genetics exam 3
Be able to describe the techniques of PCR
(PCR): Copy a specific sequence of DNA without the aid of vectors and host cell Materials: Template DNA: contains the region that needs to be amplified Oligonucleotide primers: Complementary to sequences at the ends of the DNA fragment to be amplified . Synthetic and about 15-20 nucleotides long Deoxynucleoside triphosphates (dNTPs): Provide the precursors for DNA synthesis Taq Polymerase: Thermostable enzyme that is necessary because PCR involves heating straps that inactivate most other DNA polymerases 3 Steps in the PCR cycle (occurs in thermocycler): DENTAURATION: separate DNA strands with a high temperature PRIMER ANNEALING: Lower temp that allows primers to bind to DNA PRIMER EXTENSION: Incubate at a slightly higher temperature allowing DNA synthesis to occur repeated for many cycles 20-30 cycles: 20 cycles, 2^20 DNA. 30 cycles 2^30 DNA
Compare and contrast the key differences between the Holiday model and the double strand break model (see Figure 19.25).
1. A double strand break occurs. 2. Strand degradation occurs at the double-strand break site to yield single-stranded ends. 3. Strand invasions causes D-loop formation. 4. Gap repair synthesis fills in the vacant regions 5. Branch migration and resolution can produce recombinant or non-recombinant chromosomes.
Be able to describe the Holliday model for homologous recombination (see Figure 19.24).
1. Both chromatids are nicked at identical locations. 2. The DNA strands to the left of the nicks invade the homologous chromatids and covalently link to the strands to the right of the nicks. 3. The Holliday junction migrates from left to right. This is called branch migration. It creates two heteroduplex regions. 4. Two heteroduplex regions that have a few base mismatches. 5. The strands that were initially nicked are broken. The strands are connected to create nonrecombinant chromosomes with a short heteroduplex region. Makes nonrecombinant chromosomes with a heteroduplex region. 6. The strands that were not originally nicked are broke. The strands are connected to create recombinant chromosomes with a short heteroduplex region. Makes recombinant chromosomes with a heteroduplex region.
Be able to describe the two mechanisms that account for gene conversion. 1.
1. DNA mismatch repair→ A branch migrates past a homologous region that contains slightly different DNA sequences. This produces two heteroduplexes: DNA double helices with mismatches. The mismatches can be repaired in four possible ways by the mismatch repair system. Two of these ways result in gene conversion.
Be able to describe how Dolly was created (see Figure 21.6).
1. Donor sheep mammary cell + unfertilized egg w/o nucleus fused together through electrical pulses 2. Initiate development of the egg into the embryo 3. The embryo is transferred to a surrogate 4. Lab genetically identical to the donor sheep is born
Be able to describe the two mechanisms that account for gene conversion. 2.
2. DNA gap repair synthesis→ A gene is found in two alleles, designated b and B. A double strand break occurs in the DNA encoding the b allele. Both of these DNA strands are digested away, thereby eliminating the b allele. A complementary DNA strand encoding the B allele migrates to this region and provides the template to synthesize a new double-stranded region. Following resolution, both DNA double helices carry the B allele.
Describe what a DNA microarray is,
A DNA microarray is a small silica, glass, or plastic slide that is dotted with many different sequences of DNA, each corresponding to a short sequence within a known gene. The relative location of each gene represented in the array is known.
Describe the general functions of E. coli proteins involved with homologous recombination RecBCD --
A complex of 3 proteins that tracks along the DNA and recognizes double-strand breaks. The complex partially degrades the double-stranded regions to generate single-stranded regions that can participate in strand invasion. RecBCD can create single-strand breaks that are used to initiate homologous recombination.
Be able to describe the general features of various DNA repair systems Base excision repair and nucleotide excision repair→
An abnormal base or nucleotide is first recognized and removed from the DNA, and a segment of DNA in this region is excised. Then the complementary DNA strand is used as a template to synthesize a normal DNA strand.
List the seven observations that suggest a disease is caused (at least in part) by a genetic component. Be able to calculate concordance between twins.
An individual who exhibits a diseases is more likely to have genetic relatives with the disorder than are people in the general population. Identical twins share the disease more often than nonidentical twins. The disease does not spread to individuals sharing similar environmental situations. Different populations tend to have different frequencies of the disease. The disease tends to develop at a characteristic age. The human disorder may resemble a disorder that is already known to have a genetic basis in an animal. A correlation is observed between a disease and a mutant human gene or a chromosomal alteration.
Describe a frequency distribution (see Figure 28.1).
Another way to describe quantitative traits... often fall into a normal distribution
Explain why gene knockout collections are useful.
Consider, for example, the phenotype produced by a particular gene knockout, which causes deafness in mice. Such a result suggests that the function of the normal gene is to promote hearing. Geneticists may also produce knockouts involving two or more genes to understand how the protein products of genes participate in a particular cellular pathway or contribute to a complex trait .In addition, gene knockouts in mice are used in the study of inherited human diseases.
be able to describe a few different uses of gene cloning.
DNA sequencing- Cloned genes provide enough DNA to subject the gene to DNA sequencing Site-directed mutagenesis- A cloned gene can be manipulated to change its DNA sequence Gene probes- Labeled DNA strands from a cloned gene can be used as probes for identifying RNA. Expression of cloned genes- Cloned genes can be introduced into a different cell type or different species. The expression of cloned genes has many uses: research, biotechnology, clinical trials
Explain how gene number and the environment affect the overlap between genotypes and phenotypes for polygenic traits (see Figure 28.4).
Difficult or impossible to categorize into discrete genotypic categories: 1. Number of genes controlling the trait increases 2. Influences from the environment
Describe how epigenetics can play a role in cancer.
Epigenetic modifications can also change gene expression and, therefore, can cause cancer.
Explain the concept of genetic drift including the bottleneck effect and the founder effect (see Figures 27.16, 27.77).
Genetic drift: Changes in allele frequencies in a population due to random fluctuationsBottleneck effect: A mechanism that can give rise to genetic drift; occurs when most members of a population are eliminated without any regard to their genetic composition.Founder effect: A change in allele frequency that occurs when a small group of individuals separates from a larger population and establishes a colony in a new location.
Describe how microarrays are used to characterize tumors (see Figure 24.13).
Goal: Identify those genes whose pattern of expression correlates with each other. In the study of cancer, researchers can compare cancer cells with normal cells and identify groups of genes that are turned on in cancer cells and turned off in normal cells and vice versa. Likewise researchers can compare two different types of tumors and identify groups of genes that show different patterns of expression.
List the assumptions that underlie a Hardy-Weinberg equilibrium.
In the absence of evolutionary changes, the Hardy-Weinberg equation predicts an equilibrium-unchanging allele and genotype frequencies from generation to generation-if certain conditions are met in a population.••No new mutations••No genetic drift••No migration••No natural selection••Random mating or breeding
explain how deamination is a relatively common cause of spontaneous mutations
Involves the removal of an amino group from the C base, this produces U. If not repaired, a DNA template strand has U instead of C, a newly made strand will incorporate A into the daughter cell instead of G
Describe how most cancers involve a progression of multiple mutations (see Figure 25.7).
Many cancers begin with a benign genetic alterations that, over time and with additional mutations, progresses to malignancy.
List the four common types of genetic changes that produce oncogenes.
Missense Mutation Gene Amplification Chromosomal Translocation Viral Integration
Define personalized medicine.
Personalized medicine→ The use of information about a patient's genotype and other clinical data in order to select a medication, therapy, or preventative measure that is specifically suited to that patient.
Define pharmacogenetics.
Pharmacogenetics→ The study or clinical testing of genetic variation that causes differing responses to drugs.
Define population and gene pool.
Population: A group of individuals of the same species that occupy the same region and can interbreed with one another Gene pool: All of the alleles of every gene within a particular population.
be able to describe a few different uses of microorganisms in biotechnology.
Production of medicine- antibiotics, insulin Food fermentation- cheese wine beer Biological control- control of plant diseases, insects, weeds bioremediation- cleanup of pollutents
Be able to describe the general features of various DNA repair systems Mismatch repair→
Similar to excision repair except that the DNA defect is a base pair mismatch in the DNA, not an abnormal nucleotide. The mismatch is recognized, and a segment of DNA in this region is removed. The parental strand is used to synthesize a normal daughter strand of DNA.
Describe the net results of sister chromatid exchange and homologous recombination
Sister chromatid → sister chromatids are genetically identical, so SCE does not produce a new combination of alleles. Homologous → may produce new combinations of alleles in the resulting chromosomes. Genetic recombination can occur, or the shuffling of genetic material to create a new combination of alleles that differs from the original.
Explain how insulin is made by bacteria (see Figure 21.1).
The two different polypeptides (A chain and B chain) are inserted into different plasmids and then put into bacteria (transformed). The cells are cultured. Purified B-galactosidase-insulin fusion proteins. Treat with CNBr. CNBr cleaves the peptide bond after methionine. purify A and B chains. Refolding and disulfide bond formation
Be able to describe the techniques of reverse transcriptase PCR
Used to detect and quantitate the amount of RNA in living cells. EXTRAORDINARILY SENSITIVE Materials: single-stranded RNA, primer, reverse transcriptase End product: double stranded cDNA from RNA of interest
Describe what a position effect is, and explain how two ways that it can be caused (see Figure 19.2).
a change in phenotype that occurs when the location of a gene is changed from one chromosomal site to a different one 1. movement to a position next to regulatory sequences (silencers/enhancers) 2. movement to a heterochromatic region
Explain how loss of heterozygosity can explain certain forms of familial cancers, such as breast cancer (see Figure 25.9).
a common form of allelic imbalance by which a heterozygous somatic cell becomes homozygous because one of the two alleles gets lost. This form of chromosome instability is sufficient to provide selective growth advantage and has been recognized as a major cause of tumorigenesis.
Define mutagen and distinguish between chemical and physical mutagens
an agent that causes alteration in the structure of DNA Chemical Mutagens: -Nitrous Acid Deaminates Bases -Nitrogen Mustard & EMS Alkylates Bases -Proflavin: Intercalates within DNA helix 5-Bromouracil: Functions as a base analog Physical Mutagens: X-rays--> Cause base deletions, single-stranded nicks in DNA, crosslinking, chromosomal breaks, and oxidized bases UV light--> causes formation of thymine dimers
explain how base modifiers, intercalating agents, and base analogues cause induced mutations
base modifiers-covalently modify the structure of a nucleotide & Others disrupt pairing by alkylating bases intercalating-Directly interfere with replication process base analogues-Incorporate into DNA and disrupt structure & Some tautomerize at a high rate
Explain how mutations in the coding sequence of a gene may affect polypeptide structure and function (see Table 19.1). Nonsense Mutations
base substitutions that change a normal codon to a stop codon inhibitory effect
Explain how mutations in the coding sequence of a gene may affect polypeptide structure and function (see Table 19.1). Missense Mutations
base substitutions which amino acid change does occur neutral or inhibitory effect of protein function
Describe the general functions of E. coli proteins involved with homologous recombination RuvABC--
binds to holliday junctions, RuvAB promotes branch migration, Ruv C is an endonuclease that cuts the crossed or uncrossed strands to resolve Holliday junctions into separate chromosomes.
Define biological control and bioremediation.
biological control:the use of living organisms or their products to alleviate plant diseases or damage from environmental conditions. Bioremediation: the use of living organisms or their products to decrease pollutants in the environment.
describe a couple of different types of induced mutations
caused by environmental agents chemical agents- base modifiers, intercalating agents, base analogues physical agents-x-rays & gamma rays= ionizing radiation, UV light = nonionizing
Explain what a transgenic organism is (see Figure 21.2).
contain recombinant DNA from another species that has been integrated into their genome.
Compare and contrast the different consequences of a germ-line versus a somatic mutation (see Figure 19.4).
germ-line ->passed to half of the gametes in the next generation; mutation found in whole body . somatic -> size of affected region depends on timing of mutation (earlier=larger), mutations not present in gametes
Be able to do the following types of calculations: mean, variance, standard deviation, covariance, and correlation
look at formulas
Explain how mutations outside of the coding sequence can affect gene function (see Table 19.2). Splice Recognition Sequence
may alter the ability of pre-mRNA to be properly spliced
Explain how mutations outside of the coding sequence can affect gene function (see Table 19.2). 5'-UTR/3'-UTR
may alter the ability of the mRNA to be translated; may alter mRNA stability
Explain how mutations outside of the coding sequence can affect gene function (see Table 19.2). Regulatory Element/Operator Site
may disrupt the ability of the gene to be properly regulated
Describe the general functions of E. coli proteins involved with homologous recombination RecG --
promotes branch migration of holliday junctions
Describe how radiation causes mutations and that UV light causes thymine dimers
radiation - can penetrate into biological tissues & produces chemically reactive molecules called free radicals. They alter the structure of DNA. UV - The energy in UV light causes the formation of thymine dimers, adjacent thymine bases that have become covalently linked. Thymine dimers do not base-pair properly during DNA replication, and thus can produce a mutation when the DNA strand is replicated
describe a couple of different types of spontaneous mutations
results from abnormalities in cellular/biological processes depurination- removal of purine (G/A) from the DNA, if not repaired mutation occurs (75% chance) deamination - removal of amino group from cytosine base = uracil or 5-methylcytosine = thymine tautomeric shift - temporary change in base structure (T/G=keto)(A/C=amino)
Define quantitative genetics.
study of traits that can be described numerically
Explain how mutations in the coding sequence of a gene may affect polypeptide structure and function (see Table 19.1). Frameshift Mutations
the addition or deletion of a number of nucleotides that is not divisible by 3 shifts the reading frame so that the translation of the mRNA results in a completely different amino acid sequence downstream of the mutation inhibitory effect
Define heritability and distinguish between broad sense heritability and narrow sense heritability (see Section 28.5).
the amount of phenotypic variation within a group of individuals that is due to genetic variation Broad sense heritability - h2=Vg/Vp... Different types of genetic variation may affect the phenotype Narrow sense heritability- h2= Va/Vp= Robs RExp; heritability of a trait due to the additive effects of alleles robs= phenotypic correlation between related individuals Rexp: expected correlation based on the known genetic relationship
Describe the technique of RNA sequencing (RNA Seq) and explain what type of information it yields (see Figure 23.3)
•Isolate RNA from a sample of cells. In some cases a researcher may want to focus on a subpopulation of RNA, such as mRNAs or short non-coding RNAs. The illustration below shows three different types of RNA in different colors. In an actual experiment, there would be hundreds or thousands of different RNAs. •Break the RNAs into small fragments •Attach short oligonucleotide linkers to the ends of RNAs •Synthesize cDNAs via reverse transcriptase PCR, using the RNAs as templates. The PCR primers are complementary to the linkers. •Sequence the cDNAs using a next generation sequencing technology. •Using computer technology, align the cDNA sequences along the genomic sequence. •Allows researchers to determine the pattern of RNA splicing that is found in a particular cell type under a given set of conditions.
Be able to describe the Ames test (see Figure 19.15).
1. Mix together the suspected mutagen, a rat liver extract, and a Salmonella strain that cannot synthesize histidine. 2. The suspected mutagen is omitted from the control sample. 3. Plate the mixtures onto petri dishes that lack histidine. 4. Incubate overnight to allow bacterial growth. 5. A large number of colonies suggests that the suspected mutagen causes mutation.
Describe the two key characteristics of stem cells and why they may be useful medically
1. capacity to divide 2. capacity to differentiate into one or more specialized cell types Neural:Implantation of cells into the brain to treat Parkinson's diseaseTreatment of injuries such as those to the spinal cord Skin: Treatment of burns and other types of skin disorders Cardiac: Repair of heart damage associated with heart attacks Cartilage: Repair of joints damaged by injury or arthritis Bone: Repair of damaged bone or replacement with new bone Liver: Repair or replacement of liver tissue that has been damaged by injury or disease Skeletal Muscle: Repair or replacement of damaged muscle
Explain how the work of the Lederberg's supported the random mutation theory (see Figure 19.6)
1. place bacteria on growth media 2. incubate overnight 3. press velvet cloth for replica, incubate for growth of mutant cells the new colonies did not appear due to the presence of T1, so random mutation theory is supported
Be able to describe how a trinucleotide repeat expansion occurs
1.DNA replication begins and goes just past the TNRE. 2.Hairpin formation in the newly made daughter strand causes DNA polymerase to slip off. (TNRE can form hairpin structures due to CG base pairing). 3.DNA polymerase resumes replication. 4.Hairpin spreads out. 5.DNA gap repair synthesis occurs.
Explain how a DNA microarray is used to study gene expression patterns:
A DNA microarray is used as a hybridization tool. In this experiment, mRNA was isolated from a sample of cells and then used to make fluorescently labeled cDNA. The labeled cDNAs were then layered onto a DNA microarray. The cDNAs will be complementary to some of the DNA spots in the microarray, binding to DNA in these spots. The array is then washed with a buffer to remove any unbound cDNAs and placed in a microscopy device called a laser scanner. Results in a group of fluorescent spots at defined locations in the microarray. High fluorescence intensity at a particular location means that a large amount of cDNA in the sample hybridized to the DNA at that location. Because the cDNA was generated from mRNA this technique identifies RNAs that have been made in a particular cell type under a given set of conditions.
Explain the BLAST program does (see Table 23.5).
A computer program that can start with a particular genetic sequence and then locate homologous sequences within a large database.
Be able to analyze a multiple sequence alignment to deduce important features of a gene or protein sequence (Figure 23.10).
A conserved site is a site that is identical or similar across multiple species. Conserved sites are more likely to be functionally important compared to nonconserved sites. Multiple-sequence alignment→ An alignment by a computer program of two or more genetic sequences based on their homology to one another. ••May reveal important features concerning the similarities and differences within a gene family. ••Dots are shown where it is necessary to create gaps to keep the amino acid sequences aligned. ••Amino acids that are highly conserved within a gene family are more likely to be important functionally.
Compare and contrast mutations that produce oncogenes (gain-of-function) versus those that affect tumor-suppressor genes (loss-of-function).
A gain-of-function mutation that produces an oncogene typically has one of three possible effects: ••The amount of protein is greatly increased ••A change occurs in the structure of the encoded protein that causes it to be overly active ••The encoded protein is expressed in a cell type where it is not normally expressed The role of tumor suppressor genes is to inhibit cancerous growth. Therefore, when a tumor-suppressor gene is inactivated by mutation, cancer is more likely to occur. It is a loss-of-function mutation in a tumor-suppressor gene that promotes cancer
Be able to analyze a pedigree and determine if an inheritance pattern for a disease is or is not consistent with autosomal recessive, autosomal dominant, X-linked recessive, or X-linked dominant inheritance (see Figures 24.2, 24.3, 24.4).
A geneticist must obtain data from many large pedigrees containing several individuals who exhibit the disorder and then follow its pattern of inheritance from generation to generation. Autosomal recessive→ ••Frequently, an affected offspring has two unaffected parents. ••When two unaffected heterozygotes have children, the percentage of affected children is (on average) 25%. ••Two affected individuals have 100% affected children. ••The trait occurs with the same frequency in both sexes. Autosomal dominant→ ••An affected offspring usually has one or two affected parents. ••An affected individual with only one affected parent is expected to produce 50% affected offspring (on average). ••Two affected heterozygous individuals have (on average) 25% unaffected offspring. ••The trait occurs with the same frequency in both sexes. ••For most dominant, diseases-causing alleles, the homozygote is more severely affected with the disorder. In some cases, a dominant allele may be lethal in the homozygous condition. X-linked recessive inheritance→ ••Males are more likely to exhibit the trait. ••Mothers of affected males often have brothers or fathers who are also affected. ••Daughters of affected males produce, on average, 50% affected sons. X-linked dominant inheritance→ ••Only females exhibit the trait when it is lethal to males. ••Affected mother have a 50% chance of passing the trait to daughters.
Define database
A large number of computer data files such as those containing genetic sequences, collected and stored in a single location.
List the three common ways that the function of tumor-suppressor genes is lost.
A mutation can occur specifically within a tumor-suppressor gene to inactivate its function. For example, a mutation could inactivate the promoter of a tumor-suppressor gene or introduce an early stop codon in the coding sequence. Either of these would prevent the expression of a functional protein. Tumor suppressor genes are sometimes inhibited via epigenetic changes, such as DNA methylation. DNA methylation usually inhibits the transcription of eukaryotic genes, particularly when it occurs in the vicinity of the promoter. The methylation of CpG islands near the promoters of tumor-suppressor genes has been found in many types of tumors. Many types of cancer are associated with aneuploidy, which involves the loss of addition of one or more chromosomes, so that the total number of chromosomes is not an even multiple of a set. In some cases, chromosome loss may contribute to the progression of cancer because the lost chromosome carries one or more tumor-suppressor genes.
Be able to describe the general features of various DNA repair systems Direct repair→
An enzyme recognizes an incorrect alteration in DNA structure and directly converts the structure back to the correct form.
Define assortative mating and inbreeding. Be able to calculate the inbreeding coefficient using pedigree data (see Figure 27.18).
Assortative Mating: Breeding in which individuals preferentially mate with each other based on their phenotype.Inbreeding: Mating of two genetically related individuals
explain how tautomeric shifts are relatively common causes of spontaneous mutations
At a low rate, G and T can interconvert to an enol form, and A and C can change to an imino form. Though the amounts of the enol and imino forms of these bases are small, their presence can cause a mutation because these rare forms do not conform to the AT/GC rule of base pairing. Instead, if one of the bases is in the enol or imino form, hydrogen bonding will promote TG and CA base pairs. (The shift must occur immediately prior to DNA replication).
Explain how mutations in the coding sequence of a gene may affect polypeptide structure and function (see Table 19.1). Silent Mutations
Base substitutions that do not alter the amino acid sequence of the polypeptide due to the degeneracy of the genetic code
Describe the general functions of E. coli proteins involved with homologous recombination RecA --
Binds to single-stranded DNA and promotes strand invasion, which enables homologous strands to find each other, promotes the displacement of the complementary strand to generate a D-loop.
Explain how QTLs are mapped (see Figure 28.5).
Cross 2 strains to produce f1 offspring Back cross the f1 offspring to both parental strain From the offspring of the back cross determine marker composition
explain how oxidation is a relatively common cause of spontaneous mutations
DNA bases are very susceptible to oxidation. G bases are particularly vulnerable to oxidation; one oxidized product is 8-oxoguanine (8-oxoG) Oxidized base-pairs are harmful because, for example, 8-oxoG base pairs with adenine during DNA replication, causing mutations in which a GC base pair becomes a TA base pair.
Explain what a DNA library is (see Figure 20.4)
DNA library: a collection of hybrid vectors containing a particular segment of DNA from a larger source Genomic library: starting material was chromosomal DNA cDNA library: starting material was cDNA
Define cDNA, and explain how it is made (see Figure 20.3).
DNA made from RNA that lacks introns Synthesis of cDNA. A poly-dT primer binds to the 3′ end of eukaryotic mRNAs. Reverse transcriptase catalyzes the synthesis of a complementary DNA strand (cDNA). RNaseH digests the mRNA into short pieces that are used as primers by DNA polymerase I to synthesize the second DNA strand. The 5′ to 3′ exonuclease function of DNA polymerase I removes all of the RNA primers except the one at the 5′ end (because there is no primer upstream from this site). This RNA primer can be removed by the subsequent addition of an RNase. After the double-stranded cDNA is made, it can then be inserted into a vector,
Compare and contrast the four common types of natural selection Be able to do the calculations for directional selection and heterozygote advantage.
Directional Selection: favors individuals at one extreme of a phenotypic distribution that are more likely to survive and reproduce in a particular environment. Stabilizing Selection: The extreme phenotypes for a trait are selected against, and those individuals with intermediate phenotypes have the highest fitness values. Disruptive Selection: Favors the survival of two or more different phenotypes. This type of selection typically acts on traits that are determined by multiple genes. In disruptive selection, the fitness values of particular genotypes are higher in one environment and lower in a different one.••Also known as diversifying selection••Caused by fitness values for a given genotype that vary in different environments••Typically acts on traits that are determined by multiple genes Balancing Selection: For genetic variation involving a single gene, balancing selection may arise when the heterozygote has a higher fitness than either corresponding homozygote, a situation called heterozygote advantage.
Explain the underlying causes of dominant patterns of inheritance; they are usually due to haploinsufficiency, gain-of-function, or a dominant negative mutation.
Haploinsufficiency→ The phenomenon in which a person has only a single functional copy of a gene and that single functional copy does not produce a normal phenotype. Shows a dominant pattern of inheritance. Gain-of-function→ A mutation that changes a gene product so it gains a new or abnormal function Dominant negative mutation→ A mutation that produces an altered gene product that acts antagonistically to the normal gene product. Shows a dominant pattern of inheritance
Explain the underlying cause of most recessive patterns of inheritance; it's usually due to a loss-of-function mutation.
Human recessive alleles are often caused by mutations that result in a loss of function in the encoded enzyme. In the case of Tay-Sachs disease, a heterozygous carrier has approximately 50% of the functional enzyme, which is sufficient for a normal phenotype. However, for other genetic diseases, the level of functional protein may vary due to the effects of gene regulation.
Explain the five factors that govern microevolution (see Table 27.1).
Microevolution describes changes in a population's gene pool from generation to generation.Sources of New Allelic Variation:Mutation: Consider allelic variation. Random mutations within preexisting genes introduce new alleles into populations, but at a very low rate. New mutations may be beneficial, neutral, or deleterious. For new alleles to rise to a significant percentage in a population, other evolutionary mechanisms must operate on them.Mechanisms That Alter Existing Genetic Variation:Natural Selection: This is the phenomenon in which certain phenotypes have greater reproductive success compared to other phenotypes. For example, natural selection may be related to the survival of members to reproductive age.Genetic Drift: This is a change in genetic variation from generation to generation due to random sampling error. Allele frequencies may change as a matter of chance from one generation to the next. This tends to have a greater effect in a small population.Migration: Migration can occur between two different populations. The introduction of migrants into a recipient population may change the allele frequencies of that population.Nonrandom Mating: This is the phenomenon in which individuals select mates based on their phenotypes or genetic lineage. This can alter the relative proportion of homozygotes and heterozygotes predicted by the Hardy-Weinberg equation, but does not change allele frequencies.
Explain the concept of migration and how it affects allele frequencies.
Migration between two different established populations can alter allele frequencies.Gene flow refers to the transfer of alleles or genes from one population to another, thereby changing the gene pool of the recipient population. One way this occurs is by the migration of fertile individuals from one population to another population and the successful breeding of such migrants with the members of the recipient population.In nature, it is common for individuals to migrate between populations in both directions••This bidirectional migration has two important consequences••It tends to reduce allele frequency differences between populations••It can enhance genetic diversity within a population•••New mutations in one population can be introduced to neighboring group
Describe how cancer is clonal in origin (see Figure 25.1).
Most cancers originate in a single cell. This single cell, and its line of daughter cells, usually undergoes a series of genetic changes that accumulate during cell division. In this regard, a cancerous growth can be considered to be CLONAL in origin. A hallmark of a cancer cell is that it divides to produce two daughter cancer cells. In a healthy individual, one or more gene mutations convert a normal cell into a tumor cell. This tumor cell divides to produce a benign tumor. Additional genetic changes in the tumor cells may occur, leading to malignant growth. At this malignant stage, the tumor cells invade surrounding tissues, and some malignant cells may metastasize by traveling through the blood-stream to other parts of the body where they can grow and cause secondary tumors. As a trait, cancer can be viewed as a series of genetic changes that eventually lead to uncontrolled cell growth.
Describe the two common functions of most tumor-suppressor genes (either negative regulators of cell division or are involved with maintaining genome integrity)(see Table 25.3, but don't memorize).
Negative regulators of cell division→ Some TS genes encode proteins that directly affect the regulation of cell division. Loss of function in these kinds of negative regulators has a direct effect on the abnormal cell division rates seen in cancer cells. Maintenance of genome integrity→ TS genes play a role in the proper maintenance of the integrity of the genome. The term genome maintenance refers to cellular mechanisms that prevent mutations from occurring and/or prevent mutant cells from surviving or dividing. The proteins encoded by genes that participate in genome maintenance help to ensure that gene mutations or changes in chromosome structure and number do not occur and are not transmitted to daughter cells.
Describe how Northern and Western blotting are used to detect RNA or proteins, respectively
Northern Blotting: Used to identify specific RNA sequences Uses: Specific gene is transcribed in a specific cell type, particular stage of development, Reveal if pre-mRNA is alternatively spliced Procedure: 1. RNAS is extracted from cells 2. Separated via gel electrophoresis 3. Blotted onto nitrocellulose or nylon filters RNAs that are complementary to the radiolabeled probe are detected as a dark band Western Blotting: Used to identify a specific protein in a mixture Uses: Determine if a specific protein is made in a cell type, particular stage of development Procedure: Proteins extracted from cells and separated by SDS-Page Dissolved into detergent sodium dodecyl sulfate- Then separated by polyacrylamide gel electrophoresis Samples blotted onto nitrocellulose or nylon filters Placed in solution containing primary antibodies (recognized protein of interest) Secondary antibody (w/ alkaline phosphatase) recognizes primary antibody Colorless dye is added which the alkaline phosphatase converts to black compound Protein interest indicated by dark bands
Be able to describe the general features of various DNA repair systems Homologous recombination→
Occurs at double stranded breaks or when DNA repair damage causes a gap in synthesis during DNA replication. The strands of a normal sister chromatid are used to repair a damaged sister chromatid.
Be able to describe the general features of various DNA repair systems Nonhomologous end joining→
Occurs at double-strand breaks. The broken ends are recognized by proteins that keep the ends together; the broken ends are eventually rejoined.
Describe the relationship between certain oncogenes and cell growth signaling pathways (see Figures 25.2, 25.3).
Oncogenes commonly encode proteins that function in cell-signaling pathways related to dell division. These include growth factor receptors, intracellular signaling proteins, and transcription factors.The overexpression of such oncogenes causes cell division to occur when it is not supposed to occur. The activation of a cell-signaling pathway by a growth factor: ••Epidermal growth factor binds to its receptor, leading to the activation of an intracellular signaling pathway. This pathway causes a change in gene transcription; the transcription of specific genes is activated in response to the growth factor. After these genes are transcribed and mRNAs are translated into proteins, the proteins promote the progression through the cell cycle. In other words, the cell is stimulated to divide. Mutations that alter the amino acid sequence of the Ras protein have been shown to cause functional abnormalities. The Ras protein is a GTPase, which hydrolyzes GTP to GTP +Pi. Therefore, after it has been activated, the Ras protein returns to its inactive state by hydrolyzing GTP. Certain mutations that convert the normal ras gene into an oncogene decreases the ability of the Ras protein to hydrolyze GTP. This results in a greater amount of the active GTP-bound form of the Ras protein. In this way, such mutations keep the signaling pathway turned on, thereby stimulating the cell to divide.
Describe how to clone genes into vectors (see Figures 20.1, 20.2).
Plasmids... Restriction enzymes cut DNA into pieces Recognize palindromic sequences Ex: 5'GATTC3'/ 3'CTTAAG5' Creates end that are sticky... short, single stranded regions that can base pair with complementary DNA Two different DNA sequences can hydrogen bond at sticky ends to create a recombinant DNA molecule DNA ligase creates a covalent bond 3 outcomes include recircularized vector, a recombinant vector with the gene of interest, or a recombinant vector with another gene Recircularized have a functional lacZ gene Mix DNA with Ecoli cells Plate with: IPTG- a lactose analogue that can induce lac gene expression X-Gal- a colorless compound that is cleaved by B-galactosidase into a blue dye Blue colonies: functional b galactosidase enzyme, white colonies: nonfunctional Overall: blue= no recombination, white= recombination
Define polygenic inheritance and quantitative trait loci (QTL).
Polygenic - transmission of traits that are governed by two or more genes Quantitative- the locations on chromosomes that affect the outcome of quantitative traits
Compare and contrast polymorphism at the level of organisms and the level of genes (see Figures 27.2, 27.3).
Polymorphism at the level of organisms is the variation in traits, typically we think of those traits that are observable with the naked eye.At the DNA level, polymorphism may be due to two or more alleles that influence the phenotype of the individual that inherits them. It is due to genetic variation.••At the level of a particular gene, polymorphism may involve various types of changes such as a deletion of a significant region of the gene, a duplication of a region, or a change in a single nucleotide.
Be able to describe the technique of chromatin immunoprecipitation (see Figure 23.2).
Proteins that are noncovalently bound to DNA can be more tightly attached to chromatin by the addition of formaldehyde or some other agent that covalently crosslinks the protein to the DNA. Following crosslinking, the cells are lysed, and the DNA is broken by sonication into pieces ~100-200 bp long. Next, an antibody is added that is specific from the protein of interest. To conduct a ChIP assay, a researcher must suspect that a particular proteins binds to the DNA, and previously had an antibody made that recognizes the protein. The antibodies are attached to heavy beads. The antibodies bind to DNA-protein complexes and cause the complexes to form a pellet following centrifugation. Because an antibody is made by the immune system of an animal, this step is called immunoprecipitation.The next step is to identify the DNA to which the protein is covalently crosslinked. To do so the protein is removed by treatment with chemicals that break the covalent crosslinks and amplified with PCR.If researchers already suspect that a protein binds to a known DNA region, they can use PCR primers that specifically flank the DNA region. If a PCR product is obtained, the protein of interest must have been bound to this DNA site in living cells.OR: Conduct PCR using primers that are complementary to the linkers. Incorporate fluorescently labeled nucleotides during PCR. Then denature DNA and hybridize to a microarray. Fluorescently labeled spots identify sites in the genome where the protein of interest binds.
Be able to describe the function of Rb and p53 (see Figure 25.5).
Rb: Tumor-suppressor gene found on the long arm of chromosome 13. ••Rb regulates a transcription factor called E2F, which activates genes required for cell-cycle progression. The binding of the Rb protein to E2F inhibits its activity and prevents the cell from progressing through the cell-cycle. When a normal cell is supposed to divide, cellular proteins called cyclins bind to CDKs. This activates the kinases, which then leads to the phosphorylation of the Rb protein. The phosphorylated form of the Rb proteins is released from E2F, thereby allowing E2F to activate genes needed to progress through the cell cycle. If both copies of the rb gene are rendered inactive by mutation, the E2F protein is always active and uncontrolled cell division occurs. P53: Primary role is to determine if a cell has incurred DNA damage. The event that induces p53 gene expression is double-stranded breaks in the DNA. The p53 protein acts as a transcription factor. ••When confronted with DNA damage, the expression of p53 activates genes involved with DNA repair. This may prevent the accumulation of mutations that activate oncogenes or inactivate tumor-suppressor genes. ••If a cell is in the process of dividing, it can arrest itself in the cell cycle gaining time to repair its DNA and avoiding producing two mutant daughter cells. Here, the p53 protein stimulates the expression of another gene, p21. The p21 protein inhibits the formation of cyclin/CDK complexes that are needed to advance from the G1 phase of the cell cycle to the S phase. ••Expression of p53 can initiate a series of events called apoptosis or programmed cell death.
Be able to describe different strategies for identifying a gene within a long DNA sequence (search by signal, search by content, open reading frames, etc.).
Search by signal→ In bioinformatics, an approach in which a computer program relies on known sequences such as promoters, start and stop codons, and splice sites to help predict whether or not a DNA sequence contains a protein encoding gene Search by content→ In bioinformatics, an approach in which a computer program predicts the location of a gene based on the facts that the nucleotide content of a particular region differs significantly (due to codon bias) from a random distribution. Open reading frame→ An open reading frame is a region of a genetic sequence that does not contain any stop codons. Because most proteins are several hundred amino acids in length, a relatively long RF is required to encode the,. In bacteria, such long ORFs are contained within protein-encoding genes.
Explain the basic strategies that are used to analyze DNA sequences (sequence recognition versus pattern recognition).
Sequence recognition→ In bioinformatics, the ability of a computer program to recognize particular sequences Pattern recognition→ In bioinformatics, this term refers to a program the recognizes a pattern of symbols. •Locate specialized sequences within a very long sequence. A specialized sequence with a particular meaning or function is called a sequence element or motif. A computer program is supplied with a list of predefined sequence elements and can identify such elements within a sequence of interest. •Locate an organization of sequences. This could be an organization of sequence elements. Alternatively, it could be an organization of a pattern of symbols. •Locate a pattern of symbols.
Define homology.
Similarities among various species that occur because the species are derived from a common ancestor.
explain how depurination is a relatively common cause of spontaneous mutations
The covalent bond between deoxyribose and a purine base (A/G) is unstable and undergoes a spontaneous reaction with water that releases the base from the sugar, creating an apurinic site. If an apurinic site remains in the DNA as it is being replicated, any of the four nucleotides can be added to the newly made strand.
Describe how a gene knockin is made in mice (see Figure 21.3).
The gene of interest is cloned and flanked with pieces of DNA that are homologous to a noncritical site in the mouse genome. The cloned DNA is introduced into a fertilized oocyte. The gene of interest inserts into the noncritical site by nonhomologous recombination.
Explain the concept of locus heterogeneity.
The phenomenon in which a particular type of disease or trait may be caused by mutations in two or more different genes. For rare diseases that are poorly understood at the molecular level, locus heterogeneity may obscure the pattern of inheritance.
Explain the concept of molecular pharming (see Figure 21.5, Table 21.3).
The production of medically important proteins in the mammary glands of livestock and in agricultural plants, using transgenic techniques.
Define gene editing, and describe how CRISPR-Cas technology works (see Figure 20.13)
The spacer region of the sgRNA is complementary to one of the strands of the gene to be edited (called the target gene)sgRNA binds to Cas9 and guides it to the target geneCas9 then makes a double-strand break in the gene After Cas9 cuts the DNA, two different repair events are possible1. Repair by nonhomologous end joining (NHEJ)• The region may incur a small deletion that inactivates gene2. Repair by homologous recombination repair (HRR) • If HRR is desired, donor DNA homologous to the target region that also carries the desired mutation is added
Describe the technique of dideoxy DNA sequencing (see Figures 20.10, 20.11).
Uses dideoxynucleotides (ddNTPS) to terminate the growth of a DNA strand. Chain termination ddNTPS have a 3' H instead of an OH Clone or PCR DNA to create large amounts Target DNA is cloned into the vector at a site adjacent to the primer annealing site Then add nucleotides to a single tube. ddA, ddT, ddG, ddC with a different fluorescent label. Fluorescence= last base in each strand Read thee sequence on a sequencing ladder
Describe the general functions of E. coli proteins involved with homologous recombination Single Strand Binding Protein --
coats broken ends of chromosomes and prevents excessive strand degradation
Explain how mutations outside of the coding sequence can affect gene function (see Table 19.2). Promoter
may increase or decrease the rate of transcription
Be able to describe the techniques of real time PCR
used to determine how much.. Quantitate the specific amount of RNA. Done in a thermocycler that can measure the change in fluorescence emitted by detector molecules Uses an oligonucleotide that has fluorescent reporters at one end and a quencher at the other. Quencher blocks fluorescence of the reporter Taq polymerase exonuclease activity digests the probe and separates the reporter and the quencher.. Causing fluorescence Compare cycle threshold with standards