Genetics Exam 3

Super-repressed

"ALWAYS OFF" Always epistatic in I gene: making lactose is always off- repressor cannot bind the lactose dominant to wild type inducible: everytime the wt accepts the lactose, the mutant represor binds to the operator gene and turns it off

Lac I

"Lac Regulator" makes Lac repressor Recognizes only the lac O gene- Repressor sits on O to block transcription Wt + inducible is dominant to c constituitive

monoallelic expression

-limited amount of transctription factor codes one allele at a time -genomic imprinting: DNA methylation is used to turn off one gene and not the other -histone modification: less permanent than DNA methylation

Is there any protein in eukaryotes that has a function similar to that of the σ-factor in prokaryotes? If so, identify it and explain why its function is analogous to that of σ-factor.

-sigma factor promotes transcription -no direct analog to eukaryotes -Similar to tF2D and TBP(bends DNA and sigma does not) -A bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters.

What are the two main mechanisms of epigenetic regulation of gene expression? What features of each are important for long-term or permanent repression of gene expression? Whatfeatures are more appropriate for transient (short-term) repression of gene expression?

1.Histone modification: short term- reversible and not heritable 2.DNA methylation: permanent, inherited

DNA-binding domain

A DNA-binding domain (DBD) is an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence (a recognition sequence) or have a general affinity to DNA.

constituitive:

Always on, always expressed dom inducer shows over constiruitive

araC protein

AraC activates by binding to araI. If there is no AraC bound to araI, then RNA polymerase does not bindto the promoter. No AraC function, no transcription. Imagine a mutation that disturbs the araO region. AraC will bind to araI but not araO, thus no looping. Transcription will occur whether or not arabinose is present, no the phenotype will be constitutive.AraC is BOTH an activator and a repressor. Arabinose should be called the co-activator or inducer. In the absence of arabinose, AraC binds to BOTH araI and araO, looping the DNA and repressing transcription. When arabinose is present, it binds to AraC, causing it to release araO and activate transcription via its interaction with araI. If you damage AraC via mutation, the result is a non-inducible phenotype; this shows that AraC acts as an activator. (The term "mediator" is something else entirely.)

RNAi

BLOCK VIRUSES a) Exogenous DNA: DNA from outside the cell, not originally there b) double stranded RNA (at least 21 basepairs) c) Dicer- no strand specitivity d)specitivity determine by loaded RNA e)RISC: Cut the mRNA and lock translation

anonymous DNA polymorphisms/DNA markers

DNA polymorphisms that do not affect phenotype but can be used to track specific regions of the genome

nonanonymous DNA polymorphisms

DNA polymorphisms that do affect phenotype by altering gene function, such as frameshift, nonsense, or missense mutations

The cAMP-CAP system provides positive control in the lac operon (superimposed upon negative control by the lacI gene) and in the ara operon (superimposed upon positive control by the araC gene). Based upon your understanding of these mechanisms, predict how each of the following mutations would affect expression and regulation of the lac and ara operons: A mutation that inactivated the CAP protein.

DNA will not be activated--- no protein = no activation even if cAMP is present ONE OPERATOR CONTROLS TRANSCRPTION OF MULTIPLE GENES

dideoxynucleotide

Dideoxynucleotides are chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing. They are also known as 2',3' dideoxynucleotides, and abbreviated as ddNTPs (ddGTP, ddATP, ddTTP and ddCTP). The absence of the 3'-hydroxyl group means that, after being added by a DNA polymerase to a growing nucleotide chain, no further nucleotides can be added as no phosphodiester bond can be created based on the fact that deoxyribonucleoside triphosphates (which are the building blocks of DNA) allow DNA chain synthesis to occur through a condensation reaction between the 5' phosphate (following the cleavage of pyrophospate) of the current nucleotide with the 3' hydroxyl group of the previous nucleotide. The dideoxyribonucleotides do not have a 3' hydroxyl group, hence no further chain elongation can occur once this dideoxynucleotide is on the chain. This can lead to the termination of the DNA sequence. Thus, these molecules form the basis of the dideoxy chain-termination method of DNA sequencing, which was developed by Frederick Sanger in 1977.[1] Dideoxynucleotides are useful in the sequencing of DNA in combination with electrophoresis. A DNA sample that undergoes PCR (polymerase chain reaction) in a mixture containing all four deoxynucleotides and one dideoxynucleotide will produce strands of length equal to the position of each base of the type that complements the type having a dideoxynucleotide present. That is, each nucleotide base of that particular type has a probability of being bonded to not a deoxynucleotide but rather a dideoxynucleotide, which ends chain elongation. Thus, if the sample then undergoes electrophoresis, there will be a band present for each length at which the complement of the dideoxynucleotide is present. It is now common to use fluorescent dideoxynucleotides such that each one of the four has a different fluorescence that can be detected by a sequencer; thus only one reaction is needed.

The cAMP-CAP system provides positive control in the lac operon (superimposed upon negative control by the lacI gene) and in the ara operon (superimposed upon positive control by the araC gene). Based upon your understanding of these mechanisms, predict how each of the following mutations would affect expression and regulation of the lac and ara operons: add lactose to high glucose levels

Does not do much, cell would rather use glucose Need araC bound to arabinose and the CAP-cAMP to bind

Based upon your understanding of enhancer elements, would you expect mutations in an enhancer region to be cis-dominant or trans-dominant? Explain why.

Enhancers and operators are cis-mutations

exome sequencing

Exome: set of DNA sequences that are expressed as RNA -problem: number of copies of mRNA varies from gene to gene, and low abundance mrna is hard to see -could take a stretch of oligoDT, stick it onto mRNAs, use as primers to make cDNAs (eliminating introns), massively parallel sequencing of exons; MRNa expressed genes (non heirerarchical)

inversions

Flipping the orientation to backwards

CAP protein

Helps RNA polymerase bind to the promoter CAP needs to have cyclic AMP to be bound to it, a pump pumps cyclic AMP out of the cell and it cannot bind to CAP therefore cannot bind to DNA Bends DNA around itself near the promoter region Positive control: allows transcription to occur "activator" Binding site: TTAC BINDA TO MULTIPLE SITES

Yeast two-hybrid interaction

Identify binding partners and proteins Works in the nucleus only Shows direct interactions Diagram: Key: AD activation domain Y gene set 2 BD binding domain (that will bind to enhancer) X gene set 1 2 hybrid genes consisting of piece of a yeast gene and a piece of something else. If the two hybrids molecules ineract with eacother correctly then this will turn on some gene we can see when will a yeast cell turn on this gene? when a protein in one cell interracts with a protein in another

DNAse hypersenitivity

In genetics, DNase I hypersensitive sites (DHSs) are regions of chromatin that are sensitive to cleavage by the DNase I enzyme. In these specific regions of the genome, chromatin has lost its condensed structure, exposing the DNA and making it accessible. This raises the availability of DNA to degradation by enzymes, such as DNase I. These accessible chromatin zones are functionally related to transcriptional activity, since this remodeled state is necessary for the binding of proteins such as transcription factors. Since the discovery of DHSs 30 years ago, they have been used as markers of regulatory DNA regions. These regions have been shown to map many types of cis-regulatory elements including promoters, enhancers, insulators, silencers and locus control regions. A high-throughput measure of these regions is available through DNase-Seq.[2]

histone deacetylase (HDA)

Increase pos charge, promote winding of Nucleosomes, demote transcription

Co-immunoprecipitation

Lecture 20 Experiment: -Protein X; who does it interract with in a cell? -Inject protein X into an organism's (bunny etc) cell, to make Anti-X antibodies. -Anti-x antibodiest then atached to plastic bead. -"mush" up cell and see which proteins stick -Study by elecrophoresis. -precipitating a protein by using its antibody, and anything else that comes with that protein -If proteins are known: western blot -If proteins are unknown: Mass spectometry -Drawback: shows if proteins are interacting but not HOW or in which way -Does not tell you anything about the interactions of AB or C just that they interact somehow.

GFP

Lecture 20 Green Fluorescent Protein comes from jelly fish can be inserted into other organisms and expressed

catabolite repression (CAP-cAMP)

Lecture 21 38.00 Why break down lactose if gluose is present? if you put both glucose and lactose_ lac operon will not turn on. phenomenon when the glucose prevents expression of lac operon a. glucose present only the repressor will bind to operator *No transcription bc cell has enough glucose* b. glucose present, lactose present the repressor wont bind but the CAP needs to have cyclic AMP to be bound to it, a pump pumps cyclic AMP out of the cell and it cannot bind to CAP to bind to DNA *No transcription- CAP protein SOMEWHAT required for transcription (see "CAP protein") c. lactose present only Repressor doesn't bind but CAP can now bind *Transcription begins for operon to make B-galactosidase and Y gene and A gene proteins, glucose and galactose biproducts*

Genetic interaction between mutations (e.g., extragenic suppression)

Looking at all mutations and deciding if they interact Just says genes are invlved in a process somehow, says nothing about the actual protein stuff

Non-allelic non-complementation

Non-allelic: not same loci- produce different proteins Non complementation: aa/b+b+ ' a+a+/bb = a+a/b+b mutant Would expect that result to be allelic but its not.... Proteins are forming ligamer: only some percent of proteins can occur

4. Explain the similarities and differences between the mechanisms that lead to monoalleleic expression of X-linked genes (X-inactivation) and the mechanisms that lead to monoallelic expression of autosomal genes (there are several distinct mechanisms!).

Not one difinitive method for X-inactivation

Cis- dominance b) Based upon your model, explain why Oo mutants are cis-dominant. (Consider the situation in O+/O0 and in Oc/O0.)

O+/Oo wild type Oc/Oo

miRNA

OUR CELLS a)Endogenous: encoded by genome b) hairpin loop (must have at least 21 bp, can be divided by bulge in hair loop means a nucleotide that is unpaired) c)Drosha- takes strand with buldge d)specitivity determine by loaded RNA e)RISC: Cut the mRNA and lock translation

Lac operon (lactose operon)

P: promotor gene O: operator gene Repressor protein blocks RNA polymerase and turns transcription off, lactose binds to repressor and is released from the operator- transcription can continue 1 mRNA=3 genes (Z,Y,A) *Lactose makes beta-galactosidase- makes glucose-makes ATP*

TBP

Protein that begins pre-initiation

Northern Blot

RNA

Explain the differences and similarities between RNA interference (RNAi) and gene regulation by micro RNAs. Give explicit molecular details. Consider (at least) such issues as: a) Where does the RNA come from? b) What is the size and structure of the RNA? c) How is the RNA modified in the cell? d) How does the RNA select its target gene? e) How does the RNA affect expression of a target gene?

RNAi

co-activator

Small molecule that is required to activate transc.

Genomics

Study of the structure and function of the complete genome

expression vector

WIKI- An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene.

repressible regulation

a mechanism of gene control where transcription occurs only in the absence of a corepressor. In prokaryotes, a corepressor is a small molecule that binds to a repressor, thereby altering repressor conformation so that it can bind DNA. Repressible regulation is particularly important for the regulation of anabolic pathways.

Inducible regulation:

a mechanism of gene control where transcription occurs only in the presence of a molecule called an inducer. An inducer is a small molecule that binds to a positive regulator, altering the positive regulator's conformation so that it can bind DNA. Inducible regulation is particularly important for the regulation of catabolic pathways.

SSR (simple sequence repeat)/microsatellite

a polymorphism caused by different numbers of repeating units of a short, tandemly repeated sequence (< 10 bp long) such as ACACACAC

CNV (copy number variant)

a polymorphism caused by different numbers of copies of a region of DNA >10 bp long; for example, different numbers of copies of a gene

SNP (single nucleotide polymorphism)

a polymorphism that substitutes one base pair for another

Transcription Factors

a protein that binds DNA sequence-specifically and regulates transcription. In prokaryotes, two types of transcription factors exist: positive regulators activate transcription, and negative regulators (also called repressors) inhibit transcription.

Cis-activation

activator located on same chromosome as gene

DIP/Indel

alternative terms for describing polymorphisms associated with the insertion or deletion of a few base pairs of DNA at a given locus

trans-activation

an enhancer on one chromosome can affect transcription of a gene on a different chromosome.

cAMP-CAP

cAMP= cyclic amp - nucleotide When glucose is high, cAMP is low (glucose opens pumps that sends camp out of cell High cAMP binds to CAP and makes functional protein CAP= activator that binds to DNA - bends DNA and allows rna polymerase and promotors Do not bind if glucose levels are high!!!!! CAP not requred for transcription

Enhancers sometimes act in a tissue-or-cell-specific fashion. For example, if a muscle-specific enhancer is placed near a foreign gene and this is then reintroduced into a eukaryotic organism, one observes expression of the foreign gene only in muscle cells. Is the tissue-specificity of enhancers a sufficient explanation of tissue-specific gene expression? If so, why? If not, why not?

certain cells have transcription factors: i.e. muscles have different proteins than neuron cells The sequences are the same because every cell has the same DNA. Many transcription factors code for proteins for specific tissues.

Genome

complete genetic material of an organism

Epistasis c) Based upon your model, make predictions about whether or not Oo mutations are epistatic to lacI- (constitutive) mutations or to lacI+ (inducible).

constituitive(always on) epistatic to super expressed If I+ (inducible)=super repressed -- lac o epistatic Constituitive: A switch regulatory pathway consists of a series of genes or gene products that alternate between two states, 'on' and 'off'. The components of this pathway are usually acting directly on each other as opposed to on substrates, as occurs in a substrate-dependent pathway. The activity of a switch regulatory pathway is regulated by an upstream signal that stimulates the pathway and produces the downstream response. Environmental changes, cell-cell interactions, zygote formation, and mitogenic signals are only a few of the signals that can act as initiators of a switch regulatory pathway. The downstream response can be altered gene expression, cell division, or the initiation of a developmental process such as pattern formation. Mutations in the genes encoding components of a switch regulatory pathway can lock the component into a permanently 'on' or permanently 'off' state. This has the effect of separating the downstream response from the initiating signal. Mutations that allow the response to be produced even in the absence of a stimulatory signal or despite the presence of an inhibitory signal are referred to as constitutive mutations. The isolation of constitutive mutations is a strong indicator that one is dealing with a switch regulatory pathway.

contig

contiguous- things that are next to eachother -Chromosomal sequences in common between DNA from different cells(i.e YAC) (DNA hybridization) assembled together -allows us to assemble a set of clones that ajoin eachother along a chromosome--assemble the structure of a genome

Reconsider question 4 in a different way. It is sometimes found that an enhancer on one chromosome can affect transcription of a gene on a different chromosome. Can you think of any explanation?

histone modifier and chromatin remodler get turned on by an enhancer in the nucleus and can act on genes nearby

plasmids

lecture 20: can only replicate small pieces of DNA

chromatin remodeling factor

machines that shove all of the histones out of the nucleosomes get turned on by an enhancer in the nucleus and can act on genes nearby Relieves winding in nucleosome

Lac O

make a heterozygote, phenotype constituitive: appears dominant- lac I (apears One can isolate rare mutations in the lac operator (lacO) gene that produce a non-inducible (super-repressed) phenotype ( two different sis-dominant- same DNA as the Z gene whichever of the copy is with the good Z gene is dominant) (Lac I- phenotype wild type is constituent to the mutant regardless if whether its cis or trans. Like other lacO mutations, these mutations (called Oo) are cisdominant. a) Propose a model for the molecular basis of Oo mutations. Trans-dom is product Lac I Cis-dom: signal lac O

Western blot

proteins

6. What is the distinction between a proto-oncogene and an oncogene? Provide some examples of the kinds of genes in these categories; that is, the kinds of gene products encoded by such genes.

proto-oncogene : gene that if it gets messed up could be an oncogene oncogene: cancer gene

histone modifier

put modification on the histone tails get turned on by an enhancer in the nucleus and can act on genes nearby

histone acetylase (HAT)

remodling chromosomes to unwind and increase transcription

Sigma factor

required for transctriptin

DNA polymorphisms

sequence differences between individual genomes within a species

enhancer

sequence of DNA: site on the gene that the activator binds to- transcription factor does not directly bind to polymerase... histone modifiers or chromosome Do not have to be before a promoter or gene that it activates, can be anywhere Protein binding site on a DNA 1. Just protein binding site or 2.Activation Domain Target HAT and transcription occurs

cosmid

special type of plasmid that will carry a lot of DNA -WIKIA cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. Cosmids' (cos sites + plasmid = cosmids) DNA sequences are originally from the lambda phage.[1] They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in 1978.[2]Cosmids can contain 37 to 52 (normally 45) kb of DNA, limits based on the normal bacteriophage packaging size. They can replicate as plasmids if they have a suitable origin of replication: for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication or f1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance, so that the transformed cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow.[3] Unlike plasmids, they can also be packaged in phage capsids, which allows the foreign genes to be transferred into or between cells by transduction. Plasmids become unstable after a certain amount of DNA has been inserted into them, because their increased size is more conducive to recombination. To circumvent this, phage transduction is used instead. This is made possible by the cohesive ends, also known as cos sites. In this way, they are similar to using the lambda phage as a vector, except all the lambda genes have been deleted with the exception of the cos sequence. -C-elegans cosmids Raf sequence is proto-oncogene CDS (cDNA sequence) related to human cancer gene

Arabinose (Ara) operon

two pos control activators: AraC protein and CAP protein *when Ara is absent:* protein C binds to operator and I and makes loop (bends DNA) -C protein binds to two regions (not one) *when ara is present:* - C is bound to I, lets go of O (allows for expression) -transcription goes on but needs help from cap -C protein lets got of loop allows transcription but need positive control from CAP Mutated C super-repressed = no transcription *C protein (and therefore arabinose) and cap necessary for transcription* The cAMP-CAP system provides positive control in the lac operon (superimposed upon negative control by the lacI gene) and in the ara operon (superimposed upon positive control by the araC gene).