Genetics Exam 3
Epigenetics
"Inheritance but not like we know it" Due to turning genes on or off and that pattern of on/off is passed on to progeny Due to methylation/demethylation
Name six different levels at which gene expression might be controlled
1. Alteration of DNA or chromatin structure 2. Transcription 3. mRNA processing 4. Regulation of RNA stability 5. Translation 6. Protein modification after translation
RNA Interference: 2 Major Classes
1. si RNA 2. mi RNA
Mutation Rates may differ among genes
1.4 X 10-5 = fibroblast growth factor, receptor gen (achondroplasia) 1.4 X 10-4 = Duchenne Muscular Dystrophy (dystrophin) (DHD) 1.4 X 10-6 = Huntington's Disease (HD)
Helix-Loop-Helix Motif
2 alpha helices connected by a turn Common in bacterial regulatory proteins; related motifs in eukaryotic proteins Binding site in DNA is a major groove
In RNA silencing, siRNAs and miRNAs usually bind to which part of the mRNA molecules that they control?
3' UTR
Mutation Rates: Backward Rate
A < a (Mutant > Normal
What is a constitutive gene?
A constitutive gene is not regulated and is expressed continually.
Why is the use of a heat-stable DNA polymerase important to the success of PCR?
A heat stable DNA polymerase enzyme is important to the success of PCR b/c the first step of the reaction requires that the solution be heated to b/w 90 degrees and 100 degrees C to separate the 2 DNA strands. Most enzymes are denatured at this temperature. With the use of a heat stable polymerase, the enzyme can be added at the beginning of the reaction and will function throughout multiple cycles.
How is a suppressor mutation different from a reverse mutation.
A reverse mutation restores the original phenotype by changing the DNA sequence back to the wild-type sequence. A suppressor mutation restores the phenotype by causing an additional change in the DNA at a site that is different from that of the original mutation.
How do base analogs lead to mutations?
Base analogs have structures similar to the nucleotides and if present may be incorporated into the DNA during replication. Many analogs have an increased tendency for mispairing, which can lead to mutations. DNA replication is required for the base analog-induced mutations to be incorporated into the DNA.
How do amino acids in DNA-binding proteins interact with DNA?
By forming Hydrogen bonds with DNA bases
How are microsatellites detected?
By using PCR with primers that flank the region containing tandem repeats
In the presence of allolactose, the lac repressor
Cannot bind to the operator
Why are defects in DNA repair often associated with increases in cancer?
Changes in DNA structure may not undergo repair in people with defects in DNA-repair mechanisms. Consequently, increased numbers of mutations occur at all genes, including those that predispose to cancer. This observation indicates that cancer arises from mutations in DNA.
Point Mutations -------Germ-line
Changes to individual base pairs Germ-line ------Passed on to offspring ------All cells in offspring carry the mutation
What do regulatory genes do?
Code for proteins or RNAs that affect gene expression
How do direct-repair mechanisms differ from mismatch repair and base-excision repair?
Direct-repair mechanisms return an altered base to its correct structure without removing and replacing nucleotides. Mismatch repair and base-excision repair remove and replace nucleotides
What is an enhancer? How does it affect the transcription of distinct genes?
Enhancers are DNA sequences that are the binding sites of transcriptional activator proteins. Transcription at a distinct gene is affected when the DNA sequence located b/w the gene's promoter and the enhancer is looped out, allowing for the interaction of the enhancer-bound proteins with proteins needed at the promoter, which stimulate trascription.
Difference between inducible and repressible operons?
Inducible Operon = Transcription is normally off (Not taking place); something must happen to induce transcription or turn it on Repressible Operon= Transcription is normally on (Taking place); something must happen to repress transcription (Turn is off)
In the trp operon, what happens to the trp repressor in the absence of tryptophan?
It cannot bind to the operator and transcription takes place.
Polycistronic
Multiple Genes
What is a silencer?
Regulatory sequence on DNA to which proteins bind and inhibit or decrease transcription
What are Cis-Acting Sequences?
Sites that are located upstream (5') from the transcription initiation site
si RNA
Small Interfering RNA Inhibits expression of same gene Degradation of RNA
Hot Spot
Some regions are more susceptible to change (Ex: Achondroplasia)
Briefly explain how the polymerase chain reaction is used to amplify a specific DNA sequence. What are some of the limitations of PCR?
The double-stranded template DNA is denatured by high temperature. Then, synthetic oligonucleotide primers corresponding to the ends of the DNA sequence to be amplified are annealed to the single-stranded DNA template strands. These primers are extended by a thermostable DNA polymerase so that the target DNA sequence is duplicated. These steps are repeated 30 times or more. Each cycle of denaturation, primer annealing, and extension results in doubling the number of copies of the target sequence between the primers. PCR amplification is limited by several factors. One is that the sequence of the gene to be amplified must be known, at least at the ends of the region to be amplified, in order to synthesize the PCR primers. Another is that the extreme sensitivity of the technique renders it susceptible to contamination. A third limitation is that the most common thermostable DNA polymerase used for PCR, taq polymerase, has a relatively high error rate. A fourth limitation is that PCR amplification is usually limited to DNA fragments of up to a few thousand base pairs; optimized DNA polymerase mixtures and reaction conditions extend the amplifiable length to around 20 kb.
Describe the lac operon and how it controls the metabolism of lactose
The lac operon consists of three structural genes—lacZ, lacY, and lacA. The lacZ gene encodes the enzyme β-galactosidase, which cleaves the disaccharide lactose into galactose and glucose and converts lactose into allolactose. The lacY gene encodes lactose permease, an enzyme necessary for the passage of lactose through the E. coli cell membrane. The lacA gene encodes the enzyme thiogalactoside transacetylase, whose function in lactose metabolism has not yet been determined. All three genes have an overlapping promoter and operator region in common. Upstream of the lactose operon, the lacI gene encodes the lac operon repressor, which binds at the operator region and inhibits the transcription of the lac operon by preventing RNA polymerase from successfully initiating transcription. When lactose is present in the cell, the enzyme β-galactosidase converts some of it into allolactose, which binds to the lac repressor, altering its shape and reducing the repressor's affi nity for the operator. Because the allolactose-bound repressor does not bind to the operator, RNA polymerase can initiate transcription of the lac structural genes from the lac promoter
miRNA
micro RNA Inhibit another gene somewhere else Inhibits translation
What type of bonds are formed from amino acids of DNA binding domains interacting with bases and sugar phosphate backbone?
typically form H-bonds with the bases
Briefly list some of the ways in which siRNAs and miRNAs regulate genes.
1. Through cleavage of mRNA through "silencer activity." The binding of RISCs containing either siRNA or miRNA to complementary sequences in mRNA molecules stimulate cleavage of the mRNA through "silencer activity" 2. Through binding of complementary regions with the mRNA by miRNA to prevent translation: The miRNA as part of the RISC bind to complementary mRNA sequences preventing either translation initiation or elongation which results in premature termination. 3. Through silencer-independent mRNA degradation stimultated by miRNA binding to complementary regions in the 3' UTR of the mRNA: A miRNA binds to the AU rich element in the 3' UTR of the mRNA stimulating degredation using RISC and dicer. 4. Through transcriptional silencing due to methylation of either histone proteins or DNA sequences: The siRNA bind to complementary DNA sequences within the nucleus and stimulate methylation of histone proteins. Methylated histones bind DNA more tightly preventing transcriptional factors from binding the DNA. The miRNA molecules bind to complementary DNA sequences and stimulate DNA methylases to directly methylate the DNA sequences, which results in transcriptional silencing.
DNA fragments that are 500 bp, 1000 bp, and 2000 bp in length are separated by gel electrophoresis. Which fragment will migrate farthest in the gel? a. 2000 bp fragment b. 100 bp fragment c. 500 bp fragment d. All will migrate equal distances
500 bp fragment
Mutation Rates: Forward Rate
A > a (normal > Mutant) More likely because there are more ways to alter a gene than ways of putting it back
List some important differences b/w bacterial and eukaryotic cells that affect the way in which genes are regulated.
A-----Bacterial genes are frequently organized into operons w/ coordinate regulation, and genes w/ operons can be transcribed as on a single long mRNA. Eukaryotic genes are not organized. B-----In Eukaryotic cells, DNA must be unwound from histone proteins prior to transcription occurring. Essentially, the chromatin must assume a more open configeration state, allowing for access by transcription associated factors. C------Activator and repressor molecules function in both Eukaryotes and Bacterial cells. However, in Eukaryotic cells activators appear to be more common than in bacterial cells. D-----In bacteria, transcription and translation can occur concurrently. In Eukaryotes, the nuclear membrane separates transcription from translation both physically and temporally. This separation results in a greater diversity of regulatory mechanisms that can occur at different points during gene expression.
Lac Operon
Absence of lactose Lac Z - β galactosidase Lac Y - permease Lac A - thiogalactoside transacetylase Presence of lactose
How do alkylating agents, nitrous acid, and hydroxylamine produce mutations?
Alkalating agents donate alkyl groups (either methyl or ethyl) to the nucleotide bases. The addition of the alkyl groups results in mispairing of the alkylated base and typically leads to transition mutations. Nitrous acid treatment results in the deamination of cytosine, producing uracil which pairs with adenine. During the next round of replication, a CG to AT transition will occur. THe deamination of guanine by nitrious acid produces xanthine. Xanthine can pair with either cytosine or thymine. If paired with thymine, then a CG to TA transition can occur. Hydroxylamine works by adding a hydroxyl group to cytosine, producing hydroxylaminocytosine. The hydroxylaminocytosine has an increased tendency to undergo tautomeric shifts, which allow pairings with Adenine, resulting in GC to AT transitions.
In a negative repressible operon, the regulator protein is synthesized as
An inactive repressor
Negative Inducible Operons
And inducible operons in general Polycistronic = Multiple Genes Usually code for proteins involved in catabolic (degredation) pathways
Negative Repressible Operons
And repressible operons in general Usually code for proteins invovled in biosynthetic pathways
Why is transcription a particularly important level of gene regulation in both bacteria and eukaryotes?
Because it is the first step in the process of information transfer from DNA to protein. For cellular efficiency, gene expression is often regulated early in the process of protein production.
What are Trans-Acting Factors?
Bind (typically proteins) to the cis-acting sequences and help to initiate transcription
What changes take place in chromatin structure and what role do these changes play in eukaryotic gene regulation?
Changes in chromatin structure can result in repression or stimulation of gene expression. As genes become more transcriptionally active, chromatin shows increased sensitivity to DNase I digestion, suggesting that the chromatin strucute is more open. Acetylation of histone proteins by aceteyltransferase proteins results in the destabilization of the nucleosome structure and increases transcription as well as hypersensitivity to DNase I. The reverse reaction by deaceylase stabilizes nuclesome structure and lessens DNase I sensitivity. Other transcriptional factors and regulatory proteins call chromatin remodeling complexes bind directly to the DNA altering chromatin structure without aceteylating histone proteins. The chromatin remodeling complexes allow for the transcription to be initiated by increasing accessibility to the promoters by trancriptional factors.
Give 3 important characteristics of cloning vectors.
Cloning Vectors Should Have:::::: 1. An origin of DNA replicatoin so they can be maintained in a cell. 2. A gene, such as antibiotic resistance, to select for cells that carry the vector. 3. A unique restriction site or series of sites to where a foreign DNA molecule may be inserted.
Operon
Cluster of genes that are simultaneously expressed Found in prokaryotes Consist of a single promoter upstream from several structural genes Operator = region of promoter where regulatory protein binds Associated with the operon is a regulatory protein, coded for a regulatory gene (not part of operon)
What is an operon?
Cluster of metabolically related structural genes that share one promoter and are expressed together; found in prokaryotes; consists of a promoter, an operator, and a number of structural genes
What do structural genes do?
Code for proteins that are used in metabolism or have structural roles
What do constitutive genes do?
Constantly expressed - proteins encode vital cellular functions
What is gene regulation also known as?
Control of Gene Expression
Gene Size: The larger it is, the greater the chance of error
DMD = Large coding region (higher rate) HD = Smaller coding region (smaller rate)
After DNA fragments have been separated by gel electrophoresis, how can they be visualized?
DNA molecules can be visualized by staining with a fluorescent dye. Ethidium bromide intercalates between the stacked bases of the DNA double helix and the ehtidium bromide-DNA complex fluoresces orange when irradiated with an ultraviolet light source. Alternatively, they can be visualized by attaching radioactive or chemical labels to the DNA before it is placed in the gel.
What is the purpose of the dideoxynucleoside triphosphate in the dideoxy sequencing reaction?
Dideoxynucleoside triphosphates (ddNTPs) act as a substrate for DNA Polymerase but cause termination of DNA synthesis when they are incorporated. Mixed with regular dNTPs, fluorescently labeled ddNTps generate a series of DNA fragments that have terminated at every nucleotide position along the template DNA molecule being sequenced. These fragments can be separated by gel electrophoresis. Because each of the four ddNTPs carries a different fluorescent label, a laser detector can distinguish which base terminates each fragment. Reading the fragments from shorter to longer, an automated sequencer can determine the sequence of the template DNA molecule.
Explain how gel electrophoresis is used to separate DNA fragments of different lengths.
Gel electrophoresis uses an electric field to drive DNA molecules through a gel that acts as a molecular sieve. The gel is an agarose matrix of agarose or polyacrylamide. DNA molecules are loaded into a slot or well at one end of the gel. When an electric field is applied, the negatively charged DNA molecules migrate towards the positive electrode.
Why is gene regulation important for bacterial cells?
Gene regulation maintains internal flexibility, turning genes on and off in response to environmental changes
What is gene therapy?
Gene therapy is the correction of a defective gene by either gene replacement or the addition of a wild-type copy of the gene. For this to work, enough of the cells of the critically affected tissues or organs must be transformed with the functional copy of the gene to restore normal physiology.
Mechanisms of Epigenetics
Genetic Imprinting (Phenotype depends on parent donating gene) X chromosomal inactivation DNA methylation (cytosine bases) chromatin remodeling Methylation of histone tails (Prader-willi's and Angelman's syndrome--------One parent w/ mehtylated C and the other without
Which of the following changes is a transition base substitution?
Guanine is replaced by Adenine
Leucine Zipper
Helix of leucine residues and a basic arm; two leucine residues interdigitate Common in eukaryotic transcription factors Binding site in DNA is two adjacent major grooves
Mutations
Heritable changes in DNA A. Rearrangements B. Aneuploidy, polyploidy C. Point Mutations
Briefly describe two different ways in which intragenic suppressors can reverse the effects of mutations?
Intragenic suppression is the result of second mutations w/in a gene that restore a wild-type phenotype. The suppressor mutations are located at different sites w/in the gene from the original mutation. One type of suppressor mutation restores the original phenotype by reverting the meaning of a previously mutated codon to that of the original codon. The suppressor mutation occurs at a different position than the first mutation, which is still present within the codon. Intragenic suppression may also occur at two different location within the same protein. If two regions of a protein interact, a mutation in one of these regions could disrupt that interaction. The suppressor mutation in the other region would restore the interaction. Finally, a frameshift mutation due to an insertion or deletion could be suppressed by a second insertion or deletion that restores the proper reading frame.
Zinc-Finger Motif
Loop of amino acids with zinc ion at the base Common in eukaryotic regulatory and other proteins Binding site in DNA is a major groove
RNA interference
Mechanism used by eukaryotic cells to limit invasion of foreign genes (Viruses, transposons) and control expression of their own genes (censor or inhibit)
Riboswitches
Metabolic binding RS Regulatory sequences in mRNA that when bound to small proteins form secondary structures that inhibit gene expression, typically by inhibition of translation Discovered in 2009 in human 3' UTR
Mismatch repair in E. coli distinguishes between old and new strands of DNA on the basis of...........
Methyl groups on the old strand
List at least three different types of DNA repair and briefly explain how each is carried out.
Mismatch Repair = Replication errors that are the result of base-pair mismatches are repaired. Mismatch-repair enzymes recognize distortions in the DNA structure due to mispairing and detect the newly synthesized strand by the lack of methylation on the new strand. The bulge is excised and DNA polymerase and DNA ligase fill in the gap. Direct Repair = DNA damage is repaired by directly changing the damaged nucleotide back to its original structure. Base-Excision Repair = The damaged base is excised and then the entire nucleotide is replaced. Nucleotide-Excision Repair = Repair enzymes recognize distortions of the DNA double helix. Damaged regions are excised by enzymes, which cut phosphdiester bonds on either side of the damaged region. The gap generated step is filled in by DNA polymerase.
What is the difference between a missense mutation and a nonsense mutation? Between a silent mutation and a neutral mutation?
Missence Mutation = A base substitution that changes the sequence and the meaning of a mRNA codon, resulting in a different amino acid being inserted into a protein. Nonsense Mutation = When a mutation replaces a sense codon w/ a STOP (or nonsense) codon. Silent Mutation = A nucleotide substitution that changes the sequence of a mRNA codon but not the meaning. Neutral Mutation = The sequence and the meaning of a mRNA codon are changed. However, the amino acid substitution has little or no effect on protein function.
List some of the effects and practical applications of molecular genetic analyses.
Molecular genetics has had profound effects on all fields of biology. Whole genomes have been sequenced, structures of genes elucidaded, the patterns of molecular evolution studied. Recombinant DNA technology is now used to diagnose and screen for genetic diseases, and gene therapy is used to make pharmaceutical products, such as recombinant insulin and clotting factors. Genetically modified organisms will change the lives of farmers and improve agricultural productivity and the quality of food and fiber.
Purpose of DNA Binding Proteins?
Most regulation involves the binding of proteins to DNA - these proteins either promote or inhibit transcription; possess DNA-binding domains containing specific characteristic motifs (ex: helix-turn-helix)
Control of Operon
Negative = regulatory protein is a repressor Positive = regulatory protein is an activator
Point Mutations-----Somatic
Not passed on to offspring except in asexual reproduction Can lead to cancer in an individual
Difference between Positive & Negative Control?
Positive Control = Gene regulation in which the binding of a regulatory to DNA stimulates transcription (the regulatory protein is an activator) Negative Control= Gene regulation in which the binding of a regulatory to DNA inhibits transcription (the regulatory protein is a repressor).
Histone Acetylation
Positively charged tails of nucleosomal histone proteins that interact with negative charged phosphate groups of DNA Addition of an acetal group - (C=O)CH3
DNA Methylation
Prevents transcription Makes heterochromatin Methylation of amino acid in N-terminal histone tails may induce or repress transcription
Mutation Rates
Rates can be calculated from incidence in autosomal (as frequencies) dominant conditions in which parents are unaffected
Negative Repressible
Regulatory protein is a repressor; binds to operator only when attached to another molecule
Negative Inducible
Regulatory protein is a repressor; transcription occurs when another molecule binds to it, causing the repressor to come off the operator
What is an enhancer?
Regulatory sequence on DNA to which proteins bind and enhance transcription; may be located upstream or downstream from a gene, or even within an intron in the gene
What are riboswitches?
Regulatory sequences in an RNA molecule. When an inducer molecule binds to the riboswitch, the binding changes the configeration of the RNA molecule and alters the expression of the RNA, usually by affecting the termination of transcription or affecting translation.
What normal role do restriction enzymes play in bacteria? How do bacteria protect their own DNA from the action of restriction enzymes?
Restriction enzymes cut foreign DNA, such as viral DNA, into fragments. Bacteria protect their own DNA by modifying bases, usually by methylation, at the recognition sites.
Where do restriction enzymes come from?
Restriction enzymes exist naturally in bacteria, which use them to prevent the entry of viral DNA.
What is the difference between somatic gene therapy and germ-line gene therapy?
Somatic gene therapy modifies genes only in somatic tissue, and these modifications cannot be inherited. Germline gene therapy alters genes in germ-line cells and will be inherited.
What is the purpose of Southern blotting? How is it carried out?
Southern blotting is used to detect and visualize specific DNA fragments that have a sequence complementary to a labeled DNA probe. DNA is first cleaved into fragments with restriction endonucleases. The fragments are separated by size via gel electrophoresis. These fragments are then denatured and transferred by blotting onto the surface of a membrane filter. The membrane filter now has single stranded DNA fragments bound to its surface separated by size as in the gel. The filter is then incubated with a solution containing a denatured labeled probe DNA. The probe DNA hybridizes to its complementary DNA on the filter. After washing away excess unbound probes, the labeled probe hybrized to the DNA on the filter can be detected using the appropriate methods to visualize the label.
How do Northern and Western blotting differ from Southern blotting?
Southern blotting is used to transfer DNA from a gel to a solid medium. Northern blotting is used to transfer RNA from a gel to a solid medium, and Western blotting is used to transfer protein from a gel to a solid medium.
How to insertions and deletions arise?
Strand slippage that occur during DNA replication and unequal crossing over events due to misalignment at repetative sequences have been shown to cause deletions and additions of nucelotides to DNA molecules. Strand slippage results from the formation of small loops on either the template or the newly synthesized strand, then a deletion occurs. Loops formed on the newly synthesized strand results in insertions. If during crossing over, a misalignment of the 2 strands at repetative sequences occurs, then the resolution of the cross over will result in one DNA molecule containing an insertion and the other molecule containing a deletion.
What is the difference between a structural gene and a regulator gene?
Structural genes encode proteins; regulator genes control the transcription of structural genes
What is the purpose of the Ames test? How are his- bacteria used in this test?
The Ames Test allows for rapid and inexpensive detection of potentially carcinogenic compounds using bacteria. The majority of carcinogenic compounds result in damage to DNA and are mutagens. The reversion of his- bacteria to his + is used to detect the mutagenic potential of the compound being tested.
How does the binding of regulatory proteins to enhancers affect transcription at genes that are thousands of base pairs away?
The DNA b/w the enhancer and the promoter loops out, so regulatory proteins bound
In the dideoxy sequencing reaction, what terminates DNA synthesis at a particular base?
The absence of a 3'-OH group on the ddNTP prevents the addition of another nucleotide
Most transcriptional activator proteins affect transcription by interacting with ________
The basal transcription apparatus
How is a gene inserted into a plasmid cloning vector?
The gene and plasmid are cut with the same restriction enzyme and mixed together. DNA ligase is used to seal nicks in the sugar-phosphate backbone.
How does the poly (A) tail affect stability?
The poly (A) tail stabilizes the 5' Cap, which must be removed before the mRNA molecule can be degraded from the 5' end
What feature is commonly seen in the sequences recognized by type II restriction enzymes?
The recognition sequences are palindromes, and 4-8 base pairs long
Base analogs are mutagenic because of which characteristic?
They are similar in structure to the normal bases
What are some of different processes that affect gene regulation by altering chromatin structure?
Three general processes are chromatin remodeling, the modification of histone proteins (ex methylation and acetylation of histones) and DNA methylation
Briefly explain how transcriptional activator proteins and repressors affect the level of transcription of eukaryotic genes.
Transcriptional activator proteins stimulate transcription by binding DNA at specific base sequences such as an enhancer or regulatory promoter and attracting or stabilizing the basal transcriptional factor apparatus. Repressor proteins bind to silencer sequences or promoter regulator sequences. The proteins may inhibit transcription by blocking access to the enhancer sequence by the activator protein, preventing the activator from interacting with the basal transcription apparatus, or preventing the basal transcription factor from being assembled.
What is the difference between a transition and a transversion? Which type of base substitution is usually more common?
Transition Mutations = Base substitutions in which one purine (A or G) is changed to the other purine, or pyrimidine (T or C) is changed to the other pyrimidine. Transversion Mutations = Base substitutions in which a purine is changed to a pyrimidine or vice versa. ****Although transversions would seem to be statistically favored b/c there are eight possible transversions and only four possible transitions, about twice as many transition mutations are actually observed in the human genome.
Riboswitch Features
Usually located in the 5' UTR originally described (2002) in bacteria; later found in fungi and plants 2. Most often inhibits translation
Gene Amplification
Via endoduplication Polyteny = duplicated chromosomes stay attached to the original Ex: 2N = 4 (100 pg DNA) 2N = 4 (200 pg DNA)