Genetics Exam 4

what is transcriptional regulation?
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is the lac operon an inducible operon?yeswhat are parts of the lac operon?regulatory region, structural genses, repressor gene, and permeasewhat is a regulatory region/promoter and operatorwhat are structural genes?B-gallactosidase gene (lac z), permease gene (lac Y), transacetylase gene (lac A), terminator downstreamwhat is a repressor gene?just upstream of lac operon the lacI gene is there, which stands for inhibitory, consitutive proteinwhat is permease?lacY, inserts into plas mem of bacterium, allows lactose to enter. when it enters, then B-gal will either -take some of lactose and split into gluc and galactose, glucose will enter glycolysis for ATP production and galactose can enter glycolysis also (LacZ) -take some lactose and convert into allolactose (inducer) (lacZ)when lac operon is repressed what is happeningglucose is present and lactose is absent it is off, so repressor protein (coded by lacI) binds to operator region of operon and prev RNA pol from binding or transcribing struc genes, conserving energy needswhen lac operon is induced what is happening?when glucose is absent and lactose is present, then allolactose will bind to repressor protein, changing its shape. So repressor can't bind to operator but RNA pol can bind to promoter and transcribe the genes. mRNA translated and enzymes necessary for lac metabolism are produced.B-galactosidase function (LacZ)cleaves lactose into two monosacc (glucose and galactose) important mutants: Z- (no functional B-gal)Repressor protein function (lacI)contains two binding sites, one for operator and one for allolactose, the inducer important mutants: I- (unable to bind to operator) Is- (so called super repressor unable to bind the inducer (allolactose) blocking all transcriptionPermease function (lac Y)facilitates lactose transport across the cell membrane important mutants: Y- (no functional permease)transacetylase function (lacA)protects against harmful by-products of lactose metabolism important mutants: A- (no transacetylase)in the lac operon the operators are....cis-acting (DNA sequences)in lac operon the repressors are...trans-acting (proteins), and repressors contain an allolactose binding siteFunction of operator (lacO)binds repressor protein to block transcription of operon genes important mutants: Oc- (fails to bind repressor protein, resulting in continuous constitutive transcription)function of promoter (lacP)Binds RNA pol important mutants: P- (fails to bind RNA pol or does so weakly)what happens in I- mutation in lac operon?-transcription is constitutive -repressor protein that is altered shape so no binding to operator, even when lactose is absent the operon is transcribed and mRNA is translated -so get production of 3 enzymeswhat happens in an Oc mutation in lac operon?-transcription is constitutive even w out lactose -DNA seq of operator is altered, which repressor can't recognize it, so doesn't bind -transcription of operon, and production of 3 enzymes -lactose is absentwhat happens in Is mutation in lac operon?-lactose present, normally operon would be induced, but now is repressed -super repressor has altered shape in which allolactose (inducer) can not bind, so binds anyway whether there is an inducer or not -no transcription bc it is repressedwhat is catabolite repression?when both glucose and lactose are present, glucose is the preferred sugar and lac operon is repressed (glucose takes less energy)if there are high amounts of glucose it indirectly....decreases cAMPif there is a low amount of glucose it indirectly...increases cAMP (important)why is increasing cAMP important?bc cAMP binds to catabolite activator protein (CAP)what does cAMP-CAP do?-binds to the CAP site of lac operon (upstream of promoter sequence) -interacts physically with RNA pol -increases the affinity of RNA pol for the lacPif glucose is absent what happens w CAP?cAMP levels are high, so binds to CAP. CAP-cAMP binds to CAP site upstream of promoter and stimulates binding of RNA pol and transcriptionif glucose is present what happens w CAP?-cAMP levels are low, so CAP complex can't bind to CAP site, so RNA pol can't bind or binds weakly -results in very low transcription levelwhat is the lac operon controlled by?lac repressor (negative control) and the CAP (positive control)if glucose is present, lactose absent, cAMP absent, and allolactose absent is there transcription?noif glucose is present, lactose is present, cAMP is absent, and allolactose is present is there transcription?basalif glucose is absent, lactose is absent, cAMP is present, and allolactose is absent is there transcriptionnoif gluose is absent, lactose is present, cAMP is present, and allolactose is present is there transcripion?yes, high levelwhat does the trp operon do?-encodes enzymes for synthesizing tryptophan -repressible operoncomponents of trp operon?promoter, operator, leader, attneuator, structural genes (5)if tryptophan is absent, trp operon is...ON via inactive repressorif tryptophan is present, trp operon off via: 1. corepression (trp molecule acts as corepressor) 2. attenuationif Tryptophan is present what is happening in corepression?-plenty of trp in cell, so it binds to repressor molecule and stimulate it to bind to operator -excess trp can bind acting as corepressorif tryptophan is absent what is happening?repressor proteins can not bind, so get expressionswhat is attenuation?the trp leader sequence (trpL) is a gene that encodes a peptide containing 14 amino acids called leader peptidewhat two features of trpL are important in attenuation?1. two trp codons that act as a sensor to determine if the cell has sufficient Trp to synthesize its proteins 2. the ability to form hairpin structuresif low levels of trp will trp get inserted into leader peptide?nowhen trp levels are high during attentuation what does that mean?-charged tRNA carring trp, 3-4 terminator hairpin forms in mRNA -RNA pol will not transcribe struc genes of trp operon -complete leader peptide formed, transcription will stop before structural geneswhen trp levels are low during attenuation what does that mean?-no available charged tRNA carying trp, 2-3 formation of antiterminator hairpin -leader peptide is not completely made, allow RNA pol to transcribe struc genes of operonwhat is the advantage of attenuation?allows the cells to regulate trp operon at a sensitive level, moment by moment needs for making trpwhat are traits of bacterial gene regulation?-transcription and translation happen at same time -no mRNA modification, translated as iswhat are traits of eukaryotic gene regulation?-DNA packed into chromatin which has to be modified -mature mRNA has to exit nucleus into cytoplasm where it's translated into protein -ground state is off -more coding genes, diff cell types, tight control of gene regulationwhat are eukaryotic genes controlled by>DNA sequences 1. Promoter (TATA. box and Initiator region) 2. Promoter-proximal elements (GC rich box and CCAAT box) 3. Regulatory elements (enhancers and silencers 4. Insulatorswhat is the promoter-proximal elements?-deletion analysis that indicates that they are necessary for efficient transcription -100 bp upstream of initiatorwhat does the promoter do?RNA pol binds to TATA and transcription starts at the initiatorwhat is deletion analysis?delete certain nucleotides or base pairs, decrease in transcriptionhow can effects of enhancer location and orientation be investigated?-using reporter geen constructs (lacZ, GFP) -can be located downstream or upstream and some inverted or in the geneswhen promoter is activated what does lacZ do?lacZ will be expressed nd the mRNA will translate to the B-galif we add a substrate to B-gal what happensadd X-gal which leads to a BLUE productwhat does GFP code forgreen flourscent proteinwhat happens when you shine UV light on GFP?if will flourescewhat are insulators?-DNA segments that function as a boundary between two genes -Protect a gene from the regulatory effects of a neighboring genewhat are the types of insulators?1. enhancer-blocking insulators 2. DNA loop-forming insulatorswhat do enhancer-blocking insulators do?prevent neighboring enhancers from activating a genewhat do dna loop forming insulators do?allow for enhancer-promoter interactionwhat is the diff between the initiator element and tata box?RNAP binds to TATA sequence and then initiates transcription downstream at initiatoran enhancer seqeunce...can be inverted without altering its functional abilitywhich DNA sequences may be located thousands of nucleotides away from the genes they regulate?enhancerswhat does RNA pol make?mRNA and translated into protein, located in nucleoplasmwhat is TFIID?cinoised if TATA binding protein and other TBP associated factors. Recognizes the tata box of euk structural genewhat is a mediator?multisubunit complex that mediates the effects of reg transcription factors (activators and repressors) of RNA pol IIWhat happens with RNA pol and General TFs?-Tata and Inr, TFIID comes in first to bind and other TFs come along w recruitment of RNA pol II -mediator attached to reg transcription factorswhat is required for basal transcription?only need GTF, RNA pol II to carry outwhen the gene is on what is occurring?activator/coactivator complex recruits TFIID to core promoter (TATA) and activates its function so transcription is activatedwhen the gene is off what is occuring?repressor protein inhibits the binding of TFIID to the core promoter or inhibits its function. Transcription is repressed.what is occuring for transcriptional activation via TFIID?-activator protein interacts with the mediator. enables RNA pol to form a preinitiation complex that can proceed to elongation phase of transcription -expression onwhat is occurring for transcriptional repression via TFIID?-repressor protein interacts with mediator so the transcription is repressed -expression is offwhat are regulatory transcription factors?bind to TFIID or mediator to either promote or stimey gene expressionwhat do RTFS do?control the rate of transcription of target geneswhat do GTFs do?necessary for basal transcription of all geneswhere do RTFs bindspecific DNA sequences called regulatory or response elements (REs) = same as enhancers and silencerswhere do RTF amino acid residues and DNA nucleotides interact and how?interact in the major groove of the DNA double helix via: 1. H-bond formation 2. Ionic bonds 3. Hydrophobic interactionswhat are the possible domains that RTFs possess?1. DNA-binding domain (DBD)- binds to a reg element 2. activation domain (AD)-binds to RNA pol II and/or GTFs 3. Transactivation domain (TAD)- binds to other RTFswhy is a heterodimer more advantatious?increases number of dna seq that RTF can recognizewhat is GCR and how is it modulated?an activator modulated by 1. Hormone binding (glucocorticoids) 2. Dimerizationwhat happens when glucocorticoid is present?remove the proteins out of the way so it can bind to receptor which results in a dimerwhat does the dimer do?dimer goes into nucleus and binds to target genes that binds to glucocorticoid response elementswhat is NLS?nuclear localization sequence which has glucocorticoid bind to itwhat is CREB and how is it modulated (not lipid soluble)?activator modulated by 1. dimerization 2. phosphorylationwhen will creb dimers activate transcriptionafter it has been phosphorylatedwhat does creb bind to?CRE (cAMP response element)what is HIF-1 (hypoxia inducible factor) and how is it modulated?activator 1. phosphorylation 2. dimerizationwhat happens in normal oxygen conditions (normoxia)?hormone known as HPH adds hydroxyl groups onto proline AA, signals pVHL to bind to hydroxylated prolines which recruits ubiquitin ligase, adding ubiquitin to HIF-1 alpha proteasomal degradationwhat happens in hypoxia (low oxygen)?prevent HPH to add hydroxyl groups, not hydroxylated or degraded. phosphorylated where it goes to nucleus and binds to ARNT, dimer binds to turn on sev target geneswhat does HIF-1 trigger?angiogenesis (formation of new blood vessels)what is Von Hippel-Lindau disease?-autosomal dom disease, where people develop cancerous or noncancerous emangioblastoma (cancer with lots of blood vessels) -if pVHL is absent it can't degrade themwhat happens in the yeast Gal gene system?-all activated when galactose is present, promotes metabolismin the yeast Gal system what happens if there is no galactose?-gal genes are not transcribed -Gal4p homodimer (activation domain and dna binding domain) bind to enhancer seq -gal80 homodimer binds to AD preventing gal genes from being expressedin the yeast Gal system what happens if galactose is present?-Gal3p binds to Gal80 and removes parts of it off Gal4p and Gal3 binds to Gal4 -allows transcription of Gal geneshow can RTFS be modulated?1. binding of small effector molecule (hormone) 2. protein-protein interaction 3. covalent modification (phosphorylation)what is binding of small effector molecule?hormone binds to RTF, allowing it to bind to enhancer (RE) allowing transcriptionwhat is protein-protein interaction?-required and all have this -formation of homodimer which binds to enhancer causing transcriptionwhat is covalent modification (phosphorylation)?-homodimer bound to enhancer, inactive -after phosphorylated it changes the shape a bit and allows to work as reg transcription factor to target geneswhat is functional genomics?study of function, expression, and interaction of gene productswhat is a transcriptome?sequence and expression patterns of all RNA transcripts (kinds, where, when, how much)what is a proteome?sequence and expression patterns of all proteinswhat is interactome?complete set of physical interactions bw proteins and DNA segments, b/w proteins and RNA seg, and between proteinswhat is DNA microarray used for?to study transcriptomewhat is the process for DNA microarray?1. extract mRNA 2. make cDNA (complimentary DNA) reverse transcript, label cDNA with fluorescent dyes (green=control) (red=experiment) 3. hybridize to microarray 4. laser shined on it 5. released 6. computer calc levels of hybridized probeafter DNA microarray is done what can you tell?if gene expression increased, decreased, or if they stayed the samewhat does a yeast two hybrid system do?used to study the protein-protein ineractomehow does a yeast two hybrid system work?1. The Dna seq of Gal4 binding domain (BD) fused with bait protein 2. The DNA seq of the Gal4 activator domain (AD) fused with the target protein 3. a protein-protein interaction will result in the expression of a reporter gene (lacZ)what is a ChIP assay used for (chromatin immunoprecipitation)to study the protein-DNA interactions (what DNA seq does a certain RTF bind to?)how does a ChIP assay work?1. cross link proteins to DNA (formaldehyde) 2. Break the chromatin into small pieces 3. add antibody to target protein (RTF) and purify, will precipitate out 4. reverse cross-links to sep DNA and proteins 5. amplify DNA through PCR and DNA sequence itwhat is the band shift assay used for?to study protein-DNA interactomehow does band shiff assay work?1. control with no protein introduced 2. experimental to see if RTF will bind to DNA seq 3. if there is a band shift or is slower, then that shows that protein is binding to DNA sequencewhat is DNase footprinting assay used for?to study the protein-DNA interactomehow does a DNase footprinting assay work (what DNA sequence does a certain RTF bind to)?1. label dna and add protein 2. add enzyme dnase, which cuts DNA at random places 3. if no protein is bound then cut into cut into multiple fragments 4. if protein is bound it can't cut so there is a blank spot in the test showing there is a protein bounddimerization of RTFs is accomplished by what domain?transactivation domainsGRE, CRE, and HRE are what?enhancers and RTF binding sitesGCR, CREB and HIF-1 are all modulated by?dimerizationwhat would not be used to study interaction between RTF (protein) and an enhancer (DNA seq)?DNA microarraywhat is alternative splicing?-ability to produce diff proteins from the same mRNA by splicing together different combinations of exons -1 gene results in one or more mRNA and one or more proteinWhat is the calcitonin/calcitonin gene-related peptide gene (CT/CGRP)?-alternative splicing example -have primary transcript (pre mRNA) undergoes splicing -in thyroid cells, exons 1,2,3,4 are joined together excluded 5 and 6 which translated to form calcitonin peptides -calcitonin functions to decrease blood calcium levels (if high thyroid releases it to tone it down in the blood) -in neurons exons, 1,2,3,5,6 come together skipping 4 to form CGRP, which dilates blood vesselsWhat is myotonic dystrophy?autosomal dominant condition characterized by myotonia, insulin resistance, cataracts, intellectual disability and cardiac musclesWhat is DM1?the DMPK gene has > 150 copies of a CTG repeat (trinucleotide repeat)what is DM2?CNBP gene has > 11,000 copies of a CCTG repeatwhat is myotonic dystrophy due to (alternative splicing goes wrong)?not aberrant proteins, but build up of the RNAs (cause the problems) in the nucleus, which interfere with alternative splicing regulators important in muscles and neuron functionwhat stabilizes mRNA and what happens if you lose them?by 5' cap and 3' poly-A tail. loss of one or both results in its degradationwhat are types of mRNA decay?1. Deadenylation-dependent decay 2. Deadenylation-independent decayWhat occurs for deadenylation-dependent decay?1. removal of A on poly-A tail (deadenylase) shortens tail (3'-5') 2. Enzyme exoribonuclease--> shortens poly A tail further (3'-5') 3. decapping enzyme--> remove 5' cap 4. Exoribonuclease (XRN1)--> degrade mRNA in 5'-3' directionwhat occurs in deadenylation-independent decay?1. Decapping enzyme removes 5' cap 2. Exoribonuclease (XRN1)--->degrades mRNA in 5'-3" fashion 3. Endonuclease cuts internally, mRNA breaks into multiple fragments 4. Exoribonuclease degrade unprotected fragmentswhat is small noncoding RNA (sncRNAs)?double stranded RNA molecules 20-31 nucleotides long with short 3' overhangswhat are two types of sncRNAs?1. microRNAs (miRNAs, endogenous)- internal, we produce them 2. small interfering RNAs (siRNAs, exogenous)- have their own genes that produce themwhat is RNA interference?mechanism by which sncRNA molecules guide the post-transcriptional silencing of mRNAs in a sequence specific mannerwhat is the general mechanism of mRNA silencing?roughly the same for both miRNA and siRNA, and results in a gene knockdown (decrease in expression) by either 1. triggering mRNA degradation 2. inhibiting mRNA translationwhat occurs in miRNA pathway?1. miRNA genes--> primary miRNA transcript 2. Drosha in nucleus that cuts transcript to form pre-cursor miRNA 3. Dicer cuts even further making it even shorter 4. RISC binds to miRNA, AGO (argonaute) slicer removes 1 strand and keeps the other 5. Complementary -Partial--> translation inhibition -exact--> mRNA degradation by RISCwhat does Drosha do?moves the 3' tail and 5' cap in because hair pin structures form in miRNAhow does siRNA pathway differ from miRNA?not processed by Droshawhat did Andrew Fire and Craig Mello do?showed one of first examples of RNA interferencewhat was Fire and Mello's procedure they did?1. (c) inject either mex-3 antisense RNA (abundantly expressed RNA) or a mix of (c) and injected sense: antisense dsRNA into gonads of roundworm, RNA is taken up by eggs and early embryos 2. Subject early embryos to in situ hybridization. A labeled probe is added that is complementary to mex-3 mRNA. If cells express mex-3, the mRNA in cells will bind to probe and become labeledwhat was the result of Fire and Mello's experiment?significant reduction in mex-3 mRNA expression by the sense: antisense dsRNA as compared to mex-3 antisense RNAwhat are long noncoding RNAS (lncRNAs)?dsRNA molecules >200 nucleotides longwhat do some lncRNAs function as?competing endogenous RNAs (ceRNAs) that act as "sponges" to "soak up" miRNAswhat is an example of lncRNAs?-in normal muscle differentiation, linc-MD1 acts as a ceRNA by binding to miR-133 and miR-135, allowing muscle-promoting RTFs to upregulate their target genes -in duchenne muscluar dystrophy, linc-MD1 is abnormally low, allowing miR-133 and miR-135 to prevent the translation of RTFswhat is another example of long non coding RNA?-x inactivation by the lncRNA Xist -proteins will coat the x chromosome and condense to form barr bodywhat is most efficient at changing gene expression through RNAi?dsRNA that includes the gene sequence you wish to silencewhat is epigenetics?changes in gene expression independent of changes in DNA sequencewhat are three major epigenetic mechanisms?1. Chromatin remodeling 2. DNA methylation 3. Regulation by noncoding RNAwhat is epigenetic inheritance of chemical modifications?during DNA replication, nucleosomes are dissembled in the parental strands and reassembled in the daughter molecules. The old histones are randomly distributed to the daughter molecules and guide the modification of new histones.where is eukaryotic DNA packaged?in form of chromatin, substance of chromosomes, composed of DNA and chromosomal proteinswhat are types of chromatin?1. euchromatin 2. Heterochromatinwhat is euchromatin?-less compacted, transcriptionally active -genes expressed in this regionwhat is heterochromatin?-more compacted, transcriptionally inactive -genes in those region do not get expressedwhat types of heterochromatin are there?1. constitutive- always heterochromatin (telomeres, centromere) 2. facultative- can switch b/c heterochromatin and euchromatin (x inactivation)what are histones?small, basic, positively charged proteins composes of 20-30% arginine and lysine residues that bind to DNA in the minor groove.what is a nucleosome composed of?DNA wrapped around a histone corecentromeres and telomeres are examples of what?constitutive heterochromatinwhere do activators bind?major groove of DNAwhat are open promoters?associated w constitutive genes, NDRs, enhancers, activators, and H2A.Z histones that are readily removed before transcriptionwhat are covered promoters (wrapped in nucleosome and inaccessible)?associated with regulated genes. In order for the gene to be transcribed, chromatin remodeling complexes modify nucleosomes to go from a closed chromatin state to an open chromatin state (slide 4)what does NDR stand for?nucleosome depleted regionwhat is chromatin remodeling?packaging of eukaryotic DNA into chromatin means that much of the DNA is not readily accessible to RTFs and the transcriptional machinerywhat are examples of chromatin rremodeling?1. nucleosome modificiation 2. histone modificationwhat is nucleosome modification?carried out by ATP dependent chromatin remodeling complexes (SWI/SNF) that: 1. change nucleosome position 2. evict histones 3. add histone variantswhat are histone modifications?1. acetylation 2. methylation 3. phosphorylationwhat is histone code?diff patterns of modification result in diff levels of chromatin compaction to hide or expose genes for expression on histone tailsWhat is acetylation of lysine?histone acetyltransferases (HATs) vs. histone deacetylases (HDACs)what happens in acetylation of lysine?-lysine is positively charged and adding acetyl group makes it neutral -positive charges help to charge to dna so now neutral wont bind as well, loosening histone dna Interaction -transcription can take placewhat happens in deacetylation of lysine?-remove acetyl group and go back to positive charge lysine -more compacted so gene expression will not be as much so no transcription taking placewhat is methylation of lysine and arginine?-the number of methyl groups creates diff binding site for other proteins to activate or repress gene expression -histone methyltransferases (HMTs, add methyl groups) vs. Histone demethylases (HDMs) -charge does not changewhat is phosphorylation of various amino acids?kinases (add phosphate groups) vs. phosphatases on serine or tyrosine, easy to add a phosphate groupwhat happens when you add a phosphate group to the histone?-will cause a negative charge, DNA will loosen DNA histone interaction, from repulsion -loosens dna histone interactionWhat is occurring if there is a covered promoter?1. Closed chromatin- enhancers and promoters are inaccessible in the nucleosome 2. Binding of activators- activators bind to enhancer sequences and activators recruit chromatin remodeling complexes and histone modifying enzymes (HATs) 3. Open chromatin- RNA pol II and GTFs bind to the core promoter and the pre-initiation complex formswhat is occurring during elongation?histones ahead of the open complex are covalently modified by acetylation and evicted or partially displaced. Behind the open complex, histones are deacetylated by HDACs and become tightly bound to the DNAwhat is happening during transcriptional activation of a euk gene?1. activator proteins bind to enhancer, triggering DNA bending 2. activators interact w coactivators to stimulate chromatin remodeling and histone acetylation 3. activators bind to mediator, triggering assembly of RNA pol and GRF at the promoter siteif dna is unmethylated the gene, transcription occursif dna is methylated the gene is...inactive, no transcriptiondoes DNA methylation increase or decrease gene expression?decreases typicallywhat is happening in DNA methylation?-Cs of CpG islands are methylated via DNA methyltransferases (DNMTs). -CpG islands occur near many gene promoters, and are around 1,000-2000 bp in lengthhow does DNA methylation of CpG islands inhibit transcription?1. preventing activators from binding (methyl groups block binding) 2. causing methyl-CpG-binding proteins (MECPs) to bind, which recruit HDACs, resulting in DNA compactionwhat is Rett syndrome?-a neurodevelopment disorder that includes a deceleration of head growth and small hands and feet, fatal in male, x linked dominant -gene product: methyl-CpG-binding protein-2what is parent-of-origin monoallelic expression by imprinting?-Beckwith-wiedemann syndrom and silver-russell syndrome (AD) Normal: -H19: maternal copy active, paternal copy is silent -IGF2: paternal copy active, maternal copy Is silentwhat does ICR do?acts as enhancer blocking insulator when bound by CTC binding factor and prevents IGF2 transcriptionFor people with Beckwith-wiedemann syndrome what is true for H19, IGF2 genes?H19: paternal and maternal copies are silent IGF2: paternal and maternal copies are expressed undergrowth of tissueswhat is another example of monoallelic expression by imprinting?-angelman sydrome and prader-willi syndrome Normal- -AS gene: maternal copy active in brain, paternal copy is silent -PWS gene: paternal copy active in brain, maternal copy is silentwhat is random mono-allelic expression?-X inactivation by the lncRNA Xist -when Xist is more the Tsix it creates barr body -deletion of Tsix: both remaining active, both being inactive, or one being inactive, one active -addition of Xic: no x chrom inactivationa tautomeric shift may result in...transition mutationswhat is spontaneous rather than induced, mutation?depurinationan ames test reveals that the experimental plate has about the same number of his+ revertants as the control plate. This indicates...tested substance is not mutagenicwhich conditions would account for the formation of cAMP-CAP complex and the absence of allolactose?lactose absent, glucose absentwhich mutants result in constitutive expression of lac operon?I- and Ocin the trp operon attenuation model, the formation of 2-3 hairpin would result in what?-partial translation of trpL mRNA -transcription of the trp operon structural genes -stalling of the ribosome at trp codonsThe ChIP, band shift, and DNase footprinting assays are similar in that they can be used to study what?protein-DNA interactomehow do deadenylases contribute to regulation of expressiondestabilize mRNA by removing poly-A tailthe interaction between an activator and RNA pol II is accomplished by the activator's...activation domainwhat are differences b.w RNA interference pathways of miRNA and siRNA?-processing by Drosha -initial presence of a 5' cap and poly a tail -begins in nucleusthe long ncRNA linc-MD1 acts as what in normal muscle differentiationceRNAwhat is true about an epigenetic trait?-can be passed from mother cell to daughter cell by mitosis -can be passed to next gen by meiosis -causes changes in gene expressionwhat is an example of epigenetic changes?-methylation of DNA bases -addition of acetyl groups to histones -imprinting of geneswhat is primarily responsible for genomic imprinting?methylation of cytosine in DNAwhat is a function of chromatin remodeling complexes?displacement or ejection of nucleosomesincreased acetylation of histones is typically associated with what?gene activation and euchrom formationwhat is role of dna methylation in euk?to prevent transcriptionthe expression of both the paternal and maternal IGF2 genes results in... syndrome, whereas the expression of both the paternal and maternal H19 genes results in...syndrome?Beckwith-Wiedemann;silver-russellwhat syndrome is associated w overgrowth of body tissues?Beckwith-WiedemannRecombinant DNA technology is the use of __________ techniques to isolate and manipulate vitroGene cloning is the process of isolating and making many copies of a gene in vitro or in vivo.Molecular geneticist's toolbox: - restriction enzymes - DNA polymerase - dNTPs - ligase - cloning vectors- restriction enzymes = cut DNA - DNA polymerase = synthesis of DNA - dNTPs = A,T G,C - ligase = joins DNA together - cloning vectors = (CV), harbor gene of interest for cloninggene cloning in vivo (3 steps)1. Isolate DNA (ex: chromosomes) from an organism or collection of cells. 2. Cut DNA and CV with same RE. Insert (ligate) DNA fragments into CV to make a recombinant DNA molecule. 3. Transform host cell (ex: E. coli) with recombinant CV. Host cell and recombinant CV replicate multiple times to generate identical copies of the GOI.restriction site sequences are usuallypalindromicrestriction enzymes can cut two ways : symmetrical or asymmetricalsymmetrical produce blunt ends asymmetrical cuts produce sticky endsFormation of a Recombinant DNA Moleculeinvolves restriction enzymes and DNA ligaseCloning Vectors: Cloning Vectors E. coli plasmid CVs must have: 1. 2. 3. 4.Cloning Vectors E. coli plasmid CVs must have: 1. ori to replicate within host cell 2. multiple cloning sit/MCS (poly linker) 3. antibiotic resistance gene 4. lacZ gene spanning multiple cloning siteBlue white screening (4 steps)1. Cut CV with RE. 2. Cut chromosomal DNA with same RE. 3. Add ligase to create recombinant CV. 4. Transform bacteria with recombinant CV and plate on agar containing ampicillin and X-galSelf-ligation can be prevented by ___________alkaline phosphatase.Artificial transformation via: cold CaCl2 and heat shock and electroporationsynthesis of complementary DNA (cDNA) cDNA is used for cloning genes starting with mRNA vs. Chromosomal DNA. This procedure applies only if you are interested in...cloning the coding sequence of a gene instead of all of it (introns and exons), applies to eukaryotes. basically, converting mRNA into cDNA then cloning the cDNAGene Cloning in VitroPolymerase Chain Reaction Need: target DNA, two primers, Taq polymerase, dNTPs, thermal cycler Procedure : __________________________Procedure: 1. Denature at 95oC 2. Anneal primers at 45-68oC 3. Extend primers at 72oC. 4. Repeat 20-40x.Reverse Transcriptase-PCR RT-PCR is used to detect the presence of a ________________specific mRNA.Reverse Transcriptase-PCR RT-PCR is used to detect the presence of a specific mRNA. Procedure: 1. _________________ 2. _________________1.) make cDNA from mRNA by RT 2.) amplify cDNA by PCRPCR and RT PCR are end point semiquantitative meaningit cant tell us the exact levels of DNA that have been amplified. just saying presence ofReal-Time PCR qPCR is used to detect the quantity of DNA present or RNA expressed. Procedure: 1. 2.1.) synthesize cDNA from mRNA via RT 2.) PCR with either a SYBR green or taqman probeGene Targeting OverviewGene targeting is the process of altering a genetic sequence and observing the consequence _______________in vivo.1. _______________ (transgenic) organisms are used to study the function of a gene when it is over-expressed in vivo. 2. _______________ organisms are used to study the function of a gene when it is under- expressed in vivo.1. Knock-in (transgenic) organisms are used to study the function of a gene when it is over-expressed in vivo. 2. Knockout organisms are used to study the function of a gene when it is under- expressed in vivo.Transgenic Mice Procedure: 1. 2. 3. 4.1.)Inject transgene (TG) into pronucleus of fertilized egg. TG will randomly insert into chromosome via nonhomologous recombination 2.) Implant embryos into a foster mother 3.) embryos will express TG in all cells 4.) breed to produce TG miceKO (knock out) miceMeiosis I is about to start in a cell of a plant in which 2n=20. At this stage, the cell has20 chromosomes, and a total of 40 chromatidsIf a cell has 6 pairs of chromosomes and undergoes meiosis, how many possible chromosome arrangements are there during metaphase I?64A human primary spermatocyte undergoes meiosis to produce _____ sperm cells with _____ chromosomes in each.4, 23A male has the same X chromosome as his paternal grandfather.falseFor an X-linked dominant disease, all sons of an affected female must be affected.falseA very common type of red-green colorblindness in humans is caused by a mutation in a gene located on the X chromosome. Knowing that the mutant allele is recessive to the wild type, what is the probability that the son of a normal woman whose father is colorblind is going to also be colorblind?1/2A phenotypically normal woman is heterozygous for the recessive Mendelian allele causing phenylketonuria, a disease caused by the inability to process phenylalanine in food. She is also heterozygous for a recessive X-linked allele causing red-green colorblindness. What is the probability that one of her eggs will carry the dominant allele that allows normal processing of phenylalanine and the X-linked recessive allele that causes colorblindness?1/4Cystic fibrosis is an autosomal recessive condition. If the heterozygous parents of a boy with cystic fibrosis have two more children, what is the probability that both of these children will be unaffected?9/16The ends of the X and Y chromosomes that contain similar/homologous sequences are called ______ regions.pseudoautosomalA cross occurs between a male AabbccDd and a female aabbCcDd. Assuming independent assortment, what is the probability of producing individuals that are completely homozygous?1/8A cross occurs between a male AabbccDd and a female aabbCcDd. Assuming independent assortment, what is the probability of producing individuals that are phenotypically identical to the female parent?3/16In an Electrophoresis gel, DNA fragments migrate to the ___ anode because DNA is ____.positively-charged; negatively-chargedWhich of the following is NOT true of restriction fragment length polymorphisms (RFLPs)?they are a form of SSLPsIn pea plants, round peas is a dominant trait. A true-breeding pea plant with round peas is crossed to a plant with wrinkled peas. The F1 individuals were allowed to self-fertilize. What is the probability for obtaining the following in the F2 generation? The first three plants have wrinkled peas.0.0156In pea plants, round peas is a dominant trait. A true-breeding pea plant with round peas is crossed to a plant with wrinkled peas. The F1 individuals were allowed to self-fertilize. What is the probability for obtaining the following in the F2 generation? For any seven plants, three have wrinkled peas and four have round peas.0.173The following pedigree depicts the inheritance of a rare hereditary disease affecting muscles. Only males show this disease, what is the most likely mode of inheritance of this disease?X-linked recessiveWhat is the probability that individual A(4) is heterozygous with respect to the condition in the pedigree? Mother is affected (1), granddaughter affected (2), and other daughters husband (3) is affected with unaffected daughter(4).1Which individual in this pedigree rules out X-linked dominance as a mode of inheritance? affected father (I), affected son (II-2) and unaffected son (II-3) and unaffected daughter (II-1) and affected daughter (II-4)(II-1) and (II-2)A monohybrid cross between two plants with purple flowers resulted in 1572 pyrogeny, in which 1254 had purple flowers and 318 had white flowers. Based on the X2 test,p<0.05, so the results are NOT consistent with Mendel's law of segregationIn two-point cross experiments, the following data was obtained: Gene loci: RF% a, b 14 a, c 36 a, d 4 Gene loci: RF% b, c 22 b, d 10 c, d 32 What is the likely order of the four genes?a-d-b-cIn hogs, a dominant allele B results in a white belt around the body. At a separate locus, the dominant allele S causes fusion of the two parts of the normally cloven hoof resulting in a condition known as syndactyly. A belted syndactylous sow was crossed to an unbelted cloven-hoofed boar, and in the litter there were 25% belted syndactylous 25% belted cloven 25% unbelted syndactylous 25% unbelted cloven The genotypes of the parents can be best represented as which of the following?BbSs x bbssThe advantage of performing a Southern blot analysis over an electrophoresis analysis of a RFLP is that the Southern blot proceduredetects specific, and therefore less, DNA fragmentsHuman mitochondrial diseases are primarily inherited from the mother becausethe sperm's mitochondria undergo autophagy after entering the egg.In D. melanogaster, cherub wings (ch), black body (b), and cinnabar eyes (cn) result from recessive alleles that are all located on chromosome 2. A homozygous wild-type fly was mated with a cherub, black, and cinnabar fly, and the resulting F1 females were test-crossed with cherub, black, and cinnabar males. The following progeny were produced from the testcross: ch+ b+ cn+ 750 ch b cn 753 ch+ b cn+ 102 ch b+ cn 105 ch+ b cn 40 ch b+ cn+ 41 ch+ b+ cn 4 ch b cn+ 5 What is the correct linkage map?ch (5 mu) cn (12 mu) bIn D. melanogaster, cherub wings (ch), black body (b), and cinnabar eyes (cn) result from recessive alleles that are all located on chromosome 2. A homozygous wild-type fly was mated with a cherub, black, and cinnabar fly, and the resulting F1 females were test-crossed with cherub, black, and cinnabar males. The following progeny were produced from the testcross: ch+ b+ cn+ 750 ch b cn 753 ch+ b cn+ 102 ch b+ cn 105 ch+ b cn 40 ch b+ cn+ 41 ch+ b+ cn 4 ch b cn+ 5 The interference value is0.17. DNA is acquired directly from the environment duringtransformationHow many telomeres are present in a human gamete?46The ABO blood types are an example ofcodominanceBeard growth is an example ofsex-limited inheritancePeople with cystic fibrosis display multiple symptoms such as salty sweat and mucus build up in the lungs. This is an example ofpleiotropythe abbreviation CPD corresponds to thenucleotide cytidine diphosphateIn DNA nucleotides, the base is attached to the deoxyribose sugar by a(n) _____ at the ______N-glycosidic bond; 1' carbontelomerase activity is typically observed ingerm cells and cancer cellsin the Hershey-Chase experiment of 1952, 32P and 35S were used in order to labelDNA and Protein; respectivelyIn chickens, the dominant allele C produces the creeper phenotype (having extremely short legs). However, the creeper allele is lethal in the homozygous condition. If two creepers are mated, what proportion of the living progeny will be creepers?2/3. A man with sickle cell disease (HbS HbS) has children with a woman who is heterozygous (HbA HbS). What proportion of their children will display overdominance?1/2A woman with type A blood (who had a type O father) has children with a type AB man. What is the probability that the couple will produce a child of either blood type A or AB?3/4A very rare dominant allele that causes polydactyly has a penetrance value of 60%. If two heterozygotes with polydactyly have children, what is the probability that an offspring will have polydactyly?0.45Which of the following statements about the Watson-Crick model of DNA is FALSE?it is a left-handed double helixWhich of the following statements about bacterial and eukaryotic replication is FALSE?RNA primers are removed by flap nuclease in bacteria; RNA primers are removed by Pol I in eukaryotes.A double-stranded DNA molecule is 200,000 bp (400 kilobase-pairs [kb]) long. How many nucleotides does it contain?400,000A double-stranded DNA molecule is 200,000 bp (400 kilobase-pairs [kb]) long. How long is the DNA molecule in nm?68,000 nmIn poultry, the dominant alleles for rose comb (R) and pea comb (P), if present together, give a walnut comb. The recessive alleles of each gene, when present together in a homozygous state, give single comb. What are the phenotypic ratios of offspring from a Rrpp x rrPp cross?1 walnut : 1 rose : 1 pea : 1 singleWith regard to pattern baldness in humans: BB -> bald males, bald female Bb -> bald males, nonbald females bb -> nonbald males, nonbald females A woman who is not bald (whose mother is bald) has children with a bald man (whose father is not bald). What is the probability that their first child will be a girl who will not become bald?3/8A mixed infection of an E. coli strain was performed using the h- r+ and h+ r- genotypes of the T2 phage. Progeny phage were collected after lysis and plated on two E.coli strains to obtain plaque characters important for detection of parental and recombinant types. h+ can only infect one strain of E.coli h- can infect both strains r+ shows slower lysis, forms small plaques r- shows rapid lysis plaque phenotype: cloudy, large cloudy, small cloudy, small cloudy large Number of plaques 600 480 180 180 plaque genotype h+ r- h- r+ h+ r+ h- r- What is the map distance between the h and r genes based on the information provided? HINT: determine which plaques are parental and which plaques are recombinant, then calculate the recombinant frequency.25.0 muFrom one F+ strain, three distinct Hfr strains were derived. The first three genes transferred during an Hfr x F- cross (different for each of the three Hfr) are: Hfr 1: F A D Hfr 2: F B E Hfr 3: D C E Which of the following must be the order of the genes on the bacterial chromosomal circle?A D C E B FIf the percentage of cytosine in a double-stranded DNA molecule is 14, what is the percentage of adenine?36%Coat color in Labrador retrievers is determined by two genes, b and e. B- produces black, bb produces brown, E- allows expression of the b gene, and ee does not allow expression of the b gene, resulting in a yellow coat color. A male with the genotype BbEe is crossed with a female with the genotype bbEe. What are the phenotypic ratios of their offspring?3 black : 3 brown : 2 yellowThe three bacterial genes a, b, and c are linked, but their order is unknown. A cotransduction experiment is performed and the following results are obtained. a and b are co-transduced at a frequency of 2% a and c are co-transduced at a frequency of 20% b and c are co-transduced at a frequency of 65% What can be concluded about the arrangement of the three genes on the bacterial chromosome?the order is a-c-b and c is closer to b than to aFor the pedigree below, which of the following inheritance patterns are possible? (II-3)M, (III-4)M, (III-5)F, (IV-3)M are all affectedautosomal recessive, autosomal dominant, incomplete penetranceWhen tRNAs are said to be "charged" it means that they carry a positive or negative charge.FalsePremature termination codons (PTCs) can be "read" as sense codons by nonsense suppressor mutations of tRNAs to produce functional (though mutant) proteins; otherwise a truncated protein will be synthesized.trueThe functional equivalent of the TATA binding protein (TBP) in prokaryotes isthe sigma factorWhich of the following techniques does NOT involve RNA?colony hybridizationWhich of the following is NOT an effector molecule?ActivatorWhich of the following is NOT a charactetristic of the genetic code?overlappingWhich of the following is NOT a post-translational modification?exon splicingThe enzyme β-galactosidase can convert lactose intoallolactose, glucose, galactoseThe inducer of the lac operon isallolactoseHow many nucleotides are expected for a mRNA coding for a protein with 600 amino acids?1800Which of the following mRNA codons would form a codon-anticodon base pairing interaction with the 3′-UAG-5′ tRNA anticodon?5'-AUC-3'Human insulin can be produced by bacteria using recombinant technology. To do so, one must ligate ________ into a cloning expression vector.cDNA of insulin mRNAIn eukaryotes, one gene can code for more than one mRNA molecule, which can result in the production of more than one protein. This is due toalternative splicingIf you began with a sample that contained 300 copies of double-stranded DNA, how many copies would be present after 25 cycles of PCR, assuming 100% efficiency?1 x 10^10Which one of the following conditions is required for the lac operon to be "on"?glucose absent, lactose presentA null repressor mutation (I-) of the lac operon results inconstitutive transcriptionSuppose that a bacterial operon is repressible. Which combination of effector molecules and regulatory proteins could be involved?inhibitor and activatorTemplate strand DNA and encoded RNA arecomplementary of one another with antiparallel orientationFormation of a stem-looop and a U-rich sequence near the 3′ end of RNA is characteristic ofbacterial intrinsic transcription terminationThe carboxy terminal domain (CTD) of RNA polymerase II plays a key role incapping of the 5′ end of a new transcript., addition of a poly(A) tail at the 3′ end of a transcript., splicing of exons in RNA transcripts.The role of most microRNAs within a eukaryotic cell is torepress the expression of genes by destroying mRNAs or preventing their translation.Which of the following about plasmid vectors is TRUE?Plasmid vectors must have an origin of replication., Plasmid vectors must have a multiple cloning site (polylinker)., Plasmid vectors must carry a selectable marker (drug resistance) for selectionA cDNA library is produced from ________ and is valued in the analysis of ________.a DNA copy of mRNA isolated from a group of cells; the protein-encoding open reading frame of a gene (no introns)In the creation of a knockout mouse, the targeting vector containing neoR and tk+ genes is introduced into embryonic stem cells (ESCs). Three genotypes are possible: neoR tk+, neoR tk-; and neoS tk-. Exposure to neomycin and ganciclovir screens the ESCs to identify those that have a gene knockout. Based on the information below, which correctly describes the genotype for each ESC? neo tk ESC #1 died N/A ESC #2 lived lived ESC #3 lived diedESC #1 is neoS tk-; ESC #2 is neoR tk-; ESC #3 is neoR tk+Suppose you insert a gene of interest (GOI) into a pUC19 plasmid cloning vector. You then transform E. coli with your cloning vector and plate the cells on media containing ampicillin and X-Gal to get the result shown below. The colony on the right is blue. (Circle: white spot on left and blue spot on right)The white colony contains your GOI becuase the insertion of the GOI results in a nonfunctional β-galactosidase, so that X-gal cannot be converted into a blue product.Angelman and Prader-Willi syndromes are correctly designated as46,del(15q)Prader-Willi syndrome is due to inheritance of an absent ______ gene from the ______.PWS; fatherWhich of the following histone modifications does NOT alter their charge?methylationHumans cannot produce egg yolk because of inactivation of the ______ genesVTGWhich of the following human syndromes is NOT due to aneuploidy?Cri du chatWhich of the following is NOT a factor that contributes to microevolution?random matingPseduogenes in the α-globin gene family (eg, ψα1 and ψα2) are most likelyduplication pseudogenesWhich of the following would be different between a hepatocyte and a neuron?regulatory transcription factorsWhich of the following is NOT true of inactivated X chromosomes?they are seen in the nucleus as polar bodiesWhich of the following would NOT be present in the activation of a eukaryotic gene?histone deacetylasesMale calico cats must have ______ sex chromosomes due to ______.XOXoY; meiotic nondisjunctionWhich of the following is NOT a form of epigenetic inheritance?chromatin remodelingIf you wanted to study the function of a particular amino acid in a protein, you should usetarget (site-directed) mutagenesisIf you wanted to determine the DNA sequence to which an activator binds, you should usethe chromatin immunoprecipitation (ChIP) assay.Histone acetyltransferases ______ acetyl groups on lysine residues, resulting in a ______ of DNA compaction.add; decreaseDNA elements that prevent the spreading of heterochromatin into adjacent euchromatic regions are calledbarrier insulatorsIn the presence of abundant tryptophan, the3-4 stem-loop structure forms, terminating transcriptionWhich of the following is FALSE regarding chronic myelogenous leukemia?It is due to a nonreciprocal interchromosomal transclocationWhich of the following is FALSE regarding the histones of nucleosomes?they bind to the major groove of DNAThe glucocorticoid receptor can be modulated bybinding of a hormone and dimerizationFamilial Down syndrome involvesa deletion and a nonreciprocal interchromosomal translocationSuppose that nondisjunction of one pair of homologous chromosomes occurred in meiosis I during gamete production in a man. If one of the resulting four sperm cells fertilizes a normal female egg, what is the probability that a child with a monosomic condition will be produced?50%A very unlucky individual is born with Turner syndrome, Down syndrome, Cri du chat syndrome, and Prader-Willi syndrome. How many chromosomes does this person have altogether in their somatic cells?46Calculate the gene density for each of the following organisms. Indicate the order from least complex to most complex MB #genes C.elegans 100 20,100 M. barkeri 4.8 3,600 A. Melan. 2400 21,000M. barkeri, C. elegans, A. melanoleucaThe karyotype shown below is that of a (XX, del on 5p)female with cri du chat syndrome. Which of the following mRNA codons would form a codon-anticodon base pairing interaction with the 3′-UGA-5′ tRNA anticodon?5'-ACU-3'. How many nucleotides would be expected for a mRNA coding for a protein with 300 amino acids?900A promoter mutation (P-) of the lac operon results in:no transcriptionSuppose that a bacterial operon is inducible. Which combination of effector molecules and regulatory proteins could be involved?inducer and repressorFormation of a stem-looop and a rut sequence near the 3′ end of RNA is characteristic ofbacterial rho-dependent transcription termination.A transgenic mouse is different from a knockout mouse in thatthe transgene is inserted at a random (ectopic) site in the genomeA plasmid vector has a gene for erythromycin resistance (eryR) and a gene for ampicillin resistance (ampR). The amp gene is cut with restriction enzyme, and donor DNA treated with the same enzyme is added. What genotype of cells needs to be selected to show evidence of transformation?ampS eryRWhen tRNAs are said to be "charged" it means that they carry an amino acidtrueNonsense suppressor tRNAs insert an amino acid into a growing peptide, depsite there being a premature termination codon (PTC) in the mRNA.truePCR and DNA sequencing are similar in that they both useDNA polymeraseWhich of the following RNAs is a encoded by a structural gene?mRNAKlinefelter syndrome is correctly designated as47, XXYAngelman syndrome is due to inheritance of an absent ______ gene from the ______.AS; motherIf you wanted to study the gene expression differences between a normal skin cell and a cancerous skin cell, you should usea DNA microarrayA very unlucky individual is born with Klinefelter syndrome, Patau syndrome, Cri du chat syndrome, and Angelman syndrome. How many chromosomes does this person have altogether in their somatic cells?48The Karyotype below shows an individual with 47 ,XYYman with Jacob's syndromeCri du chat syndrome is correctly designated as46, del (5p)A very unlucky individual is born with Jacobs syndrome, Down syndrome, Cri du chat syndrome, and Angelman syndrome. How many chromosomes does this person have altogether in their somatic cells?48The karyotype below shows that there is an extra pair on chromosome 18Edward's syndromeHumans cannot produce vitamin C because of inactivation of the ______ genesGULOTurner syndrome is correctly designated as46, XThe activator CREB can be modulated byphosphorylation and dimerizationSuppose that nondisjunction of one pair of homologous chromosomes occurred in meiosis II during gamete production in a man. If one of the resulting four sperm cells fertilizes a normal female egg, what is the probability that a child with a monosomic condition will be produced?25%A very unlucky individual is born with Turner syndrome, Edward syndrome, Cri du chat syndrome, and Prader-Willi syndrome. How many chromosomes does this person have altogether in their somatic cells?46The karyotype below shows an individual with only one x chromosome and no y chromosomefemale with turner syndrome