EXAM3

What types of epigenetic changes are often seen in cancer?

-histone deacetylase inhibitors as cancer drugs -DNA methylation inhibitors -HDAC inhibitors

List the steps in gel electrophoresis

1) Pouring the gel, 2) Preparing your samples, 3) Loading the gel, 4) Running the gel (exposing it to an electric field) and 5) Staining th

Mechanisms of transcription factors

1. Transcription activators may help recruit TFIID to the promoter, or to help recruit RNA polymerase to the promoter. They may also change the enzymatic activity of RNAP. 2. Different domains of a transcription factors may act independently, which means one domain can perform its normal function when it is physically separated from other domains of the protein. 3. Transcription activators usually bind to enhancer far away from the promoter, but relay its action by DNA looping 4. One gene is usually regulated simultaneously by multiple transcription activators and repressors to achieve optimal level of transcription in a specific cell and specific condition. This type of regulation is called combinatorial regulation. 5. The function of a transcription activator is often dependent on other proteins (e.g. mediators, insulators, co-activators). A transcription activator may interact with a co-activator to activate transcription, or it may interact with a co-repressor to inhibit transcription.

Structure of transcription factors

1. Transcription factors have two major domains: a DNA-binding domain and an activation or suppression domain. The DNA- binding domain interacts with specific DNA sequences, the activation or suppression domain activates or suppresses transcription. 2. Examples of DNA-binding domains: homeodomain, zinc- finger, leucine zipper, and helix-loop-helix. A DNA-binding domain contain the domain-specific amino acid sequence. Transcription factors are usually classified by the DNA-binding domain. 3. Examples of transcription regulatory (such as activation) domains: acidic domain, basic domain, and Q-rich domain. Activation domains allow transcription factor to interact with other proteins, such as TFII factors or various subunits of RNAPII to activate transcription.

Describe how siRNAs and microRNAs can affect gene expression - how are the mechanisms the same? How do they differ?

Gene silencing mediated by miRNA The major difference between siRNAs and miRNAs is that the former inhibit the expression of one specific target mRNA while the latter regulate the expression of multiple mRNAs. All cells in a single organism carry the exact same genome, so how do we end up with so many varieties of tissues and organs? Scientists know that transcription of many genes in eukaryotic cells is repressed, or "silenced," but in some cases, genes are transcribed into mRNA that never gets translated. Various post-transcriptional mechanisms are in place to add another level of control over the already complex systems that regulate eukaryotic gene expression. These mechanisms are the result of small, noncoding pieces of RNA called siRNA (small inhibitory RNA), or interference RNA, and miRNA (microRNA), or antisense RNA. siRNAs begin as small, double-stranded RNA molecules (about 20 base pairs in length), generated by the cleavage of dsRNA by an enzyme called Dicer, a member of the RNase III family. siRNAs have two nucleotide overhangs at each 3' end. miRNAs, on the other hand, originate as small hairpin-shaped precursor molecules that are cut to size by a Dicer enzyme. siRNA and miRNA inhibit translation by two different mechanisms while working in association with a protein, forming a ribonucleoprotein complex called RNA-induced silencing complex (RISC). The proteins in RISC unwind siRNA and remain bound to a single antisense strand, which then binds to mRNA in a sequence-specific manner, at which time a protein component of RISC called Slicer cuts the mRNA in the middle of the binding region. The cut mRNA is recognized by the cell as being abnormal and is subsequently destroyed. In the case of miRNA, a microRNA-induced silencing complex (miRISC) associates with the mature miRNA, and the complex binds to mRNA

Compare and contrast the terms physical map and genetic map

Genetic mapping and physical mapping are ongoing studies, which will advance more in the future. While genetic mapping gives the outline of a chromosome, physical mapping gives the details. Information from both the maps are combined together to study the chromosomes. - A genetic map identifies genes by their mutant phenotypes; a physical map identifies genes by their wild-type functions. - A genetic map gives the order of genes on a chromosome; a physical map provides the recombination frequencies between the genes. - A genetic map is linear; a physical map is circular. - A genetic map shows recombination frequencies between genes; a physical map shows distances in terms of a physical measurement such as base pairs of DNA sequence - A genetic map gives the relative order and nucleotide distances between genetic loci; a physical map shows the banding patterns and other cytological features of chromosomes. A genetic map shows recombination frequencies between genes; a physical map shows distances in terms of a physical measurement such as base pairs of DNA sequence

Describe the consequences of somatic and germ-line mutations

Germline mutations are changes to your DNA that you inherit from the egg and sperm cells during conception. Somatic mutations are changes to your DNA that happen after conception to cells other than the egg and sperm. Mutations can lead to genetic conditions that affect your health Eukaryotic organisms have two primary cell types --- germ and somatic. Mutations can occur in either cell type. If a gene is altered in a germ cell, the mutation is termed a germinal mutation. Because germ cells give rise to gametes, some gamete s will carry the mutation and it will be passed on to the next generation when the individual successfully mates. Typically germinal mutations are not expressed in the individual containing the mutation. The only instance in which it would be expressed is if it negatively (or positively) affected gamete production. Somatic cells give rise to all non-germline tissues. Mutations in somatic cells are called somatic mutations. Because they do not occur in cells that give rise to gametes, the mutation is not passed along to the next generation by sexual means. To maintain this mutation, the individual containing the mutation must be cloned. Cancer tumors are a unique class of somatic mutations. The tumor arises when a gene involved in cell division, a protooncogene, is mutated. All of the daughter cells contain this mutation. The phenotype of all cells containing the mutation is un controlled cell division. This results in a tumor that is a collection of undifferentiated cells called tumor cells.

Define the terms "in cis" and "in trans" as applies to protein and DNA components of an operon

Define and give examples of cis-acting and trans-acting regulatory elements. Operators and promoters work in cis Repressors work in trans A protein that, when bound to a cis-acting regulatory DNA element such as an operator or an enhancer, activates transcription from an adjacent promoter. Activator A protein that binds to a cis-acting element such as an operator or a silencer, thereby preventing transcription from an adjacent promoter. Repressor Cis-acting factors are mechanisms that affect gene expression only on the same chromosomal allele, while trans-factors act equally on both alleles. Transcription factors and long noncoding RNAs are a classic example of trans-acting factors. Compare and contrast cis- and trans-genetic effects that can lead to epigenetic defects. they both have to do with alternations in regulatory regions but trans has more to do with mutations in proteins while cis has more to do with repeating expansions for example in fragile X syndrome it repeats expansion in FMR1 promoter leading to CpG island methylation.

List the steps in PCR

Denaturation - 97deg C: As in DNA replication, the two strands in the DNA double helix need to be separated. The separation happens by raising the temperature of the mixture, causing the hydrogen bonds between the complementary DNA strands to break. This process is called denaturation. Annealing - around 54deg C: Primers bind to the target DNA sequences and initiate polymerisation. This can only occur once the temperature of the solution has been lowered. One primer binds to each strand. Extension: New strands of DNA are made using the original strands as templates. A DNA polymerase enzyme joins free DNA nucleotides together. This enzyme is often Taq polymerase, an enzyme originally isolated from a thermophilic bacteria called Thermus aquaticus. The order in which the free nucleotides are added is determined by the sequence of nucleotides in the original (template) DNA strand. (Steps repeated 30-40 times) The result of one cycle of PCR is two double-stranded sequences of target DNA, each containing one newly made strand and one original strand. The cycle is repeated many times (usually 20-30) as most processes using PCR need large quantities of DNA. It only takes 2-3 hours to get a billion or so copies.

List the steps in Southern Blotting

(1) Cleave genomic DNA with restriction enzymes (2) Perform gel electrophoresis (3) Transfer fragmented DNA from gel to nitrocellulose membrane (4) Hybridize DNA of interest with labeled probe (5) Expose membrane to film and develop

Explain the triplet nature of the genetic code (give evidence)

+ and - mutational changes; the triplet code due to the sequence of three nucleotides that code for an amino acid in a protein. Then four bases of nucleotides (AGCU); start codon (AUG) and stop codon (UAA, UAG, UGA)

Discuss three ways how epigenetics can influence human disease.

-epigenetic inheritance. because what affects the mother affects the fetus and what affects the fetus affects the reproductive cells. -in autoimmune dieseases -environment and genotype is linked to phenotype and disease.

Which of the following DNA repair mechanisms is most prone to error? A) mismatch repair B) base excision repair C) SOS repair D) nucleotide excision repair E) All DNA repair systems are equally prone to error.

C) SOS repair

How does epigenetics contribute to cancer?

DNA methylation can silence tumor suppressor. can cause genome instability.

Describe the role of methylation in gene silencing and genomic imprinting

Genomic imprinting is a process of silencing genes through DNA methylation. The repressed allele is methylated, while the active allele is unmethylated. This stamping process, called methylation, is a chemical reaction that attaches small molecules called methyl groups to certain segments of DNA what are three functions of cytosine methylation in mammals -for the silencing of transposable elements -control of imprinted genes -X chromosome inactivation what is the problem with the statement "cytosine methylation is associated with gene silencing"? its wrong because it is mainly associated with gene silencing in CpG context. however there are exceptions in ESCs. compare and contrast the following two methods to assay DNA methylation; Bs-seq and nanpore sequencing. Bs-seq used to detect methylated cytosines in genomic DNA. they are treated with sodium bisulfate. unmethylated cytosine is turned into thymidine. locations of methylated cytosine can be found by comparing treated and untreated strands. What is a CpG island? - ~1kb, CpG-rich - promoter associated - often unmethylated - regulatory function -correlated with silencing.

List the steps in modern sequencing techniques.

Step 1- Nucleic Acid Extraction and Isolation. ... Step 2- Library Preparation. ... Step 3- Clonal Amplification and Sequencing. ... Step 4 -Data Analysis Using Bioinformatics.

tertiary structure refers to the three-dimensional structure of an entire polypeptide chain

Tertiary structure is the overall shape of the protein. Most proteins (e.g. lysozyme, hemoglobin and insulin) have a compact, globular tertiary structure. Some proteins are fibrous. Fibrous proteins like collagen (tendons, cartilage) and keratin (hair, feathers, horns, hoofs, etc.) have the alpha helix formation over their entire length. Other fibrous proteins like fibroin (the structural protein of silk) are dominated by beta sheets. Tertiary structure is influenced by ionic bonds between opposite charged R-groups, hydrogen bonds between R-groups bearing opposite partial charges, and hydrophobic interactions resulting from the tendency of nonpolar R-groups to stay close together in an aqueous solution. Another important bond affecting tertiary structure occurs in proteins that contain the amino acid cysteine. Where two cysteine monomers are close together, the sulfur of one cysteine bonds to the sulfur of the other, forming strong covalent bonds known as disulfide bridges.

Define polycistronic

The coding pattern of prokaryotes, in which one mRNA may code for multiple proteins.

Distinguish between the levels of protein structure

The shape of a protein can be described by four levels of structure: primary, secondary, tertiary and quaternary.

General transcription factors

also called TFII factors, theyare required for transcription of all or most genes. TFII factors are required for basal transcription (relatively low level of transcription). Higher level of transcription are activated by transcription activators. Basal transcription can be suppressed by transcription repressor

DNA damage can occur

during DNA replication, spontaneously, or due to exposure to certain chemicals or radiation.

The most common form of spontaneous replication error is

tautomeric shift.

Define 'reading frame'

A group of several codons that, taken together, provide the code for an amino acid A sequence of bases in messenger RNA (or deduced from DNA) that encodes for a polypeptide.

Describe the formation of heterochromatin, relationship to gene silencing

At centromeres, heterochromatin formation is directed by RNA interference (RNAi) a naturally occurring process in the nucleus of eukaryotic cells that silences gene expression

What is a human disorder affecting chromatin in cis? Make sure to explain what is meant by "in cis".

Fragile X syndrome- repeat expansion in FMR1 promoter leading to CpG island methylation. cis effects regulatory sequence (promoter) mutation.

List the important features of a plasmid vector and the function of each.

Important features of the plasmid are .... (1) a sequence for the initiation of DNA replication, which allows the plasmid to replicate in the bacteria (ori) (2) a promoter sequence for initiating transcription of the inserted gene (pBAD) (3) the gene of interest (rfp) (4) a gene encoding a protein for antibiotic resistance, which allows for identification of bacteria that have taken in the plasmid (kanR / ampR). Ori - orgin of replication. RFP - red fluorescent protein . Amp-R - selectable marker. Ara-C - binds promoter together so we can get the transcription of gene interest.

Compare and contrast the terms genetic mosaic and chimera

Mosaicism generally starts as one or a small group of mutant cells while chimerism generally involves a more massive input of genetically different cells. Mosaicism and chimerism refer to one organism with two or more distinct populations of cells. Mosaics start with the same genome but chimeras is a fusion of two different genomes. They differ in the mechanisms by which each is prevented.

Explain how next generation sequencing is different than previous methods of sequencing

NGS allows you to screen more samples cost-effectively and detect multiple variants across targeted areas of the genome—an approach that would be costly and time-consuming using Sanger sequencing.

A protein's primary structure is defined as the amino acid sequence of its polypeptide chain

Primary structure is the unique and linear sequence of amino acids in a protein. It is the sequence in which amino acids are added to a growing polypeptide during translation. With 20 different amino acids, the number of primary sequences is almost infinite. It is the primary structure that determines how (and where) the polypeptide will fold to give a protein its shape. Thus, primary structure determines the higher levels of protein structure. Small changes in primary structure can result in large changes in protein shape and function.

quaternary structure is the three-dimensional arrangement of the subunits in a multisubunit protein.

Quaternary structure occurs in proteins that are made up of more than one polypeptide chain. Combining different polypeptides leads to a greater range of biological activity. Collagen, for example, is made of three subunits intertwined into a triple helix, and hemoglobin is made of four heme groups, each a different polypeptide. An influence on the quaternary structure of some proteins is the presence of a prosthetic group: a small molecule that is not a peptide but that tightly binds to the protein and plays a crucial role in its function. For example, the four heme groups on a hemoglobin protein are prosthetic and they function to carry oxygen. Proteins with prosthetic groups are called conjugated proteins.

Explain how an Ames test can be used to identify mutagens

The Ames test uses bacteria as a very sensitive biological indicator of whether or not a substance can cause a change in DNA sequence The Ames test is used to identify mutagenic, potentially carcinogenic chemicals. The Ames test is used to determine the potential mutagenicity of test compounds by screening for new mutations in which of the following organisms? Salmonella typhimurium

List common sources of DNA damage.

Endogenous sources of DNA damage include hydrolysis, oxidation, alkylation, and mismatch of DNA bases; sources for exogenous DNA damage include ionizing radiation (IR), ultraviolet (UV) radiation, and various chemicals agents. Explain how each of the following can lead to mutations: depurination, deamination, free radicals & oxidative damage, ionizing radiation (X-rays and gamma-rays), UV light Depurination- loss of nitrogenous base leading to site without purine Deamination- amino acid group in cytosine or adenine is converted to uracil and adenine converted to hypoxanthine free radical UV light- creates pyrimidine dimers, two identical pyrimidines that distort DNA conformation Ionizing radiation- energy of radiation varies intensely w wavelength shorter wavelengths have more energy X-rays and gamma rays causes ionization of molecules and DNA double strand breaks free radicals- stable molecules transformed into free radicals (one or more unpaired electrons) by radiation directly and indirectly affect DNA alter purines and pyrimidines, break phosphodiester bonds, and produce deletions translocations and fragmentations Oxidative damage - due to by products of normal cellular processes exposure to high energy radiation

Define epigenetics and give examples

Epigenetics is the study of how your behaviors and environment can cause changes that affect the way your genes work. Unlike genetic changes, epigenetic changes are reversible and do not change your DNA sequence, but they can change how your body reads a DNA sequence. Explain how PRC1 and PRC2 function using the "writer-reader-eraser" model for epigenetic marks. PCR2 acts as the writer because it has a 4 molecule complex and E(Z) on its own is not functional - it needs ESC and SU(Z)12 for HMT function. (ESC, SU(Z), P55, E(Z)) PRC1 acts as the reader because PC has a chromo-domain (reader) PRC1 also acts as the eraser because it removes the PRC2 and replaces it with another PRC1 molecule. What is genomic imprinting, and how does it fit the definition of epigenetics we use? Genomic imprinting is the epigenetic phenomenon by which certain genes are expressed in a parent-of-origin- specific manner in a diploid cell affects only a subset of genes occurs in mammals and plants results in monoallelic, parental specific expression pattern.

List the specific components of the lac operon and the function of each protein encoded, outcomes of mutations described; explain the process of negative induction

The lactose operon of E. coli encodes the enzyme b-galactosidase which hydrolyzes lactose into galactose and glucose. The lac operon contains three cistrons or DNA fragments that encode a functional protein. The proteins encoded by cistrons may function alone or as sub-units of larger enzymes or structural proteins. The Z gene encodes for b-galactosidase. The Y gene encodes a permease that facilitates the transport of lactose into the bacterium. The A gene encodes a thiogalactoside transacetylase whose function is not known. All three of these genes are transcribed as a single, polycistronic mRNA. Polycistronic RNA contains multiple genetic messages each with its own translational initiation and termination signals. The activity of the promoter that controls the expression of the lac operon is regulated by two different proteins. One of the proteins prevents the RNA polymerase from transcribing (negative control), the other enhances the binding of RNA polymerase to the promoter (positive control). The protein that inhibits transcription of the lac operon is a tetramer with four identical subunits called lac repressor. The lac repressor is encoded by the lacI gene, located upstream of the lac operon and has its own promoter. Expression of the lacI gene is not regulated and very low levels of the lac repressor are continuously synthesized. Genes whose expression is not regulated are called constitutive genes. In the absence of lactose the lac repressor blocks the expression of the lac operon by binding to the DNA at a site, called the operator that is downstream of the promoter and upstream of the transcriptional initiation site. The operator consists of a specific nucleotide sequence that is recognized by the repressor which binds very tightly, physically blocking (strangling) the initiation of transcription. The

Explain how retrotransposons differ from other mobile genetic elements

Transposons are mobile genetic elements that can multiply in the genome using a variety of mechanisms. Retrotransposons replicate through reverse transcription of their RNA and integration of the resulting cDNA into another locus. How do transposons differ from retrotransposons? Transposons may or may not leave a copy behind at the original site, whereas retrotransposons always leave a copy behind at the original site. Transposons move by means of a DNA intermediate, whereas retrotransposons move by means of an RNA intermediate.

Deamination is a spontaneous DNA mutation that converts cytosine to

Uracil

Double-stranded break repair mechanisms

are error prone and may join nonhomologous ends of DNA together or use homologous DNA to repair the damage.

List all levels at which gene expression can be regulated in eukaryotes

gene expression is regulated at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels.

Chimerism

in genetics, the presence of cells of different origin in an individual, whether by mutation, transplant, or some other process; Two or more genetically different cell lines within a single individual derived from different zygotes Presence of two or more chromosomal complements found in same tissue of individual; Can happen with organ transplant (donor cells different from recipient); twins (may start out as two or more zygotes but absorbed by remaining fetus) Divergent genotypes usually found in all across genome; leads to dilemma in sex determination Artificial: blood transfusion, organ, stem cell and bone marrow transplantation Twin/multiple gestation: via trans placental passage of second cell line Naturally occurring: chimerism XX/XY highly frequent, increase with IVF birth. (Fetal-maternal; blood sharing and twin; whole body/dispermic; tumor; germ cell) Tetragametic: via fusion of two fertilized zygote Before transplantation tests: HLA, red cells phenotype, peripheral blood and skin fibroblast karyotyping, FISH

What is the "two-hit" model for cancer?

many cancers are initiated by mutations in genes who's normal function is to prevent bad cell growth (tumor-suppressor genes) these mutant alleles are usually recessive to normal alleles, so a tumor will only grow when both alleles are nonfunctional.

Explain how bacteria are able to identify incorrectly incorporated bases.

the re-replication of highly repetitive DNA sequences

Describe steps of the DNA repair mechanisms discussed in lecture.

Exposure to chemicals, ultraviolet radiation, and other mutagens, such as tobacco smoke, can damage DNA. DNA damage typically occurs on one strand of double‑stranded DNA and can result in bulges or bends in the DNA. Cells can use nucleotide excision repair to remove and replace sections of a damaged DNA strand that alters the shape of the DNA. Nucleotide excision repair often takes place prior to DNA replication, correcting the deformed DNA sequence before the DNA is copied. Nucleotide excision repair can also be performed if the bulge is detected during DNA replication. After a mistake is detected, a nuclease enzyme makes cuts upstream and downstream of the DNA damage on one strand of DNA. The cut DNA segment, which contains the damaged area and several bases on either side, is then removed. The undamaged DNA strand has the correct sequence and is left intact. A second enzyme, DNA polymerase, uses the undamaged DNA strand as a template to synthesize the correct sequence on the damaged DNA strand. The orange DNA in the illustration is the newly synthesized segment. A third enzyme, DNA ligase, joins the new sequence to the existing DNA upstream and downstream of the new sequence. Damaged DNA is not repaired by copying a correct sequence from one chromosome in a homologous pair and using it to replace an incorrect sequence on the other chromosome in the pair. Not only would it be difficult to identify the specific sequence needed, but chromosomes in a homologous pair often contain different alleles of a gene. DNA damage cannot be repaired by simply deleting an incorrect DNA sequence from a strand of DNA. A cell requires many genes to function, and removing a damaged section of DNA and joining the remaining ends together would result in the loss of genetic information. Many different enzymes proofread DNA, both du

Define constitutive expression

Expression of a gene that is transcribed at a constant level. Supplement. For example, the expression of housekeeping genes to produce proteins such as actin, GAPDH and ubiquitin.

Define merodiploid/merozygote

Formed by recombination during a genetic exchange process, these merodiploids are thus true merozygotes, defined as bacterial cells containing a second copy of part of the chromosome. Merozygote is a state when a cell, usually bacterial, is temporarily partial diploid as result of DNA transfer processes like conjugation

Define forward, reverse, and suppressive mutations

When a mutation changes the wild type normal genotype to a mutant type, as is more often the case, the event is called a forward mutation. This is in contrast to reverse mutations in which the mutant genotype changes to the original wild type. Reverse mutations could occur in different ways. In a true reverse mutation, the original base pair sequence of the wild type may be restored. Thus if a GC pair of the wild type sequence is replaced by an AT pair to produce a forward mutation, a true reverse mutation could again substitute a GC pair in that position. Sometimes a different base pair may be inserted at the site of the altered pair which had produced the forward mutation. Thus when GC is replaced by AT, the reversion may be due to substitution by CG instead of GC. This produces a reverse phenotype even though its sequence differs from the wild type in a single base pair. Sometimes an apparently reverse mutation is due to a second suppressor mutation which suppresses the effect of the primary mutation so that the phenotype appears like the wild type. There may be intragenic suppression when the second mutation occurs within the gene carrying the first mutation but in a different site. Or suppression may be intergenic (extragenic) when the second mutation lies in a different gene. In both types of suppression, the second suppressor mutation produces functional products of the gene which carries the first or primary mutation. For example suppose gene A is not able to produce A protein due to a mutation. A suppressor mutation in the same or in a different gene could result in the production of A protein, thereby reversing the mutation in gene A. Scientists who study mutation use the most common genotype found in natural populations, called the wild type, as the standard against which to compare a mutant allele. Mu

Which of the following refers to a mutation that causes a change from mutant phenotype to wild type phenotype. forward mutation reversion (reverse) mutation second site reversion (suppressor) mutation nonsense mutation a, b and c are correct. b, c and d are correct. b and d are correct

b and c are correct Reversion mutations are key to understanding the Ames test, which starts out with bacteria that cannot make histidine and therefore cannot grow on medium that lacks histidine (for example, minimal medium). It then selects for rare mutations that convert the his- ("histidine minus") mutants to his+ (histidine plus) by plating millions of cells on a plate that lacks histidine. Only the rare his+ reverse mutants ("revertants") can grow on medium lacking histidine. We'll do lots of work with the Ames test as a way of helping you understand mutagens and mutation frequency.

Describe the following types of operons: negative inducible, positive inducible, negative repressible, positive repressible

Negative inducible operons: The control at the operator site is negative. Molecule binding is to the operator, inhibiting transcription. Such operons are usually off and need to be turned on, so the transcription is inducible. Negative repressible operons: The control at the operator site is negative. But such transcription is usually on and needs to be turned off, so the transcription is repressible. Positive inducible regulation, the default state of gene transcription is "off." The regulatory protein alone cannot bind to the operator site to turn it on, but when the effector is present, it binds to the regulatory protein and the resulting molecular complex binds to the operator, and turns gene transcription on.

List the steps in northern blotting

RNA isolation (total or poly(A) RNA) Probe generation. Denaturing agarose gel electrophoresis. Transfer to solid support and immobilization. Prehybridization and hybridization with probe. Washing. Detection. Stripping and reprobing (optional)

Describe the function of bacterial restriction enzymes and how they are used in recombinant DNA techniques.

Restriction enzymes cut DNA strands at points on restriction sites. Used in recombinant DNA technology since they cut the DNA in a staggered manner, creating sticky ends that bond with complementary sticky ends of other fragments. Bacteria protect their DNA by modifying their own recognition sequences, usually by adding methyl (CH3) molecules to nucleotides in the recognition sequences .

secondary structure is the local spatial arrangement of a polypeptide's backbone (main chain) atoms

Secondary structure describes regions where the polypeptide is folded into localized shapes. There are two types of secondary structure (alpha helix and Beta pleated sheet). The alpha helix is a delicate coil formed by hydrogen bonding between a hydrogen atom on one amino acid and an oxygen atom on the fourth amino acid away. The beta sheet results from hydrogen bonding between different polypeptide chains or between different sections of the same polypeptide.

Construct a simple genetic map given data from double and single RE digests.

1) To start, focus on the single enzyme digests. These tell you how many cut sites (also known as cleavage sites) each enzyme has. In plasmids, an enzyme that has one cut site will produce a single fragment. Such one-cut enzymes are extra useful if you're working with an unknown plasmid as the size of the single fragment is also the size of the plasmid. Enzymes with more cut sites will produce multiple fragments. For example, an enzyme that cuts the plasmid at two sites will produce two fragments. 2) Add the fragment lengths produced by each single enzyme digestion to double check your experiment and make sure that your chosen conditions allowed for complete digestions. Every single enzyme digest should add up to the same final plasmid size. Digestions that sum to a different number are either the result of incomplete digestions or may have two fragments of the exact same size. (Two fragments of the same size will look like a single band on an electrophoresis gel). Summing fragments across all the single enzyme digestions also lets confirm plasmid size even in the absence of a single cut site enzyme. 3) Once you know the size of your plasmid, use a pencil to draw a circle with the total bp number next to it. Take a second to imagine zooming in on this circle's line to a focus where each point along the circumference is a box with a nucleotide base inside of it and a position number. The 'box' at the very top of the circle is given the position number 0. Just to the left of it is the final nucleotide which is at position 0+N where N equals the total size of your plasmid. 4) Begin by mapping the single enzyme digest with the most bands. (Choosing the most bands isn't required but it tends to make life easier.) Arbitrarily assign the first restriction enzyme cut site of this enzyme to the zero box at the to

Define allosteric binding site

Allosteric regulation, broadly speaking, is just any form of regulation where the regulatory molecule (an activator or inhibitor) binds to an enzyme someplace other than the active site. The place where the regulator binds is called the allosteric site. These enzymes, which include some of our key metabolic regulators, are often given the name of allosteric enzymes. Allosteric enzymes typically have multiple active sites located on different protein subunits. When an allosteric inhibitor binds to an enzyme, all active sites on the protein subunits are changed slightly so that they work less well.

Explain how autonomous and non-autonomous transposons differ

Autonomous and Nonautonomous Transposons Both class 1 and class 2 TEs can be either autonomous or nonautonomous. Autonomous TEs can move on their own, while nonautonomous elements require the presence of other TEs in order to move. An autonomous element has the genes that are necessary for transposition. For example, a cut-and-paste transposon that was autonomous would also have the transposase gene. A nonautonomous element does not have all the genes that are necessary for transposition. However, if a cell contains an autonomous element and a nonautonomous element of the same type, the nonautonomous element can move. For example, if a Drosophila cell contained two P elements, one autonomous and one nonautonomous, the transposase expressed from the autonomous P element could recognize the nonautonomous P element and catalyze its transposition.

Explain how mutation in non-coding regions can affect gene expression

By altering one of these regions, a variant (also known as a mutation) in noncoding DNA can turn on a gene and cause a protein to be produced in the wrong place or at the wrong time. Alternatively, a variant can reduce or eliminate the production of an important protein when it is needed. Explain the difference in effects for mutations in coding regions vs noncoding regions of the genome. How can mutations in coding regions cause a change in phenotype? How can mutations in noncoding regions cause a change in phenotype? Is it possible for either of these types of mutations to not cause a change in phenotype? In coding regions, mutations can change the amino acid sequence of the polypeptide. The phenotype would change because the amino acid would change resulting a new or altered polypeptide In noncoding regions, mutations can change gene expression or alter genome function in other ways. Phenotype would be altered because if gene expression changes then a visible change can occur yeah if the amino acid doesnt code for something use and yes if the gene is not affecting ones visible charactersitics

Indicate which repair mechanisms are error prone.

In translesion replication, the DNA polymerase shifts from template directed synthesis to catalyzing the incorporation of random nucleotides. These random nucleotides are usually mutations (i.e. in three out of four times), hence this process is also designated error-prone repair.

given an mRNA sequence, provide the corresponding proteins sequence using the genetic code chart 5′-AUG-UCU-UCG-UUA-UCC-UUG-3′

Met-Ser-Ser-Leu-Ser-Leu

Define structural, functional, and comparative genomics.

Structural genomics involves the physical nature of genomes and includes the sequencing and mapping of genomes. Functional genomics involves studying the expression and function of the genome. Genomics can also involve the investigation of interactions between genes and between genes and the environment. Structural genomics is the field of genomics that deals with structures of genome sequences. Understanding the genome structure involves constructing genome maps, sequencing genes, annotating gene features, and comparing genome structures. Functional genomics deals with the study of gene expression and the function of genes in a genome. It involves studying gene functions at the whole genome level using high-throughput methods. Comparative genomics involves the comparison of genomes from different species that can provide insights into evolutionary relationships, functional elements, and genetic variations among species. It uses various tools that help to identify and understand the similarities and differences in the genomes of various species.

Compare and contrast the mapping/sequencing methods used in the Human Genome Project and that of Celera Genetics and describe characteristics of the human genome

With the genomes of many species fully sequenced, scientists can study whole sets of genes and their interactions, an approach called GENOMICS. The sequencing efforts that feed this approach have generated, and continue to generate, enormous volumes of data. The need to deal with this ever-increasing flood of information has spawned the field of BIOINFORMATICS the application of computational methods to store and analyze biological data. The Human Genome Project fostered development of faster, less expensive sequencing techniques Celera: Shooting at Random and Organizing Later Before the IHGSC had completed the first phase of the Human Genome Project, a private biotechnology company called Celera Genomics also entered the race to sequence the human genome. Led by Dr. Craig Venter, Celera proclaimed that it would sequence the entire human genome within three years. As outlined in Figure 4, Celera used two independent data sets together with two distinct computational approaches to determine the sequence of the human genome (Venter et al., 2001). The first data set was generated by Celera and consisted of 27.27 million DNA sequence reads, each with an average length of 543 base pairs, derived from five different individuals. The second data set was obtained from the publicly funded Human Genome Project and was derived from the BAC contigs (called bactigs); here, Celera "shredded" the Human Genome Project DNA sequence into 550-base-pair sequence reads representing a total of 16.05 million sequence reads. The company then used a whole-genome assembly method and a regional chromosome assembly method to sequence the human genome.

Describe mechanisms of chromatin remodeling, indicating effects on transcription

chromatin not only serves as a way to condense DNA within the cellular nucleus, but also as a way to control how that DNA is used. In particular, within eukaryotes, specific genes are not expressed unless they can be accessed by RNA polymerase and proteins known as transcription factors. In its default state, the tight coiling that characterizes chromatin structure limits the access of these substances to eukaryotic DNA. Therefore, a cell's chromatin must "open" in order for gene expression to take place. This process of "opening" is called chromatin remodeling, and it is of vital importance to the proper functioning of all eukaryotic cells. In recent years, researchers have discovered a great deal about chromatin remodeling, including the roles that different protein complexes, histone variants, and biochemical modifications play in this process. Various molecules called chromatin remodelers provide the mechanism for modifying chromatin and allowing transcription signals to reach their destinations on the DNA strand. Understanding the nature and processes of these cellular construction workers remains an active area of discovery in genetic research. Currently, investigators know that chromatin remodelers are large, multiprotein complexes that use the energy of ATP hydrolysis to mobilize and restructure nucleosomes. Recall that nucleosomes wrap 146 base pairs of DNA in approximately 1.7 turns around a histone-octamer disk, and the DNA inside each nucleosome is generally inaccessible to DNA-binding factors. Remodelers are thus necessary to provide access to the underlying DNA to enable transcription, chromatin assembly, DNA repair, and other processes. Just how remodelers convert the energy of ATP hydrolysis into mechanical force to mobilize the nucleosome, and how different remodeler complexes select which nucleosom

Give an example of eukaryotic transposons

two types of eukaryotic transposons - classical - retrotransposons (LTR and non LTR) Give a description of classical transposons like prokaryotic transposons flanked by IR autonomous or nonautonomous encode at least one gene (if autonomous) DDE active site code for a transposase target site duplication structure: direct repeats (target duplication)-IR-gene (transposase)-IR-direct repeat DIfference between classical transposon and prokaryotic transposon classical may be autonomous or nonautonomous and only the autonomous ones carry the transposase gene. the nonautonomous do not code for the gene. describe the 2 examples of classical transposons 1. AC/DC: barbara mclitock disovered it, first identified transposon in any system, it is responsible for the different colored kernels on the corn . due to a nonreplicative transposon. AC is activatorfconsidered the autonomous transposon with all the parts, and DS has multiple parts but they are a set of related yet nonautonomous transposons (dissociator) . thousands of copies of the ds are found in the corn genomes but generally no copies of the Ac are found in the modern corn genomes. Gene C codes for the pigment purple gene which was what was occurring for a while but hten on one of the cells the DS came on and AC was on there too so AC helped the DS move in trans into the C gene so now you have a mutant C which makes colorless kernals. The DS then jumps out with the help of AC regenerating the normal C but it makes purple again but this time with pigments. depending on when exactly the mutation occurs, the kernal can look different. Overall, Ds requires trans-acting protein functions that are supplied by the Ac. 2. P elements in Drosophila: The P element encodes both a transposase as well as a repressor of transposition Sperm contains p elements but none are found in

Describe how epigenetic mechanisms can contribute to the two hits in the "two-hit" model for cancer.

well if there is a gene thats been "hit" with methylation and only silences one of the alleles than the tumor suppressor will continue to work and there will be no tumor but if there is a second "hit" and methylation occurs on the other allele than the tumor suppressor gene will be silenced and the tumor will rise.

Define homolog, ortholog, and paralog

A homologous gene (or homolog) is a gene inherited in two species from a common ancestor. While homologous genes can be similar in sequence, similar sequences are not necessarily homologous. Orthologous are homologous genes where a gene diverges after a speciation event, but the gene and its main function are conserved. If a gene is duplicated in a species, the resulting duplicated genes are paralogs of each other, even though over time they might become different in sequence composition and function. Homology refers to two structures or sequences that evolved from a single ancestral structure or sequence Orthologous structures or sequences in two organisms are homologs that evolved from the same feature in their last common ancestor but they do not necessarily retain their ancestral function. The evolution of orthologs reflects organismal evolution — molecular systematics has, therefore, traditionally been concerned with comparing orthologous sequences. In contrast, homologs whose evolution reflects gene duplication events are called paralogs. For example, the beta chain of hemoglobin is a paralog of the hemoglobin alpha chain and of myoglobin as they evolved from the same ancestral globin gene through repeated gene-duplication events

Define operon and list the structural components

An operon is a cluster of functionally-related genes that are controlled by a shared operator. Operons consist of multiple genes grouped together with a promoter and an operator. Operons are present in prokaryotes (bacteria and archaea), but are absent in eukaryotes. In some situations multiple operons are controlled by the same regulatory protein Operons are regions of DNA that contain clusters of related genes. They are made up of a promoter region, an operator, and multiple related genes. The operator can be located either within the promoter or between the promoter and the genes. RNA polymerase initiates transcription by binding to the promoter region. The location of the operator is important as its regulation either allows or prevents transcription of the genes into mRNA. the bacterium Escherichia coli contains a number of genes clustered into operons and regulons: the Lac operon which is involved in lactose degradation, the Trp operon which is involved in tryptophan biosynthesis, and the His operon which is involved in histidine biosynthesis. These operons are turned on when the gene products are needed. Operons can be under negative or positive control. Negative control involves turning off the operon in the presence of a repressor; this can be either repressible or inducible. A repressible operon is one that is usually on but which can be repressed in the presence of a repressor molecule. The repressor binds to the operator in such a way that the movement or binding of RNA polymerase is blocked and transcription cannot proceed. An inducible operon is one that is usually off. In the absence of an inducer the operator is blocked by a repressor molecule. When the inducer is present it interacts with the repressor protein, releasing it from the operator and allowing transcription to proceed. Repressible operons

List the steps necessary for inserting DNA into a plasmid vector, describe expected results if a given step is altered or skipped.

Bacteria can take up foreign DNA in a process called transformation. Transformation is a key step in DNA cloning. It occurs after restriction digest and ligation and transfers newly made plasmids to bacteria. After transformation, bacteria are selected on antibiotic plates. Bacteria with a plasmid are antibiotic-resistant, and each one will form a colony. Colonies with the right plasmid can be grown to make large cultures of identical bacteria, which are used to produce plasmid or make protein. 1. Specially prepared bacteria are mixed with DNA (e.g., from a ligation). 2. The bacteria are given a heat shock, which causes some of them to take up a plasmid. [The basic answer is that a heat shock makes the bacterial membrane more permeable to DNA molecules, such as plasmids. It appears that the heat shock causes the formation of pores in the bacterial membrane, through which the DNA molecules can pass.] 3. Plasmids used in cloning contain an antibiotic resistance gene. Thus, all of the bacteria are placed on an antibiotic plate to select for ones that took up a plasmid. 4. Bacteria without a plasmid die. Each bacterium with a plasmid gives rise to a cluster of identical, plasmid-containing bacteria called a colony. 5. Several colonies are checked to identify one with the right plasmid (e.g., by PCR or restriction digest). 6. A colony containing the right plasmid is grown in bulk and used for plasmid or protein production.

Describe the role of bioinformatics in the study of genomic data

Bioinformatics, as related to genetics and genomics, is a scientific subdiscipline that involves using computer technology to collect, store, analyze and disseminate biological data and information, such as DNA and amino acid sequences or annotations about those sequences. The use of computer database and computer algorithms to analyze proteins, genes, and the complete collection of DNA that comprises an organism (the genome) Research, development, or application of computational tools and approaches for expanding the use of biological, medical, behavioral, or health data including those to acquire, store, organize, analyze, or visualize such data Bioinformatics focuses on the analysis of molecular sequences, genomics and functional genetics and two closely related disciplines. The goal of genomics is to determine and analyze the complete DNA sequence of an organism, which is the genome. DNA encodes genes that are expressed as RNA transcripts that can be translated into protein. Functional genomics describes the use of genome-wide assays to study gene and protein function. 1. The first perspective on bioinformatics is the cell. The central dogma. Collection of DNA (genome), RNA (transcriptome), and the protein sequence (proteome). Understand how to study both individual genes and proteins and collections of thousands go genes/proteins 2. From the cell we focus on the individual organisms. Each organism change across different stages of development. Gene expression varies in a disease state. Use of DNA microarrays or RNA-seq to measure expression of thousands of genes 3. The largest scale is the tree of life. Grouped into three major branches of bacteria, arches, and eukaryotes. Fundamental unity of life at the molecular level. Through DNA sequence analysis we learn how chromosomes evolve and are sculpted (through c

Explain how an expression vector differs from a regular plasmid vector.

Cloning vectors are the DNA molecules that carry a specific gene of interest into the host cell and its main purpose is to make numerous copies of the inserted gene. A typical cloning vector consists of an origin of replication, a selectable marker, a reporter gene, and restriction sites. Expression vectors are associated with the actual expression of the gene into mRNA and protein in the target organism. Therefore, the expression vectors not only contain all the elements of a typical cloning vector, but also contain all the regulatory sequences, such as promoter, ribosomal binding site, transcription initiation site, translation initiation site, which are essential for getting maximum expression. cloning vectors and expression vectors are essential tools in genetic research, each serving a unique purpose. Cloning vectors facilitate DNA replication and amplification, while expression vectors enable gene expression and protein production. Choosing the right vector depends on your research goals, and understanding their differences is crucial for success.

Mosaicism

Condition in which regions of tissue within a single individual have different chromosome constitutions. Mosaicism is a condition in which presence of two or more chromosomal complements found in the same tissue of an individual where cells derived from the same genetic origin/same zygotes genotype. Mosaicism can arise when there is a mutation in early development so there can be group of cells that behave differently, can happen on a gene level or whole chromosomes. PATTERN OF INHERITANCE: Both autosomal dominant and recessive. Non-disjunction, premature centromere division, anaphase lag in meiosis or mitosis Can be complete or partial Germinal: asymptomatic; only germ cells affected; could be transmitted to progeny; DMD, Turner Syndrome, Hemophilia Somatic: symptomatic; somatic cell affected; not inherited; cancer, Down Syndrome

Describe role of hormones, enhancers, promotors, insulators, activators, co-activators, transcription factors, etc. in the regulation of eukaryotic gene expression

Genes are organized to make the control of gene expression easier. The promoter region is immediately upstream of the coding sequence. The purpose of the promoter is to bind transcription factors that control the initiation of transcription. In some eukaryotic genes, there are regions that help increase or enhance transcription. These regions, called enhancers, are not necessarily close to the genes they enhance. They can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or may be thousands of nucleotides away. Enhancer regions are binding sequences, or sites, for transcription factors. When a DNA-bending protein binds, the shape of the DNA changes. This shape change allows for the interaction of the activators bound to the enhancers with the transcription factors bound to the promoter region and the RNA polymerase. Like prokaryotic cells, eukaryotic cells also have mechanisms to prevent transcription. Transcriptional repressors can bind to promoter or enhancer regions and block transcription. Like the transcriptional activators, repressors respond to external stimuli to prevent the binding of activating transcription factors.

List the known types of histone modifications, indicating effects of each type on transcription

Histone sequences are highly conserved. The diagram in Figure 2 shows a typical chromatin fiber, with the blue cylinders representing histones. Extending from each of the histones is a "tail," called the N-terminal tail because proteins have two ends--an N terminus and C terminus. Here, the C terminus forms a globular domain that is packaged into the nucleosome. The other end of the histone is more flexible and capable of interacting more directly with DNA and the different proteins within the nucleus. Specifically, histone modification involves covalent bonding of various functional groups to the free nitrogens in the R-groups of lysines in the N-terminal tail. Early research has linked differing levels of acetylation and methylation on the histones to altered rates of DNA transcription (Turner, 2005). While the most common additions are acetylation and methylation of lysine residues, many more types of modifications have also been observed, including phosphorylation, a common posttranslational modification. The different types of modifications, which have been called the "histone code," are put in place by a variety of different enzymes, many of which have yet to be fully characterized. Thus, the story of the remodeling machinery continues to be told through a variety of experiments, and much remains to be revealed.

List the steps in Sanger sequencing (be able to read results)

How is Sanger sequencing different from PCR? Sanger sequencing differs from PCR in two important ways: 1. Sanger sequencing uses dideoxynucleotides in addition to deoxynucleotides, whereas PCR uses only deoxynucleotides. 2. In Sanger sequencing, only one primer, either forward or reverse, is used, whereas PCR uses both the primers. What is Sanger sequencing used for? Sequencing is used to study genomes that allow the identification of genetic mutations, and their associations with diseases. Sanger sequencing has applications in the areas of medicine, forensics, and evolutionary biology. How does the Sanger sequencing work? The major technique involved in the Sanger sequencing method is polymerase chain reaction, or PCR. Like PCR, in vitro DNA replication takes place in the Sanger sequencing method, utilizing the reagents that are used in a PCR reaction. What are the 4 basic components of the Sanger sequencing reaction? The 4 basic components of the Sanger sequencing reaction are as follows: 1. DNA template 2. A primer 3. DNA polymerase 4. Dideoxynucleotides and deoxy nucleotides What are the 3 basic steps of sequencing DNA? The 3 basic steps of sequencing DNA are mentioned below: 1. Chain termination PCR 2. Size separation of DNA fragments by gel electrophoresis 3. Interpreting sequencing data

Define the terms genome, transcriptome, and proteome

Human genome is made up of DNA (deoxyribonucleic acid), a long, winding molecule that contains the instructions needed to build and maintain cells. These instructions are spelled out in the form of "base pairs" of four different chemicals, organized into 20,000 to 25,000 genes. For the instructions to be carried out, DNA must be "read" and transcribed - in other words, copied - into RNA (ribonucleic acid). These gene readouts are called transcripts, and a transcriptome is a collection of all the gene readouts present in a cell. The transcriptome is constructed by the process called transcription, in which individual genes are copied into RNA molecules. Construction of the proteome involves translation of these RNA molecules into protein. A proteome is the complete set of proteins expressed by an organism. The term can also be used to describe the assortment of proteins produced at a specific time in a particular cell or tissue type. The proteome is an expression of an organism's genome. However, in contrast with the genome, which is characterized by its stability, the proteome actively changes in response to various factors, including the organism's developmental stage and both internal and external conditions. The study of the proteome is called proteomics, and it involves understanding how proteins function and interact with one another. For instance, many proteins fold into elaborate three-dimensional structures, and some form complexes with each other to perform their functions. In addition, proteins undergo modifications, which may occur either before or after translation. The proteome can be studied using a variety of techniques. For example, two-dimensional gel electrophoresis can be used to separate proteins by their sizes and by their charges. The proteome can also be studied using another laboratory techni

Describe other ways by which base pairing can be altered (incorporation of base analogs, alkylating agents, etc.)

Induced mutations are induced by known factor- such as-physical (ionizing irradiation, ultraviolet light), chemical and biological mutagens (bacteria and viruses). Induced mutations occurs by at least three different mechanisms. They are- By replacing nitrogenous base with base analogs By base alteration-altering a base so that it specifically mispair with another base By distortion of DNA molecule- damaging a base so that it no longer pair with another base Some chemical compounds are sufficiently similar to the normal nitrogenous bases of DNA known as base analogs and they can incorporated into DNA in place of normal bases. These analogs can base pair with other nitrogenous bases but they induce insertion of incorrect nucleotide during replication causing mutation. Uracil is halogenated in the carbon-5 position to give 5-bromouracil, 5-chlorouracil, and 5-iodouracil which can be incorporated into DNA in the place of thymine. 5-bromouracil (5-BU) bromine is formed by bromination at the carbon-5 position of uracil. The resulting structure of 5-bromouracil is similar to thymine but thymine has CH3 group at C5 . 5-Bromouracil is most effective analog to thymine because size of bromine has same van der Waals radius as the methyl group in thymine. 5-bromouracil is highly mutagenic and it pairs with Adenine in normal condition. In 5-BU, the bromine atom is not in a position in which it can hydrogen-bond during base pairing, so the keto form of 5-BU pairs with adenine. However the frequency of tautomeric shift of 5-bromouracil is much higher than Thymine. It changes from keto form to either enol form or an ionized from amino form to keto form when protonated. Now, 5-bromouracil form hydrogen bond with Guanine instead of complementary base Adenine which result in base pair transition from T=A to C=G in subsequent replicat

List the steps of translation in E. coli, indicate the importance of the Shine-Dalgarno sequence

Initiation: the ribosome gets together with the mRNA and the first tRNA so translation can begin. Small ribosomal subunit attaches directly to certain sequences in the mRNA known as Shine-Dalgarno sequences, which come just before start codons and 'point them out' to the ribosome. Bacterial genes are often transcribed in groups called operons so one bacterial mRNA can contain the coding sequences for several genes. The Shine-Dalgarno sequence marks the start of each coding sequences, letting the ribosome find the right start codon for each gene Elongation: amino acids are brought to the ribosome by tRNAs and linked together to form a chain. Our first, methionine-carrying tRNA starts out in the middle slot of the ribosome, called the P site. Next to it, a fresh codon is exposed in another slot, called the A site. The A site will be the "landing site" for the next tRNA, one whose anticodon is a perfect (complementary) match for the exposed codon. Termination: the finished polypeptide is released to go and do its job in the cell. Termination happens when a stop codon in the mRNA (UAA, UAG, or UGA) enters the A site. Stop codons are recognized by proteins called release factors, which fit neatly into the P site (though they aren't tRNAs). Release factors mess with the enzyme that normally forms peptide bonds: they make it add a water molecule to the last amino acid of the chain. This reaction separates the chain from the tRNA, and the newly made protein is released.

Define the term "mutagen"

Mutagen An environmental agent that alters DNA and causes mutations. this process is called mutagenesis, and the resulting mutations are called induced mutations. Chemical mutagen definition Chemicals that cause mutations if cells are exposed to them at high frequencies or for prolonged periods of time. Chemical mutagens cause a change in DNA that alters the function of proteins, as a result, cellular processes are impaired. Naturally occurring mutagen definition Are mutagenic agents that are present at normal levels within natural environments, and may cause mutations. These mutagens can be divided into two groups: biological mutagens and non-biological naturally occurring mutagens. Physical mutagen definition Physical mutagens include heat and ionising radiation. Direct heat often has combined action with chemical and naturally occurring mutagens.

Describe how spontaneous point mutations (base substitutions and insertions/deletions) occur via altered base pairing (recognize tautomers!) and strand slippage

Mutations that involve changes in one or a few nucleotides are known as point mutations because they occur at a single point in the DNA sequence. They generally occur during replication. If a gene in one cell is altered, the alteration can be passed on to every cell that develops from the original one. Point mutations include substitutions, insertions, and deletions In a substitution, one base is changed to a different base. Substitutions usually affect no more than a single amino acid, and sometimes they have no effect at all. In this example, the base cytosine is replaced by the base thymine, resulting in a change in the mRNA codon from CGU (arginine) to CAU (histidine). However, a change in the last base of the codon, from CGU to CGA for example, would still specify the amino acid arginine. Insertions and deletions are point mutations in which one base is inserted or removed from the DNA sequence. If a nucleotide is added or deleted, the bases are still read in groups of three, but now those groupings shift in every codon that follows the mutation Insertions and deletions are also called frameshift mutations because they shift the "reading frame" of the genetic message. Frameshift mutations can change every amino acid that follows the point of the mutation and can alter a protein so much that it is unable to perform its normal function Replication errors can also involve insertions or deletions of nucleotide bases that occur during a process called strand slippage. Sometimes, a newly synthesized strand loops out a bit, resulting in the addition of an extra nucleotide base. Other times, the template strand loops out a bit, resulting in the omission, or deletion, of a nucleotide base in the newly synthesized, or primer, strand. Regions of DNA containing many copies of small repeated sequences are particula

In a pedigree, differential between a maternally and paternally imprinted inheritance pattern

Paternal imprinting favors the production of larger offspring, and maternal imprinting favors smaller offspring. Often maternally and paternally imprinted genes work in the very same growth pathways. This conflict of interest sets up an epigenetic battle between the parents -- a sort of parental tug-of-war. We know now that one of the reasons that mammalian gynogenetic and androgenetic diploids cannot be made is because of an epigenetic phenomenon called genomic imprinting. The expression of an imprinted gene depends upon the parent (maternal or paternal) that transmits it. Epigenetic means "outside the genes". Epigenetic inheritance describes a variant condition that does not involve a change in DNA sequence, yet is transmitted from one somatic cell generation to the next during development and growth of an organism. Maternal imprinting means that the allele of a particular gene inherited from the mother is transcriptionally silent and the paternally- inherited allele is active. Paternal imprinting is the opposite; the paternally-inherited allele is silenced and the maternally-inherited allele is activ

Define point, silent, conservative missense, non-conservative missense, nonsense, transition, and transversion mutations

Point mutations are changes to one base in the DNA code and may involve either: The substitution of a base (e.g. ATG becomes ACG) The insertion of a base (e.g. ATG becomes ATCG) The deletion of a base (e.g. ATG becomes AG) The inversion of bases (e.g. ATG becomes AGT) Base substitutions may create either silent, missense or nonsense mutations, while insertions and deletions cause frameshift mutations [Point mutations that occur in DNA sequences encoding proteins are either silent, missense or nonsense.] Effects of Point Mutations Silent mutations occur when the DNA change does not alter the amino acid sequence of the polypeptide This is possible because the genetic code is degenerate and certain codons may code for the same amino acid Missense mutations occur when the DNA change alters a single amino acid in the polypeptide chain Sickle cell anaemia is an example of a disease caused by a single base substitution mutation (GAG → GTG ; Glu → Val) Nonsense mutations occur when the DNA change creates a premature STOP codon which truncates the polypeptide Cystic fibrosis is an example of a disease which can result from a nonsense mutation (this may not be the only cause though) Frameshift mutations occur when the addition or removal of a base alters the reading frame of the gene This change will affect every codon beyond the point of mutation and thus may dramatically change amino acid sequence Silent: If abase substitution occurs in the third position of the codon there is a good chance that a synonymous codon will be generated. Thus the amino acid sequence encoded by the gene is not changed and the mutation is said to be silent. Missence: When base substitution results in the generation of a codon that specifies a different amino acid and hence leads to a different polypeptide sequence. Depending on the

List the goals of the Human Genome Project

Primary goals were to discover the complete set of human genes and make them accessible for further biological study, and determine the complete sequence of human genomes The first one is to sequence and determine the genes within the human genome. Second is to identify genes that are related to diseases or disorders. Third is to identify the factors that cause DNA variation in humans. This is by identifying the key regions where single-base DNA differences occur. are related to diseases or disorders. Here's a breakdown of this step: 1. The project aims to identify specific genes within the human genome that are associated with diseases or disorders. 2. By sequencing and determining the genes in the human genome, scientists can analyze the genetic information and look for patterns or variations that may be linked to certain conditions. 3. This information can help in understanding the genetic basis of diseases and disorders, which can lead to improved diagnosis, treatment, and prevention strategies. 4. Additionally, the project aims to identify the factors that cause DNA variation in humans. This involves identifying key regions where single-base DNA differences occur, which can pro

Compare and contrast shotgun sequencing and primer walking of long clones

Primer walking is an example of directed sequencing because the primer is designed from a known region of DNA to guide the sequencing in a specific direction. In contrast to directed sequencing, shotgun sequencing of DNA is a more rapid sequencing strategy. 1. Primer Walking: relies on successive synthesis of primers based on the progressive attainment of new sequence information - reiteration of the process allows technicians to walk along a long DNa molecule, designing new primers every 600-800 bases - the speed with which it is sequenced is limited by its reiterative nature 2. Shotgun sequencing; relies on parallel, redundant sequencing of fragmented target DNA - random, overlapping pieces od DNA are used to form a library of sequences; computer algorithms are sued to assemble a contagious sequence ( contig) - this is the more efficient way to sequence DNA - WGS: DNA representing the entire genome is fragmented into smaller pieces and a large number of fragments are chosen at random and sequenced Clone by clone sequencing : each chromosome is first broken into overlapping closets that are then arranged in linear order to produce a physical map of the genome each clone in the map is then sequenced separately - this relies on the availability of specific genetic resource and thus only applicable to some model organisms This is a technique from the "old time" of genome sequencing. The underlying method for sequencing is the Sanger chain termination method which can have read lenghts between 100 and 1000 basepairs (depending on the instruments used). This means you have to break down longer DNA molecules, clone and subsequently sequence them. There are two methods possible. The first is called chromosome (or primer) walking and starts with sequencing the first piece. The next piece of DNA (which is and then uses

Compare and contrast genomic and cDNA libraries - also explain how to screen these libraries.

The DNA library is a collection of DNA fragments that have been cloned into vectors to recognize and isolate desired DNA fragments. There are two types of libraries - cDNA and genomic libraries. The genomic DNA libraries comprise large DNA fragments. On the other hand, cDNA libraries are constituted by cloned, reverse-transcribed mRNA. As a result, they are devoid of the DNA sequences relative to the genomic areas which are not expressed such as the introns. DNA Library (Complementary DNA library) cDNA - It is a DNA copy of an mRNA molecule generated by reverse transcriptase, a DNA polymerase that can use either RNA or DNA as their template These are prepared using mRNA as templates, their starting material They are representative of only those genes of the genome that are expressed given specific conditions cDNA does not have introns, and hence can be expressed in prokaryotic cells Genomic DNA library Genomic DNA is the chromosomal DNA of an entity representative of the collection of its genomic content. They are different from that of complementary DNA, mitochondrial DNA or bacterial plasmid DNA. These are directly prepared from the genomic DNA, and represent the complete genome of an entity For their construction, ligases and restriction endonucleases are vital As it carries introns, they are incapable of expression in the prokaryotes. Moreover, prokaryotes lack the machinery for processing introns What it is: cDNA library = Collection of clones having complementary DNA to the mRNA of an entity Genomic DNA library = Collection of clones having the complete genomic DNA of an entity What does it contain? cDNA library: Represents genes expressed in a particular cell at a given period of time Genomic DNA library: Represents all genes Size cDNA library: Smaller compared to genomic DNA library genomic

Explain the basic process of modern whole genome shotgun sequencing and define associated terms

The method involves randomly breaking up the genome into small DNA fragments that are sequenced individually. A computer program looks for overlaps in the DNA sequences, using them to reassemble the fragments in their correct order to reconstitute the genome. Traditional and next-generation WGS sequencing. The first method, used to sequence the first human genome, relied on the cloning of DNA in microbial cells and employed the dideoxy sequencing technique. We will refer to this approach as "traditional WGS sequencing." Methods in the second group are generally cell-free methods that employ new techniques for sequencing and are designed for very high throughput (referring to the number of reads per machine per unit time). We will refer to this group of methods as "next-generation WGS sequencing." The traditional WGS approach begins with the construction of genomic libraries, which are collections of these short segments of DNA, representing the entire genome. To generate a genomic library, a researcher first uses restriction enzymes, which cleave DNA at specific sequences, to cut up purified genomic DNA. The resulting fragments have short single strands of DNA at both ends. Each fragment is then joined to the DNA molecule of the accessory chromosome. the resulting pool of recombinant DNA molecules is then propagated, typically by introducing the molecules into bacterial cells. Next, the genome fragments in clones from the shotgun library are partially sequenced. The sequencing reaction must start from a primer of known sequence. After sequencing, the output is a large collection of random short sequences, some of them overlapping. These sequence reads are assembled into a consensus sequence covering the whole genome by matching homologous sequences shared by reads from overlapping clones (sequence contigs)

List the specific components of the Tryptophan operon; explain the process of repression and attenuation

The trp operon, found in E. coli bacteria, is a group of genes that encode biosynthetic enzymes for the amino acid tryptophan. The trp operon is expressed (turned "on") when tryptophan levels are low and repressed (turned "off") when they are high. The trp operon is regulated by the trp repressor. When bound to tryptophan, the trp repressor blocks expression of the operon. Tryptophan biosynthesis is also regulated by attenuation (a mechanism based on coupling of transcription and translation). The trp repressor does not always bind to DNA. Instead, it binds and blocks transcription only when tryptophan is present. When tryptophan is around, it attaches to the repressor molecules and changes their shape so they become active. A small molecule like trytophan, which switches a repressor into its active state, is called a corepressor. When there is little tryptophan in the cell, on the other hand, the trp repressor is inactive (because no tryptophan is available to bind to and activate it). It does not attach to the DNA or block transcription, and this allows the trp operon to be transcribed by RNA polymerase.'' Like regulation by the trp repressor, attenuation is a mechanism for reducing expression of the trp operon when levels of tryptophan are high. However, rather than blocking initiation of transcription, attenuation prevents completion of transcription. When levels of tryptophan are high, attenuation causes RNA polymerase to stop prematurely when it's transcribing the trp operon. Only a short, stubby mRNA is made, one that does not encode any tryptophan biosynthesis enzymes. Attenuation works through a mechanism that depends on coupling (the translation of an mRNA that is still in the process of being transcribed)

Distinguish between insertion sequences and transposons, name elements present

The two major classes of transposable elements are defined by the intermediates in the transposition process. One class moves by DNA intermediates, using transposases and DNA polymerases to catalyze transposition. The other class moves by RNA intermediates, using RNA polymerase, endonucleases and reverse transcriptase to catalyze the process. Among the most thoroughly characterized transposable elements are those that move by DNA intermediates. In bacteria, these are either short insertion sequences or longer transposons. An insertion sequences, or IS, is a short DNA sequence that moves from one location to another. They were first recognized by the mutations they cause by inserting into bacterial genes An insertion sequence encodes a transposase enzyme that catalyzes the transposition. The amount of transposase is well regulated and is the primary determinant of the rate of transposition. Transposons are larger transposable elements, ranging in size from 2500 to 21,000 bp. They usually encode a drug resistance gene or other marker besides the functions required for transposition (Figure 9.10.B.). One type of transposon, called a composite transposon, has an IS element at each end (Figure 9.10.C.). One or both IS elements may be functional; these encode the transposition function for this class of transposons. The IS elements flank the drug resistance gene (or other selectable marker). It is likely that the composite transposon evolved when two IS elements inserted on both sides of a gene. The IS elements at the end could either move by themselves or they can recognize the ends of the closely spaced IS elements and move them together with the DNA between them. If the DNA between the IS elements confers a selective advantage when transposed, then it will become fixed in a population.

Identify which DNA repair mechanism would be used to repair different types of damage.

Three examples (A, B, and C) of DNA damage and repair are shown. Determine which image represents base excision repair, which represents nucleotide excision repair, and which represents end joining. A) A strand of DNA is broken across both strands. The two pieces are aligned and DNA ligase connects thems. B) A double strand of DNA has a damaged base on the top strand. It is removed by DNA glycosylase, creating an abasic site. Endonuclease removes a section of the top strand containing the abasic site and and the two nucleotides to the left. DNA polymerase and DNA ligase replace the removed section, adding the correct base. C) A double strand of DNA has a damaged nucleotide on the top strand. Endonuclease separates the two strands and helicase removes a section of the top strand. DNA polymerase and DNA ligase replace the removed section, adding the correct base. In base excision repair, modified bases are removed and replaced. (B) In nucleotide excision repair, a segment containing a damaged nucleotides is removed and replaced. (C) In end joining, double-strand breaks are repaired. (A) Exposure to chemicals, ultraviolet radiation, and other mutagens, such as tobacco smoke, can damage DNA. DNA damage typically occurs on one strand of double‑stranded DNA and can result in bulges or bends in the DNA. Cells can use nucleotide excision repair to remove and replace sections of a damaged DNA strand that alters the shape of the DNA. Nucleotide excision repair often takes place prior to DNA replication, correcting the deformed DNA sequence before the DNA is copied. Nucleotide excision repair can also be performed if the bulge is detected during DNA replication. After a mistake is detected, a nuclease enzyme makes cuts upstream and downstream of the DNA damage on one strand of DNA. The cut DNA segment, which contains t

Types of transcription factors

Transcription factors are usually classified by DNA-binding domains Zinc finger: bind zinc ion by specific C or H residues (e.g. Sp1, Gal4, GR) Homeodomain: a special type of helix-turn-helix domain, found in transcription factors that regulate organ development (e.g. Ant, Ey, etc) bZIP (basic Leucine Zipper): containing Leu-rich alpha helix DNA-binding domain and basic activation domain, usually act as dimer (e.g. GCN4, Fos, Jun) bHLH (basic helix-loop-helix): in bHLH, the basic region binds DNA, the HLH region acts as the DNA-binding and dimerization domain (e.g. Myc, MyoD). bHLH transcription factors often bind the E box DNA sequence (CANNTG)

Describe unique structural characteristics of tRNAs and processes in which they are involved (function, charging, and 'wobble')

Transfer RNAs (tRNAs) are structural RNA molecules and, depending on the species, many different types of tRNAs exist in the cytoplasm. Bacterial species typically have between 60 and 90 types. Serving as adaptors, each tRNA type binds to a specific codon on the mRNA template and adds the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually "translate" the language of RNA into the language of proteins. As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. The tRNA molecule interacts with three factors: aminoacyl tRNA synthetases, ribosomes, and mRNA. Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the single-stranded RNA molecule hydrogen bond with each other (Figure 11.4.3 ). This shape positions the amino-acid binding site, called the CCA amino acid binding end, which is a cytosine-cytosine-adenine sequence at the 3' end of the tRNA, and the anticodonat the other end. The anticodon is a three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing. An amino acid is added to the end of a tRNA molecule through the process of tRNA "charging," during which each tRNA molecule is linked to its correct or cognate amino acid by a group of enzymes called aminoacyl tRNA synthetases. At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids. During this process, the amino acid is first activated by the addition of adenosine monophosphate (AMP) and then transferred to the tRNA, making it a charged tRNA, and AMP is released. Typically, whereas the first two positions in a codon are important for determining which amino acid will be incorporated into a growing polypeptide, the third position, called the wobb

List the steps of translation in eukaryotes, indicate the importance of the 5'-methyl cap, poly-A tail, and Kozak sequence

Translation is mediated in part by the ribosome54. In eukaryotes, the small ribosomal subunit and associated factors first assemble at the mRNA cap structure then scan along the 5' UTR until a start codon is found. Choosing the correct start codon is critical as it determines the reading frame and thus the polypeptide sequence! During the scanning process, ribosomal proteins within the small ribosomal subunit search for then interact with specific nucleotides both upstream and downstream of the start codon The most frequent nucleotides she found at each position from -6 to +4 is called the Kozak consensus sequence: GCCACCATGG.