. Most mitochondria are present at the base of sperms mid-piece, which is used for propelling the sperm cells - the mid-piece and tail is usually lost during fertilization. The two strands of mtDNA are differentiated by their nucleotide content - heavy (H-) and light (L-) strands: the heavy strand is guanine-rich strand, and the light strand is cytosine-rich. The heavy strand encodes 28 genes, and the light strand encodes the remaining 9 genes. Mitochondrial disease is a group of disorders caused by dysfunctional mitochondria. It sometimes (in 15% of the time) caused by mutations in the mtDNA that affect mitochondrial function. Other causes of mitochondrial disease are mutations in genes of the nuclear DNA, whose gene products are imported into the mitochondria, as well as acquired mitochondrial conditions. More than 100 different rearrangements and about 100 different point mutations have been identified in mtDNA - 64 cause human disorders 🡪 clinical features are mostly combination of neurological signs and myopathic signs. Mitochondria are about 10 times more susceptible to DNA damage than nuclear DNA: no large amounts of repair systems as nuclear DNA and free radicals of the respiratory chain damage the mtDNA. The diseases that result from mutations in mtDNA show distinctive pattern of inheritance due to three unusual features of mitochondrial chromosome: Replicative segregation - at each stage in the duplication of mitochondria (DNA replication 🡪 DNA segregation to duplicate mitochondria 🡪 organelle segregation to daughter cells) the process appears to be stochastic - random distribution of each copy - there is no control over which particular copies are replicated, so that in any cycle some mtDNA molecule may replicate more times than others. Hetroplasmy vs Homoplasmy - a cell can have some mitochondria that have a mutation in the mtDNA and some that do not. This is termed heteroplasmy. In the other hand, Homoplasmy refers to a cell that has a uniform collection of mtDNA: either completely normal or completely mutant mtDNA. In conjugation with replicative segregation - in cells where hetroplasmy is present, each daughter cell may receive different proportions of mitochondria carrying normal and mutant mtDNA. Uniparental inheritance - as already said, when organelle genes inherited genes from only one parent, it referred to as uniparental inheritance. Entities that undergoing uniparental inheritance may be expected to be subject to Muller's ratchet - the accumulation of deleterious mutations. Mitochondrial bottleneck concept refers to the term that cause to reduction of polymorphism and specifies population. According to bottleneck concept - mtDNA in an embryo might be drastically different from that of its mother - wide variety of mtDNA genotypes in the maternal pool, which is represented by the bottle. When the embryo mtDNA gene pool is generated, each oocyte receives a small subsampling of mtDNA molecules in differing proportions (which may be completely different proportions that the maternal mtDNA). Consequently, when oocyte get a high degree of mutations, a rare or mutated allele can begin to proportionally dominate in certain oocytes (whereas in others will be negligible). Mitochondrial function diseases: Most mitochondrial proteins are encoded by nuclear genes, thus diseases are mostly follow AR inheritance. Disorders resulting from mutations in these genes tend to breed true (phenotypic traits passes down from organism to its offspring). From the other hand, the disorders resulting from mutations in mtDNA are extremely variable owing to the phenomenon of heteroplasmy. Clinical features of mitochondrial diseases are mainly a combination of neurological signs - encephalopathy, dementia, neuropathy and seizures - together with myopathic signs - hypotonia, weakness, cardiomyopathy with conduction defects. Other symptoms may include deafness, diabetes mellitus, retinal pigmentation and acidosis. Mitochondrial diseases may relate to the following functions of mitochondria: oxidative phosphorylation, Citric acid cycle, Beta-oxidaion, Urea cycle, Apoptosis triggering. Conservative estimate of the incidence of mitochondrial diseases is a 11.5/10000. Threshold hypothesis and Aging- a healthy person at young age has 100% working mitochondria which gradually decreases with age, and at around 100 years of age falls to a level at which they can manifest difficulties. The most sensitive tissue is brain damage, followed by heart, endocrine organs and the kidneys. Due to heteroplasmy characteristic of the mitochondria - different level of power supply found in different sections of tissue (replicative segregation). The mitochondria produce energy for the cell via the respiratory chain, whereby free radicals are released as a waste product of cell respiration. These oxidative oxygen compounds then damage the mtDNA 🡪 accumulation of mtDNA damage during lifetime 🡪 threshold value - damages the cell and eventually leads to apoptosis 🡪 one of the causes of neurodegenerative diseases is an accelerated death of certain types of cell. MELAS disorder - Mitochondrial Encephalomyopathy, Lactic Acidosis, Stroke-like episodes - one of the commonest mitochondrial disorders. Short stature may be a feature. Stroke-like episodes mark out this particular disorder. They may manifest as vomiting, headache or visual disturbance. Diabetes mellitus and a hearing loss may also occur. Kearns-Sayre syndrome is a condition that affects many parts of the body, especially the eyes. The features of Kearns-Sayre syndrome usually appear before age 20, and the condition is diagnosed by a few characteristic signs and symptoms. People with Kearns-Sayre syndrome have progressive external ophthalmoplegia, which is weakness or paralysis of the eye muscles that impairs eye movement and causes drooping eyelids. People with Kearns-Sayre syndrome may also experience muscle weakness in their limbs, deafness, kidney problems, or a deterioration of cognitive functions (dementia). Affected individuals often have short stature. In addition, diabetes mellitus is occasionally seen in people with Kearns-Sayre syndrome. When the muscle cells of affected individuals are stained and viewed under a microscope, these cells usually appear abnormal. The abnormal muscle cells contain an excess of structures called mitochondria and are known as ragged-red fibers Maternally inherited diabetes and deafness Leber's hereditary optic neuropathy - inherited form of vision loss. Vision loss is typically the only symptom of LHON Chronic progressive external ophthalmoplegia (CPEO) occurs together with other changes in Kearns-Sayre syndrome or alone, heredity most often autosomal dominant or autosomal recessive cause: point mutations of nuclear genes, eg POLG , TWNK , RRM2B or SLC25A4 , whose protein products are involved in mtDNA replication and nucleotide metabolism in mitochondria. As a result of their incorrect function, mutations (especially deletions) accumulate in the mtDNA. It can also be caused by a single large mtDNA deletion similar to Kearns-Sayre syndrome (see below) or by a mtDNA point mutation, eg in the MT-TL1 gene encoding leucine tRNA clinical picture: ptosa , ocular myopathy with onset between 18 and 40 years of age, or generalized myopathy, exercise intolerance, dysphagia, ragged red fibers , hearing disorders, etc. Pearson syndrome cause: large deletion in mtDNA in the range of 1000-10000 nucleotides, most often 4997 nt clinical picture: anemia / pancytopenia, pancreatic and hepatic dysfunction in childhood, survivors progress to Kearns-Sayre syndrome Leigh syndrome cause: Mutations in one of more than 75 different genes. About 20% of those affected are mutations in mtDNA, in others mutations in nuclear DNA that encode mitochondrial proteins - most often a disorder of complex I (> 25 known genes in mtDNA and nDNA), then complex IV (eg SURF1 gene ), pyruvate dehydrogenase or coenzyme Q10 formation proteins . The most common mtDNA mutation causing this syndrome is the m.8993T> G substitution in the MT-ATP6 gene , whose protein product is part of ATP synthase mtDNA mutations causing high heteroplasmy Leigh syndrome can cause neurogenic weakness with ataxia and retinitis pigmentosa (NARP) at lower levels of mutated molecules the threshold effect of mtDNA mutation is 31% clinical picture: The first symptoms usually include vomiting, diarrhea and dysphagia. Furthermore, degeneration of the basal ganglia , hyperlactacidemia, muscle weakness, convulsions, progressive motor impairment, deepening psychomotor retardation and irregular breathing, ophthalmoparesis, nystagmus, atrophy of the optician . Manifestations before the first year of life, progressive course, death within a few months or. years usually due to respiratory failure. OMIM # 256000 NARP = neuropathy, ataxia, and retinitis pigmentosa cause: Mutation in the MT-ATP6 gene , whose protein product is part of ATP synthase, most often substitution m.8993T> G. With heteroplasmy higher than ~ 90%, it causes Leigh's syndrome clinical picture: neurodegeneration, muscle weakness, ataxia and retinitis pigmentosa OMIM # 551500 MERRF cause: Most often mtDNA mutations in the MT-TK gene (tRNA for Lys), specifically m.8344G> A, then MT-TL1 , MT-TH or MT-TS1 clinical picture: Myopathy, ataxia, myoclonic epilepsy and ragged red fibers . Furthermore, sensorineural deafness, or atrophy n. opticians or progressive dementia. OMIM # 545000

what is structural genomics? what is the physical mapping of DNA? how is this different than genetic mapping? what are the methods used? what is restriction mapping? (68)

Structural genomics is concerned with sequencing and understanding the content of genomes. Often, one of the early steps in characterizing a genome is to prepare genetic and physical maps of its chromosomes which provide information about the relative locations of genes, molecular markers and chromosome segments. Physical maps are based on the direct analysis of DNA, and they place genes in relation to distances measured in number of base pairs (kilo or mega bases). Physical maps generally have higher resolution and are more accurate than genetic maps. Physical map is analogous to a neighborhood map that shows the location of every house along a street, whereas a genetic map is analogous to a highway map that shows the locations of major towns and cities. A number of techniques exist for creating physical maps, including restriction mapping, which determines the positions of restriction sites on DNA: STS - sequence-tagged site mapping, which locates the positions of short unique sequences of DNA on a chromosome. It is a short (200-500) DNA sequence that has a specific single occurrence in the genome and whose location and base sequence are known. It can be easily detected by PCR (using specific primers) thus they are really useful for constructing physical maps because they serve as landmarks on the physical map of a genome. When the STS loci contain polymorphism - they become even more valuable genetic markers because those loci can be used to distinguish between individuals. FISH - Fluorescent in situ hybridization - by which markers can be visually mapped to locations on chromosomes. It makes use in fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity and used to locate and localize specific DNA sequences on chromosomes. DNA sequencing (Sanger) Somatic Cell Hybridization (Hybridoma) - Mouse cell + Human cell Reminder - Genetic maps, also known as linkage maps, provide a rough approximation of the locations of genes relative to the location of other known genes. In order to construct genetic maps, we need to develop genetic markers with known location on chromosomes - the closer two markers are, the more likely they are to be passed on to the next generation together. The quality of the genetic maps are largely depends upon the number of genetic markers on the map. individual organisms heterozygous at two or more genetic loci are crossed. The frequency of recombination between loci is determined by examining the progeny. If the recombination frequency between two loci is higher than 50%, then the loci are located on different chromosomes or are far apart on the same chromosome (means - there is high chance of recombination to occur, the genes are not linked). If the recombination frequency is less than 50%, the loci are located close together on the same chromosome (said to be "same linkage group"). For linked genes, the rate of recombination is proportional to the physical distance between the loci, measured in "percent recombination" (Centimorgans- cM). In Summary, both genetic and physical maps provide information about the relative positions and distances between genes, molecular markers and chromosome segments. Genetic maps are based on rates of recombination and are measured in percent recombination; one centimorgan - cM - defined as the distance between chromosome positions - loci - for which the expected average number of chromosomal crossover in a single generation is 0.01 - it is equal to recombination frequency of 1% - when distances become higher, the likelihood of a recombination increase. From the other hand, Physical maps are based on the physical distances and are measured in base pairs. Restriction mapping determines the relative position of restriction sites on a piece of DNA. When a piece of DNA is cut with a restriction enzyme and the fragments are separated by gel electrophoresis, the number of restriction sites in the DNA and the distances between them can be determined by the number and position of bands on the gel (EcoRI - 2 restriction points, BamHI - 1), but this information doesn't tell us the order to the precise location of the restriction sites. To map restriction sites, a sample of the DNA is cut with one restriction enzyme, and another sample is cut with a different restriction enzyme. A third sample is cut with both restriction enzymes together. The DNA fragments produced by these restriction digests are then separated by gel electrophoresis, and their sizes are compared. Overlap in size of fragments produced by the digests can be used to position the restriction sites on the original DNA molecules. DNA-sequencing method The most detailed physical maps are based on direct information from DNA sequencing - Sanger method - dideoxy method. The method relies on the use of a special substrate for DNA synthesis. Normally, DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs), which have an OH group on the 3'-carbon atom, connected to the phosphate part of the next nucleotide by phosphor-di-ester bond. In the sanger method, a special nucleotide - dideoxyNTPs (ddNTP) is used as substrate - it is identical with dNTPs except that they lack a 3'-OH group - they are incorporated into the growing DNA chain, but when incorporated - no more nucleotides can be added, because there is no 3'-OH group to form a phosphor-di-ester bond 🡪 ddNTP terminate DNA synthesis. Any DNA fragment to be sequenced must first be amplified by PCR. Copies of the target DNA are isolated and split into four parts - each part is placed in different tube, to which are added: Primer complementary to one end of the target DNA strand All four dNTPs - the normal precursors of DNA synthesis A small amount of one of the four types of ddNTP - which will terminate DNA synthesis as soon as it is incorporated into any growing chain DNA Polymerase

characteristics of tumor growth? what are the 3 main types of tumors? what is a malignant tumor? how do they develop? risk of transmission? difference b/t malignant and benign? (109)

The growth of tumor cells is caused by uncontrolled cell division and impediment to their wear. Tumor cells also have an increased ability to survive. Tumor cells are characterized by the acquisition of resistance to apoptosis (their programmed death). In the case of tumor growth, the regulation of the G1-phase to S-phase transition is mainly affected . A characteristic feature of the transformed cell is the continued division. Their requirements for the presence of hormones and growth factors are reduced(needed for a normal cell) coming from outside. Some transformed cells have the ability to stimulate autocrine, ie after the elimination of a specific substance, it affects the cell. These are mainly specific growth factors. There is also a loss of the ability to stop growth. Aberrant regulation of the cell division cycle is one of the key points of tumor growth. The tumor cell tends to deviate from the physiological mechanism of cell division control. There are basically 3 main types of tumors: sarcomas - from mesenchymal tissue, carcinomas - from epithelial tissue, hematopoietic and lymphoid malignancies - leukemias and lymphomas. A more accurate classification includes the site of origin, tissue type, or, for example, clinical degree or rate of progression. malignant tumor what is it specific phenotypic characteristics nonrandom accumulation of somatic mutations high individual cumulative risk of cancer early onset presence of other cancer types 50% risk of transmission clinical importance carriers indentification early diagnositics prevention therapy hereditary most frequent genetic disease majority caused by accumulation of somatic mutations in 5 to 10% of tumors - germline mutations - hereditary tumors 1850 deaths / year how does it develop caused by genetic imbalance cells imbalance in tissue homeostasis oncogenesis gain of function mutation of one allele tumor suppressors loss of function of both alleles (Knudson's theory) inactivated tumor suppressors, oncogenes formation, loss of DNA repair proteins caused by various mechanisms modification of DNA structure = mutation normally tumor suppressors that are involved in DNA repair processes types of mutations single, indel, CNV (lose or amplify genetic material) somatic changes hypermethylization - loss of heterozygosity incidence of malignant tumors out of 94,000 1/3 of deaths in Europe is caused by cancer newly diagnosed cases in a time period (1 year) basaliomas - 30% do not destroy host hereditary malignant tumors cancer predispostion - tumor supressors (majority) mutant allele is inherited 50% probability to transmit mutant allele sporadic cancer dominant form of cancer development somatic mutations hereditary Neoplasm is an abnormal new growth of cells that usually grow more rapidly than normal cells and will continue to grow if not treated. The term neoplasm can refer to benign or malignant (=cancerous) growth. A tumor is a common used term for a neoplasm - it simply refers to a mass. The terms tumor and cancer are not the same - tumor is not necessarily a cancer. The word tumor simply refers to a mass, even a collection of fluid would meet the definition of a tumor. A cancer, from the other hand, is particularly threatening type of tumor. Tumor can be either benign or malignant: Benign tumors are non-malignant/non-cancerous tumors. It is usually localized, and doesn't spread to other parts of the body. Most of the benign tumors respond well to treatment; however, if left untreated, they can grow large and lead to serious disease because of their size. Malignant tumors are cancerous type of tumors. They are often resistant to treatment, may spread to other parts of the body and sometimes recur even after they were removed. Thus, cancer is another word for a malignant tumor - which also called malignant neoplasm. There are four fundamental features by which benign and malignant tumors can be distinguished, relying on their growth characteristics: Differentiation and anaplasia Differentiation of the tumors refers to the extent to which they resemble their normal ancestors morphologically and functionally; Anaplasia is a condition in which the cells have poor cellular differentiation, losing the morphological characteristics of mature cells. Benign tumors are usually well-differentiated and closely resemble the normal cells from which they divided; Malignant cells have a wide range of differentiation; they may be highly different from their parent cells and said to be anaplastic. They may lose the structural and functional characteristics of normal cells. Rate of growth: Benign tumors - mostly increase in size slowly over the span of months to years; they may increase in size in higher rates under the influence of high levels of hormones (such as high levels of estrogen during pregnancy) or under the influence of high blood supply which will increase the rate of growth. Malignant tumors - there is a wide variation in the rate of growth; generally, the less the cells resemble the normal cells (the more anaplastic) 🡪 the higher the growth rate. Some grow slowly for years and then enters a phase of rapid growth; others grow relatively slowly and steadily. Rapidly growing malignant tumors often contain central areas of ischemic necrosis, due to inadequate blood supply which cannot keep pace with the oxygen need of the expanding mass of cells. Local invasion and metastasis - whereas benign neoplasm remains localized at its site of origin and are usually encapsulated or discretely defined, cancers grow (malignant tumors) and spread from the point of origin to many body parts through the lymph or blood. When the tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, and its cells are similar to those in the original primary tumor. The property of metastasis is the most reliable in identifying between benign and malignant tumors. With venous invasion into the tumor site by angiogenesis, the tumor cells follow the venous flow. Since all portal area drainage flows to the liver, and all caval blood finally flows to the lungs, the liver and the lungs are most frequently involved as secondary metastatic sites. Breast cancer that metastasized to the lungs is called metastatic breast cancer and not long cancer. Cancer hallmarks characterize tumor growth: SARCOMA - self-sufficiency in growth signals, doesn't go through apoptosis, resistance to anti-growth factors, continuously dividing - limitless replicative potential, metastasis and angiogenesis. As the tumor grows, the canceric cells evolve as well - over a period of time, many tumors become more aggressive and acquire greater malignant potential - Tumor Progression. This progression occurs due to the rapid mutation rate in canceric cells and their rapid replication rate. They become less vulnerable to the body's immune system and may be less responsive to therapy over time.

how does the number of protein-coding genes compare to non-protein coding genes? what is made up of the non-protein coding genes? what are the types ? (48)

The human genome consists of 3*10^9 base pairs, subdivided into 23 chromosomes. The genes in the genome that are coding for proteins are only 2% of the total genome - around 25,000 protein-coding genes. All the other genes are non-coding DNA sequences. Repetitive sequences sometimes referred to as Junk DNA. Some of the "junk" DNA shows evolutionary conservation, and might play a role in the regulation of gene expression. Gene contains in its translated area portion within the protein that are coding sequence - exons, and translated, but non-coding regions - introns. In addition, it contains the untranslated region at the 3' end (polyadenylation cap) and the 5' end tail. Enhancers and Silencers are regulatory elements - DNA sequences that influence on gene expression but are not transcripted, i.e. not part of the mRNA. Non coding sequences of DNA: Eukaryote and also human DNA contains large portion of noncoding sequences. More than half the DNA is in unique or Non-repetitive sequence, while at least 30% of the genome consists of repetitive sequences. Noncoding RNAs are functional RNA molecules that are not translated into protein. Examples of noncoding RNA include ribosomal RNA, transfer RNA, microRNA. MicroRNAs are predicted to control the translational activity of approximately 30% of all protein-coding genes. Introns are non-coding sections of a gene, which transcripted into the precursor mRNA sequence, but ultimately removed by RNA splicing during the processing to mature messenger RNA. Many introns appear to be mobile genetic elements. Pseudogenes are DNA sequences, related to known genes, that have lost their protein-coding ability. Pseudogenes arise from retrotransposition (processed) or genomic duplication (non-processed) of functional genes, and become "genomic fossils" that are nonfunctional due to mutations or absence that prevent the transcription of the gene, such as within the gene promoter region. Although not fully functional, pseudogenes may be functional similar to other kinds of non-coding DNA, which can perform regulatory functions. "Processed pseudogene" - Those referred to as Retrotransposed pseudogenes. Retrotransposition, such as in the case of SINEs and LINEs, occurs when a DNA is transcribed into mRNA, and some portion of the mRNA transcript of the gene is spontaneously reverse transcribed back into DNA and inserted into the genome. In this way, the genome becomes much larger, because it contains "non-functional" copies of DNA. Once these pseudogenes are inserted back, they do contain a Poly-A tail and usually have had their introns spliced out. However, because they are derived from an RNA products, processed pseudogenes also lack the upstream promoters of normal genes, which are necessary for the initiation of the transcription of the gene. Thus, they are considered "dead on arrival" - becoming non-functional immediately upon the retro-transposition event. "Non-processed pseudogene" also known as duplicated pseudogenes - a copy of a functional gene may arise as a result of gene duplication event which occurred during homologous recombination (in cell division), which didn't occur correctly and results in mutations that cause the copy to lose the original gene's function. They have all the same characteristics as genes but are non-functional. Unitary pseudogenes - can result from various mutations (such as indels [insertion-deletion] and non-sense mutations) - which prevent a gene from being normally transcribed, and thus the gene may become less functional or even deactivated. The difference between Unitary to Non-processed pseudogene is that the gene wasn't duplicated in this instance. Normally, such a pseudogene probably wouldn't become fixed in a population, but various population effects such as genetic drift or bottleneck can lead to fixation. Telomeres are regions of repetitive DNA at the end of a chromosome, which provide protection from chromosomal deterioration during DNA replication. Cis-regulatory elements are sequences that control the transcription of a nearby gene. Cis-elements may be located in 5' or 3' untranslated regions or within introns. They contain the HRE (hormone-responsive elemnt) specific for hormone, to which activated nuclear receptor binds and enhancer/repress initiation of transcription. Trans-regulatory elements control the transcription of a distant gene. Promoters - is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5' region of the sense strand). Promoters can be about 100-1000 base pairs long. The other types of non-coding DNA are DNA sequences with high copy numbers, called repetitive sequences. If the copies of a sequence lie adjacent to each other in a block, or an array, we are speaking about tandem repeats, while if the repetitive sequences dispersed throughout the genome as single units flanked by unique sequence are interspersed repeats. Tandem Repeats - found in a block along the same chromosome: Tandem repeats are made of successive identical repeat units. They vary in length of repeat unit (e.g. dinucleotide repeat - ACACACACAC) as well as number of the repeat units. The largest repeats, which tend to be composed from large repeat units are called satellites. Satellite DNA - 10-15% of the repetitive DNA sequences in human genome; range between 100 to 500 base pairs for each repeat unit. They consist of large series of tandemly repeated non-coding DNA sequences, and are clustered around the centromere and telomers of the chromosome. These sequences can be separated from the whole genes with density-gradient centrifugation - thus named "satellites". Mini satellite DNA - are shorter tandem repeats, in the range of 50-100 base pairs for each repeat unit. Found in the sub-telomeric regions (can be in the centromere region as well) of the chromosomes. They are often highly polymorphic as to the number of repeat units in a repeat (the length of the region) and used as genetic markers. Thus, they are referred to as VNTR - variable number of tandem repeats - useful for DNA Fingerprint technique (each repeat unit composed of 50 bases, and the polymorphism is regarding how many times it is repeated). Certain mini satellites are hypothesized to have regulatory functions. Usage of mini-satellites in DNA fingerprinting - the pattern and length of the VNTR's is highly different between individuals - i.e. highly polymorphic. Only identical twins show an indistinguishable pattern. Due to these facts, detection of a number of minisatellite polymorphisms was one of the first methods of DNA matching in forensic. Obtaining of DNA sample from a crime area and matching the minisatellite of the sample with possible suspected criminals or with database. Microsatellites - most common form of repetitive sequences. Sometimes referred to as short tandem repeats (STRs) are short sequences of DNA, normally of length 2-5 base pairs, that are repeated numerous times in a head-tail manner, i.e. the 16 bp sequence of "gatagatagatagata" would represent 4 head-tail copies of the tetramer "gata". The polymorphisms in STRs are due to the different number of copies of the repeat element that can occur in a population of individuals. They represent 0.5% of the genome and are highly polymorphic thus very often used as genetic markers. Trinucleotide Expansion: Unstable transmission of some repetitive sequences, called expansion. E.g. expansion of CAG trinucleotide in a coding sequence of the gene for Huntington disease leads to a production of an abnormal protein Huntingtin, and causes clinical manifestation of Huntington disease. Huntington disease is a progressive neurodegenerative disorder with late and variable age at onset causing to dementia and progressive disorders of movement. It is AD trait and caused by CAG expansion in the HD gene - 99% of normal genes contain less than 30 CAG triplets, but mutated genes in patients with HD contain always 40 or more of the CAG repeats. Interspersed repeats - transposable elements: Depending on their length, moderately repetitive sequences are classified as LINEs and SINEs. Both types appear to be retrotransposons - arrive through an RNA intermediate by the action of reverse transcriptase that transcribed an RNA template into DNA. Interspersed repeats are repeated DNA sequences located at dispersed regions in a genome. They are also known as mobile elements or transposable elements. Transposable elements are segments of DNA that can move from one location in the genome to a target sequence in another. After many generations, such sequence (the repeat unit) could spread over various regions. LINEs stands for Long Interspersed Nuclear Elements - compose approximately 21% of the human genome. 6000-7000 base pairs long, interspersed in many copies in different regions through the genome. SINEs stands for short interspersed nuclear elements - are typically less than 500bp long and have no protein coding potential. The main SINE family in humans if formed by Alu elements; the amount of Alu elements in the human genome account for about 11% of its mass. Microsatellite repeat sequences, which consist of 1-5 base pairs (ACACACACAC) and repeated up to 5 times, can be both dispersed (interspersed) as well as grouped in tandem repeats. These microsatellite sequences most commonly are found as dinucleotide repeats of AC on one strand, and TG on the opposite strand. The AC repeat sequences occur at 50,000-100,000 locations in the genome. At any locus, the number of these repeats may vary on the two chromosomes (e.g. chromosome 1 that arrive from dad can have 30 repeats, while its homologue that arrive from the mother has only 25) - thus, providing heterozygosity of the number of copies of a particular microsatellite number in an individual. This is a heritable trait, thus, such repeats are useful in constructing genetic linkage maps: most genes are associated with one or more microsatellite markers, so the relative position of genes on chromosomes can be assessed, as can the association of a gene with a disease. Large number of family members can be screened for a certain microsatellite polymorphism. Association of a specific polymorphism with a gene in affected family members, and the lack of this association in unaffected members - may be the first clue about the genetic basis of a disease.

genetic control of the immune response? what cells have a major role? (75)

The immune response is how your body recognizes and defends itself against bacteria, viruses, and substances that appear foreign and harmful. The immune system protects the body from possibly harmful substances by recognizing and responding to antigens. Antigens are substances (usually proteins) on the surface of cells, viruses, fungi, or bacteria. Nonliving substances such as toxins, chemicals, drugs, and foreign particles (such as a splinter) can also be antigens. The immune system recognizes and destroys, or tries to destroy, substances that contain antigens. Your body's cells have proteins that are antigens. These include a group of antigens called HLA antigens. Your immune system learns to see these antigens as normal and usually does not react against them. Control of Immune response by MHC MHCs are cluster of genes found on chromosome 6, and their product is an integral membrane protein found on the surface of all body's cells. They serve as indicators for T-cells to distinguish between "self" and "non-self" peptides. The human MHC is also called the HLA (human leukocyte antigen) complex. MHC molecules can be classified into two types: Class I - found on the surface of every nucleated cell. Each MHC molecule on the cell surface displays a molecular fraction of a protein - called an epitope - which gives the body's immunity the ability to recognize it as "self" cell - thus do not generate any immune reaction. In case of viral infection or tumor cells, different peptides are joined to the proteins generated from the MHC genes, and thus they aren't recognized as "self" anymore - CD8+ T-cells (cytotoxic) elicit immune response. The body's T-Cells learn to recognize self from non-self during their maturation in the thymus - in a "selection mechanism" of the thymus: they are exposed to self-MHC molecules that are normally found in the body, and in case they trigger an immune response against them - they destroyed; thus, prevent auto-immune responses. MHC II molecules are a class of MHC normally found only on antigen-presenting-cells such as dendritic, mononuclear phagocytes (macrophages), some endothelial cells and B-lymphocytes. The antigens presented by class II peptides are derived from extracellular proteins (not cytosolic as in MHC I). Loading oh MHC class II molecule occurs by phagocytosis; extracellular proteins are endocytosed, digested in lysosomes, and the resulting epitope peptide fragments are loaded onto MHC class II molecules prior to their migration to the cell surface. Because class II MHC is loaded with extracellular proteins, it is mainly concerned with presentation of extracellular pathogens (e.g. bacteria that might be infecting a wound or the blood). Class II molecules interact mainly with immune cells, like T-helper (CD4) which then triggers an appropriate immune response. Control of immune response by antibody (question 76) Control through action of Cytokines Cytokines are polypeptide products of many types of cells, principally activated lymphocytes and macrophages. They function as mediators of inflammation and immune responses and are important in cell signaling. Their release has an effect on the behavior of cells around them. Cytokines act through receptors, modulate the balance between humoral and cell-based immune response and regulate the maturation, growth and responsiveness of particular cell populations. Cytokines can be classified to several groups according to their biological activities and function: Cytokines which affect cells in the innate immune response Cytokines which affect cells of the adaptive immune Cytokines which stimulate hematopoiesis Each cytokine has a matching cell-surface receptor; when binding to this receptor, subsequent cascades of intracellular signaling alter the cell functions. This may include the upregulation or downregulation of several genes and their transcription factors, resulting in: Production of other cytokines An increase in the number of surface receptors for other molecules Suppression of their own effect Genetic control through action of Growth Factors: During immune response, or after its termination, there is a need for increased proliferation of leukocytes and monocytes. The production of WBC's is regulated and maintained by hematopoetic growth factors, cause a cascade within the progenitor WBCs which activates specific transcription factors and DNA-binding molecules that determine the specific differentiation pathway for the cell.

what is Sanger sequencing? how it is done? (54)

The most widely used approach for DNA sequence analysis is Sanger sequencing. This method, takes advantage of certain chemical analogues of the four nucleotides known as dideoxy nucleotides - ddA, ddC, ddG, ddT - when incorporated into a growing strand of DNA, they do not allow the enzyme DNA polymerase to attach the next base complementary to the original template, and therefore terminate the growing DNA chain.

transcription in prokaryotes? steps? major enzymes? structure of RNA pol? differences b/t eukaryotic transcription? (88)

The structure of RNA polymerase (the signals that control transcription) and the RNA modification is different in prokaryotes from eukaryotes. RNA Polymerase is an enzyme that recognizes a nucleotide sequence - the promoter region - at the beginning of a length of DNA that is to be transcribed. It next makes a complementary RNA copy of the DNA template strand, and recognizes the end of the DNA sequence when reach the termination region. RNA is synthesized from 5'-end to 3'-end, antiparallel to its DNA template strand. Same principles of DNA to RNA coupling as occur in eukaryotes (A turns to U, T turns to A, G to C, C to G). The RNA, then, is complementary to the DNA template (non-coding, antisense, minus, template) strand and identical to the coding (coding, sense, plus, non-template) strand, with U replacing the T. Important to note, that regions of both strands can serve as templates for transcription. However, for a given gene, only one of the two strands can be the template. The strand to be used is determined by the location of the promoter for that gene. Transcription by RNA polymerase involves a core enzyme (five enzymes subunits - alpha and omega - required for enzyme assembly, betta-tag for template binding, and betta for 5'🡪3' RNA polymerase activity) and several proteins. Holoenzyme - "sigma factor" + the core enzyme complex - enables RNA polymerase to recognize promoter regions on the DNA. There is no need of special transcription factors in order to recognize the promoter and initiate transcription. Steps: Transcription unit extends from the promoter to the termination region; initial product of transcription is termed the primary transcript. Initiation - recognition and binding of the RNA polymerase holoenzyme to a region the promoter region of the DNA (which isn't transcribed). Promoter contains characteristic consensus sequences: -35 sequence - 5'-TTGACA-3' - 35 bases to the left of the transcription start site, the initial point for the holoenzyme. Prinbow box - similar to eukaryotic "TATA" box - the holoenzyme moves and covers a second consensus sequence - 5'-TATAAT-3' - centered at about -10, where DNA unwinding initiates. Unwinding converts the close complex to open complex - formation of transcription bubble. Elongation - once the promoter region has been recognized by the holoenzyme, unwinding of the DNA continues. Unwinding generates supercoils, relieved by DNA topoisomerases. Elongation phase begins when the transcript exceeds ten nucleotides in length - sigma is then released, and the core enzyme is able to leave the promoter. Like DNA polymerase, RNA polymerase uses nucleoside triphosphates as substrates and transcription is always in the 5'🡪 3' direction. RNA polymerase doesn't require a primer. Termination - termination signal is reached can be either spontaneous (intrinsic) or dependent upon the participation of rho factor. Spontaneous - sequence in the DNA template generates a sequence in the RNA that is self-complementary, allows the RNA to fold back on itself forming "hair pin". Rho-dependent termination - participation of additional protein - rho - binds a C-rich "rho recognition site" near the 5' end of the new RNA, and using ATPase activity, moves along the RNA until it reaches the paused RNA polymerase at the termination site, separates the RNA-DNA 🡪 releasing the RNA. Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis.

origin and evolution of species? what are the theories? what is the prebiotic chemical evolution theory? theory of natural selection? what are the components? what are some other types of speciation? (121)

There are two old theories regarding the emergence of life on Earth: The Creationist theory - Creationism - a religious belief, according which the universe and life originated from divine creation - the universe, earth, life and human being created by god - based on reading of religious texts (e.g. The Bible, the Quran). This includes a literalist interpretation of the genesis creation narrative and the rejection of the scientist theory of evolution. The Panspermia theory - according which, the origin of life isn't from earth, rather - from space - life may cause its distribution in the universe. The life exists throughout the universe, distributed by meteoroids, asteroids etc. in the form on transporting microorganisms (i.e bacteria) through space to the earth 🡪 if met with ideal conditions on a new planet's surfaces, the organisms become active and the process of evolution begins. Today's theories based on the central dogma: Stage I - origin of biological monomers (e.g. amino acids); we now know that these basic building blocks are common throughout space and early Earth would have had them all. Stage II - Formation of larger polymers Stage III - evolution from molecules 🡪 cells Prebiotic chemical evolution - In 1920s, Haldane and Oparin proposed concepts on which modern scientific ideas about the origin of life are based - some kinds of molecules could persist in the lifeless environment of early earth better than others, and would therefore become more common over time. This gradual selection of molecules was termed "prebiotic chemical evolution". They proposed that the molecules became increasingly more complex and eventually gave rise to life. Prebiotic chemical evolution occurred in 3 stages: Prebiotic synthesis and accumulation of small organic molecules formed a pool of building blocks. How does the synthesis occurred? - in 1953 another two smart people simulate the first stage of prebiotic evolution in lab - they noted that the atmosphere of early earth probably contained methane, ammonia, hydrogen and water vapor - but no oxygen. They simulated early Earth's atmosphere by mixing the gases and adding an electrical charge to simulate lightning. Simple organic molecules appeared after a few days. Additional organic molecules arrived from space, when meteorites crashed into the earth's surface; analysis of present-day meteorites recovered from impact craters on Earth has revealed that some meteorites contain relatively high concentrations of amino acids and other simple organic molecules. Small organic molecules combined to form larger molecules Progressively more complex molecules eventually gave rise to living organisms The processes occurred under natural selection. Species Evolution - question 119 Species is the basic unit of biological classification, and defined as a set of individuals who are able to reproduce fertile offspring. Speciation is formed by RIM's - Reproductive isolating mechanisms - occurs when a population becomes reproductively isolated from other populations of the species. The gene pools of the two separated populations begin to diverge in genetic composition. When a population is sufficiently different from its ancestral species and there is no genetic exchange possible between them 🡪 speciation occurs. We distinguish between external (geographical) and internal mechanisms (barriers) that lead to speciation: Internal mechanisms includes prezygotic barriers - temporal, ecological, behavioral, physical-anatomical and gametic isolation; and postzygotic barriers - zygote mortality and non-viability of offspring (offspring dies before able to reproduce), hybrid sterility (horse + donkey = mule, which is usually infertile). External mechanisms include - Allopetric, Parapetric, Peripetric and Sympatric (polymorphism 🡪 disruption selection 🡪 speciation) speciation. The theory of evolution by natural selection (formulated by Darwin's in his book - Origin of species 1859), is the process by which organisms change over time as a result of changes in heritable physical or behavioral traits. Changes that allow an organism to better adapt to its environment will help it survive and have more offspring. Darwin's process of natural selection has four components: Variation. Organisms (within populations) exhibit individual variation in appearance and behavior. These variations may involve body size, hair color, facial markings, voice properties, or number of offspring. On the other hand, some traits show little to no variation among individuals—for example, number of eyes in vertebrates. Inheritance. Some traits are consistently passed on from parent to offspring. Such traits are heritable, whereas other traits are strongly influenced by environmental conditions and show weak heritability. High rate of population growth. Most populations have more offspring each year than local resources can support leading to a struggle for resources. Each generation experiences substantial mortality. Differential survival and reproduction. Individuals possessing traits well suited for the struggle for local resources will contribute more offspring to the next generation. Due to the selection, together with genetic drift, the evolution and development of living systems occurred. The goal of evolution is producing a large gene variability which in any natural disaster or changing conditions - selection will act only against part of the population. Carriers of advantageous mutations survive and their positive mutations will pass on to next generations - Natural selection. Other important types of specializations are: Stasipatric speciation , which arises from a sudden big change, causing a reproductive barrier. Since then, the species cannot interbreed and continue to evolve separately. An example is the formation of a reproductive barrier by Robertson translocation . Extinction speciation , which arises from the extinction of a part of a large population and the subsequent separation of a marginal population and the interruption of gene flow to it. Further speciation division _ Speciation can be further divided into: cladogenesis - splitting of individual species gradually from one ancestor, anagenesis (phylogenetic speciation) - the emergence of changes in one non-cleaving species, syngenesis - the emergence of one species from two originally different ones. We further divide it into:symbiogenesis - two species that originally lived in the form of symbiosis, which gives rise to a multicellular organism (for example, the formation of eukaryotes by the inclusion of originally prokaryotic mitochondria ),interspecific hybridization - an individual is created by crossing two very related species (for example, different species of jumpers). This individual ( klepton ) can further reproduce only with members of the parental generation.

what are direct diagnostics of hereditary diseases? what are some advantages? what are the methods used testing deviations from standard sequences? what are some of their advantages? (66)

This method of diagnostic search for a specific nucleotide sequence of DNA causing the disease, therefore, it is essential to know the exact sequence of DNA segment that you're looking for and its localization on the chromosome. By using probes - oligonucleotides and anneal with the target sequence - you can reveal the disease. Advantage of this method is that no family history is required to detect affected person SSCP Method - Single Strand Conformation Polymorphism Analysis. It is defined as a conformational difference of single-stranded nucleotide sequence of identical length that induced as a consequence of difference in the sequence. A single nucleotide change in a particular sequence as seen in dsDNA cannot be distinguished by electrophoresis because the physical properties of the double strands are almost identical for both alleles. But, after amplification of DNA by PCR, the amplified region is going through denaturation to ssDNA and applied to non-denaturing polyacrylamide gel. ssDNA undergoes a 3D folding (similar to tRNA) and may assume a unique conformational state based on its DNA sequence. Electrophoresis is apply, and the mobility of ssDNA in course of electrophoresis depends then on particular conformation - the difference in shape between two ssDNA strands with different sequences can cause them to migrate differently on an electrophoresis gel, even though the number of nucleotides is the same. By this mean, SSCP used to be a way to discover new DNA polymorphisms apart from DNA sequencing. Even a small change in sequence of nucleotides may lead to completely different spatial structure of the folded ssDNA 🡪 different mobility in electrophoresis 🡪 different location of the band representing the DNA sequence. SSCP, thus, is able to distinguish even a single nucleotide mutation TGGE and DGGE - temperature gradient electrophoresis and Denaturing gradient gel electrophoresis are forms of electrophoresis which use either a temperature or chemical gradient to denature the sample as it moves across an acrylamide gel. DGGE - electrophoresis with polyacrylamide gel with continuously growing concentration of denaturing agents is used in this method. The technique is based on the fact that the less H-bonds DNA contains 🡪 the more susceptible is it to denaturation (denature quicker). Both strands segregate each from other much easier in regions rich in A-T pairs while C-G rich regions are more stable (contains 3 H-bonds, A-T only 2). The velocity of movement of the examined DNA in the electric field corresponds to its molecular weight until the strands start to separate. Then, the denatured single strands are much slower and finally stop in the gel. The more susceptible the examined DNA to denaturing - means it will denature quicker - the closer to the start it will stop. This method makes it possible to discern differences in DNA sequences or mutations of various genes. Further analysis is accomplished through PCR amplification. TGGE is a similar technique, based on the same principle - gradually increasing temperature of gel is used, instead of increased concentration of denaturing agents, in order to denature dsDNA. heteroduplex analysis (see next card) PTT - Protein Truncation test - detects mutations at the protein level that lead to premature translation termination (truncated protein - usually inactive). The appropriate genomic DNA or mRNA is isolated, amplified by PCR, and used as a template for in vitro transcription and translation. The size of the resulting protein is compared with that of a wild type protein by means of polyacrylamide gel electrophoresis. Sanger Sequencing - based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. In order to perform the sequencing there is a need in ssDNA template, DNA primer, DNA polymerase, normal deoxynuelosidetriphosphates (dNTPs - A, T, C, G) and modified di-deoxyNTPs (which terminate DNA strand elongation). These chain-terminating nucleotides lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated (which is radioactively or fluorescently labeled for detection). The DNA sample is divided into four separate sequencing reactions, containing all four of the standard dNTPs + DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (in low amount, allowing for enough fragments to be produced while still transcribing the complete sequence). Each mixture will produce fragments that include different lengths of fragments, each terminates with the specific ddNTPs added to this reaction.

what are the transportation rules? important exceptions? how do you prevent rejection? transplantations rules for hematopoeitic cells? (79)

Tissue transplanted between genetically identical members of the same strain is permanently healed . Syngeneic transplantation is successful. Tissue transplanted between members of two inbred strains that differ in alleles of one or more histocompatibility systems is destroyed by the recipient . Allogeneic transplantation is unsuccessful. An individual of strain A (aa) responds with an immune response against the antigenic product of strain B (bb) and vice versa. Tissue from individuals of both parental strains transplanted into members of the F1 hybrid generation is permanently adhered , while tissue from F1 hybrid individuals is abraded by members of both parental strains . Due to the codominance of histocompatibility genes, the alloantigenic equipment of the F1 hybrid includes antigenic products of different alleles of both parental strains. The F1 hybrid is thus genetically reactive to the parent antigens, but on the contrary, its tissue elicits an immune response in both parent strains. The tissue of members of the F2 hybrid generation and all subsequent generations is permanently added by F1 hybrid recipients . This is because in future generations only different combinations of alloantigens of the parental strains (unless a locus is mutated) can appear, all of which are contained in the F1 hybrid genotype . The F1 hybrid of two inbred strains is a universal recipient of grafts of both parental strains and all types of offspring of their crosses. The tissue of the individuals of both parental strains is abraded by some members of the F2 hybrid generation and partly survives . In the example where parental inbred strains differ in alleles of a single histocompatibility locus, we see that 75% of parental grafts survive on F2 hybrid recipients exceptions: failed skin transplantation from males to females of the same inbred strain - caused by expression of a male transplant antigen encoded by the H-locus on the Y chromosome (anti-HY immune response) unsuccessful skin transplantation of a male parental strain to an F1 female - response against HY antigen failed skin transplantation of both sexes of both sexes of the same parental strain from which the father came, to F1 males - response against HX antigen of strain A failed skin transplantation of both sexes of both sexes of the same parental strain from which the father came, to F1 males - response against HX antigen of strain B (see presentation) prevention The selection of appropriate donors and recipients within the histocompatibility system is a matter of course. The problem remains the fact that in serological examinations we are able to assess only the main antigens (MHC, HLA). However, there are also minor antigens. These may elicit immune responses in seemingly unsuitable patients. The only exceptions in this regard are identical twins. Immunosuppressive treatment coverage is also a matter of course. This is especially important as a prevention of the host's response to the graft. The main thing is the suppression of T C -cells. At the same time, however, we must take into account the increased risk of infection of these patients. We verify the suitability of the donor and recipient both by serological tests and by PCR (polymerase chain reaction). Serological testing of major antigens takes only a few hours. Transplantation of Hematopoetic stem cells - has different transplantation rules: Transplantation occurs in cases of hematopoetic malignancies (bone marrow cancers, such as leukemia) or nonhematopoetic malignancies - such as aplastic anemias, immunodeficiency and severe thalassemias. The same rules of tissue transplantation apply in this case as well - the necessity to match donor with HLA genes similar as possible to HLA of the host. Immunodepression, such as chemotherapy or irradiation which destroy the malignant cells is applied. The differences in this form of transplantation occurs due to graft versus host cases: Unlike in normal tissue transplant, where it is only necessary to ensure that the acceptor of the transplant possesses all of the donor's antigen (AA can donate to AB; AB can't donate to AA because it contains antigens that are lack in AA blood type) - in hematopoetic stem cells transplantation it is crucial to make sure that the donor also has all the antigens that present in the host blood. Why? - The reason is that T-cells presents in the donor graft perceive the recipient's body tissues as foreign and react against it. Thus, donor with AA blood - its T-cells will react against the AB recipient blood (attack of the GRAFT to the HOST): If AA donates to AB - AB won't reject AA, but AA hematopoetic cells of the donor won't recognize antigen B and will cause immune reaction. These reactions can be prevented by HLA complete matching or by depleting the donor T-cells before marrow transplant.

what is transcription? how is transcription regulated in eukaryotes? what are the roles of transcription factors? what are their structures? how are they classified? (49)

Transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby orchestrating gene activity. A single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus mount a response. Some examples of this include producing the mRNA that encode enzymes to adapt to a change in a food source or producing the gene products involved in cell cycle specific activities. The regulation of transcription is a vital process in all living organisms. It is orchestrated by transcription factors and other proteins working in concert to finely tune the amount of RNA being produced through a variety of mechanisms. Eukaryotes have three RNA polymerases, known as Pol I, Pol II, and Pol III. Each polymerase has specific targets and activities, and is regulated by independent mechanisms. There are a number of additional mechanisms through which polymerase activity can be controlled. These mechanisms can be generally grouped into three main areas: Control over polymerase access to the gene. This is perhaps the broadest of the three control mechanisms. This includes the functions of histone remodeling enzymes, transcription factors, enhancers and repressors, and many other complexes Productive elongation of the RNA transcript. Once polymerase is bound to a promoter, it requires another set of factors to allow it to escape the promoter complex and begin successfully transcribing RNA. Termination of the polymerase. A number of factors which have been found to control how and when termination occurs, which will dictate the fate of the RNA transcript. All three of these systems work in concert to integrate signals from the cell and change the transcriptional program accordingly. While in prokaryotic systems the basal transcription state can be thought of as nonrestrictive, eukaryotes have a restrictive basal state which requires the recruitment of other factors in order to generate RNA transcripts. This difference is largely due to the compaction of the eukaryotic genome by winding DNA around histones to form higher order structures. This compaction makes the gene promoter inaccessible without the assistance of other factors in the nucleus, and thus chromatin structure is a common site of regulation. The general transcription factors (GTFs) are a set of factors in eukaryotes that are required for all transcription events. These factors are responsible for stabilizing binding interactions and opening the DNA helix to allow the RNA polymerase to access the template, but generally lack specificity for different promoter sites. A large part of gene regulation occurs through transcription factors that either recruit or inhibit the binding of the general transcription machinery and/or the polymerase. This can be accomplished through close interactions with core promoter elements, or through the long distance enhancer elements. Once a polymerase is successfully bound to a DNA template, it often requires the assistance of other proteins in order to leave the stable promoter complex and begin elongating the nascent RNA strand. This process is called promoter escape, and is another step at which regulatory elements can act to accelerate or slow the transcription process. Similarly, protein and nucleic acid factors can associate with the elongation complex and modulate the rate at which the polymerase moves along the DNA template. Regulation at the level of chromatin state: In eukaryotes, genomic DNA is highly compacted in order to be able to fit it into the nucleus. This is accomplished by winding the DNA around protein octamers called histones, which has consequences for the physical accessibility of parts of the genome at any given time. Significant portions are silenced through histone modifications, and thus are inaccessible to the polymerases or their cofactors. The highest level of transcription regulation occurs through the rearrangement of histones in order to expose or sequester genes. These are achieved by epigenetics changes (see epigenetics). Regulation through transcription factors and enhancers: Transcription factors are proteins that bind to specific DNA sequences in order to regulate the expression of a given gene. The power of transcription factors resides in their ability to activate and/or repress wide repertoires of downstream target genes. Transcription factors function through a wide variety of mechanisms. Often they are at the end of a signal transduction pathway that functions to change something about the factor, like its subcellular localization or its activity. Post-translational modifications to transcription factors located in the cytosol can cause them to translocate to the nucleus where they can interact with their corresponding enhancers. Enhancers Enhancers, or cis-regulatory elements (CRE), are non-coding DNA sequences containing multiple activator and repressor binding sites. Enhancers range from 200-1000 base pairs in length and can be either proximal, 5' upstream to the promoter or within the first intron of the regulated gene, or distal, in introns of neighboring genes or intergenic regions far away from the locus. Through DNA looping, active enhancers contact the promoter dependently of the core DNA binding motif promoter specificity. Promoter-enhancer dichotomy provides the basis for the functional interaction between transcription factors and transcriptional core machinery to trigger RNA Pol II escape from the promoter. operator - a regulatory region on DNA that lies between the promoter and the start of transcription. The binding of an active repressor to the operator blocks the transcription and thus the expression of structural genes. silencer - a DNA sequence that binds inhibitory factors promoter - part of DNA near the gene (RNA sequences of bases that have specific binding points for RNA polymerase), which is involved in the regulation of its expression (eg TATA box, CAT box) promoter strength - the ability of the promoter to initiate transcription lies in the affinity of the promoter region for RNA polymerase. The frequency of transcription of the adjacent gene depends on the strength of the promoter. Strong promoters have sequences in the region of -35 and -10 (Pribnow box) identical to the conventional sequence, the more they differ from the conventional sequence, the weaker the promoter. Regulation of basal transcription GTFs (general transcription factors) - necessary for the course of transcription many of them do not bind to DNA, but are part of a preinitiation complex that reacts directly with RNA polymerase II most common: TFIIA, TFIIB, TFIID (includes a subunit called TATA binding protein (TBP) - binds specifically to the TATA box sequence), TFIIE, TFIIF and TFIIH Cell development they regulate cell differentiation and determination based on signals The TF Hox family is important for proper body composition TF encoded SRY (Sex-determining region of Y) - determining the sex of a person Response to extracellular signals part of the signaling cascade (activation x suppression) eg estrogen signaling : TF is part of the estrogen receptor, which after activation travels to the nucleus, where it regulates the transcription of certain genes The answer to the external environment TFs also regulate signaling cascades of exogenous origin Heat shock factor (HSF) - activates genes that allow survival at higher temperatures Hypoxia inducible factor (HIF) - survival in an oxygen deficient environment Cell cycle control hl. TFs, which are oncogenes (eg myc) and tumor suppressors (eg p53 ) - role in cell growth and apoptosis Transcription factors consist of the following domains: The DNA-binding domain (DNA-binding domain, the DBD) binds to specific DNA sequences, so-called responsive elements (enhancer, promoter ) adjacent to regulated genes, examples: lambda repressor-like srf-like (serum response factor) GCC box Zn2 / Cys6 basic helix-loop-helix homeodomain proteins - role in the regulation of development, binds to homeobox DNA sequences that encode other TFs (short nucleotide sequence , identical in different genes and organisms, role in the expression of relevant genes) multi-domain Cys2His2 zinc fingers basic leucine zipper (bZIP) Trans-activating domain (TAD) contains binding sites (so-called AFs - activation functions ) for other proteins that act as co-regulators of transcription The ligand binding domain (ligand binding Domanin, signal sensing domain, SSD) may not be present responds to external signals and transmits them to the rest of the transcription complex - the result of up / down regulation of gene expression Transcription factor binding sites (responsive elements) Transcription factors usually react with their binding sites with the help of hydrogen bonds and Van der Waals forces . Due to the nature of these chemical interactions, most TFs bind to DNA specifically . However, not all binding site bases need to actually react with TF. In addition, some of these interactions may be weaker than others. TFs are able to bind several related sequences, each with a different strength (e.g., although the major binding region for the TATA binding protein (TBP) sequence is TATAAAA, TBP can also bind to TATATAT or TATATAA). However, binding to all of these compatible sequences is unlikely, as DNA accessibility and a number of cofactors also interact. So it's still hard to guess where TF will end up. Mechanical classification Transcription factors pre-initiation complex (general transcription factors) - TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH - are ubiquitous and react with a promoter (often a TATA box) structural genes important in the development of vertebrates and invertebrates Upstream transcription factors (UTF) - upstream - towards the 5´ part, proteins that bind to the regulatory part of the RNA polymerase I promoter at position -110 to -180, the presence is not necessary to initiate transcription, but multiplies its efficiency (it can also repressive) Inducible transcription factors - same as UTF, but need to be activated or inhibited Functional classification Constitutive - present (and active) in the cell at all times - general transcription factors, Sp1, NF1, CCAAT Conditionally active - their activation required Developmental (cell-specific) TF - expression is strictly controlled, but after expression they do not require further activation - GATA, HNF, PIT-1, MyoD, Myf5, Hox, Winged Helix Signal-dependent TF - need an external signal to activate extracellular ligand-dependent nuclear receptors intracellular ligand-dependent - activated by small ic. molecules - SREBP, p53, single nuclear receptor cell membrane receptor-dependent cascades with a second messenger causing TF phosphorylation resident nuclear factors - are in the nucleus regardless of the activation state - CREB, AP-1, Mef2 latent cytoplasmic factors - in the cytoplasm in an inactive form, after activation translocated to the nucleus - STAT, R-SMAD, NF-kB, Notch, TUBBY, NFAT

types of twins and why are they important in genetic studies? what does concordance refer to and why is this relevant to twin studies? (15)

Twin studies reveal the importance of environmental and genetic influences for traits, phenotypes, and disorders. Twin research is considered a key tool in behavioral genetics and in content fields, from biology to psychology. Twins are a valuable source for observation because they allow the study of environmental influence and varying genetic makeup. Twin studies reveal the absolute and relative importance of environmental and genetic influences on individuals in a sample. Twins can be monozygotic, identical twins - meaning that they can develop from just one zygote (single egg-single sperm) that will then split and form two embryos - have the same genetic makeup (coefficient of relationship = 1), must be the same sex. Their epigenetic equipment, such as DNA methylation, is not completely identical - and these differences increase throughout life. (exception to being identical - rare mutations). Frequency of monozygotic twins in most ethnic groups is about 4:1000, little tendency to run in families. Twins can be dizygotic - which have the same coefficient of relationship that normal siblings share (r=0.5); meaning that they develop from two different eggs, each are fertilized by separate sperm. They can be of the same sex or of different sexes; the only difference between dizygotic twins and other siblings is that dizygotic twins are the same age and shared the same uterine environment. Frequency of dizygotic is widely vary in populations; in America - 7:1000 births; Nigeria - 40:1000. The rate of twinning also varies with maternal age, and dizygotic twinning tends to run in families. "Identical" or monozygotic twins share nearly 100% of their genes, which means that most differences between the twins (such as height, susceptibility to boredom, intelligence, depression, etc.) is due to experiences (e.g. external factors) that one twin has but not the other twin. Incidence of MZ twins - 1/300 "Fraternal" or dizygotic (DZ) twins share only about 50% of their genes, the same as any other sibling. Twins also share many aspects of their environment (e.g., uterine environment, parenting style, education, wealth, culture, community) because they are born into the same family. The presence of a given genetic trait in only one member of a pair of identical twins (called discordance) provides a powerful window into environmental effects. concordance If both members of a twin pair have a trait - the twins are said to be concordant; if only one member of the pair has the trait, the twins are said to be discordant. Concordance, then, is the percentage of twin pairs that are concordant for a trait. Because identical twins have 100% of their genes in common and dizygotic twins have on average only 50% in common, genetically influenced traits should exhibit higher concordance in monozygotic twins. Important to conclude that the hallmark of genetic influence on a particular trait is higher concordance in monozygotic twins compared with dizygotic twins - high concordance in monozygotic twins by itself doesn't signal a genetic influence - because twins usually share the same environment (home, friends, school) so high concordance may be related to common environment (not just common genes). Thus, if the high concordance is due to environmental factors - dizygotic twins should have high concordance as well. Any discordance among monozygotic twins must be due to environmental factors, because they are genetically identical.

what is fitness? how is this affected by selection?

ability to survive and reproduce w = 1 - s s = selection coefficient (range from 0 to 1)

what are the genetic aspects of aging and death? what are the causes? (97)

aging is the process of becoming older; it is time-related deterioration of the physiological functions necessary for survival and fertility. In humans, ageing represents the accumulation of changes in a human being over time - encompassing physical, psychological and social change. It is important to know the changes that accompany old age it order to offer adequate medical preventive care for the elderly. The maximum life span is a characteristic of the species - it is the maximum number of years a member of that species has been known to survive (e.g. human -121). However, the life expectancy in reality is not characteristic of species - rather it is characteristic of populations. Each population has different life expectancy due to genetic, environmental and other factors. Basically, ageing is an individual process. Thus, life expectancy is usually defined as the age at which half the population still survives. It is a statistical measure of the average time an organism in population is expected to live, based on their sex and other demographic factors. Till recently, people didn't die as a result of ageing - but as a consequence of infectious diseases and parasites. Only recently, people began exhibiting the phenotypic features for ageing - Reduce in Immune system functions Causes of ageing: It is unclear what exactly causes ageing, though there are some theories regarding certain effects on ageing - Oxidative damage as a result of normal metabolism- one theory sees our metabolism as the cause of our aging. According to this theory, aging is a by-product of normal metabolism: about 2-3% of the oxygen atoms taken up by the mitochondria are reduced insufficiently to ROS - Reactive Oxygen Species - including superoxide ion, hydroxyl radicals and hydrogen peroxide. These can oxidize and damage cell membranes, proteins and nucleic acids. Evidence for this theory: Drosophila fruit-fly that overexpresses enzymes that destroy ROS lives 30-40% longer than do normal flies. Mice which has lack in certain protein - the lack gave them resistance to ROS However, results in mammals aren't easy to interpret - there is no clear evidence that ROS inhibitors work in mammals as well. General genetic instability - another theory states that with time, accumulation of genetic errors causes ageing; point mutations increase in number, and the efficiencies of the enzymes encoded by our genes decrease. Mutations that increased significantly the process of aging are mutations in DNA sequences coding for: Parts of organelles involved in protein synthesis and functions (RER, ribosomes, golgi, RNA's) Enzymes responsible for DNA repair and synthesis Mitochondrial genome damage - the mutation rate in mitochondria's DNA is 10-20 times faster than the nuclear DNA mutation rate. These mutations may cause defects in energy production, over-production of ROS by faulty electron transport (and being more sensitive to ROS damage), and induce apoptosis. Increase in number of mitochondria defects 🡪 increase in ROS 🡪 increase in defects in mitochondria Telomere shortening - telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. It is believed that each cell division, the telomeres shorten. When telomeres become too short, the cells deteriorate and die, or simply cease multiplying when the telomere reaches certain critical point. Telomere shortening below 2.5 kilobytes ceases to divide and may be induced apoptosis. The length of telomeres is therefore referred to "molecular clock". However, stem cells and cancer cells has enzyme called telomerase which elongate these telomeres and prolonged their life-span. Thus, it has been suggested that telomere-dependent inhibition of cell division might serve primarily as a defense against cancer rather than as a kind of molecular clock. Molecular genetic evidence demonstrates that age is programmed by telomere length . Chromosome telomeres are made up of a large number of short repeats that are species-specific. In humans, the telomere repetitive sequence is TTAGGG with a telomere length of 5 - 15 kb. The telomere is terminated by a single-stranded section; telomere bases are methylated, which allows the formation of a specific hairpin in which methylated guanosines pair. Telomere The structure of the telomere prevents the cleavage of DNA by deoxyribonucleases, the fusion of DNA molecules in the genome and allows DNA replication without loss of terminal sequences. The complex enzyme telomerase (RNA dependent DNA polymerase) is capable of elongating telomeres . It is active only: in germline cells, bone marrow stem cells, stimulated T and B lymphocytes and in tumor-transformed cells . In differentiated somatic cells, telomerase is not expressed and telomere length is reduced by about 100 bases with each cell division. Telomere shortening below 2.5 kb is critical for the cell, ceases to divide, and apoptosis can be induced . Cells with longer telomeres are able to divide more than cells with shorter telomeres. Glycation - sugar molecules binds to protein or lipid molecules, forms advanced glycation end products - AGE's - which cause protein fibers to become stiff and malformed.

What is non-Mendelian inheritance? what are the Mendelian laws? what are some types of non-Mendelian inheritance? (29)

any pattern of inheritance in which traits do not segregate in accordance with Mendel's laws - law of uniformity - law of segregation - law of independent assortment Mendel's laws of inheritance only describe the basic genetic makeup - monogenic autosomal inheritance with complete dominance and complete penetrance and expressivity. Each parent contributes one of two possible alleles for a trait - if the genotypes of both parents in a genetic cross are known - Mendel's laws can be used to determine the distribution of phenotypes expected for the population of offspring. He refers to genes inherited without taking into account non-allelic interactions such as Epistasis (effect of one gene being dependent on the presence of modifier genes), multifactorial inheritance which takes into account the environmental factors, polygenic inheritance in which more than one gene effect on the phenotype (additive cumulative affect) .Sex-linked inheritance: males has only one X chromosome, and they can inherit it only from their mothers; in addition, only males can inherit Y chromosome - which violate the segregation rule in which for each trait there are two alleles. As a consequence, the principle of dominance doesn't apply here - and males said to be hemizygous for the genes located on their sex chromosomes, both X and Y. Gene interactions - Epistasis - one allele affects the expression of another allele which found in completely different loci, possible even completely different chromosome. It is an example for gene interaction in which one gene may silence another gene (mask), such as in the gene for hair coat in Labradors dogs - when the gene for yellow hair is found in the loci for the yellow hair, the gene for black or brown hair, which is found in another loci, is silenced and dog will be anyway yellow, doesn't matter which allele he has in the black/brown loci. Epistasis doesn't show Mendelian pattern of inheritance since Mendelian characteristics do not depend on other genes, and show a direct correlation between the genotype and the phenotype. Polygenic and multifactorial inheritance: Additive polygenic characteristics - the phenotype is composed of many genes, each have additive contribution to the resulting phenotype. Multifactorial characteristic - consists of polygenic inheritance and is affected by environmental factors. These two types of inheritance are not Mendelian since Mendelian characteristic shows a direct correlation between a phenotype and genotype, and in both cases one genotype can produce many phenotypes (due to other effector such as other genes or environmental factors). Linkage - linked genes are genes that have a tendency to be inherited together - which clearly violate the 2nd rule of independent assortment (each pair of allele's segregates independently of each other pair of alleles during gamete formation. This law applies only to genes (allele pairs) located on different chromosomes). Regarding linkage-inheritance, there is higher frequency of closely located genes in the chromosomes to be inherited together, i.e. closer the genes 🡪 more "linked" 🡪 lower recombination frequency. Extra-nuclear inheritance - Mitochondrial inheritance is not Mendelian since the origin of mtDNA is only maternal; in addition, there could be variations in the DNA between each mitochondrion in the cells (segregation of mitochondria in cellular division occur independently - question 65). Mosaicism - Individuals who possess cells with genetic differences from the other cells in their body are termed mosaics - these differences can result from mutations that occur in different tissues and at different periods of development. Mutations that occur early on in development will affect a greater number of cells and can result in an individual that can be identified as a mosaic strictly based on phenotype. In mosaicism, the genetically different cell types arise from a single zygote, and occurred due to mutations or epigenetics. Chimerism - like mosaicism, refer to animal which has more than one genetically-distinct population of cells, which occurred due to formation of zygote from two different germ lines - instead of becoming dizygotic twins, they merged to form one individual which has 2 different DNA sets.

what are tumor-supressor genes? general characteristics? (mutation, alleles, role in a cell) functions? two major types? relevant hypotheses? common TSGs? relavant cancers involved in TSG? (112)

genes that code for proteins that cause stability of genome via DNA repair, antimitotic, and proapoptotic loss of function mutation 2 alleles tumor suppressor genes inhibit cancer and are also termed "recessive oncogenes" - means both alleles must be mutated before the inhibition of cell division is removed. Because it is the failure of their function that promotes cell proliferation, they cannot be identified by adding them to cells and looking for cancer. TSG can be divided into two groups: Gatekeepers - control cell growth and prevent tumor development by regulating the transition of cells through checkpoint in the cell cycle (Rb, CDKi such as p21, p27, p57, p14, p53) Caretakers - protect the integrity of the genome by encode proteins involved in detecting and repairing mutations and by take care for proper dis-junction during mitosis. Caretakers TSG are also components of apoptosis (Bak-Bax, TBID, Caspases). Thus, Tumor-suppressor genes encode for proteins that either have a repressive effect on the regulation of the cell cycle or promote apoptosis. Functions: Repression of genes essential for the continuing of the cell cycle Coupling the cell cycle to DNA damage - damaged DNA 🡪 cell shouldn't divide (ATM) Damage repaired? 🡪 cell cycle continues, damaged cannot be repaired? 🡪 apoptosis (P53) Some proteins involved in cell adhesion - prevent tumor cells from blocking the "contact inhibition" mechanism. DNA repair proteins are usually classified as tumor-suppressors as well, as mutations in their genes increase the risk of cancer (e.g. mutations in BRCA that accounts for double-strand breaks or mutator form of genes that is the mutation form of gene that encode for DNA repair protein). Loss of function of caretakers permits mutations to accumulate in oncogene and gatekeeper genes 🡪 cancer. Principles of tumor suppressor genes "Two-hit" hypothesis - TSG mutations, unlike oncogenes, are recessive - both alleles that code for a particular protein must be affected before an effect is manifested. Two-hits must occur. Loss of heterozygosity - LOH - if an organism inherits one defective copy of the TSG (germline mutation of one of his parents) thus he is heterozygous for the cancer-causing mutation - he doesn't have cancer. But if somatic mutation occurs, i.e. inactivation or loss of the one remaining allele occurs, the tumor-suppressor products completely eliminate - "loss of heterozygosity"; common mechanism is deletion on the chromosome that carried the normal copy. Haplo-insufficiency - appearance of the trait in an individual cell or organism that is heterozygous for a normally recessive trait; it occurs because of dosage effects. Even though the heterozygote produces product, it produces only half as much of the product should be encoded by the tumor-suppressing gene, which normally is sufficient - but it less than the optimal amount 🡪 in some cases, may cause cancer. Similar to oncogenes development - changes in chromosome number and structure may also affect tumor-suppressor genes by disrupt their normal function or bring together parts of different genes - creating a fusion protein (chimeric genes); translocation into new location which change TSG expression may also be cause for cancer, point mutation in critical region that encodes for TSG (either regulatory region/coding region). Genes controlling the cell cycle - Briefly, the cell cycle is regulated by cyclins, whose concentration oscillates during the cell cycle, and cyclin-dependent kinases (CDKs) which have constant concentration. Cyclins bind to CDKs 🡪 activated protein kinases that initiate cell cycle. Genes that encode cyclins referred to as oncogenes, and factors that inhibit formation of activated CDKs are often tumor-suppressor genes. Examples The first tumor-suppressor protein discovered was the Retinoblastoma protein (pRb) - it plays a vital role in the negative control of the cell cycle and in tumor progression. It is responsible for the G1 Restriction point, blocking S-phase entry and cell growth. The retinoblastoma family includes three members which collectively referred to as "pocket proteins". They repress gene transcription required for transition from G1 to S phase, by directly binding to the transactivation domain of E2F and by binding to the promoter of these genes as a complex with E2F. Phosphorylation of pRb by CDK 4,6-Cyclin D complex is maximal at the start of S-phase, and lowest after mitosis and entry into G1: in its hypophosphorylated form it suppresses cell proliferation, while when it is phosphorylated - it releases E2F. Loss of pRb functions may induce cell cycle deregulation and so lead to a malignant phenotype. Gene inactivation of pRb through chromosomal mutations is one of the principal reasons for retinoblastoma tumor development. Functional inactivation of pRb by viral onco-protein binding is also common (human papilloma virus - cervix cancer) - HPV that incorporated its genes into the cellular genome produce analog protein to E2F, which occupies pRb and prevents its functional role in inhibition of E2F. In retinoblastoma cancer cells - there is total absence of pRb. In the familial form of retinoblastoma, individuals inherit a mutant gene form an affected parent. A subsequent mutational event - Loss of Heterozygosity - inactivates the normal allele and resulting in Rb development - apparently dominant mode of inheritance. In other words, the risk of developing cancer is dominantly inherited. In the sporadic forms of Retinoblastoma, which are rare, two somatic mutational events must occur. TP53 gene encode for P53 tumor-suppressor protein. P53 has been described as "the guardian of the genome" because of its role in conserving stability by preventing genome mutation by its major factor in the activation of apoptosis. TP53 gene is the most frequently mutated of all the known cancer genes - 20-25% of breast and more than 50% of bladder, colon and lung cancer have been found to have TP53 mutations. It has many mechanism of anticancer function and plays a role in apoptosis, genomic stability and inhibition of angiogenesis: It can activate DNA repair proteins when DNA is damaged It can arrest growth by holding the cell cycle at the G1/S regulation point when DNA damage recognized. P53 acts by promoting the expression of p21 which is a potent inhibitor of kinases that bind to CDKs. It can initiate apoptosis if DNA damage proves to be irreparable (activation of Bak - pro-apoptotic factor, which also inhibits Bcl-2) Plays a role in the duplication of the centrosome, which is required for proper formation of the spindle and chromosome segregation - if p53 is mutated the centrosome may undergo extra duplications 🡪 unequal segregation of chromosomes. Li-Fraumeni syndrome - inherited mutations in TP53; members of families with this syndrome, inherited as an AD trait, highly susceptible to developing a variety of malignancies at an early age - sarcomas, breast cancer, adrenal carcinomas. Lynch Syndrome - Hereditary non-polyposis Colon cancer - mutations in MMR (mismatch repair genes) ATM - a serine/threonine protein kinase that is recruited and activated by DNA-double strand breaks. It phosphorylates several key proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. In double-strand breaks, it phosphorylates and activates BRCA genes. When ATM is activated by DNA damage - it signals to MDM2 to release and activate P53 (mdm2 is ubiquitin ligase enzyme). ATM, thus, causes activation and phosphorylation of P53. P53, in turn, cause enhance transcription of protein P21 - which is CDKI - CDK inhibitor, which inhibits Cyclin D - CDK 4,6 Complex (the first and most important committed\limiting step of cell cycle) from being activated. Following activation of p21, Cyclin D - CDK4,6 complex doesn't phosphorylate Retinoblastoma protein which doesn't release E2F - cause cell cycle arrest. Moreover, P53 activates DNA repair mechanisms to repair the damaged DNA. In case the DNA damage is too severe - it activates apoptosis through Bax. MDM2 - which is proto-oncogene rather than TSG - is a type of ubiquitin ligase (E3 enzyme family - accounts for ubiquitination of proteins), means that when associated with p53 and perform ubiquitination - it leads to proteasome-mediated degradation of p53 (reminder - ubiquitination of protein is the process in which the cell mark proteins for degradation, which identified and performed by proteasome). In most common human sarcomas, amplification of MDM2 gene is observed - causes to suppression of P53 and its tumor-suppressor functions. APC Protein - Mutations in genes that encode parts of the spindle apparatus (of mitosis) may contribute to abnormal segregation and lead to chromosome abnormalities - APC is a tumor-suppressor gene that is often mutated in colon cancer cells. It has several functions, one of which is to interact with the ends of the microtubules that associate with the kinetochore - part of the spindle-assembly checkpoint that monitors the proper assembly of the mitotic spindle and block the onset of anaphase till all the microtubules attached properly in the kinetochore.

what is the threshold effect of polygenetic inheritance? (3)

susceptibility to a genetic defect can be inherited polygenically if a certain number of recessive alleles cause disease

what is genetic engineering? what is the importance? what are libraries? how are they constructed? (55)

the deliberate modification of the characteristics of an organism by manipulating its genetic material. genetic engineering purposefully constructs cells, resp. organisms, about such combinations of genes in genomes that do not exist in nature. This opens up new approaches for deeper analysis of separate genomes and at the same time completely new possibilities in biotechnological processes. genetic engineering works mainly with bacteria and yeasts , two main experimental approaches - genetic and cellular engineering genetic engineering (gene manipulation)it is possible to create completely new DNA moleculesconstruction of chimeric DNA molecules in vitro from DNA fragments of isolated cells of completely different species, genera, families and even kingdomsit is a combination of their genes into contiguous nucleotide sequences by cloning DNA, we can obtain rare cellular proteins in large quantities; so-called expression vectors have been constructed for the production of proteins - they contain suitable regulatory sequences and a promoter in close proximity to the site into which the insert with the coding sequence is cloned; the promoter together with the regulatory sequences ensures the production of a large amount of mRNA that can be translated within the cell By cloning DNA, one particular sequence can be selected from a million others and an infinite number of copies can be made DNA fragments can be joined in vitro by DNA ligase to give rise to a DNA molecule that does not occur in nature the first step in cloning is the insertion of the desired fragment into a DNA molecule that is capable of replication - e.g., a plasmid or viral genome; the generated recombinant DNA molecule is then inserted into a rapidly dividing host cell - such as a bacterium - and replicates during each cell division a set of cloned fragments that represent the complete genome of an organism = genomic library - is often maintained in the form of bacterial clones, where each clone carries a different cloned DNA fragment cloned genes can be permanently integrated into the genome of a cell or organism using genetic engineering techniques importance: enabled the recognition of complete and accurate sequences of a number of eukaryotic genes, incl. regulatory sequences significantly complemented the construction of phylogenetic "trees" of animal and plant species at the same time, gene manipulations started a new era of biotechnology , enabled the production of a new generation of vaccines (against hepatitis A and B , against the flu , foot - and - mouth disease, etc.) gene "supplemented" bacteria, resp. yeast, today they produce human hormones : insulin , somatostatin, somatotropin and a number of brain hormones (enkephalins and endorphins) Extremely pure AMK, enzymes , alkaloids, steroids, gibberillins and other biologically active substances can also be obtained from bacteria with recombinant DNA in practically unlimited amounts in medicine, gene therapy for hereditary diseases can be used in perspective Libraries - a collection of clones with different fragment of DNA derived from the total DNA or RNA of a cell. If the library is large enough, it should contain all of the sequences found in the original DNA source. Complementary DNA (cDNA) libraries - contains complementary DNA copies of the mRNA population present within a particular tissue. They are preferable to genomic libraries as a source of cloned genes because it contains the final mRNA type (after post-transcription modifications), e.g. only the exons of a gene (thus - direct representation of the coding sequence of a gene, without the introns or promoter sequences). Cloning of cDNAs relies on the enzyme reverse transcriptase, an RNA-dependent DNA polymerase that can synthesize a single-stranded cDNA fragment complementary to an mRNA template. The single-stranded cDNA is then used as the template for DNA polymerase in PCR, which converts the single-stranded molecule to a double-strand molecule, which can then be ligated into a suitable vector to create a cDNA library representing all of the original mRNA transcripts found in the starting cell or tissue. Once a library is made, the next step is to identify the clone carrying a sequence of interest among the millions of other clones carrying other fragments in the library - a process which is called "library screening", performed by nucleic acid hybridization. Hybridization reaction proceeds by mixing single-stranded nucleic acids, allow base-pairing. Only those strands that are correctly base paired can form a stable double-stranded nucleic acid. One sequence (the target, desired, sequence) in a mixture of nucleic acids is tested for its ability to form stable base pairing with a DNA or RNA fragment of known sequence (the "probe") - which has been tagged with a radioactive tracer to allow the probe to be subsequently detected and then isolated.

what is allele frequency? how are they represented? how are they determine?

the number of times an allele occurs in a gene pool, compared to the total number of alleles in that pool for the same gene p + q = 1

what is pleiotropy? (1)

the production by a single gene of two or more apparently unrelated effects. CF

What is heritability? what are the equations to estimate it? what is phenotypic variance and equation? what are the components of genetic variance? what is the difference between broad-sense and narrow-sense heritability? what is the Holzinger index of heritability equation? importance of its assessment in medicine? (9)

the proportion of variation among individuals that we can attribute to genes Heritability is a statistic used in genetics that estimates how much variation in a phenotypic trait in a population is due to genetic variation among individuals in that population. In other words, it measures the fraction of phenotype variability that can be attributed to genetic variation. Heritability increases when genetics are contributing more variation or because non-genetic factors are contributing less variation - what matters is the relative contribution of each factor. The extent of dependence of phenotype on environment can also be a function of the genes involved. Matters of heritability are complicated because genes may canalize a phenotype, making its expression almost inevitable in all occurring environments. Individuals with the same genotype can also exhibit different phenotypes through a mechanism called phenotypic plasticity (the ability of an organism to change its phenotype in response to changes in the environment), which makes heritability difficult to measure in some cases. To determine how much of phenotypic differences in a population are due to genetic and environmental factors, we should have quantitative measure of the phenotype under consideration. Consider a population of wild plants that differ in size - all the different sizes is the phenotypic variance, and it is represented by VP. In summary, the total phenotypic variance can be demonstrated by: VP = VG + VE + VGE VG is genetic variance VE is environmental variance VGE is the genetic-environmental variance genetic variance composed of: VG = VA + VD + VI Additive genetic variance - VA - comprises the additive effects of genes on the phenotype - Dominance genetic variance - VD - when some genes have a dominance component - thus not additive variance - the effect of allele depends on the identity of the other allele at that locus - thus we must add only the dominant allele to the genetic variance. Genic interaction variance - VI - Multi-genic interaction - Epistasis- the effect of one gene being dependent on the presence of one or more 'modifier' genes - VP = VA + VD + VI + VE + VGE H2 is called the broad-sense heritability. This reflects the ratio of all the genetic contributions to a population's phenotypic variance including additive, dominant and multi-genic interaction with the total phenotypic variance. H^2 = Vg/Vp - H2 can range from 0 to 1, when it equals 0, variance phenotype fully dependent on environmental factors; when it equals 1 - environmental factors have no influence at all, and the phenotypes depends on genetic factors. A heritability value between 0 and 1 indicates that both genetic and environmental factors influence the phenotypic variance. h^2 = Va/Vp The additive genetic variance primarily determines the resemblance between parents and offspring - thus we are more interested in the proportion of VA and the VP. If all of the phenotypic variance is due to additive genetic variance, then the phenotypes of the offspring will be exactly intermediate between those of the parents, but if some genes have dominance, then offspring may be phenotypically different from both parents (Aa X Aa 🡪 aa). Use of the broad-sense heritability H2 is generally restricted to discussions of clones (such as identical twins or asexual propagates of an individual). While H2 also gives the total fraction of variation in a trait due to differences in genotypic values, for sexually reproducing species only variation in breeding values is (easily) converted into selection response. Hence, h2 rather than H2 is a better measure for sexual species of the fraction of (easily) usable genetic variance. According to the analyzes, the kinship coefficient r is: parents - children : r = h 2 parent - child: r = ½ h 2 own siblings (for any child of the given parent): r> ½ h 2 siblings do not own : r = ¼ h 2 The third option for evaluating heredity is the use of the twin method , in which we evaluate the concordance (identity) and discordance (difference) of monozygotic and dizygotic twins in a given trait. We estimate heritability according to the Holzinger index of heritability : H = ( K mz - K dz ) / (1 - K dz ) where K mz is the relative representation of concordant pairs in the MZ twin group and K dz is the relative representation of concordant pairs in the DZ twin group. importance If the heritability is equal to one, it means that the variance of the phenotype is caused only by variation of the genetic information. Therefore, the disease cannot be solved by intervention. If the heritability value is less than one, it means that the expression of the sign can be changed by changing external factors. Such interventions include, for example, lifestyle changes, diet changes or medications. An example is the treatment of multifactorial hereditary obesity (heritability 0.4-0.8) - although there is a significant genetic component (predisposition), changing the ratio of energy intake and expenditure (physical activity, caloric restriction, or pharmacological or bariatric treatment) can be successfully to treat obesity in patients with "unfavorable" allele combinations.

what is incomplete dominance (1)

A blending of traits. Red+White=Pink. neither allele is completely dominant

what is genetic mapping? what is the basis for constructing a genetic map? what is determined by genetic mapping? what are the methods? (19)

Allows scientists to find the location of gene alleles that affect diseases or inherited traits on the 22 human autosomes and in the 2 sex chromosomes Genetic maps, also known as linkage maps, provide a rough approximation of the locations of genes relative to the location of other known genes. By observing a trait that segregate within a family and linking this trait with certain markers, we can estimate the location of the gene in reference to the location of the marker. This type of analysis is mainly used for Mendelian inheritance in which there is an almost direct relation between the genotype and the phenotype. In order to construct genetic maps, we need to develop genetic markers with known location on chromosomes - the closer two markers are, the more likely they are to be passed on to the next generation together. The quality of the genetic maps are largely depends upon the number of genetic markers on the map. Individual organisms heterozygous at two or more genetic loci are crossed. The frequency of recombination between loci is determined by examining the progeny. If the recombination frequency between two loci is higher than 50%, then the loci are located on different chromosomes or are far apart on the same chromosome (means - there is high chance of recombination to occur, the genes are not linked). If the recombination frequency is less than 50%, the loci are located close together on the same chromosome (said to be "same linkage group"). For linked genes, the rate of recombination is proportional to the physical distance between the loci, measured in "percent recombination" (Centimorgans- cM). The basis of mapping is to determine the number of chromosomes , the position of genes on given chromosomes and in what order the genes are located (gene distance). Exact sequence determination is possible using genome mapping and genome sequencing methods . Two point linkage analysis - in case the location of the trait is completely unknown, at least one marker for each chromosome is required. If we observe that certain marker, with known location, segregates with the desired trait more often than would be expected by chance alone 🡪 low recombination fraction 🡪 we can deduce that the marker is closely related to the trait. However, recombination fraction based on probability - thus we cannot simply by observing the family pedigree determine whether a trait and marker are linked or not. In small groups, such as families, there is a higher chance that several chromosomes segregated together 🡪 the recombination frequency alone is not reliable enough to deduce whether genes are linked. For this reason, we use the "LOD" score method which helps us to determine linkage: We calculate both the probability for having a linkage (recombination fraction) and the probability that chance occurred - independent assortment between the two genes - and the genes are inherited together.We then calculate the ratio between these two probabilities, and its logarithm is the LOD score; if its higher than 3 - usually considered as convincing evidence for linkage between the genes. Two-point linkage, thus, may be used in order to decide roughly in which region a gene for specific desired trait is located in the chromosome; in order to be more accurate and specific - its better to use Multipoint linkage analysis, in order to place the disease in a more precise location on the chromosome. For this process, we require polymorphic markers that are located closer to each other on the region found in the two-point methods. The result for this study is analyzed by a computer that calculates the most probable locations for the trait

what are some examples of autosomal recessive inheritance? (13)

Cystic fibrosis - 1:2500 - presence of mutations in both copies of the gene for the CFTR - cystic fibrosis transmembrane conductance regulator protein which involved in production of sweat, digestive fluids and mucus - when it is not functional (two recessive alleles) - secretions which are usually thin instead become thick (usually screening for infant at birth takes place). Long-term issues include difficulty breathing and coughing up mucus, lung infections. Poor growth, clubbing fingers and toes, infertility in males. PKU Phenylketonuria - frequency in the population 1:10000 - inborn error of metabolism which involve impaired metabolism of amino acid Phenylalanine to tyrosine. It is caused by absent phenylalanine hydroxylase enzyme activity. Left untreated, may cause intellectual disability, seizures, behavioral problems and mental disorders, lighter skin. Babies who born to mothers who have PKU may have heart problems, small head and low birth weight. Wilson disease - Copper accumulates in tissues - this manifested as neurological or psychiatric symptoms and liver disease, due to mutations in the Wilson protein gene. Liver related symptoms include vomiting, weakness, fluid builds up in the abdomen, yellowish skin and itchiness. Brain related symptoms include tremors, brown ring on the edge of cornea, muscle stiffness, trouble speaking, and personality changes. Congenital Adrenal hyperplasia - CAH - resulting from mutations of genes for enzymes mediating the biochemical steps of production of cortisol from cholesterol by the adrenal glands; most of these conditions involve excessive or deficient production of sex steroids and can alter development of primary or secondary sex characteristics in some affected infants, children or adults. Glycogen storage diseases - result of defects in the processing of glycogen synthesis or breakdown within muscles, liver and other cell types. Hurler syndrome - buildup of glycosaminoglycans (mucopolysaccharides) due to a deficiency of enzyme responsible for the degradation of mucopolysaccharides in lysosomes 🡪 buildup of heparin sulfate and dermatan sulfate occurs in the body. Tay-Sachs diseases - rare AR genetic disorder increased disease prevalence in Ashkenazi jews, causes a progressive deterioration of nerve cells and mental and physical abilities that begin around 7 months of age, usually results in death by the age of four. The disease occurs when harmful quantities of cell membrane sphingolipids accumulate in the brain's nerve cells. Hemoglobinopathy is a kind of genetic defect that results in abnormal structure of one of the globin chains of the hemoglobin molecule. Hemoglobinopathies are inherited as single-gene disorder (monogenic); in most cases, they are inherited as autosomal co-dominant traits; e.g. sickle cell disease. Albinism - complete or partial absence of pigment in the skin, hair and eyes due to absence or defect of tyrosinase - a copper-containing enzyme involved in the production of melanin.

what are the methods of chromosomal examination? steps to prepare cytogenetic analysis for peripheral blood? what is a karyotype and what are they used for? (31)

A chromosomal karyotype is used to detect chromosome abnormalities and thus used to diagnose genetic diseases, some birth defects, and certain disorders of the blood or lymphatic system. Any tissue with living nucleated cells that undergo division can be used for studying human chromosomes. Most commonly circulating lymphocytes from peripheral blood are used, although samples for chromosomal analysis can be prepared using skin, bone marrow, chorionic villi, or cells from amniotic fluid (amniocytes). In the case of peripheral (venous) blood, a sample is added to small volume of nutrient medium containing substance which stimulates T lymphocytes to divide. The cells are cultured for about 3 days, during which they divide and a drug calls colchicine is then added. This drug is useful for preventing formation of the spindle, thereby arresting cell division during metaphase, the time when the chromosomes are maximally condensed and therefore most visible. Hypotonic saline is then added, which causes the cells to lyze and results in spreading of the chromosomes, which are then fixed, mounted on a slide and stained ready for analysis. G-banding - Several different staining methods can be used to identify individual chromosomes but G (Giemsa) banding is used most commonly. Giemsa, a DNA-binding dye gives each chromosome a characteristic and reproducible pattern of light and dark bands, approximately 400-500 bands per haploid set, corresponds to 6000-8000 kilobases of DNA. The next stage in chromosome analysis involves first counting the number of chromosomes present in a specified number of cells, sometimes referred to as metaphase spreads, followed by careful analysis of the banding pattern of each individual chromosome in selected cells. The banding pattern of each chromosome is specific and can be shown in the form of ideal karyotype ideogram. The cytogeneticist analyzes each pair of homologues chromosomes. The number of stripes depends on the degree of condensation of chromosomes. In metaphase chromosomes there are 400-450 stripes. The prometaphase karyotype is identifiable around 850 stripes. Bright stripes correspond to euchromatin form, rich in guanine and cytosine, whereas darkly stained heterochromatin region comprising the DNA rich in adenine and thymine. R-banding - R-banding is a cytogenetics technique that produces the reverse of the G-band stain on chromosomes. R-banding is obtained by incubating the slides in hot phosphate buffer, then a subsequent treatment of giemsa dye. Resulting chromosome patterns shows darkly stained R bands, the complement to G-bands. Darkly colored R bands are guanine-cytosine rich, and adenine-thymine rich regions are more readily denatured by heat. The technique is useful for analyzing genetic deletions or chromosomal translocations that involve the telomeres of chromosomes. AgNOR staining - This staining method is used to track variation of the secondary constriction of acrocentric chromosomes. Nucleolar organizer regions (NORs) are defined as nucleolar components containing a set of argyrophilic proteins, which are selectively stained by silver methods. After silver-staining, the NORs can be easily identified as black dots exclusively localized throughout the nucleolar area, and are called "AgNORs". Silver nitrate is selectively precipitated in nucleolar organizer regions. C-banding - Constitutive heterochromatin domains are regions of DNA found throughout the chromosomes of eukaryotes. The majority of constitutive heterochromatin is found at the pericentromeric regions of chromosomes, but is also found at the telomeres and throughout the chromosomes. Constitutive heterochromatin is composed mainly of high copy number tandem repeats known as satellite repeats, minisatellite and microsatellite repeats, and transposon repeats. In humans these regions account for about 6.5% of the total human genome, but their repeat composition makes them difficult to sequence, so only small regions have been sequenced. special methods: SCEs Sister chromatid exchanges (SCEs) involve breakage of both DNA strands, followed by an exchange of whole DNA duplexes. This occurs during the S phase and is efficiently induced by mutagens that form DNA adducts or that interfere with DNA replication. The formation of SCEs has been correlated with recombinational repair and the induction of point mutations, gene amplification and cytotoxicity. To allow for a differential staining that enables the researcher to distinguish both chromatids, BrdU (bromo-deoxy-uridine) is added to the culture medium for the duration of two complete cell cycles. Chromatids in which only one strand of DNA incorporated BrdU show a normal dark Giemsa staining, whereas those with two substituted strands, stain less darkly. If an exchange occurred, this can be seen as the dark part changes to the other arm: "harlequin chromosomes". FISH - Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity. It is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH using DNA probes labeled with a fluorescent dye to detect specific DNA segments. Fragile-X - A number of fragile sites are known in the human karyotype - areas of frequent fractures. They appear on metaphase chomosomes after exposure to certain substances or during growth in deficient media. Mostly their presence is not related to the pathological phenotype. An exception is the Xq27.3 fragile site (FRAXA) in patients with fragile X syndrome . Xq27.3 is a folate sensitive site, which can be visualized after culturing in a medium with low folic acid content, or in the presence of folic acid antagonists FudR (5´-fluoro 2´-deoxyuridine) or methotrexate. Cytogenetically, under optimal laboratory conditions (and with a little luck), the presence of fra Xq27.3 can be diagnosed in both affected men and female carriers. Fragile X syndrome is conditioned by the expansion of the triplet repeat CGG in the first exon of the FMR1 gene (Fragile X Mental Retardation 1). At present, cytogenetic diagnostics is no longer used for FRAXA diagnostics, and direct DNA diagnostics focused on the number of repeats in a given gene are preferred. In the Xq28 region there are other folate sensitive fragile sites; FRAXE - associated with milder mental retardation and perhaps benign FRAXF.

what are some examples of AD inheritance? (12)

Achondroplasia - common cause of dwarfism; occurs as a sporadic mutation in 80% of cases (usually associated with advanced paternal age), or it may be inherited as an AD genetic disorder. People with achondroplasia have short stature with an average adult height of 131 cm for males and 123 cm for females. Apert syndrome - congenital disorder characterized by malformations of the skull, face, hands and feet (varied number of fingers and toes are fused together - syndactyly); premature fusion of certain skull bones, prevents the skull from growing normally and affects the shape of the head and face. Familial hypercholesterolemia - frequencies in a population of 1:500; high cholesterol levels, high levels of LDL. Early CV diseases; arteries are tend to be blocked by plaques (atherosclerosis), disturbing blood flow, most commonly coronary artery diseases - heart attacks, angina pectoris (chest pain). Marfan syndrome - Connective Tissue disorder - misfolding of fibrillin which form elastic fibers in CT, involve defects of the heart valves and aorta. People with Marfan syndrome tend to be unusually tall - long limbs and long, thin fingers and toes. Huntington Chorea - frequencies in population is 1:10000 - neurodegenerative genetic disorder, death of brain cells, that affects muscle coordination and leads to mental decline (till dementia) and behavioral symptoms; usually late onset - symptoms appears at 30-50 years of age. Expansion of CAG triplet repeats in the gene coding for Huntingtin protein results in an abnormal protein which gradually damages cells in the brain. Myotonic dystrophy - a long term disorder that affects muscle functions - muscle loss and weakness. Muscles often contract and are unable to relax. Other symptoms may include cataracts (clouding of the lens in the eye which leads to decrease in vision), intellectual disability and heart conduction problems and sterility in men. Neurofibromatosis - 1:3500 - group of conditions in which tumors grow in the nervous system which are usually non-cancerous. The tumors involve supporting cells (glia) in the nervous system rather than neurons. Osteogenesis imperfecta - congenital bone disorder, results in bones that break easily due to lack of collagen type I. Other symptoms may include a blue color to the whites of eye, short height, loose joints, hearing loss, breathing problems and problems with the teeth. Polycystic kidney - growth of numerous cysts in the kidneys - may be expressed as onset disease but can also manifest in the childhood. Polydactyly - aka as hyperdactyly; supernumerary fingers or toes (opposite to oligodactyly - fewer fingers or toes) Brachydactyly - shortness of the fingers and toes relative to the length of other long bones and other parts of the body.

what are the types of receptors involved in cell signaling? general scheme for cAMP pathway? (23)

Ionotropic receptors - open allows ions such as sodium, potassium, calcium or chloride to pass through the membrane in response to the binding of a chemical messenger - ligand - such as neurotransmitter - allows the ions move along its electrochemical gradient and results in a change in membrane potential. This type of cell signaling occurs in "excitable membranes" - nervous system and muscles. Enzyme-linked receptors - also known as catalytic receptor - these receptors have one extracellular domain which bind to a ligand, transmembrane alpha-helix dimer, and intracellular cytoplasmic part - which is linked with specific enzyme. E.g. Receptor tyrosine-kinase: ligand such as insulin or some growth factors such as FGF connects to receptor which has intra cellular domain linked with tyrosine-protein-kinase enzyme. When activated, it has an intrinsic tyrosine activity - it phosphorylates its tyrosine residue (auto-phosphorylation - phosphorylates itself). When the tyrosine residues are phosphorylated - become active - and leads to activation of RAS signaling molecule, which in turn activates the next protein in the cascade 🡪 finally, there is change in gene expression. Activated Ras proteins phosphorylate and thus activate a cascade of three types of MAP kinases . By binding the first MAP-kinase (called Raf) to the activated Ras protein, it is phosphorylated and thus activated. This then catalyzes the serine / threonine phosphorylation of another MAP kinase and this enzyme activates another (third) MAP kinase. Activation of the last MAP kinase in the cascade by phosphorylation of MAP kinases requires phosphorylation of both threonine and tyrosine . Thus, after entering the nucleus, the activated third MAP kinase first phosphorylates the regulatory protein , which is bound to a short DNA sequence in the regulatory region of the early response genes - the myc, jun and fos genes . This causes them to transcribe. Late response gene products are involved in the regulation of cell proliferation. These include, for example, the main components of the cell cycle control system - cyclins and cyclin-dependent protein kinases. G-protein-coupled receptor - also known as 7-transmembrane domain receptors (passing through the membrane 7 times) and are the largest family of cell surface receptors and nearly a third of all drugs target this type of receptor. They detect molecule outside the cell and activate internal signal transduction pathways 🡪 cellular response. GPCRs associate with G-proteins that are composed of 3 different subunits - alpha, beta, and gamma. The subunits attached to the lipid molecules of the membrane. When ligand binds, the receptor activates the attached G-protein by causing the exchange of GTP to GDP 🡪 activated G-protein then dissociated into a G-alpha (G-alpha bound to GTP is active 🡪 can diffuse along the membrane surface to activate target proteins) and betta-gamma complex which can also able to diffuse along the inner membrane and affect protein activity. General scheme: Ligand binds to membrane receptor (GPCR) 🡪 activated the G-protein 🡪 activated G-alpha activates adenylyl cyclase 🡪 formation of cAMP 🡪 Protein kinase A 🡪 phosphorylation cAMP - Cyclic adenosine monophosphate is a second messenger important in many biological process - is a derivative of ATP and used for intracellular signal transduction. cAMP and its associated kinases function in biochemical processes - including the regulation of glycogen, sugar and lipid metabolism. It works by activating protein kinase A (PKA) which is normally inactive. When protein kinase is activated, it can affect many proteins in the cell.

what is extranuclear inheritance? what are the types? associated diseases? (28)

Mendel's principles of segregation and independent assortment are based on the assumption that genes are located on chromosomes in the nucleus of the cell. However, not all the genetic material of a cell is found in the nucleus. Some characteristics are encoded by genes located in the cytoplasm (about 2% of the total cell's genes) -and inherited by mean of cytoplasmic inheritance. Cytoplasmic inheritance is the transmission of genes that occur outside the nucleus and known to occur in cytoplasmic organelles such as mitochondria and chloroplasts (in plants). Mitochondria - function to transform energy as a result of cellular respiration, Chloroplasts - function to produce sugars via photosynthesis in plants. The genes located in mitochondria and chloroplasts are very important for proper cellular function - their genomes replicate independently of the DNA located in the nucleus (which replicates only during cell division - whereas the mitochondria and chloroplasts genes replicate out of the cell division). The mitochondria and chloroplast genes replicate in response to a cell's increasing energy needs, which adjust during the cell's lifespan. - The genes contained in the mtDNA molecule encode respiratory chain proteins , ATPase complex units, NADH- dehydrogenase complex subunits , two ribosomal RNAse genes and 22 genes for transport RNA molecules . Chondrinogens, or genes bound to mitochondria, are inherited by so-called cytoplasmic inheritance . Uniparental inheritance - occurs in extranuclear genes when only one parent contributes organelle DNA to the offspring. Classical example of uniparental gene transmission is the maternal inheritance of human mitochondria - the mother's mitochondria are transmitted to the offspring at fertilization via the egg. The father's mitochondrial genes are not transmitted to the offspring via the sperm (most mitochondria located in the mid-piece of the sperm - which propel the sperm in the way to the fallopian tube and usually doesn't enter the oocyte and disappear in fertilization). Mitochondrial inheritance (Also question 65), although being passed from only one parent, shows great phenotypic variation between the woman and her offspring, as well as between siblings. In most persons, the mtDNA from different mitochondria is identical (homoplasm). If a mutation occurs in the mtDNA of an individual, there will be two populaions of mitochondrial DNA - heteroplasmy. Mitochondria segregate during meiosis and mitosis independently and unevenly - resulting in variations in mitochondrial DNA between cells, tissue and individuals The molecular genetic basis of mitochondrial pathologies can be either deletions of the 5 kb region between the ND5 genes (NADH-dehydrogenase subunit 5) ATPase 8 (ATPase subunit 8) or point mutations (mostly changing the sense of codon reading - missense or substitution). Kearns-Sayre syndrome - progressive external ophthalmoplegia , retinal pigment degeneration, heart and cerebellar disorders. Mitochondrial pathology caused by point mutations: LHON ( Leber's Hereditary Optic Neuropathy ) - blindness in men under 25 years of age; Penetrance disease is 3-4 times higher in men than in women. MELAS (Mitochondria Myopathy, Encephalopathy, Lactic Acidosis, Stroke-like episodes) - the first symptoms start between 5.-15. one year of life, single nucleotide substitution in the leucine tRNA gene (position 3243). MERF (Myoclonic Epilepsy with Ragged red Fibers) - manifestation 5.-12. year of an individual's life.

what are genetic maps, how are they created, and what is their importance? (20)

One of these tools is genetic mapping. Genetic mapping - also called linkage mapping - can offer firm evidence that a disease transmitted from parent to child is linked to one or more genes. Mapping also provides clues about which chromosome contains the gene and precisely where the gene lies on that chromosome. Genetic maps have been used successfully to find the gene responsible for relatively rare, single-gene inherited disorders such as cystic fibrosis and Duchenne muscular dystrophy. Genetic maps are also useful in guiding scientists to the many genes that are believed to play a role in the development of more common disorders such as asthma, heart disease, diabetes, cancer, and psychiatric conditions. To produce a genetic map, researchers collect blood or tissue samples from members of families in which a certain disease or trait is prevalent. Using various laboratory techniques, the scientists isolate DNA from these samples and examine it for unique patterns that are seen only in family members who have the disease or trait. These characteristic patterns in the chemical bases that make up DNA are referred to as markers. These markers are extremely valuable for tracking inheritance in several generations in the family. One type of DNA marker is microsatellite, found throughout the genome and consists of a specific repeated sequence of bases highly polymorphic between individuals. DNA markers don't, by themselves, identify the gene responsible for the disease or trait; but they can tell researchers roughly where the gene is on the chromosome. If a particular gene is close to a DNA marker, the gene and marker will likely stay together during the recombination process, and they will likely be passed on together from parent to child. If each family member which inherited a particular disease or trait also inherits a particular DNA marker, it is very likely that the gene responsible for the disease lies near that marker. The more DNA markers there are on a genetic map, the more likely it is that at least one marker will be located close to a disease gene. Genes that are on the same chromosome are said to be 'linked' and the distance between these genes is called a 'linkage distance'. The smaller the distance the more likely two genes will be inherited together. The same concept of studying how traits are passed on was applied to develop the first human genome map. If two (or more) characteristics were seen to be frequently inherited together in a family, it suggested that the genes for the two characteristics were close together on a particular chromosome. While genetic maps are good at giving the bigger picture, they have limited accuracy and therefore need to be supplemented with further information gained from other mapping techniques, such as physical mapping. Thus, by determining the frequency of recombinant (how many times the genes weren't inherited together) - it is possible to obtain a map presenting the distances between the genes. The units in genetic maps are in cM - centimorgans, where 1 cM = 1% of recombination frequency. Two test cross: If the distance between A and B is 5cM, and the distance between A and C is 3 cM - then B and C should be either 8cM or 2cM apart. Limitations for construction of genetic map by two test cross: The theoretical maximum distance that can be measured in genetic maps in 50cM - since non-homologues chromosomes which are the farther apart genes can be from each other has 50% chance to be segregated together. Therefore, we cannot distinguish between genes on different chromosomes and genes located far apart on the same chromosome. Distant genes have higher chance for double cross to occur between them - both ends either "crossed" or not crossed - thus the two genes "moved" (or stayed) to the other chromosome during recombination - so instead of giving a high recombination frequency between the genes (that will correspond that long distance between them) - we may observe low recombination frequency - and this can mislead in estimating the physical distance between the genes. Crossing over doesn't occur in an even manner: Some chromosomal regions are more likely to go through crossing over than others. Crossing over occurs more frequently during gametogenesis of oocyte then that of a sperm. For the reason listed above, genetic linkage may not show an exact proportion regarding the actual physical distance between two genes. However, it gives us a rather valid information about the proportions of recombinant and non-recombinant gametes that will be produced - for example, if by constructing genetic map we know that A and B genes are located on the same chromosome with 20 cM (means there is 20% recombination frequency) - we can assume that one in every five gametes will have recombination at these loci and the genes won't be inherited together. Importance: Genetic maps enable us to understand gene function; after sequencing the human genome many genes has been discovered with unknown function. By gene maps we are capable to relate the phenotype of a gene to its location - and "fix a piece in the puzzle". When we discover the sequence that has a role in certain diseases - the ability to investigate the root cause of disease - may one day allow medical researchers to develop strategies to avoid the environmental conditions that serve as triggers to disease, formulate customized drugs and techniques for gene therapy. Example for importance of genetic linkage analysis: Breast cancer is common type of cancer - 1 in 12 women in western societies; Mutations in the tumor suppressor gene BRCA1 and BRCA2 Linkage analysis (family studies) showed that in these families the tendency to develop breast cancer mapped to the long arm of chromosome 17 🡪 leading to identification of the BRCA1 gene.

What are post-transcriptional modifications of RNA? (44)

Post-transcriptional modification or Co-transcriptional modification is the process in eukaryotic cells where primary transcript RNA is converted into mature RNA. The primary transcription of both prokaryotic and eukaryotic tRNA and rRNA are post-transcriptionally modified by cleavage of the original transcripts by ribonucleases. In contrast, prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic mRNA is extensively modified. rRNA - the precursor - pre-rRNAs are cleaved by ribonucleases to yield intermediate rRNA, which are further processed by exonucleases and modified at some bases and riboses - to produce final rRNA. In Eukaryotes, rRNA genes are found in long, tandem arrays and their synthesis occur in the nucleolus, with post-transcriptional modification and ribosomal protein components addition facilitated by snoRNA (small-nucleolar RNA). tRNA - also made from long precursor molecules and modified by intron removal by nucleases. In addition, a -CCA sequence added by nucleotidyltransferase to the 3'-terminal end of tRNA (to which AA binds). A notable example is the conversion of precursor messenger RNA (pre-mRNA) into mature messenger RNA (mRNA) that occurs prior to protein translation. The process includes three major steps: Capping: Addition of a 7'-methylguanosine triphosphate (methyl attached to the guanosine nitrogen base) attached to the 5'-terminal end of the mRNA through a 5 🡪 5 triphosphate linkage that is resistant to most nucleases. Methylation of the terminal guanine occurs only in the cytosol, and is catalyzed by 7'-guanine-methyltransferase (S-adenosylmethionine is the source of the methyl group). A 7-methylguanosine cap is added to the 5' end of the pre-mRNA while elongation is still in progress - The 5' cap protects the mRNA from degradation and is used as a recognition signal for ribosomes to bind to the mRNA. 3' end tailing: The poly-A tail is a long chain of adenine nucleotides that is added to a messenger RNA (mRNA) molecule during RNA processing to increase the stability of the molecule - it is not transcribed from the DNA, rather, it is added after transcription. First, the 3' end of the transcript is cleaved to free a 3' hydroxyl. Then an enzyme called poly-A polymerase adds a chain of adenine nucleotides to the RNA. This process, called polyadenylation, adds a poly-A tail that is between 100 and 250 residues long. The poly-A tail makes the RNA molecule more stable and prevents its degradation. Additionally, the poly-A tail allows the mature messenger RNA molecule to be exported from the nucleus and translated into a protein by ribosomes in the cytoplasm. RNA Splicing - This process is vital for the correct translation of the genomes of eukaryotes because the initial precursor mRNA produced during transcription contains both exons (coding or important sequences involved in translation), and introns (non-coding sequences). RNA splicing is the process by which introns, regions of RNA that do not code for protein, are removed from the pre-mRNA and the remaining exons connected to re-form a single continuous molecule. The splicing reaction is catalyzed by a large protein complex called the spliceosome assembled from proteins and snRNA (small-nuclear RNA) molecules that recognize splice sites in the pre-mRNA sequence. The binding of snRNA + its associated proteins (spliceosome) facilitate splicing by forming base pairs with consensus sequences at each end of an intron, creates loop, which brings two neighboring exons near each other: "Branch Site A" - adenine nucleotide OH group found in the intron attacks phosphate at the 5' end of the intron - the "splice donor site". The newly freed OH of exon 1 attacks the phosphate at the splice acceptor site (at Exon 2), forming a phosphodiester bond that joins exons 1 and 2 together, while release intron - degraded. Many pre-mRNAs, including those encoding antibodies, can be spliced in multiple ways to produce different mature mRNAs that encode different protein sequences. This process is known as alternative splicing, and allows production of a large variety of proteins from a limited amount of DNA.

what is the law of segregation? (1)

during the production of gametes the two copies of each hereditary factor segregate so that offspring acquire one factor from each parent two alleles for a heritable character segregate during gamete formation and end up in different gametes

what occurs with abnormalities in chromosome number? what are the types? what causes them and what are some clinical presentations? (34)

number chromosomal abnormalities deviations from the normal number of chromosomes (46) may apply to individual chromosomes entire haploid set may also multiply do not structurally alter chromosomes many are incompatible with life aneuploidy when there is one extra like in trisomy (2n + 1) or one less (2n - 1) such as monsomy trisomies caused by nondisjunction - separation of homologous chromosomes in the 1st meiotic division or a chromatid in the 2nd meiotic division risk of error depends on age of mother age of 35 is at twice the population risk then increases dramatically with age risk is associated with meitotic division in embyronic development, then is stopped and continues past puberty risk of father starts from age 50 if it occurs during meiotic division and the abnormal gamete is fertilized the error occurs in all cells of the individual if postzygotic during the mitotic division of the zygote - a mosaic is formed that contains two or more cell lines with different karyotypes autosomal examples trisomies down syndrome trisomy 21 most congenital presentations is mental retardation, CHD, muscle hypotension, eyes with skin folds, large creeping tongue in newborns, moon face trisomic cells are more sensitive to the effects of mutagens and carcinogens a large percentage are aborted can have a mosaic form - due to postzygotic loss of Ch 21 from the trisomic cell or during normal division of the zygote can be diagnosed in pregnancy also can be a balanced Robertsonian translocation Edwards syndrome trisomy 18 flexion deformity of fingers, overlapping fingers small head, small jaw, intellectual disability average survival time is 2 months Patau syndrome trisomy 13 microcephaly, sloping forehead, cyclopia (anophthalmia), cleft lip and palate heart defects usually die within the first month of life monosomy formed by nondisjunction and fertilization of an abnormal (nullisomic) gamete or by the delay in anaphase for the chromosome to move can occur in meisosi or post-zygotically during mitotic division only X monosomy is viable autosomal X monosomy leads to spontaneous abortions aneuploidy of gonosomes Turner syndrome 45, X short stature, ovarian dysgenesis associated with sterility skin fold may also be due to structural aberrations fertile is the critical area on the long arms is maintained, then individuals are fertile mosaics are common Klinefelter 47, XXY infertility, hypogonadism - Klinefelter's syndrome (KS) is due to the presence of an excessive X chromosome in a man. It is most often caused by karyotype 47, XXY , variants with more X chromosomes (48, XXXY or 49, XXXXY) are also possible, which have a more pronounced manifestation. There are also mosaic forms. The main symptoms are: infertility (azoospermia), hypogonadism , average to tall, long limbs, sparse hair, gynecomastia. In the case of karyotype 49, XXXXY, the patient is retarded and the phenotype resembles Down's syndrome, with the difference that the patient with KS is in contrast to the patients with high DS. superfemale 47,XXX - X chromosome trisomy , also called three X syndrome (and formerly "Superfemale syndrome"). As the name suggests - it is caused by karyotype 47, XXX , it is also possible to occur in a mosaic. Very rarely, karyotype 48, XXXX or 49, XXXXX can also occur, these cases have a different and more pronounced manifestation. Syndrome 47 itself, XXX does not have a clear clinical picture, some women are examined for infertility . Decreased fertility would be in the case of mosaic 45, X / 47, XXX. Otherwise, woman 47, XXX may be fertile, but some of her gametes may be abnormal. There may be minor psychosocial problems, such as learning difficulties, and an increased tendency to schizophrenia. supermale 47, XYY - This syndrome is caused by the presence of two or more Y chromosomes in the karyotype, most often directly by karyotype 47, XYY . This syndrome used to be called "Supermale" - a term no longer used today. Men may be taller , men with two Ys were previously thought to be more prone to aggression, but this has not been confirmed. There are completely normal men in the population with two Ys in the cells. polyploidy increase in the chromosomal set triploid - 3n = 69 chromosomes lethal genetic constitution f ertilization disorder - dispermia (fertilization of an egg with two spermatozoa) also caused by the fusion of an unreduced gamete (caused by nondisjunction) and a normal gamete phenotype depends on whether the supernumerary set is of paternal or maternal origin situation is caused by genetic imprinting ? tetrapoloid 4n = 92 chromosomes caused by endoreduplication (division of chromosomes without division of the cell)

what are some other methods for genetic mapping? (20)

probe-mapping If we know the protein product of a gene, we can try a molecular probe . We will assemble a sequence of nucleotides that encode the frequency of the gene under investigation and we can try to hybridize to each other . A radiolabeled mRNA strand is used in the DNA - RNA modification . The isotope-labeled base hybridizes to the portion of DNA where the structural gene is responsible. sequencing methods - Sanger - Maxam and Gilbert

what are interactions of non-allelic genes? how do these affect the phenotypic cleavage ratios? what are the types? (4)

these occur when the development of a single character is due to two ore more genes affecting the expression of each other in various ways can change the ratios and can present as deviations from the patterns described by Mendel simple interaction (9:3:3:1) complementary factor (9:7) epistasis - recessive and dominant (9:3:4), (12:3:1) inhibitory factor (13:3) duplicate genes - noncumulative dominance (15:1) - cumulative dominance (9:6:1) - cumulative w/o dominance (1:4:6:4:1)

what is Turner's syndrome? what are the characteristics and incidence? (39)

45, X The absence of a Barr body - only one X chromosome. Incidence of liveborn female infants is 1:2500. Turner syndrome is being detected during the second trimester as a result of routine ultrasonography, showing either generalized edema or swelling localized to the neck. At birth many babies with turner syndrome look entirely normal. Others show the residue of intrauterine edema with puffy extremities and neck webbing - extra skin. low posterior hair-line, short fourth metacarpals, widely spaced nipples Differences in social cognition Internal organs problems - kidney, heart defects, high BP Short stature becomes apparent by mid-childhood (without GH treatment, average adult height is 145cm), ovarian failure - nonfunctioning, infertility.

what is a karyotype? what is the process to prepare one? what six characteristics of the chromosomes are observed? what does a normal karyotype contain? what is a karyogram and what are the groups of chromosomes? (33)

A karyotype is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. The term is also used for the complete set of chromosomes in a species or in an individual organism. Karyotype also refers to the test that detects the chromosomes appearance and measures the number. Karyotypes describe the chromosome count of an organism and what these chromosomes look like under a light microscope. Attention is paid to their length, the position of the centromeres, banding pattern, any differences between the sex chromosomes, and any other physical characteristics. The study of whole sets of chromosomes is sometimes known as karyology. The chromosomes are depicted (by rearranging a photomicrograph) in a standard format known as a karyogram or idiogram: in pairs, ordered by size and position of centromere for chromosomes of the same size. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. Thus, in humans 2n = 46. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). So, in normal diploid organisms, autosomal chromosomes are present in two copies - one from each parent. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. staining The study of karyotypes is made possible by staining. Usually, a suitable dye, such as Giemsa, is applied after cells have been arrested during cell division by a solution of colchicine usually in metaphase or prometaphase when most condensed. In order for the Giemsa stain to adhere correctly, all chromosomal proteins must be digested and removed. For humans, white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. observations: Differences in absolute sizes of chromosomes. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family. Differences in the position of centromeres. These differences probably came about through translocations. Differences in relative size of chromosomes. These differences probably arose from segmental interchange of unequal lengths. Differences in basic number of chromosomes. These differences could have resulted from successive unequal translocations (rearrangement of parts between two non-homologous chromosomes) which removed all the essential genetic material from a chromosome, permitting its loss without penalty to the organism or through fusion. Differences in number and position of satellites. Satellites are small bodies attached to acrocentric chromosome by a thin thread, also termed second constriction (first constriction is the centromere). Differences in degree and distribution of heterochromatic regions. Heterochromatin stains darker than euchromatin because it is packed tighter. Human karyotype: The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes (also known as allosomes, hetero-chromosomes, idio-chromosomes, heterotypical chromosomes). Normal karyotypes for females contain two X chromosomes and are denoted 46,XX; males have both an X and a Y chromosome denoted 46,XY. Any variation from the standard karyotype may lead to developmental abnormalities. Karyogram autosomes are numbered be size from largest (chromosome 1) to the smallest (chromosome 22). According to the shape and size of the chromosomes, the karyogram is divided into seven groups - A to G: Group Chromosome Characteristic The acrocentric chromosomes of the D and G groups have satellite stems on the short arms ( NOR regions - from the English N ucleolus O rganizer R egion - organizer of the nucleolus; they contain repeated copies of genes for ribosomal RNA ) and satellites. However, these areas of acrocenters are very variable.

what are gonosomal aneuploidies? what causes them? (39)

Aneuploidy = loss (monosomy) or residence of 1 or more chromosomes in the cell genome . Causes: disorder of division ( nondisjunction ) of homologous chromosomes in I. maturation division or chromatid in II. maturation division.

what is autosomal dominant inheritance? what are the types? what are the typical characteristics found on a pedigree? what are some deviations? (12)

Dominant-recessive - Black and brown provide a clear example of a dominant-recessive relationship among alleles. Every dog has two genes at the black/brown locus. If both genes are for black, or if one is for black and one is for brown, the dog is black. If both genes are for brown, the dog is brown. BB cannot be distinguished from Bb without genetic tests or breeding tests. incomplete dominance (=Intermediate) - animal carrying two identical alleles shows one phenotype, the animal carrying two different identical alleles shows a different phenotype, and the animal carrying one copy of each of the alleles shows a third phenotype, usually intermediate between the two extremes but clearly distinguishable from either. Codominant - The dividing line between intermediate inheritance and co-dominant inheritance is fuzzy. Co-dominance is more likely to be used when biochemistry is concerned, as in blood types. Co-dominance means that both alleles at a locus are expressed. (e.g. ABO blood group, AB codominance. MN blood group - M N codominance) Sex-limited Please, don't confuse sex-limited inheritance with sex-linked inheritance. A classic example of a sex-limited trait in dogs is unilateral or bilateral cryptorchidism, in which one or both testicles cannot be found in their usual position in the scrotum. Since a female dog has no testicles, she cannot be a cryptorchidic - but she can carry the gene(s) for cryptorchidism, and pass them to her sons. Likewise, genes affecting milk production are not normally expressed in a male. Sex-linked - Every mammal has a number of paired chromosomes that are similar in appearance and line up with each other during gamete production (sperm and eggs). In addition, each mammal has two chromosomes that determine sex. These are generally called X and Y in mammals. Normal pairing of chromosomes during the production of gametes will put one or the other in each sperm or ovum. In autosomal dominant inheritance, only one copy of a disease allele is necessary for an individual to be susceptible to expressing the phenotype. With each pregnancy, there is a one in two (50%) chance the offspring will inherit the disease allele. All affected individuals will have at least one parent who carries the disease allele - doesn't skip generations Across a population, the proportion of affected males should be equal to the proportion of affected females - both sexes are effected equally Male to male transmission can be observed - father to son. Crossing of two heterozygotes - probability of 75% birth of an affected child (25% will be homozygote dominant), 25% will be homozygote recessive - thus unaffected. Crossing of homozygous recessive with heterozygous - 50% probability of affected. deviations: Incomplete penetrance - allele are phenotypically show fewer carriers than we expected (genotypically contains the allele, but phenotypically doesn't show it) Variable expressivity - variable degree of manifestation of the character Change in phenotype as a result of exposure to external factor or due to effect of other genes (polygenic inheritance/multifactorial inheritance) Diseases with late onset - such as polycystic kidney disease and Huntington's Some diseases - such as Achondroplasia - may be Sporadic cases - new mutation Vertical transmission - can be observed in each generation

what is the Holliday junction model? (26)

Homologous chromosomes are both nicked at identical location 🡪 invade to the homologous chromosomes 🡪 formation of holiday junction at the point at which one chromosome strand pass from one DNA molecule to the other. Then, The holiday junction moves along the chromosome in a process called "Branch Migration". The exchange of nucleotide strands and branch migration produce a structure termed "Holiday intermediate". The invading strands base pairing with no complementary strands - formation of Heteroduplex DNA - which means that there are mismatches in the DNA bases. predicts noncrossover or crossover recombinant DNA - depends on whether cleavage is in the vertical or horitzontal plane

What is Klinefelter syndrome? what are the characteristics? what is the incidence? (39)

47 XXY Incidence of 1:1000 male live births - additional X chromosome. In childhood the presentation may be with clumsiness or mild learning difficulties, particularly low verbal skills: Verbal IQ 10-20 below unaffected siblings and controls. May show self-obsessed in behavior Adults tend to be slightly taller with long lower limbs 30% show gynecomastia - breast enlargement All are infertile because of the absence of sperm in their semen (azoospermia), with small, soft testes. Increased incidence of leg ulcers, osteoporosis, carcinoma of the breast. Treatment with testosterone from puberty is beneficial for the development of secondary sexual characteristics and the long-term prevention of osteoporosis. Equal chance for the inherited additional X chromosome from mother or from father. Maternally derived cases are associated with advanced age.

what are ring chromosomes? how do they form? (35)

A ring chromosome is formed when a break occurs on each arm of a chromosome leaving two 'sticky' ends on the central portion, that reunite as a ring. The two distal chromosomal fragments are lost, may cause serious effects. Ring chromosomes are often unstable in mitosis so that is common to find a ring chromosome in only a proportion of cells, the other cells in the individual are usually monosomic because of the absence of the ring chromosome centromere has divided transversely rather than longitudinally

what is the complementary factor of non-allelic genes? what's an example? Phenotype fission ratios? (4)

In complementation, or double recessive epistasis, both genes must complement and have a dominant expression to produce a different phenotype. if present alone, they remain unexpressed only when they are brought together will they be expressed in sweet peas, both the genes C and P are required to synthesize anthocyanin pigment causing purple colour. But absence of any one cannot produce anthocyanin causing white flower. So C and P are complementary to each other for anthocyanin formation. *Interaction of more than two complementary genes are possible. Phenotypic fission ratios: F2 generation: 9: 7 Bc - F1 x P ( aabb ): 1: 3

what are inversions? what are the types? (35)

Two-break rearrangement involving a single chromosome in which a segment is reversed in position (inverted). If the inversion segment involves the centromere it is termed a pericentric inversion, if it involves only one arm of the chromosome it is known as a paracentric inversion. Inversions are balanced rearrangements that rarely cause problems in carriers unless one of the breakpoints has disrupted an important gene. A pericentric inversion involving chromosome number 9 occurs as a common structural variant or polymorphism, and isn't thought to be of any functional importance. However, other inversions which aren't cause any problem to carriers, can lead to significant chromosome imbalance in offspring. An individual who carries a pericentric inversion can produce unbalanced gametes if a crossover occurs within the inversion segment during meiosis I, when an inversion loop forms as the chromosomes attempt to maintain homologous pairing at synapsis. A crossover within the loop will result in two complementary recombinant chromosomes, one duplication of the distal non-inverted segment, and deletion of the other end of the chromosome; the other having the opposite arrangement.

what is down syndrome? what is the risk? what are the clincal presentations?

aka Trisomy 21 - 47, XX or XY + 21 The overall birth incidence is approximately 1:800. There is a strong association between down syndrome incidence and advancing maternal age (1:1500 for 20 years old mom, 1:30 for 45 years old). The additional chromosome is maternal in origin in more than 90% of cases, as a result of non-disjunction in maternal meiosis I. Robertsonian translocations account for approximately 4% of all cases, one-third of which a parent is found to be a carrier. - simple trisomy of chromosome 21 (95%) - translocation form of trisomy 21 (4-5%) - mosaicism of trisomy and normal line (1%) - individuals generally have milder manifestations of Down syndrome Recurrence risk: If trisomy has already occurred there is more likely that it will occur again. In translocation cases, the recurrence risks vary from around 1-3% for male carriers up to 10-15% for female carriers, with the exception of very rare carriers of 21q21q translocation, for whom the recurrence risk is 100%. Prenatal diagnosis can be offered based on analysis of chorionic villi or cultured amniotic cells. Hypotonia - low muscle tone, reduced muscle strength Facial characteristics: Upward sloping palpebral fissures Small ears Protruding tongue Flattened nose and face Single plamar creases are found in 50% of the children, in contrast to 2-3% of the general population, IQ - ranging from 25-75, average 45. Social skills are well-advanced, generally happy kids. Adult height is 150cm. In the absence of a severe cardiac anomaly, which leads to early death in 15-20% of cases, average life expectancy is 50-60 years. Most affected adults develop Alzheimer disease.

what is FISH? steps to prepare cell? types of probes and their functions? (32)

fluorescence in situ hybridization FISH This diagnostic tool combines conventional cytogenetics with molecular genetic technology. It is based on the unique ability of a portion of single-stranded DNA (a probe) to combine with its complementary target sequence on a metaphase chromosome. preparation: In fluorescent in-situ hybridization (FISH), the DNA probed is labeled with a fluor-chrome which after hybridization with the patient's sample, allows the region where hybridization has occurred to be visualized using a fluorescence microscope. Cells arrested in metaphase, treated to make them swell. The chromosomes are treated in-situ, to make the DNA strand to denature Special DNA probes put in the sample; they are small pieces of single-stranded DNA with the sequence of the gene of interest. Probes are able to hybridize only with the complementary sequence - able to localize the loci of the specific gene of interest. Excess probes washed away. Either the probes are fluorescent or the probes going through chemical modification that allows fluorescent substance to bind them. The sample is viewed under fluorescent microscope. Different types of FISH probes: Centromeric probes - consist of repetitive DNA sequences found in an around the centromere of a specific chromosome. They were the original probes used for rapid diagnosis of the common aneuploidy syndromes (trisomy's 13, 18, 21). Chromosome-specific unique-sequence probes are specific for a particular single locus. Unique-sequence probes are particularly useful for identifying tiny submicroscopic deletions and duplications. Telomeric probes - a complete set of telomeric probes was been developed for all 24 chromosomes - i.e., autosomes 1-22 plus X Y). Using these, a method has been devised that enables the simultaneous analysis of the subtelomeric region of every chromosome by means of only one microscope slide per patient. This provided to be useful technique for identifying tiny subtelomeric abnormalities. Whole-chromosome paint probes - cocktail of probes obtained from different parts of a particular chromosome. When this mixture of probes is used together in a single hybridization, the entire relevant chromosome fluoresces. Chromosome painting is extremely useful for characterizing complex rearrangements, such a subtle translocations, and for identifying the origin of additional chromosome material.

What is epistasis? (1)

the interaction of genes that are not alleles, in particular the suppression of the effect of one such gene by another. one gene affects the phenotype of another because the two gene products interact; the phenotypic expression of a gene at one locus alters that of a gene at a second locus.

what are somatic and germ cell line mutations? how does the timing of when a mutation occurs affect the mutant sector? What would be the consequences of a somatic mutation in a cell of a fully developed organism? what are the major differences and outcomes? (36)

Genes and chromosomes can mutate in either somatic or germinal tissue, and these changes are called somatic mutations and germinal mutations, respectively. If a somatic mutation occurs in a single cell in developing somatic tissue, that cell is the progenitor of a population of identical mutant cells, all of which have descended from the cell that mutated. A population of identical cells derived asexually from one progenitor cell is called a clone. Because the members of a clone tend to stay close to one another during development, an observable outcome of a somatic mutation is often a patch of phenotypically mutant cells called a mutant sector. The earlier in development the mutation event, the larger the mutant sector will be. Mutant sectors can be identified by eye only if their phenotype contrasts visually with the phenotype of the surrounding wild-type cells. it is not passed on to offspring in the early stages of zygote development - a mosaic is formed, similar syndromes occuras in gametic chromosomal aberrations postnatally usually mean the development of tumors numerical and structural aberrations for example, Philadelphia chromosome - t (9; 22) in CML , t (8; 14) translocation in Burkitt 's lymphoma, etc. In diploids, a dominant mutation is expected to show up in the phenotype of the cell or clone of cells containing it. On the other hand, a recessive mutation will not be expressed, because it is masked by a wild-type allele that is by definition dominant to the recessive mutation. A second mutation could create a homozygous recessive mutation, but this event would be rare. If the mutation is in tissue in which the cells are still dividing, then there is the possibility of a mutant clone's arising. If the mutation is in a postmitotic cell—that is, one that is no longer dividing—then the effect on phenotype is likely to be negligible. Germinal mutation: A germinal mutation occurs in the germ line, special tissue that is set aside in the course of development to form sex cells. If a mutant sex cell participates in fertilization, then the mutation will be passed on to the next generation. An individual of perfectly normal phenotype and of normal ancestry can harbor undetected mutant sex cells. These mutations can be detected only if they are included in a zygote. E.g. X-linked hemophilia mutation in European royal families is thought to have arisen in the germ cells of Queen Victoria or one of her parents. The mutation was expressed only in her male descendants. Before a new heritable phenotype can be attributed to mutation, both segregation and recombination must be ruled out as possible causes. This requirement is true for both somatic and germinal mutations. can be numerical or structural

what are genetic methods of association analysis? what are they used for? what are the different types? what are the advantages and disadvantages of each? what are the applications? (17)

Genetic association is when one or more genotypes within a population occur together with a phenotypic trait, more often than would be expected by chance occurrence. The genetic association studies are used primarily for the detection of genetic tendency to multifactorial disease - i.e. genes that if found in the genome of certain individual - their presence increase (or decrease) the risk (the probability) that he will suffer from certain disease. Such associated genes are present significantly more in the group of patient with the disease (cases) compared to the healthy group, general population - controls. case-control studies (retrospective) - use subjects who already have a disease, trait or other condition and determine if there are characteristics of these patients that differ from those who do not have the disease or trait. In genetic case-control studies, the frequency of alleles or genotypes is compared between the cases (have disease) and controls (healthy). A different in the frequency of an allele or genotype of the polymorphism under test between the two groups indicates that the genetic marker may increases risk of the disease or likelihood of the trait. The strength of association between the disease and the marker allele is measured by relativity to the presence of the marker in the general population - RR - relative risk: RR = % of infected people having the marker allele/% of healthy people in population having the marker allele If RR = 1, it means that there is no relation between the marker allele to the disease; if RR is much bigger than 1 (means that there is much more infected people which having the marker allele in compare to the general population) - so association between the marker allele to the disease occurs. Advantages relatively fast, cheap, possibility of fast repetition suitable for the study of rare diseases suitable for chronic diseases and for diseases with long latency the possibility of monitoring even more risk factors for one disease Disadvantages the need to rely on human memory - ie the problematic retrospective assessment of exposure to the suspicious factor high risk of selection bias (systematic selection errors) - a clear definition of the source population is required, both the monitored and the control group study cohort - The incidence of the disease (consequence) is compared here. Here we move from cause to effect, looking for an answer to the question of whether exposure to a suspected factor (cause) causes the disease. E.g. Determining the relationship between smoking and lung cancer, where the studied group consists of smokers (exposed group) and the control group consists of non-smokers (unexposed group). Advantages accuracy, reliability objectivity - they can also assess multiple consequences of a single exposure Disadvantages financial and time consuming are not suitable for the study of rare diseases study SNPs The human genome project aimed to describe the entire structure of DNA. One of the great benefits of this project was the discovery of millions of variants of DNA segments, most of which were SNPs. The merger of several pharmaceutical companies, technology companies and academic centers created an international SNP consortium, which focused on compiling detailed SNP maps of the human genome and publishes its results in public places. Thanks to this step, individual SNPs can be used by independent laboratories for further research and patent issues do not have to be resolved. The determination of thousands of DNA samples from different patients used in typical studies using rapid sequencing devices must be well reproducible and should be kept to a minimum. Then the genotypes (SNPs) of individual patients are compared with their phenotype (clinical manifestations) using sophisticated statistical software. Association studies focused on SNPs research are divided into two types: direct testing of SNP functional manifestations for the given disease use of SNP as a marker for imbalance (LD) LD is generally defined by measuring the association between two genetic markers, so that they can be used to identify disease-related areas. family-based association studies - Standard linkage analysis requires information regarding the mode of inheritance, gene frequencies and penetrance. For multifactorial disorders, this information is not usually available. A possible solution is to look for regions of the genome that are identical by descent in affected sibling pairs. If affected siblings inherit a particular allele more or less often than would be expected by chance, this indicates that the allele or its locus is involved in some way in causing the disease. database formation

What is X-linked recessive inheritance? what are some examples? what are some deviations to this mode of inheritance? (14)

Since males are hemizygous for X-linked genes (they have only one X chromosome), any male with one copy of an X- linked recessive disease allele is affected. Females are usually carriers because they only have one copy of the disease allele. Affected males are related through carrier females. For a carrier female, with each pregnancy there is a one in two (50%) chance her sons will inherit the disease allele and a one in two (50%) chance her daughters will be carriers. Affected males transmit the disease allele to all of their daughters, who are then carriers, but to none of their sons. Women are affected when they have two copies of the disease allele. All of their sons will be affected, and all of their daughters will be unaffected carriers. Hemophilia A, B - impairs the body's ability to control blood clotting (lack in clotting factor). Color blindness (aka daltonism) Duchenne muscular dystrophy - muscle weakness usually begin around the age of four in boys and worsens quickly. May result in trouble standing up, most are unable to walk by the age of 12. Usually inherited from a person's parents, while one third of cases are due to a new mutation for the protein dystrophin, which is important to maintain the muscle fiber cell membrane. Becker's muscular dystrophy - slowly progressive muscle weakness of the legs and pelvis related to Duchenne in that both result from a mutation in the dystrophin gene; it's a less severe type of Duchenne. Deviations: partial manifestation of the disease in carriers; female X-inactivation - a process by which one of the copies of the X chromosome present in female is inactivates in order to prevent them from having twice as many X chromosome gene products as males - the choice of which X chromosome will be inactivated is random. The inactive X-chromosome is more condensed - heterochromatin form; forms a discrete body within the nucleus called a "Barr body".

what is the etiology of chromosomal abberrations? what are the types? what is a predisposing factor? (37)

can either be numerical or structural Numerical aberrations - see question 34: The main cause of aneuploidies - trisomy, monosomy - is a non-disjunction during meiosis of maternal or paternal gametes formation (or, less frequent, non-disjunction of zygote 🡪 mosaicism). Non-disjunction is an error in the distribution of homologous chromosomes in the first meiotic division, or error in separation of sister chromatids during second meiotic division. The main cause for non-disjunction is advanced maternal age. This may be associated with the prolonged meiotic arrest of maternal oocytes, potentially lasting for more than four decades; Inadequate hormonal activity and other factors associated with aging may contribute to error in the distribution of chromosomes (non-disjunction) as well, especially for the first meiotic division. Age of the father doesn't have pronounced effect. It is found, though, that with age, there is a decrease in the frequency of recombination of chromosomes. Because chiasmata stabilize the bivalents in late prophase in its meiotic division, if recombination doesn't occur, bivalent may disintegrate properly into univalent. Another reason for Aneuploidy - numerical abnormality is Anaphase lag. Anaphase lag described a delayed movement during anaphase, where one homologous chromosome or one chromatid fails to connect to the mitotic spindle apparatus, or is slowly drawn to its pole thus fails to be included in the reforming nucleus. Instead, the chromosome forms a micronucleus in the cytoplasm and is lost from the cell. The lagging chromosome isn't incorporated into the nucleus of one of the daughter cells - resulting in one normal daughter cell and one with monosomy. Anaphase lag is one of the reasons for either aneuploidy or mosaicism. structural aberrations Structural chromosome aberrations are a result of fractures, may be as result of exogenous or endogenous factors (see question 35, 56, 57). Factors that have the ability to produce chromosome breakage are called clastogenic factors (mutagenic agent giving rise to or inducing disruption or breakages of chromosomes, leading to sections of the chromosome being deleted, added, or rearranged). DSB- double strand break, in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. Endogenous factors - It may be a result of oxidative metabolism, action of topoisomerase during DNA replication and repair, DNA recombination (crossing over) etc. Exogenous factors that has clastogenic effects include radiation (ionizing, UV), chemical agents (alkylating agents, analogues of bases, heavy metals etc..). DBS can be induced directly in the case of ionizing radiation. DNS may occur indirectly (UV rays, free radicals, chemical substances) when the primary DNA damage elicit enzymatic reparations in a single strand, causes double-stranded DNA breaks. A predisposing factor for chromosome breaks and structural rearrangements may be transposable elements (transposons) which, when integrated into the genome , represent regions that are not homologous, which may activate repair mechanisms and induce breaks and submicroscopic interstitial deletions. Another predisposing factor for the occurrence of fractures are repeated (repetitive) genome-scattered sequences , created by retrotransposition and fixed in the genome ( LINEs = l ong in terspersed e lement s , SINE = s hort in terspersedand lements). These sequences form single-stranded loops that recognize the DNA repair mechanism, which can also lead to small deletions. After genome, scattered repeats represent regions of homology between different chromosomes, which in the case of breaks at these sites can lead to Robertson or reciprocal translocations, as well as inversely repetitive sequences can cause submicroscopic inversions. The presence of these scattered repetitive sequences is the reason for the non-random distribution of breaks, so-called "hot spots" in the formation of chromosomal aberrations.

what is gametogenesis? what are the types? where do they occur? (27)

Gametogenesis is a biological process by which diploid precursor cells (primordial germ cells) undergo cell division and differentiation to form mature haploid gametes. The production of gametes in males is called spermatogenesis, and in female - oogenesis.

What is the genealogical method? what are the uses? what are the limitations? what is penetrance, expressivity, consanguinity, coefficient of inbreeding (F), incomplete dominance, codominance, hemizygous? (11) make sure you can draw a pedigree with the appropriate symbols

Genealogy (Gene - origin, lineage; logy - knowledge) is the study of families by tracing their lineages and history. It is used for basic research, the genetic patterns of transmission of a certain character between family members. Use of diagrams and symbols to record kin connections (trying to reconstruct family trees). used for basic research, the genetic patterns of transmission of a certain character between family members. Genealogy is practical application in the field of consultancy in clinical genetics. Genealogy uses: Mitochondrial DNA and direct maternal lineages - mtDNA tests only the region that is hypervariable of the mtDNA, it may include SNPs needed to assign people to a maternal haplogroup (haplotype - group of gene that are inherited together from a single parent, haplogroup is group of haplotypes share a common ancestor with mutual SNPs). Direct paternal lineages - Y-chromosome is present only in males; Y-chromosome STR analysis (short tandem repeats) will reveal a haplotype, which should be similar among all male descendants of a male ancestor (paternity check; STR analysis also widely used in forensic analysis) Human migration - genealogical DNA testing methods are also in use on a longer time scale to trace human migratory patterns; e.g. they determined when the first humans came to north America and what path they follow In genetic genealogy, we make a use in DNA testing in combination with traditional genealogical and historical records to infer relationships between individuals. In DNA testing we determine the level and type of the genetic relationship and by the traditional pedigree, we depict graphically (pedigree) basic information about the occurrence of the selected character in a certain family; the proband is the person who led to the selection of a family for genealogical analysis. Penetrance - the frequency, which expressed as a fraction (or percentage), of people who are phenotypically affected, among those of a certain genotype. A autosomal dominant disorder in which only part of the people carrying the mutant allele display the abnormal phenotype - the trait is said to show incomplete penetrance (If all the people with the mutant allele show the abnormal phenotype - the trait is said to have complete (full) penetrance. Expressivity - variations in a phenotype among individuals carrying a particular genotype. Consanguinity - blood relation - is the property of being from the same kinship as another person; i.e the quality of being descended from the same ancestor as another person. Coefficient of inbreeding (F) - measures the probability that two genes at any locus in an individual are identical by descent from the common ancestor of the two parents. It measures the likelihood of genetic effects due to inbreeding to be expected based on a known pedigree; it holds only for Mendelian inheritance. Its definition and value holds regardless of whether the organism's genome actually contains such a gene. Therefore, the coefficient of inbreeding is a statistical value derived from the individual's pedigree and cannot be verified or measured exactly by looking at the individual's genome. Incomplete dominance - Incomplete dominance occurs when the phenotype of the heterozygous phenotype is distinct from and often intermediate to the phenotypes of the homozygous phenotypes. For example, the snapdragon flower color is either homozygous for red or white. When the red homozygous flower is paired with the white homozygous flower, the result yields a pink snapdragon flower. The pink snapdragon is the result of incomplete dominance. Codominance - Codominance is a relationship between two versions of a gene. Individuals receive one version of a gene (allele), from each parent. If the alleles are different, the dominant allele will be expressed, while the effect of the other allele - the recessive, is masked. In codominance, however, neither alleles are recessive and the phenotypes of both alleles are expressed. For example, in the ABO blood group system, chemical modifications to a glycoprotein (the H antigen) on the surfaces of blood cells are controlled by three alleles, two of which are co-dominant to each other (IA, IB) and dominant over the recessive i at the ABO locus. Thus IA and IB alleles are each dominant to i (IAIA and IAi individuals both have type A blood, and IBIB and IBi individuals both have type B blood, but IAIB individuals have both modifications on their blood cells and thus have type AB blood, so the IA and IB alleles are said to be co-dominant). Hemizygous: In a diploid organism, a hemizygous individual has only one copy of a particular gene (instead of the normally two copies - such as in the 22 autosomal chromosomes, which always come in homologous pairs, contain the same loci for same genes, with different variations - alleles). For example, all cytogenetically normal male humans are hemizygous for all X and Y loci.

for meiosis what are the disturbances? how it is regulated? (25)

Nondisjunction is the failure of homologous chromosomes or sister chromatids to separate properly during cell division. There are two forms of non-disjunction failure - failure to separate in meiosis I and in meiosis II. Regulation of meiosis - cell cycle of meiosis is similar to that of mitosis: As in the mitotic cycle, these transitions are regulated by combinations of gene regulatory factors, cyclin-Cdk complex and APC (anaphase promoting complex - ensures chromosomes are properly attached to the mitotic spindle before separation). First major regulatory transition occurs in late G1; second major transition occurs at the entry into metaphase I and is done by the meiotic check point - it blocks the entry into metaphase I if the recombination is not efficiently processed. To achieve proper segregation, each pair of homologous chromosomes must be linked to each other to maintain a certain level of tension between them. Such tension is supposed to help the assembly of spindles and generally depends on meiotic recombination.

what is Morgan's law? (1)

Number of linkage group is equal to the number of homologous pairs. genes are stored on a chromosome in a linear order genes of one chromosome form a linking group frequency of crossover is proportional to the distance of genes linkage is measured in cM Genes are always stored on a chromosome in a linear fashion . The genes of one chromosome form a linking group . The number of binding groups of an organism is identical to the number of pairs of homologous chromosomes of the respective organism. Gene exchange can take place between genes of a homologous chromosome pair via cross-over . The crossing-over frequency is proportional to the distance of the genes. These laws form the so-called chromosome theory of inheritance .

what is XXX and XYY syndromes? what are their characterisitics and incidence? (39)

XXX Females 0.1% (1:1000) of all females have a 47,XXX karyotype No physical abnormalities, but can show a mild reduction of between 10-20 points in intellectual skills and oppositional behavior. Additional X chromosome is of maternal origin, arises from an error in meiosis I. Reduced fertility, irregular maturation, inconstant psychomotor retardation XYY Males 1:1000 incidence in male, it is found that 2-3% of males who are in institutions because of learning difficulties or antisocial criminal behavior has XYY karyotype. Most 47, XYY men have neither learning difficulty nor a criminal record, may show emotional immaturity and impulsive behavior. Fertility is normal. Physical and stature appearance is usually higher than average - 180cm. Intelligence is mildly impaired, 10-20 IQ points below average. Additional Y chromosome must arise either as a result of non-disjunction in paternal meiosis II or as a post-zygotic event.

What is crossing over and when does it occur? How does it work (draw mechanism) and what is the importance? (26)

Recombination is the exchange of genetic information between DNA molecules. * does not occur in sex chromosomes The process of crossing over takes place during pachytene phase of prophase I - in which regions of chromosomes are exchanged and genes are shuffled between homologous chromosomes (homologous recombination). It is the basis for intra-chromsomal recombination - in which new combinations of alleles are created. Before crossing-over take place, synapsis process must occur - during synapsis (zygotene phase of prophase I) the parental and maternal homologous chromosomes pair with each other - form a structure called a "tetrad" or "bivalent". During the pairing of homologous chromosomes a complex of RNA and proteins called Synaptonemal complex assemble, and helps to hold the bivalent together, contribute to its stability 🡪 can remain associated throughout the long prophase of meiosis I (which is extremely prolonged - years - in female gametes production). By the time prophase I ends, the synaptonemal complex dissociate and allow the homologous chromosome to separate along most of their length - except for some parts which remain held together in a structure called "Chiasma" - thus, at the end of Prophase I - the chromosomes attached together only in the chiasma part, which corresponds to a crossover between non-sister chromatids. Most bivalents contain more than one chiasma - indicating that more than one crossover point exist between homologous chromosomes. importance - As said, recombination due to crossing over is an extremely important genetic process which is the major source of the genetic variation in sexually reproductive species. It helps to produce individuals with genetic material which differs from both of their parents. Thus, the recombination of genes is important for evolution - if due to crossing over a new beneficial character achieved - by the process of natural selection the appropriate chromosome is passed down to future generations.

what is x-linked dominant inheritance? what are some examples? (14)

As in autosomal dominant inheritance, only one copy of a disease allele on the X chromosome is required for an individual to be susceptible to an X-linked dominant disease. Women are affected 2x more than men because illness may inherit from both parents (whereas men cannot inherit the mutant allele from their dad) Both males and females can be affected, although males may be more severely affected because they only carry one copy of genes found on the X chromosome. Some X-linked dominant disorders are lethal in males. When a female is affected, each pregnancy will have a one in two (50%) chance for the offspring to inherit the disease allele. When a male is affected, all his daughters will be affected, but none of his sons will be affected. Vertical mode of inheritance - victim has at least one parent affected (no skipping generation - directly from parent to child) Vitamin D resistant rickets - bone deformity leading to fractures include short stature and genu varum due to impaired metabolism of vitamin d, phosphorous or calcium. Defective mineralization or calcification of bones before epiphyseal closure. Incontinentia pigmenti - skin abnormalities - loss of melanin from the epidermis, accumulation in melanophages in the upper dermis. Adults usually have lines of light-colored skin (hypopigmentation) on their arms and legs. Blistering rash at early infancy, development of wart-like skin growths

what is autosomal recessive inheritance? what does a pedigree look like? what are the deviations? (13)

Two copies of a disease allele are required to expressing the phenotype. appears in children of unaffected parents, may skip generations both males and females affected Increases likelihood of diseases in consanguineous marriages Sometimes called horizontal transmission because there is no expression seen in previous generations by the ancestors and relatives who carry the mutation; rather, the mutation travels silently (unobserved) within the family and is expressed by siblings in a single generation. Deviations: Genetic heterogeneity (capacity of various genes to cause the same trait independently - which is different from polygenic inheritance which is a trait that determined by two or more genes) - Albinism - subject to different genes, the offspring of two recessive homozygotes (affected) may be pigmented Deafness - subject to 10 different genes that may effect on the phenotype - the offspring of two deaf can also hear normally Pseudo-dominance - inheritance of a recessive trait mimics a dominant pattern; for example, in the case of loss of the dominant allele due to deletion or of a deficiency mutation in the dominant allele in one homologue - the recessive allele will be observed.

genetic aspects of population? what is a population? What is a gene pool? what does it mean if only one allele exists for a particular locus in the population? what are the phenotypic and genotypic ratios?

population - group of individuals of the same species that live in the same area and interbreed, producing fertile offspring population's genetic makeup is described by its gene pool - consists of all copies of every type of allele at every locus, in all members of the population - if only one then the allele is said to be fixed in the gene pool - and all individuals are homozygous for that allele if there are two or more alleles for a particular locus in a population, individuals may either be heterozygous or homozygous

what's epistasis regarding the interaction of non-allelic genes? What's an example? what are the types? how would the phenotypic ratios differ? (4)

The phenotypic expression of a gene at one locus alters that of a gene at a second locus. recessive - recessive homozygote suppresses the expression of all genotypes at the other locus F2 generation: 9: 3: 4 Bc - F1 x P ( aabb ): 1: 2: 1 the dominant allele (E) results in the deposition of either black or brown pigment (depending on the genotype of the first loci - BB Bb - black, bb - brown). But if the Lab is homozygous recessive for the second locus (ee), then the coat is yellow, regardless of the genotype at the black\brown locus - golden labs. In this case, the gene for pigment deposition (E/b) is said to be epistatic to the gene that codes for black or brown pigment (B/b). dominant - the dominant allele of one gene is involved in expressing the resulting phenotype regardless of the genotype in the 2nd locus - agouti rats F2 generation: 12: 3: 1 Bc - F1 x P ( aabb ): 2: 1: 1

what are the factors that affect the phenotype of an individual? (gene-environment interaction) (7)

factors that affect an individual's phenotype genetic factors environmental factors gene-environment interaction Environmental factors can either regulate or repress some parts of an organism's genetic program (through its regulatory systems) ; however, they can also modify it - affect the resulting form of the character. Certain pathological forms of some traits of the organism arise mainly on the basis of external factors , the genotype affects them only to a small extent. In general, the phenotype is influenced by genotype and environmental influences. Rate, which is the character in its intended form of inherited quantitatively expressed as the heritability (heritability). Characters monogenic determined (ie. Qualitative characteristics) environment are much less controllable , while the largest impact environmental factors have on the characteristics of polygenic inheritance (ie. Quantitative) for quantitative traits we are often interested in what are the relative shares of hereditary component and environmental factors in the variance (variance) of phenotypic traits , then the relative share of genetic factors in the overall variance of traits is called heritability H^2 = V G / V P V G - phenotype variance caused by genetic factors V P - total variance of the phenotype value H^2 can theoretically take values from 0 to 1 , if it is equal to 0, the phenotype variance is fully dependent on environmental factors , at H^2 = 1, on the other hand, environmental factors have no effect and all observed variance depends on genetic factors. During development, environmental factors may affect gene expression and differentiation - there is a tight relation between the gene expression of a cell and the environment in which it is found (e.g. woman that smoking during pregnancy, or exposes to other mutagens). Most quantitative characteristics usually show polygenic inheritance pattern; in addition - they are influenced by environmental factors. One genotype, under differential environmental conditions, is able to produce a range of phenotypes. This range of phenotypes is known as the norm of reaction, and its extremes are limited by the genotype - e.g. skin color - a person pigmentation may be highly affected by exposure to sun, but its genotype will never allow him to be completely black. Threshold model - environmental factors that effect on the genotype may "accumulate" till the threshold reached. These factors, together with the genetic factor, contribute to the susceptibility of individual to certain disease. If enough of the susceptibility factors are present, the threshold is reached and the disease develops. Environment may also lead to change in genotype of the person - formation of mutation; exposure to mutagens in the environment can result in alternation of the DNA - may change gene expression.

what is oogenesis? when does it occur? what are the results? draw a schematic (27)

Oogenesis Diploid primordial germ cells within the ovaries divide mitotically to produce oognia. Like spermatogonia, oognia can undergo repeated rounds of mitosis or they can enter into meiosis; the oognia arranged in clusters surrounded by a layer of flat epithelial cells known as follicular cells. Initiation of oognia mitotic division begins at the end of the 2nd month and by the 5th month of female fetus development - the number of oognia reaches its maximum number - 7 million - and from that point, most of the cells go through apoptosis. The remaining cells are called primordial follicles - consist of primary oocyte which is arrested in diplotene phase prophase I, and surrounded by follicular cell. Arrest may be up to 50 years! Maturation during puberty Primary oocytes remain in Prophase I and do not finish their first meiotic division till puberty - apparently because of OMI - oocyte maturation inhibitor - a substance which is secreted by follicular cells. At the beginning of puberty, approximately 400,000 primoridal follicles (include primary oocyte) are present; eventually, fewer than 500 will be ovulated. At puberty, each month 15-20 follicles begin to mature. Each menstrual cycle, these primary oocyte that continue to develop continues meiosis I. At the end of meiosis I - in-equal cytoplasmic division during cytokinesis produce the first polar body (which usually discarded) and the secondary oocyte - which now contains haploid cells, still with two sister chromatids in each chromosome. Immediately after meiosis I, the haploid secondary oocyte initiates meiosis II. The process is also halted at the metaphase II stage until fertilization. At ovulation, secondary oocyte with 23 chromosomes and two sister chromatids (46) is waiting for fertilization to occur - when fertilization occurred, meiosis II has completed, and mature egg is formed (as well as second polar body) - ready for forming zygote with the sperm.

structure of eukaryotic chromosomes? what is cytogenetics? draw a chromosome. what is a chromosome? what are the types of chromatin? what does chromosome pattern tell us about the cell's activity? (30)

A chromosome is a packaged and organized structure containing most of the DNA of a living organism. DNA is not usually found on its own, but rather is structured in long strands that are wrapped around protein complexes called nucleosomes that consist of proteins called histones. The DNA in chromosomes serves as the source for transcription. Chromosomes are the factors that distinguish one species from another and that enable the transmission of genetic information from one generation to the next. Their behavior at somatic cell division in mitosis provides a means of ensuring that each daughter cell retains its own complete genetic complement. Similarly, their behavior during gamete formation in meiosis enables each mature ovum and sperm to contain a unique single set of parental genes. Cytogenetics - the study of chromosomes and cell division. Chromatin is the chromosomal material in a largely uncoiled state. Two types of chromatin can be distinguished under the EM or LM: Heterochromatin - tightly packed form of DNA, it has been associated with several functions, from gene regulation to the protection of chromosome integrity. Some of these roles can be attributed to the dense packing of DNA. Euchromatin is the lightly packed form of chromatin (DNA, RNA and protein) that is rich in gene concentration, and is often (but not always) under active transcription. Euchromatin comprises the most active portion of the genome within the cell nucleus. 92% of the human genome is euchromatic. The remainder is called heterochromatin. Chromatin is composed mainly of coiled strands of DNA bound to basic proteins called histones and to various non-histone proteins. The basic structural unit of chromatin and histones is the nucleosome which has a core of eight small histones (two copies of each histones - H2A H2B H3 H4), around which is wrapped DNA with about 150 base pairs. Each nucleosome also has a larger linker histone (H1) that binds both wrapped DNA and the surface of the core. DNA bound to nucleosomes is then folded further in the next order of chromatin organization (30nm fiber). Higher orders of chromatin coiling into microscopically visible stained structures, the chromosomes, which are especially important during the condensation of chromatin for mitosis and meiosis. 8 Histones with DNA wrapped = Nucleosome 🡪 Chromatin 🡪 Chromosome The chromatin pattern of a nucleus is a guide to the cell's activity. Generally cells with lightly stained nuclei are more active in protein synthesis then those with condensed, dark nuclei. In light stained nuclei with much euchromatin (=chromatin loose and not dense, ready for transcription for later protein synthesis, and thus light stained) and few heterochromatin, more DNA surface is available for transcription of RNA

what are deletions? what are the types? what are some clinical outcomes? what are insertions? (35)

A deletion involves loss of part of a chromosome and results in monosomy for that segment of the chromosome. A very large deletion is usually incompatible with survival, and as a general rule any deletion resulting in loss of more than 2% of the total haploid genome will have a lethal outcome. Large chromosomal deletion can be visualized under the light microscope, such deletion syndromes include Wolf-Hirschnhorn and Cri Du Chat, which involve loss of material from the short arms of chromosomes 4 and 5, respectively. Submicroscopic microdeletions were identified with the help of high-resolution prometaphase cytogenetics by FISH method, and include Prader-Willi and Angelman syndrome. Microscopically visible deletions of the terminal portions of chromosomes 4 and 5 cause the Wolf-Hirschhorn (4p-) and cri-du-chat (5p) syndromes. In both conditions, severe learning difficulties is usual, often with failure to thrive. Cri-du-chat syndrome derives its name from the characteristic cat-like cry of affected neonates - a consequence of underdevelopment of the larynx. Both conditions are rare, with estimated incidences of approximately 1:50,000 births. Another symptoms are severe mental retardation, microcephaly, motor disorders, growth retardation, congenital heart diseases. Patients with Wolf-Hirschhorn syndrome are retarded, have cleft lip and palate, microcephaly, cardiac malofrmations and hypospadias (opening of the urethra is on the underside of the penis instead of at the tip An insertion occurs when a segment of one chromosome becomes inserted into another chromosome. If the inserted material has moved from elsewhere in another chromosome then the karyotype is balanced (translocation). Otherwise, an insertion causes an unbalanced chromosome complement.

what are translocations? what causes them? what are the types? (35)

A translocation refers to the transfer of genetic material from one chromosome to another. A reciprocal translocation is formed when a break occurs in each of two chromosomes with the segments being exchanged to form two new derivative chromosomes. Usually, in reciprocal translocation, the chromosome number remains at 46 and, if the exchanged fragments are of roughly equal size, a reciprocal translocation can be identified only by detailed chromosomal banding studies or FISH (fluorescence in-situ hybridization, see question 32). For unknown reason, a particular balanced reciprocal translocation involving the long arms (q) of chromosomes 11 and 22 is relatively common. The overall incidence of reciprocal translocation is 1:500. Robertsonian translocation is a particular type of reciprocal translocation in which the breakpoints are located at, or close to, the centromeres of two acrocentric chromosomes. The total chromosome number is reduced to 45. Because there is no loss or gain of important genetic material (except of the rRNA), this is a functionally balanced rearrangement. The overall incidence of robertsonian translocations is 1:1000, most common being fusion of the long arms of chromosomes 13 and 14 (13q14q). The problem with these balanced robertsonian translocations, is that they are carriers of potentially risk for trisomy of their offspring. For example, a carrier of a 14q21q translocation can produce 6 types of gametes: Segregation at meiosis: balanced reciprocal translocation has different behavior in meiosis, when they can segregate to generate significant chromosome imbalance, lead to pregnancy loss or birth of infant with abnormalities. Problems arise at meiosis because the chromosomes involved in the translocation cannot pair normally to form bivalents. Instead, they form a cluster known as pachytene quadrivalent. in a case of 2:2 segregation - the quadrivalent can be segregate in different ways: If alternate chromosomes segregate to each gamete, the gamete will carry a normal balanced haploid complement, the embryo will either have normal chromosomes or carry the balanced rearrangement. However, if adjacent chromosomes segregate together, it will result in the gamete acquiring an unbalanced chromosome complement: If the gamete inherits the normal number 11 chromosome (A) and the derivative number 22 chromosome (C) - then fertilization will result in an embryo with trisomy for the distal long arm of chromosome 11 (C - yellow) and monosomy for distal part of long arm of chromosome 22 (C - purple). In a case of 3:1 segregation - three chromosomes segregate to one gamete, with only one chromosome in the other gamete. E.g. if chromosome 11 (A), 22 (D) and the derivative 22 (C) segregate together to a gamete - this will result in the embryo being trisomic for the material present in the derivative 22 chromosome - tertiary trisomy. From the other hand, the gamete that will be formed from the B derivative, will lack the information of long arm distal part of chromosome 11, as well as information of chromosome 22.

what are the cellular junctions? what do they allow for? what are the specialized cellular junctions? (21)

Adhesive connection (contact) Zonula adhaerens - band adhesive connection of lateral surfaces, clamps into actin filaments. Desmosome - a point anchor connection, clamps into intermediate filaments. Hemidesmosome - connection of the basal part of the cell with the extracellular matrix. Tight junction Zonula occludens - connection around the perimeter of the cell, prevents paracellular diffusion. Communication connection Nexus (gap junction) - allows selective diffusion of molecules and fast communication between neighboring cells. Free apical surface Microvilli - cytoplasmic protrusions, their dense complex forms a so-called brush border. Stereocilia - long immobile microlicles (eg in the middle ear ). Cilia - are elongated moving protrusions (eg in the fallopian tube ). Basolateral surface It participates in the cohesion of epithelial cells, including special intercellular connections and connections to the basement membrane (eg basal labyrinth ).

What is cell signaling? how can it be classified? what are the types? what are the methods? (23)

Cell signaling is part of a complex system of communication that governs basic activities of cells and coordinates cell actions. The ability of cells to perceive and correctly respond to their environment is the basis of development, tissue repair, immunity and normal tissue homeostasis. Errors in cellular information processing are responsible for diseases such as cancer, autoimmunity and diabetes. based on the type of signal: Mechanical signals are the forces exerted on the cell and the forces produced by the cells - can be sensed and responded by the cells. Biochemical signals are the biochemical molecules such as proteins, lipids, ion and gases - and they are further classified based on the distance between signaling and responder cells - short (all types except of endocrine) or long distances (endocrine): Intracrine - signals are produced by the target cell that stay within the cell Autocrine - signals are produced by the target cell 🡪 secreted 🡪 affect the target cell itself via receptors (in some cases, such as immune cells, autocrine cells can target cells close by if they are the same type of cells) Juxtacrine - signals target adjacent, touching, cells and transmitted along cell membranes via protein or lipid components integral to the membrane. Direct contact Paracrine - signals target cells in the vicinity of the emitting cell; Short distance contact Synaptic - form of paracrine - this signaling is specific for the nervous system - nerve cell produces a chemical signal - neurotransmitter - that is transmitted to other nerve cell via synaptic cleft (speed - 100 m/s) Endocrine - signals target distant cells - endocrine cells produce hormones that travel through the blood to reach all parts of the body. Large distance contact. Direct contact - Some cell-cell communication requires direct cell-cell contact; sometimes through gap junctions (nexus) that connect the two cells cytoplasm (e.g. cardiac muscle cells). Signaling molecules are molecules that capable of signal transmission - can be categorized according to their chemical nature into several groups: Lipophilic molecules - steroid hormones, thyroid hormones, fatty acid derivatives, retinoids Peptide(protein) molecules - peptide hormones (e.g. insulin, vasopressin, glucagon), growth factors, cytokines Amino acid derivatives - hormones (e.g. adrenaline), neurotransmitters (e.g. GABA, glutamate, glycine), mediators (e.g. histamine) Small organic molecules and ions - NO, CO

what is polygenic inheritance? what are the deviations from the Mendelial pattern? How is variance calculated? hereditary equation? (3)

Characteristics that are influenced by more than one gene (follows Gaussian, bell curve, example: skin color, height (180 genes)) Polygenic inheritance refers to the phenomenon by which multiple different allele pairs have a similar and additive effect on a given trait (e.g. skin color). - these vary in the population by gradations - quantitative characters The inheritance of characters determined by a single gene deviates from simple Mendelian pattern when (I) alleles are not completely dominant or recessive, (II) when a particular gene has more than two alleles (ABO), or (III) when a single gene produces multiple phenotypes (pleiotropy). Vp = total phenotypic variance = Vg + Ve + Vge Vg = genetic variance Ve = environmental variance Vge = an interaction between them (e.g. - exposure to sun causes melanocytes in the skin to produce more melanin) The heritability is determined by: Vg/Vp = H^2

what are the repair mechanisms for double-stranded breaks? (37)

DSBs are repaired in two ways: Homologous recombination requires the presence of homologous sequences, ie the presence of a sister chromatid (i.e. in the S or G2 phases of the mitotic cell cycle ), or the presence of a homologous chromosome (the way in which meiotic recombination takes place) Non-homologous joining of broken ends takes place mainly in the G0, G1 phases of the cell cycle, ie without the presence of a homologous template, but can take place in all phases of the cell cycle. Somatic recombination of immunoglobulin genes, T cell receptor genes and isotype switching takes place in this way. This second method of joining fractures is more error-prone because it uses short homologies that are near the broken ends of chromosomes and modification of the broken ends, which can lead to submicroscopic deletions or insertions at the site of the joints when joining them. Both methods - homologous recombination and non-homologous joining of broken chromosome ends - can lead to flawless DSB elimination as well as mutations and chromosomal aberrations due to faulty repair.

what is a genotype? what is genotypic variation and what can contribute to it? what is the importance of variation? what are mutations? what are the types? what is recombination? (6)

Genotype is the set of all genes of an individual, the genetic constitution of an individual. genome is the genetic material of an organism. It consists of DNA (or RNA in RNA viruses). The genome determines the scope and degree of phenotypic characters of the individual. Genotypic variation: Meiosis and random fertilization generate genetic variation among offspring of sexually reproducing organisms due to independent assortment of chromosomes, crossing over in meiosis I, and the possibility of any sperm fertilizing any egg. Variations are simply differences in genetic sequence Variation can be seen at every genetic level: In the DNA In the genes In the chromosomes In the proteins I n the function of proteins This abundance of genetic variation provides the raw material on which natural selection works. If the traits achieved by particular combinations of alleles are better suited for a given environment, organisms possessing those genotypes will be expected to thrive and leave more offspring, ensuring the continuation of their genetic complement. gnetic variation is important because a population has a better chance of surviving and flourishing than a population with limited genetic variation. Genetic diversity also decreases the occurrence of unfavorable inherited traits. caused by mutations The phenotype of an organism can be affected by small-scale changes involving individual genes. Random mutations are the source of all new alleles, which can lead to new phenotypic traits. Large-scale chromosomal changes can also affect an organism's phenotype. Physical and chemical disturbances, as well as error during meiosis, can damage chromosomes in major ways or alter their number in cell. nondisjunction aneuploidy - monosomic, trisomic polyploidy alterations in chromosomal structure - deletion - duplication - inversion - translocation Recombination is the rearrangement of DNA material between already existing alleles (forms of the gene) to form new alleles. This process created new individuals with different combinations of alleles from a combination of their parents. This happens as a result of crossover.

what is the importance of heritability in medicine? what are its limitations? (9)

Heritability allows us to statistically predict the phenotypes of offspring on the basis of their parent's phenotype; the greater the value for heritability, the greater the role of genetic factors. If heritability equals one - means that the variance phenotype caused only by genetic factors; therefore - we know that certain disease occurs due to genetic factors. If the value of the heritability is less than one - we know that there is significance of environmental (external) factors relate to the disease - such information, should lead to interventions in order to changing life style, diet or medication administration. E.g. treatment of multifactorial hereditary obesity - change of diet and lifestyle will definitely change the phenotype of obesity. It also has some limitations: It doesn't indicate the degree to which a characteristic is genetically determined It's impossible to calculate heritability for individuals There is no universal heritability for a characteristic - the value of heritability for a characteristic is specific for a given population in a given environment. Even when heritability is high, environmental factors may influence a characteristic

how can heritability be calculated? 3 ways (9)

Heritability by elimination of variance components: one way of calculating the broad-sense heritability is to eliminate one of the variance components. We could make VG equals to 0 by raising genetically identical individuals, causing VP to be equal to VE: we might raise highly inbred identically homozygous individuals in a defined environment and measure their phenotypic variance to estimate VE. We could then raise a group of genetically variable individuals, and measure their phenotypic variance - VP. Using the VE calculated on the genetically identical individuals (this time, we know how much variance is caused by environmental factors), we could obtain the genetic variance of the variable individuals: VG (genetically varying individuals) = VP (genetically varying individuals) - VE (genetically identical individuals). The values - .41 - indicates that 41% of the variation in spotting of guinea pigs was due to differences in genotype. Heritability by parent-offspring regression: estimating heritability by comparing the phenotypes of parents and offspring. When genetic differences are responsible for phenotypic variance, offspring should resemble their parents more than they resemble unrelated individuals. To calculate the narrow-sense heritability in this way, we first measure the characteristic on a series of parents and offspring. The data are arranged into families, and the mean parental phenotype is plotted against the mean offspring phenotype. Each data point in the graph represents one family; the value on the x (horizontal) axis is the mean phenotypic value of the parents in a family, and the value on the y (vertical) axis is the mean phenotypic value of the offspring for the family. If genes and environment both contribute to the differences in phenotype, both heritability and the regression coefficient will lie between 0 and 1. The regression coefficient therefore provides information about the magnitude of the heritability. A complex mathematical proof demonstrates that, in a regression of the mean phenotype of the offspring against the mean phenotype of the parents, narrow-sense heritability (h2) equals the regression coefficient (b). Heritability and degrees of relatedness: A third method for calculating heritability is to compare the phenotypes of individuals having different degrees of relatedness. This method is based on the concept that the more closely related two individuals are, the more genes they have in common. Monozygotic (identical) twins have 100% of their genes in common, whereas dizygotic (nonidentical) twins have, on average, 50% of their genes in common. If genes are important in determining variability in a characteristic, then monozygotic twins should be more similar in a particular characteristic than dizygotic twins. By using correlation to compare the phenotypes of monozygotic and dizygotic twins, we can estimate broad-sense heritability. A rough estimate of the broad-sense heritability can be obtained by taking twice the difference of the correlation coefficients for a quantitative characteristic in monozygotic and dizygotic twins: H^2 = 2(rMZ - rDZ) rMZ equals the correlation coefficient among monozygotic twins and rDZ is the correlation coefficient among dizygotic twins. This calculation assumes that the two individuals of a monozygotic twin pair experience environments that are no more similar to each other than those experienced by the two individuals of a dizygotic twin pair.

steps of eukaryotic transcription? (44)

Initiation Transcription begins with the binding of RNA polymerase, together with one or more general transcription factor, to a specific DNA sequence referred to as a "promoter" to form an RNA polymerase-promoter "closed complex" (called a "closed complex" because the promoter DNA is fully double-stranded). RNA polymerase, assisted by GTFs, then unwinds approximately 14 base pairs of DNA to form an RNA polymerase-promoter "open complex" (called an "open complex" because the promoter DNA is partly unwound and single-stranded) that contains an unwound, single-stranded DNA region of approximately 14 base pairs referred to as the "transcription bubble." RNA polymerase, assisted by one or more general transcription factors, then selects a transcription start site in the transcription bubble and catalyzes bond formation to yield an initial RNA product. Elongation During elongation, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome. One strand of the DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy. RNA Polymerase is reading from 3'🡪5' and synthesis complementary RNA strand from 5'🡪3' - an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one fewer oxygen atom) in its sugar-phosphate backbone). Termination RNA synthesis will continue along the DNA template strand until the polymerase encounters a signal that tells it to stop, or terminate, transcription. The termination can be: Spontaneous - a sequence on the DNA template generates a sequence on the newly formed RNA which is self-complementary - the RNA folds back on itself forming a loop - hair pin. Roh (ρ) depended termination - ρ protein is a ATPase with helicase activity; it binds a recognition site near the 3' end and moves on the newly formed RNA until it reaches the RNA polymerase. The helicase than separates the RNA from the DNA, releasing the RNA.

what is mosaicism? how does it form? (35)

Mosaicism - can be defined as the presence in an individual, or in a tissue, of two or more cell lines that differ in their genetic constitution but are derived from a single zygote - they have the same genetic origin. Chromosome mosaicism usually results from non-disjunction in an early embryonic mitotic division with the persistence of more than one cell line. If, for example, the two chromatids of a number 21 chromosome failed to separate at the 2nd mitotic division in a human zygote, this would result in the four-cell zygote having two cells with 46 chromosomes, one with 47 (trisomy 21) and one with 45 (monosomy 21). The cell line with 45 chromosomes would probably not survive, so that the resulting embryo would be expected to show approximately 33% mosaicism for trisomy 21 - it accounts for 1-2% of all clinically recognized cases of down syndrome. Mosaicism can also exist at a molecular level if a new mutation arises in a somatic or early germline cell division.

What is aneuploidy? what are some autosomal syndromes? (38)

Numerical abnormalities involve the loss or gain of one or more chromosomes, referred to as Aneuploidy. A nother case is the addition of one or more complete haploid complements, known as polyploidy (triploidy, tetraploidy). Loss of a single chromosome results in monosomy, whereas gain of one or two homologous chromosomes is referred to as trisomy or tetrasomy . Aneuploidy - monosomy, trisomy, tetrasomy Polyploidy - triploidy, tetraploidy Trisomy: The presence of an extra chromosome is referred to as trisomy. Most cases of Down syndrome are due to the presence of an additional number of 21 chromosome; hence, down syndrome is often known as trisomy 21. Other autosomal trisomies compatible with survival are Patau syndrome (trisomy 13) and Edwards syndrome (trisomy 18). Most other autosomal trisomies result in early pregnancy loss, with trisomy 16 being a particularly common finding in first-trimester spontaneous miscarriages. The presence of an additional sex chromosome - X or Y - has only mild phenotypic effects. Trisomy 21 is usually caused by failure of separation of one of the pairs of homologous chromosomes during anaphase of maternal meiosis I. This failure of the bivalent to separate is called non-disjunction. Less often, trisomy can be caused by non-disjunction occurring during meiosis II, when a pair of sister chromatids fails to separate. Either way, the gamete receives two homologous chromosomes - disomy; if subsequent fertilization occurs, a trisomic conceptus results. Studies using DNA markers have shown that most children with an autosomal trisomy have inherited their additional chromosome as a result of non-disjunction occurring during one of the maternal meiotic divisions. Non-disjunction can also occur during an early mitotic division in the developing zygote. This results in the presence of two or more different cell lines, a phenomenon known as mosaicism. The most favored explanation for the cause of non-disjunction is that of an aging effect on the primary oocyte.

duplicated genes and their non-allelic interaction? what are the types and their phenotypic ratios? (4)

Sometimes a character is controlled by two non-allelic genes whose dominant alleles produce the same phenotype whether they are alone or together. In Shepherd's purse (Capsella bursa-pastoris), the presence of either gene A or gene B or both results in triangular capsules; when both these genes are in recessive forms, the oval capsules produced Noncumulative dominated Each of the genotypes in which there is at least one gene with a dominant allele leads to the realization of the same phenotypic expression. Only recessive homozygotes in both loci are phenotypically different . Phenotypic fission ratios: F2 generation: 15: 1 Bc - F1 x P ( a1a1a2a2 ): 3: 1 Cumulative dominated A certain phenotype ("maximum") depends on the presence of a dominant allele in both interacting genes (the number of dominant alleles does not affect the phenotypic expression). A different phenotypic expression ("lower") manifests in the presence of the dominant allele (one or both) in only one of the interacting genes. And recessive homozygotes in both genes show a completely different ("third") manifestation of the trait. (For example, AB- black coat, A-bb or aaB- brown coat, aabb light coat.) Phenotypic fission ratios: F2 generation: 9: 6: 1 Bc - F1 x P ( aabb ): 1: 2: 1 Cumulative without dominance The intensity of the expression depends on the total number of dominant alleles, regardless of their order in the participating loci. Phenotypic fission ratios: F2 generation: 1: 4: 6: 4: 1 Bc - F1 x P ( aabb ): 1: 2: 1

what is the function of DNA? (41)

The DNA carries the genetic information in the cell and is capable of self-replication and synthesis of RNA. The sequence of nucleotides determines individual hereditary characteristics. The main role of DNA molecules is the long-term storage of information. We can refer to DNA as recipe, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but there are other DNA sequences that have structural purposes, or are involved in regulating the use of this genetic information. Genes carry biological information that must be copied accurately for transmission to the next generation each time a cell divides to form two daughter cells. DNA encodes information through the order, or sequence, of the nucleotides along each strand. Each base—A, C, T, or G—can be considered as a letter in a four-letter alphabet that spells out biological messages in the chemical structure of the DNA. Organisms differ from one another because their respective DNA molecules have different nucleotide sequences and, consequently, carry different biological messages. Genes contain the instructions for producing proteins. The DNA messages must therefore somehow encode proteins. The properties of a protein, which are responsible for its biological function, are determined by its 3D structure, and its structure is determined in turn by the linear sequence of the amino acids of which it is composed. Chargaff rule - in any sample of DNA double strand, the amount of adenine is equal to amount of thymine, and amount of guanine is always equal to cytosine

what are the major post-translational modification in eukaryotes? (45)

Trimming (Proteolysis) - many proteins are initially made as large, precursor molecules that aren't functionally active (zymogen form). Endoproteases remove portions of the protein chain, activate the molecule. Some precursor proteins are cleaved in the ER or the Golgi apparatus; others are cleaved in developing secretory vesicles. Covalent attachments - proteins may be activated or inactivated by the covalent attachment of a variety of chemical groups: Phosphorylation - catalyzed by one of a family of protein kinases (to hydroxyl side chain of serine, threonine or tyrosine) and may be reversed by the action of cellular protein phosphatases. It may increase or decrease the functional activity of the protein. Glycosylation - proteins that are destined to become part of a plasma membrane or to be secreted from a cell have carbohydrate chains added by O or N glycosidic bonds (depends on the AA the glycosylation performed on - Arginine - N glycosylation in RER, Serine/Threonine - O glycosylation in Golgi) occurs in the golgi or ER (glycoproteins formation). Methylation - add methyl group to increase hydrophobicity of amino acid (e.g. methylation of Histones) Acetylation - add acetyl group to decrease polarity (e.g. histones acetylation - opens the chromatin to increase access to DNA) Protein folding - proteins must fold to assume their functional state. May be either spontaneous or facilitated. Protein degradation - proteins that are defective or destined for rapid turnover are often marked for destruction by ubiquitin.

What is Patau syndrome? what is Edward's syndrome? what are the clinical presentations? (38)

Trisomy 13: 47, XX/XY +13 Trisomy 18 - 47, XX or YY +18 The very severe conditions share many features in common. Incidence for Patau is 1:15000, Edwards 1:5000 most infants dying in first days or weeks of life, though most cases are detected prenatally, often leading to termination. In the unusual event of longer term survival, there are severe learning difficulties. Cardiac abnormalities in 90% of cases Both disorders occur more frequently with advanced maternal age, the additional chromosome being of maternal origin. Approximately 10% of cases are caused by mosaicism (non-disjunction during mitosis of zygote) or unbalance rearrangements, particularly Robertsonian translocations in Patau syndrome Edwards severe psychomotor retardation and failure congenital malformations of the kidneys and heart microcephaly and prominent headers receding chin, low-set malformed auricles characteristic holding of fingers in clenched fists with crossing the second finger over the third and the fifth over the fourth nails tend to be hypoplastic Patau - severe mental and growth retardation severe CNS malformations, holoprosencephaly eye malformations that may merge into one or even a complete absence craniofacial dysmorphia with severe cleft palate and lip earlobes malformed and low set congenital heart and urogenital defects present postaxial polydactyly of the upper and lower limbs

How can heritability be quantified in twins? what are the types of twin studies? (15)

Twins-reared-together - both of the twins grow up in the same family - exposed to the same external influences; in the case of monozygotes we can then recognize their genetic susceptibility to disease; the probability that just one of the twins becomes ill is very low as compared with dizygotic twins. Twins-reared-apart - this type of studies are based on the comparisons of twins, who were adopted into different families and the conditions in which they grew up weren't the same - we can evaluate the role of environment on the disease. If no or very few cases of a disease are found in only one of the twins, then it can be assumed that the trait has a high value of heritability - meaning that it is determined primarily by genetic factors. If just one of the twins suffers from the genetic disease - we assume the influence of Threshold effect - some critical limits were crossed (e.g. physical, chemical, biological factors). Co-twin control studies - if both of the twins have some health problem, we offer the treatment to only one of them, and then we compared the results and effectiveness of the cure; it is not ethical to try with the drug therapy. Example for Co-twin control studies - there was a study few years ago, when the doctor tried to teach one of the twin to climb the stairs, whereas the second didn't practice it. However, both of them were able to climb the stairs after few weeks, the trained one did it a week earlier; it shows that the environmental influence could help them to learn something fast H = (Kmz - Kdz)/(1 - Kdz) where Kmz is the relative representation of concordant pairs in the group of MZ twins and Kdz is the relative representation of concordant pairs in the group of DZ twins.

What are Robertsonian translocations? what happens to the chromosomal count? what gametes can be produced from someone who is a carrier? draw it out (35)

Two telocentric/nearly telocentric chromosomes combine to make one larger, more metacentric chromosome. (Loses a bit of DNA, usually not noticeable). Robertsonian translocation is a particular type of reciprocal translocation in which the breakpoints are located at, or close to, the centromeres of two acrocentric chromosomes. In Robertsonian translocations, which results from the breakage of two acrocentric chromosomes (13, 14, 15; 21, 22) at or close to their centromeres, with subsequent fusion of their long arms - centric fusion. The short arms of each chromosome are lost, this being of no clinical importance as they contain genes only for rRNA, for which there are multiple copies on the various other acrocentric chromosomes. The total chromosome number is reduced to 45. Because there is no loss or gain of important genetic material (except of the rRNA), this is a functionally balanced rearrangement. The overall incidence of robertsonian translocations is 1:1000, most common being fusion of the long arms of chromosomes 13 and 14 (13q14q). The problem with these balanced robertsonian translocations, is that they are carriers of potentially risk for trisomy of their offspring. For example, a carrier of a 14q21q translocation can produce 6 types of gametes:

what is the cell cycle? what 3 things must happen for a cell to reproduce successfully? what are the phases? (22)

a sequence of mutually coordinated processes that lead from one cell division to the next cell division Its genetic information must be copied The copies of genetic information must be separated from one another Cell must divide G 1 phase - lasts 9 hours, the cell grows; course: doubling of cell mass, intensive synthetic processes - RNA , proteins. The cell grows, a supply of nucleotides is formed, and enzymes are synthesized for future replication of nuclear DNA main control point of the cycle is here S phase - 10 hours, DNA synthesis ; nuclear DNA replication (doubling of DNA), simultaneous rapid coupled synthesis of histones (H2A, H2B, H3, H4 & H1) to form new nucleosomes and chromatin strand 3´ → 5´ strand replication: DNA polymerase, leading replication ("leading strand") 5´ → 3´ strand replication: DNA polymerase, discontinuous replication (lagging strand, Okazaki fragments) Telomerase: synthesizes DNA at the ends of a chromosome at the end of the chromatids joined at the centromere site ; double gene gene G 2 phase - 4.5 hours, the cell prepares for division. mitosis (with cytokinesis) dependent on the completion of DNA replication in S phase course: synthesis and activation of proteins (for chromosome condensation, mitotic apparatus formation and destruction of the nuclear envelope), ends with the onset of mitosis here lies the 2nd control node of the cell cycle - it decides whether the cell actually enters mitosis Some cells go into a resting phase after mitosis, the so-called G 0 . From there, after various lengths of time, they can re-enter the cell cycle - activation - or remain at rest. This applies, for example, to neurons or cells of the ocular lens..

what is dihybridism? what is a dihybrid cross? what is the phenotypic ratio produced from two heterozytoes? how does this support independent assortment draw the chart (2)

analysis of two different hereditary traits or from those that appear according to two different genes, regardless of whether it is the same character. This assumes that, in the behavior of the inheritance, two pairs of genes are involved. a cross that examines the inheritance of two different traits the individual can either be homozygous or heterozygous for each trait F1 plants are dihybrids (inidividuals that are heterozygous for each trait) Mendel identified his second law of inheritance (law of independent assortment) by following two characters at the same time - such as seed color (yellow and green) and seed's shape (round and wrinkled). Mendel knew that the allele for yellow seeds is dominant and the allele for green seeds is recessive (Y, y). For the seed-shape character, the allele for round is dominant (R), and the allele for wrinkled is recessive (r). He made a cross between two true-breeding pea varieties that differ in both of these characters - cross between a plant with yellow-round seeds (YYRR) and a plant with green-wrinkled seeds (yyrr). The F1 plants will be dihybrids - individuals heterozygous for the two characters being followed in the cross - YyRr. The F1 plants, of genotype YyRr, exhibit both dominant phenotypes - yellow seeds with round shape. One hypothesis said, wrongly, that the character transmitted as a package (most be YY, RR) - but according to Mendel's second law (after experiments on F1 generation gametes and F2 offspring) - he discovered the idea of independent assortment. The gametes of F1 generation are composed of dihybrid cross of two heterozygotes - YyRr, to produce four types of gamets: YR (yellow-rounded), Yr (yellow-wrinkled), yR (green-rounded), yr (green-wrinkled) in a ratio of 9:3:3:1. Reminder: law of independent assortment: genes are packaged into gametes in all possible allelic combinations. Two or more genes assort independently - that is, each pair of alleles segregates independently of each other pair of alleles during gamete formation.

what is CGH? steps? advantages, disadvantages? (32)

comparative genomic hybridization In this test, we hybridize to two differently labeled groups of probes on a standard chromosome. One color indicates the standard chromosomal equipment, ie the same as the chromosome to which we hybridize. The second color marks the DNA from the examined tissue. Under normal conditions, both labeled samples hybridize equally to the sample chromosome. If a given region of a chromosome is duplicated in a tissue of interest, it is more likely to adhere to the sample than labeled standard DNA. Therefore, in this case, the color of the test sample shines more brightly in the area. If there is a deletion in a given region, the DNA of the test tissue will not hybridize in that region. Therefore, the color of standard chromosomal equipment will predominate in the fluorescence microscope. Comparative genomic hybridization is a molecular cytogenetic method for analyzing copy number variations (CNVs - defined as a phenomenon in which sections of the genome are repeated and the number of repeats in the genome varies between individuals in the human population) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions (a portion of a whole chromosome). The process involves the isolation of DNA from the two sources to be compared, most commonly a test and reference source, independent labelling of each DNA sample with fluorophores of different colors (usually red and green), denaturation of the DNA so that it is single stranded, and the hybridization of the two resultant samples in a 1:1 ratio to a normal metaphase spread of chromosomes, to which the labelled DNA samples will bind at their locus of origin. Using fluorescence microscope and computer software, the differentially colored fluorescent signals are then compared along the length of each chromosome for identification of chromosomal differences between the two sources. A higher intensity of the test sample color in a specific region of a chromosome indicates the gain of material of that region in the corresponding source sample, while a higher intensity of the reference sample color indicates the loss of material in the test sample in that specific region. A neutral color (yellow when the fluorophore labels are red and green) indicates no difference between the two samples in that location. CGH is only able to detect unbalanced chromosomal abnormalities. This is because balanced chromosomal abnormalities such as reciprocal translocations, inversions or ring chromosomes do not affect copy number, which is what is detected by CGH technologies. CGH does, however, allow for the exploration of all 46 human chromosomes in single test and the discovery of deletions and duplications, even on the microscopic scale which may lead to the identification of candidate genes to be further explored by other cytological techniques.

what is a disease? What are multifactorial disorders? what is polygenic inheritance vs. multifactorial inheritance? what are some examples? how can the risk be calculated? (10)

A disease is a pathophysiological response to internal or external factors. A disorder is a disruption to regular bodily structure and function. A syndrome (e.g. down syndrome) is a collection of signs and symptoms associated with a specific health-related cause. A trait is a specific characteristic of an organism. Traits can be determined by genes or the environment, or more commonly by interactions between them. The genetic contribution to a trait is called the genotype. The outward expression of the genotype is called the phenotype. Polygenic inheritance involves expression of a phenotype that is being determined by many genes at different loci. Each gene exerts an additive effect and the total effect is accumulative. Multifactorial inheritance is the type of inheritance followed by traits that are determined by multiple factors - both environmental and genetic. Environmental factors interact with many genes to generate a normally distributed susceptibility. So, multifactorial inheritance is the extension of the polygenic inheritance model just in that external factors influence the final shape of the character; the proportion of the genetic information on the total variation in phenotype is called Heritability. A mutation resulting in disease is often recessive - both alleles must be mutated for the disease to be expressed phenotypically - however, in the case of multifactorial inheritance - a disease may also be the result of the expression of mutant alleles at more than one locus. When more than one gene is involved, the disease referred to as multifactorial inheritance (or polygenic inheritance, if there are no environmental factors involved). Some diseases such as myocardial infraction, congenital birth defects, cancer, diabetes, mental illnesses and Alzheimer diseases cause along with morbidity or premature mortality in 2 out of 3 individuals during their lifetime. Many of these diseases show clustering among families. However, their inheritance pattern doesn't follow that of single gene disorders (Mendelian pattern of inheritance); rather, these kind of diseases are thought to result from complex interactions between genetic and environmental factors (i.e. multifactorial inheritance pattern). The reason why these diseases show the clustering among families is that family members share a significant portion of their genetic information, and further more - they are often exposed to the same environmental triggers. Family members experience the same gene-gene interactions and gene-environment interactions that may trigger, accelerate or in some cases even protect against the disease process. In family where the multifactorial hereditary disease has never seen before - the risk is equal to the risk of population. In the case of families with documented occurrence of this disease, the risk can be calculated by Edward's formula; f - relative frequency of the disease in the population, n is the number of 1st degree relatives with the disease. : r = (f)^1/2 * n examples Retinitis Pigmentosa - the simplest example of a polygenic trait, causing retinal degeneration. Two rare mutations in two loci encoding for proteins for photoreceptors - when heterozygotes for only one of the loci the patient do not develop the disease, but heterozygotes for both mutations do develop it. No environmental factor influences the course of disease. Alzheimer disease - fatal neurodegenerative disease, the most common cause of dementia among elderly. There are 3 rare AD forms of Alzheimer's but the common form doesn't show mendelian inheritance - it shows family cluster, and the risk increased 3-5 fold for first degree relatives. Monozygotic concurrence is 50%. There is an increased risk after head and brain trauma - which is environmental factor. Common medical problems such as heart disease, diabetes and obesity do not have a single genetic cause - they are likely associated with the effects of multiple genes in combination with lifestyle and environmental factors. Mental illness - affects about 4% of the population, for example - Schizophrenia (1%). Hypothyroidism Colon Cancer Breast Ovarian Cancer High blood pressure Arthritis the reason why these diseases show the clustering among families is that family members share a significant portion of their genetic information, and further more - they are often exposed to the same environmental triggers.

structure and function of the eukaryotic cell what are its components, membrane-bound and non-membrane bound? components of plasma membrane? function and characteristics? basic functions? (21)

Cell is the smallest structural and functional unit of the organism able to live by itself, formed from other cells by cell division - proliferation The cell composed of nucleus and cytoplasm, contains organelles limited by the plasma membrane. The organelles can be classified to membranous and non-membranous organelles. Membranous organelles: RER and SER - Rough and Smooth endoplasmic reticulum. RER - protein synthesis by polyribosomes which are destined to secretion out of the cytosol. SER - phospholipid and steroid hormones synthesis, detoxification of toxins, Calcium release (abundant in muscle cells, sarcoplasmic reticulum) Golgi apparatus - modifies and packs proteins synthesized in the RER. It is of particular importance in processing proteins for secretion Lysosomes - site of intracellular digestion; they are cellular organelles that contain acid hydrolase enzymes that break down waste materials and cellular debris. They can be described as the stomach of the cell. Mitochondria - aerobic respiration and production of ATP; in addition, it accounts for cellular differentiation and apoptosis, maintaining control of the cell cycle and cell growth. It contains mtDNA and ribosomes. Peroxisomes - contain enzymes involved in lipid metabolism. Nucleus - The Nucleus contains a masterplan for all cell structures and activities encoded in the DNA of the chromosomes. It also contains the molecular machinery to replicate its DNA and to synthesize and process all types of RNA. Macromolecular transfer between the nuclear and cytoplasmic compartments is regulated, and the proteins needed for the nucleus to function imported from the cytoplasm. The nucleus has oval shape and usually found in the center of the cell. Its main components are the nuclear envelope, chromatin consisting of DNA and associated proteins, and a specialized region of chromatin called the nucleolus. Nuclear Envelope composed of two membrane layers; At sites where the inner and outer membranes of the nuclear envelope fuse, the resulting lipid-free spaces contain nuclear pore complexes - nucleoporins - which regulates most bidirectional transport between the nucleus and the cytoplasm. heterochromatin vs. euchromatin Non-membranous organelles: Free floating Ribosomes - protein synthesize for the cell use Centrosome - composed of pair of centrioles, organizing center for microtubule. Cytoskeleton - Microtubules, Actin and Intermediate filaments All eukaryotic cells are enveloped by a limiting membrane composed of: Lipids - Phospholipids and Cholesterol, glycolipids Proteins Chains of oligosaccharides (small number of simple sugars, monosaccharides) linked to phospholipid and protein molecules. Asymmetric Fluid Mosaic Membrane phospholipids consist of two non-polar hydrophobic tails, linked to a charged polar hydrophilic head group - stability to the membrane - Amphiphilic characteristic. Cholesterol is also present (1:1 ratio with phospholipids), insert among the phospholipid fatty acid and restricting their movement. Integral transmembrane proteins - transfer of chemical substances by channels, transporters or pumps. Peripheral proteins - exhibit looser association with one of the two membranes surfaces (inner our outer surfaces). The distribution of membrane proteins is different in the two surfaces of the cell membranes - asymmetric characteristic. The external surface of the cell shows a carbohydrate rich region called the glycocalyx, a layer which made of carbohydrate chains linked to membrane's proteins and lipids. The glycocalyx has a role in cell recognition and attachment to other cells and to extracellular molecules. The membrane characterized by fluid mosaic appearance because of the combination of the fluid nature of the lipid bilayer, and the mosaic appearance of the membrane proteins. Cell membrane functions: Selective barrier which regulate the passage of materials into and out of the cell in order to keep constant ion concentration - regulates the intracellular environment. Carry out number of specific recognition and regulatory functions Interactions of the cell with its environment including adhesion, cell to cell signaling. Intercellular connections Desmosome See the Cell Connections page for more information . Adhesive connection (contact) Zonula adhaerens - band adhesive connection of lateral surfaces, clamps into actin filaments. Desmosome - a point anchor connection, clamps into intermediate filaments. Hemidesmosome - connection of the basal part of the cell with the extracellular matrix. Tight junction Zonula occludens - connection around the perimeter of the cell, prevents paracellular diffusion. Communication connection Nexus (gap junction) - allows selective diffusion of molecules and fast communication between neighboring cells.

what is chromosomal examination and what are the indications? (40)

Chromosomal examination is one of the basic methods of clinical genetics. The main objective of this examination - the patient - doing so in order to exclude numerical or structural chromosomal abnormalities. The examination of karyotype may be by standard banding techniques (e.g. Giemsa G-band) or by any of the methods of molecular cytogentics (e.g. - FISH method). Prenatal diagnositics: Pregnancy women older than 35 years Pregnancy found in higher risk of congenital chromosomal aberrations due to family history, single parent after cancer treatment, irradiation, pregnancy after assisted reproduction (fertility medication). Pregnancy, whose progress could by hindered by severe environmental factors with clastogenic effect (e.g. post-exposure to ionizing radiation) Determine gender of the fetus in the case of a family of major genetic sex-related diseases Postnatal diagnostics: Investigating the karyotype of born individuals - baby, child or adult. Material for testing is usually peripheral blood (leukocytes) or skin fibroblasts. The main indications include: Suspected chromosomal aberrations - phenotype corresponding to one of the typical syndromes, multiple congenital malformations, psychomotor and mental retardation etc. In the case of recurrent spontaneous abortions or frequent abortions in the family's history Chromosomal defects in the family history People with disorders of sexual development; examination of infertility among infertile couple; disorders of spermatogenesis in men. Karyotype examination by donors of oocytes and sperm. Karyotype examination of cells from solid tumors or blood elements with haematological malignancies. Has both diagnostic and prognostic (predict the likely course of a medical condition) significance, may also decide appropriate therapy. Examination of acquired chromosomal aberrations among people exposed to clastogenic environment (Chemicals, ionizing radiation). Preimplantation diagnostics: Preimplantation genetic diagnostics (performed within the In Vitro Fertilization program ) is rarely used in case of high risk of congenital chromosomal aberrations (especially in persons with balanced chromosomal aberrations, who are really at risk of developing an unbalanced aberration in the offspring). The material to be examined is most often blastomeres of a developing embryo.

what are gene duplications? what are dicentric chromosomes? (35)

Gene Duplications Any duplication of a region of DNA that contains a gene. It can arise as products of several types of errors in DNA replication and repair machinery. dicentric chromosome is an abnormal chromosome with two centromeres. It is formed through the fusion of two chromosome segments, each with a centromere, resulting in the loss of acentric fragments (lacking a centromere) and the formation of dicentric fragments.

what is mitosis? what are centromeres and the centrosome? what are the stages? how do the chromosome number change during mitosis? how it is regulated? what disturbances can occur? draw a schematic (24)

M-phase is the part of the cell cycle in which the copies of the cell's chromosomes - sister chromatids - separate and the cell undergoes division. Mitosis occurs only in Eukaryotic cells, in prokaryotic cell which lack a nucleus the division occurs by binary fusion. Human have a total of 46 chromosomes - 22 pairs of homologous autosomal chromosomes, and two additional sex chromosomes - X X or X Y. It is important to mention that the 22 pairs of homologous chromosomes contain the same genes but code for different traits in their allelic forms. Centromeres - constricted region of the chromosome to which spindle fibers attach - centromere sequences in the DNA are the binding sites for the kinetochore to which the fiber spindle attach. Kinetochore is a protein structure which assembles on the centromere and links chromosomes to microtubule from the mitotic spindle. Centrosomes - MTOC - microtubule organizing center - duplicated during S phase, composed of 2 centrioles. Centrioles are involved in the organization of the mitotic spindle and in the completion of cytokinesis. *Aster microtubules - attach the mitotic spindle to the cell membrane. Prophase - chromosome condensation, mitotic spindle formation - the chromosomes (composed of 2 identical chromatids) condense and becoming visible under a light microscope. The mitotic spindle - microtubules that move the chromosomes in mitosis, forms. The spindle grows out from pair of centrosomes that migrate to opposite sides of the cell. The centrosome is composed of two microtubules structures - Centrioles - oriented at right angles to each other which are duplicated during S phase of the cell cycle. Prometaphase - disintegration of the nuclear membrane - it marks the start of prometaphase, in which the microtubules enter the nuclear region and make contact with each chromosome - anchor to the kinetochore of one of the sister chromatids while a microtubules from the opposite centrosome attaches to the other sister chromatid. Metaphase - chromosomes arranged in the metaphase plate. Spindle-assembly checkpoint occurs in metaphase - it delays the onset of anaphase until all chromosomes are aligned on the metaphase plate and kinetochores are attached to spindle fibers from opposite poles; unless the chromosomes are not properly aligned, the checkpoint blocks the destruction of cyclin B. Anaphase - sister chromatids separate and move toward opposite spindle poles. After the chromatids have separated, each is considered a separate chromosome. Telophase - arrival of the chromosomes at the spindle poles; nuclear membrane re-forms around each set of chromosomes, producing two separate nuclei within the cell. Chromosomes relax and lengthen. Cytokinesis - cytoplasm divides - at the cytokinesis furrow, action-myosin contractile ring is formed and drives the cleavage process. The key components of this ring are filamentous protein actin and motor protein myosin. Counting chromosomes and DNA molecules At certain times, chromosomes are un-replicated; at other times, each possesses two chromatids. Chromosomes sometimes consist of a single DNA molecule, and sometimes of doubled DNA molecules. Chromosomes are counted by the number of centromeres - thus during S phase, number of chromosomes do not change (46) even though there is formation of sister chromatids - but the DNA molecules is duplicated (92). - in anaphase - number of chromosomes = number of DNA molecules Errors Errors can occur during mitosis - especially during early embryonic development in human. Multinucleated cells - occurs when its nucleus has divided normally, but the cytoplasm didn't divide (Cytokinesis failure) - such cells look giant and have several nuclei. Syncytial cells - several cells fuse together Increased rate of mitosis - this is typical in tumors non-disjunction regulation: G2/M check-point composed of cyclin B and CDK1 - it helps prepare the duplicated chromosomes for segregation and induces the assembly of the mitotic spindle. M-Cdk complexes accumulate during the cell cycle till threshold in which high enough concentration leads to start M-phase. From metaphase to anaphase - anaphase promoting complex is a complex of many proteins which function in triggering the transition from metaphase to anaphase by tagging specific proteins for degradation.

what are abnormalities in chromosome structure? what are the types? how do they form? (35)

Structural chromosome rearrangements results from chromosome breakage with subsequent reunion in a different configuration. They can be balanced or unbalanced. In balanced rearrangements the chromosome complement is complete, with no loss or gain of genetic material. Consequently, balanced rearrangements are generally harmless with the exception of rare cases in which one of the breakpoints damages an important functional gene. However, carriers of balanced rearrangements are often at risk of producing children with an unbalanced chromosomal complement. When a chromosome rearrangement is unbalanced, the chromosomal complement contains an incorrect amount of chromosome material and the clinical effects are usually serious. translocations - reciprocal - Robertsonian deletions duplications dicentric chromosomes insertions inversions ring chromosomes isochromosomes mosaicism spontaneous - by inducing mutagenic effects (ionizing radiation, chemicals) this creates chromosomal or chromatid breaks exposed ends of DNA tend to reconnect to the original or another free end due to the complementarity of terminal bases - the emergence of abnormal structures and a complex number of complex structural rearrangements stable aberration - the altered chromosome contains centromeres and telomeres it is passed regularly to daughter cells unstable abberation - centromere (acentric fragment) or telomere (ring chromosome) is lost segregation into daughter cells is not regular , leading to elimination or further changes in these structures congenital the phenotypic effect varies from type to type we distinguish between balanced and unbalanced changes balanced - there is a normal amount of genetic material in the cells there was no loss or dwelling of part of the chromosomal equipment carriers usually have no phenotypic manifestations , except in rare situations where a significant functional gene is damaged by a chromosomal break there is a serious risk to their offspring - gametes with unbalanced chromosomal equipment may form translocation, inversion, advertising unbalanced - a change in the genome in the sense of the absence or dwelling of a certain part of the genetic material this condition usually has serious clinical consequences deletion, duplication, ring chromosome, isochromosome acquired - we determine when testing mutagenic and genotoxic effects of chemicals and the environment on humans we use the methods of classical cytogenetic analysis to evaluate the number of chromosome breaks and rearrangements we evaluate in preparations from short-term (48 hours) cultured peripheral blood lymphocytes - we find structural and numerical deviations in 100-200 mitoses in practice we test exposure to various pollutants at selected workplaces (chemicals in health care, poisons and cytostatics in laboratories and clinical wards) - we evaluate the so-called group risk for employees of one workplace as the average value of CHD in the group normal finding is the occurrence of aberrant mitoses up to 2%, higher values ​​are the reason for tightening safety measures in the workplacethe limit values are 2-5% - a reason for repeating the examination with a time lagvalues above 5% indicate high mutagenic exposure or increased susceptibility of the individual

what is genetic linkage? what is the recombination fraction, how do you calculate it? what is Morgan's number? what are the practical uses ? what is the relevance of bond strength and what does that tell us about the type of linkage? (5)

The principle that genes/alleles that are physically close to each other on a chromosome tend to not get separated in meiosis and recombination. They are typically inherited together. some alleles are often more inherited in the same combination as they were in the genotype of the parents - this combination creates the haplotype linkage is a deviation from Mendelian genetics (goes against law of independent assortment) Genes located in the same chromosome tend to stay together during inheritance, this tendency is called linkage; Genes are arranged in a linear fashion in the chromosomes; The linkage is broken down by the process of crossing over occurring during meiosis; The intensity of linkage between two genes is inversely related to the distance between them in the chromosome; Coupling and repulsion phases are two aspects of linkage (haplotypes) Theta - is a measure of the distance separating two loci - an indication of the likelihood that a crossover will occur between them. If two loci are not linked, then theta equals 0.5 - because on average, genes at unlinked loci (or different chromosomes) will segregate together during 50% of all meiosis. If theta equals 0.05, this means that on average the alleles will segregate together 19 times out of 20 (recombination occur only 1 to 20), spans from 0 to 0.5 Morgan's number is the proportion of recombinants in the total number of offspring Centimorgans is the unit of measurement for genetic linkage, is known as map unit (m.u) or centimorgan - cM. If two loci are 1 cM apart, a crossover occurs between them once in 100 meioses (theta = 0.01). Centimorgans are a measure of the genetic, or linkage, distance between two loci. This is not the same as physical distance, which is measured in base pairs (kb - kilobases - 1000 bp) used for the contrstruction of genetic maps and used is for indirect diagnostics For the construction of genetic maps , there are problems with the use of recombination fraction as a measure of distance (it is not additive, does not reflect the interaction of chiasms, ...), so many mathematical transformations of rivers have been introduced . fraction and introduced a unit of map distance - Morgan (M), or more often centimorgan (1cM = 1 / 100M). only for small binding distances , 1 cM corresponds to 1% recombination, the maximum value of θ is 0.5, ie 50%, while the length of the chromosome after summing up the individual sections in genetic mapping can be as much as 120 to 150 cM In the case of genetic linkage , the registration of the AaBb genotype may mean two different haplotypes, ie AB / ab or Ab / aB the first possibility when the dominant or recessive alleles of both loci on the same chromosome are dominant is called cis binding or coupling the second possibility, the combination of one dominant and one recessive allele on one chromosome is called trans or repulsion binding Writing specific haplotypes is very important to correctly determine which offspring is recombinant and which is non-recombinant in order to calculate the recombinant fraction: Determine the number of recombinant offspring - count the proportion of offspring who exhibit traits that are recombinant; that is, contain alleles from each parent. For example, say you are breeding a particular plant, and count 40 offspring with a recombinant trait and 60 non-recombinant offspring. Add the recombinant and non-recombinant offspring. Using the example above, adding the two categories (40 and 60) gives 100. Divide the number of recombinant offspring by the sum of the recombinant and non-recombinant offspring. In this example, dividing 40 by 100 gives 0.4. This is the recombinant fraction. Bond strength: the strength of linkage bond between genes can be determined by the distance between two genes, i.e., the greater the distance, lower will be the linkage strength. The linkage is broken down by the process of crossing over occurring during meiosis. Complete linkage: When genes are so closely associated that they are always transmitted together, do not undergo crossing over, linkage between them is considered complete. Incomplete linkage: Complete linkage between genes on the same chromosome is rare. As a rule, linkage is not complete, and the gene pairs in most linkage groups, assort at least partially independent of each other.

what is transcription in eukaryotes? what are the steps? what enzymes are involved? what are some common promoter sequences? where does it occur? (44)

Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA (mRNA) by the enzyme RNA polymerase. Both DNA and RNA are nucleic acids, which use base pairs of nucleotides as a complementary language. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes the nucleotide uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. Transcription is divided into initiation, promoter escape, elongation and termination. Eukaryotic transcription involves separate polymerases for the synthesis of rRNA, tRNA, mRNA (in contrast to single RNA polymerase composed of 5 subunits that found in prokaryotic cells - 2 alpha + omega, betta, betta-tag, and with different sigma factor for each gene). In addition, eukaryotic transcription depends on transcription factors, which bind to distinct sites on the DNA, either within the core promoter region, or in different distance from it (cis acting elements). For TFs to recognize and bind to their specific DNA sequences, the chromatin structure in that region must be relaxed (eurchromatic) by chromatin remodeling to allow access to DNA. Most actively transcribed genes are found in a relatively relaxed form of chromatin (euchromatin), while inactive segments found in highly condensed heterochromatin. Chromatin remodeling based on covalent modification of histones, e.g. acetylation (Histone acyltransferase), which introduce acetyl group on positively charge histones. Histones, the proteins that responsible for eukaryotic DNA organization (4 pairs of histones - nucleosome - connected by linker H1), have large amount of Arginine and Lysine amino acids which are basic amino acids, i.e. positively charged in physiological pH and thus, naturally, there are attracted to the negatively charged phosphate groups of the nucleotides, leads to heterochromatin form. Following acetylation of the histones - the positive charge is masked, and the DNA becomes less compacted. RNA polymerase, together with one or more general transcription factor, binds to promoter DNA. The promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5' region of the sense strand) RNA polymerase and its transcription factors create a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides. RNA polymerase adds RNA nucleotides (which are complementary to the nucleotides of one DNA strand) - Elongation, which also involves a proofreading mechanism that can replace incorrectly incorporated bases. RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand. Hydrogen bonds of the RNA-DNA helix break, freeing the newly synthesized RNA strand. The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex. TATA box: in some genes transcribed by RNA polymerase II (mRNA), TATAA (nearly identical to that of the prinbow box of prokaryotic), is found about -25 (upstream) bases of the transcription start site. In the majority of genes no TATA box is present. Instead, different core promoter elements are present - "Inr" (initiator) or DPE (downstream promoter elements - +25). All the promoters are found on the same strand of DNA as the gene being transcribed - thus they are "cis-acting". These sequences serve as binding sites for proteins known as general transcription factor - which in turn, interact with each other and with RNA Polymerase II. General transcription factors - GTFs - are the minimal requirements for recognition of the promoter, recruitment of RNA Pol II to the promoter, and initiation of transcription. General transcription factors encoded by different genes and synthesized in the cytosol. In contrast to the prokaryotic polymerase holoenzyme, eukaryotic polymerase II doesn't itself recognize and bind the promoter. Instead, it needs GTFs - e.g. TFIID - which recognizes and binds TATA box. TFIIF -brings the polymerase to the promoter. TFIIH, has helicase activity, melts the DNA, and in addition - it has kinase activity, which phosphorylates polymerase and activates it. TFIID - recognize promoter regions TFIIF - takes polymerase to the promoter region TFIIH - helicase activity - creating of transcription bubble, and kinase activity - phosphorylates and activates polymerase II. Beyond the general transcription factors - there are regulatory elements and transcription activators: Upstream of the promoter are additional consensus sequences. Proximal regulatory elements, such as CAAT and GC boxes and distal regulatory elements such as different kinds of enhancers elements. Proteins known as transcriptional activators bind these regulatory DNA elements: Transcriptional activators that bind to promoter proximal elements regulate the frequency of transcription initiation. Transcriptional activators that bind to promoter distal elements mediate the response to signals, such as hormones, and regulate which genes are expressed at a given point in time. Enhancers are special DNA sequences that increase the rate of initiation of transcription by RNA Polymerase II - they can be located both down or upstream of the transcription start site, be close/far from the promoter and occur on either strand of the DNA. Enhancers contain the response elements (HRE) - that binds transcriptional activators. By bending or looping the DNA, these enhancer-binding proteins can interact with other transcription factors bound to a promoter and with RNA Polymerase II - thereby, stimulating transcription. Similarly, silencers exist - which also may bind to specific transcription factors and reduce gene expression. Thus, typical protein-coding eukaryotic gene, has binding sites for many such factors. The stretch of DNA transcribed into an RNA molecule is called a transcription unit and encodes at least one gene. If the gene encodes a protein, the transcription produces messenger RNA (mRNA); the mRNA will in turn serve as a template for the protein's synthesis through translation. Alternatively, the transcribed gene may encode for either non-coding RNA (an RNA molecule that is not translated into a protein, also known as non-messenger RNA. The DNA sequence from which a functional non-coding RNA is transcribed is often called an RNA gene): it may be ribosomal RNA (rRNA), transfer RNA (tRNA) or some other types of less common RNA's. Overall, RNA helps synthesize, regulate, and process proteins; it therefore plays a fundamental role in performing functions within a cell. Within the DNA molecule, regions of both strands can serve as templates for transcription. For a given gene, however, only one of the two DNA strands can be template - determined by the location of the promoter of that gene. The template strand is the antisense strand of DNA (also known as non-coding strand), and similarly to replication, it is read by RNA polymerase from the 3' end to the 5' end during transcription (3' → 5'). The complementary RNA is created in the opposite direction, in the 5' → 3' direction, matching the sequence of the sense strand with the exception of switching uracil for thymine. This directionality is because RNA polymerase can only add nucleotides to the 3' end of the growing mRNA chain. This use of only the 3' → 5' DNA strand eliminates the need for the Okazaki fragments that are seen in DNA replication. This also removes the need for an RNA primer to initiate RNA synthesis, as is the case in DNA replication. The non-template sense strand of DNA is called the coding strand, because its sequence is the same as the newly created RNA transcript (except for the substitution of uracil for thymine). Eukaryotes polymerase - there are three distinct classes of RNA polymerase in the nucleus of eukaryotic cells - each class recognizes particular types of genes. RNA Polymerase I - synthesizes the precursor of rRNA (ribosomal RNA), RNA Polymerase II - synthesizes the nuclear precursors of mRNA that are subsequently translated to produce proteins. Polymerase II also synthesizes small non-coding RNAs, such as snoRNA (small-nucleolar RNA) and snRNA (small-nuclear RNA) and miRNA (microRNA). RNA Polymerase III - tRNA.

what is array CGH? what does it measure? advantages, disadvantages? (32)

a molecular cytogenetic technique for the detection of chromosomal copy number changes on a genome. Array CGH compares the patient's genome against a reference genome and identifies differences between the two genomes, and hence locates regions of genomic imbalances in the patient, utilizing the same principles of competitive fluorescence in situ hybridization as traditional CGH. Here we compare the number of copies of the locus on the examined and standard genome, not on the whole chromosome, but in individual sections according to the wells in the chip. Array CGH is based on the same principle as conventional CGH: DNA from the sample to be tested is labeled with a red fluorophore (Cyanine 5) and a reference DNA sample is labeled with green fluorophore (Cyanine 3). Equal quantities of the two DNA samples are mixed and cohybridized to a DNA microarray of several thousand evenly spaced cloned DNA fragments. After hybridization, digital imaging systems are used to capture and quantify the relative fluorescence intensities of each of the hybridized fluorophores. The resulting ratio of the fluorescence intensities is proportional to the ratio of the copy numbers of DNA sequences in the test and reference genomes. If the intensities of the flurochromes are equal on one probe, this region of the patient's genome is interpreted as having equal quantity of DNA in the test and reference samples; if there is an altered Cy3:Cy5 ratio this indicates a loss or a gain of the patient DNA at that specific genomic region.

what is the law of uniformity basic genetics... what is a homozygote? what is a heterozygote? what are alleles? (1)

crossing any two homozygotes w/ different alleles (AA and aa) yields identical heterozygous F1 offspring dominant alleles are expressed exclusively in a heterozygote, while recessive alleles are expressed only in recessive homozygotes trait is not lost, just masked by the dominant trait cross of F1 gives 3:1 phenotype (1:2:1 genotype) homozygote - state of having identical pair of alleles for a specific gene heterozygote - state of having inherited different form of alleles of a particular gene allele - one of two or more versions of a gene gene - the basic physical and functional unit of hereditary

what is genetic analysis? what are the different types? what are some methods in experimental and human genetics? (18)

genetic analysis deals with the description of hereditary traits in terms of geneological and molecular biology diagnose inherited diseases, cancers associated with genetic predisposition, changes in gene copy number and DNA mutations geneaological examination assesses inheritance of a given trait transmission to future generations based on Mendel's laws, gene linkage, and non-Mendelian inheritance cytogenetic, molecular examines the cause of the problem PCR, q-PCR, DNA sequencing karyotype formation CGH, micro-array, FISH (see question 32) direct vs. indirect diagnostics direct examines the presence of the disease analyses the known causes and features of the problem Direct analysis refers to those procedures that detect the specific disease-causing mutations or foreign DNA sequence. These assays require that the mutation and/or the gene sequence of interest is known. Allele-specific oligonucleotide probes, DNA sequencing, and a wide array of polymerase chain reaction (PCR) mediated procedures are examples of direct analysis methods. indirect examines traits that are not directly related to the disease but inherited together in a given case - based on gene linkage Polymorphic markers or gene sequences closely associated with the disease-causing gene are used to assess whether an individual has inherited the gene responsible for the disease phenotype. linkage analysis based on tracking the inheritance of polymorphic markers in a family with a genetic disease. methods to analyze human genetics basic means is to determine the pedigree starts with the proband with the help of genealogical marks and family relationships and the possible occurrence of the disease are recorded to construct a genealogial scheme need to also determine the form of hereditary disadvantages relies on the human factor and the memory of patients ambiguities, undiagnosed illnesses, death of relative for unspecificed reasons does not provide the actual occurrence - only in terms of genetic factors use of experiments model organisms short-life cycle non-specific growth requirements tecniques to manipulate the genome Model organisms are used to study diseases that would be unethical to analyze in humans . The conclusions from the study of these organisms are then applied to other species, although with some limitations. This method of analysis is made possible by the common developmental paths that the organisms have gone through during the evolutionary process. There is therefore a certain genetic, and therefore morphological-physiological similarity between them. The model organism should have a short life cycle and non-specific growth requirements. At the same time, there must be techniques that are capable of manipulating its genome. One of the first models used in molecular biology was E. coli . Others include viruses (bacteriophages), eukaryotes such as fungi ( Saccharomyces ), plants (lotus, tobacco, rice) or smaller animals (fruit flies - Drosophila melanogaster or various worms - were often used in the past ). From vertebrates, the model organisms are guinea pigs, mice, rats and others. The advantage of model organisms is the ability to produce a large number of offspring (5-15 offspring per litter in rats, 3-5 times a year) and short lifespan. Laboratory rats ( Rattus norvegicus ) are bred in special facilities, where the gradual crossing of related individuals results in the generation of offspring with the same genome ( inbred strain ). The experiments on them are then comparable, as mentioned above. If we wanted to look for a similar equivalent in humans, they would be identical twins. Although similar "human tests" took place during World War II, they are ethically unacceptable today.

what is simple interaction of non-allelic genes? what's an example? phenotypic ratio produced? (4)

where two non-allelic genes affect the same character - dominant allele of each of the two factors produces separate phenotypes when they are alone - when present together they create a new phenotype - the absence of both - gives another phenotype 9:3:3:1 The inheritance of comb types in fowls is the best example where R gene gives rise to rose comb and P gene gives rise to pea comb; both are dominant over single comb; the presence of both the dominant genes results in walnut comb. R : rose P: pea comb PR : walnut comb neither: single