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Unformatted text preview: Name Last, First MCB121, Page 1 of 13 Gasser, Burgess MCB121 W11, Practice Final Exam
Instructions: There are 11 pages in this exam including the cover sheet, please count them before you start to make sure all are present. Write your name on each page of the exam. Write your answers in the space provided below each question. If you need more space use the back of the page and indicate clearly that you have continued your answer on the back. Do not use additional paper. Abbreviations Ala A Arg R Asn N Asp D Cys C Gln Q Glu E Gly G His H Ile I Amino acid Abbreviations Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Amino acid Alanine Arginine Asparagine Aspartic acid Cysteine Glutamine Glutamic Acid Glycine Histidine Isoleucine Question Value Score 1 2 3 4 5 6 7 8 9 10 11 12 13 14 23 24 23 5 13 12 8 16 6 14 12 8 20 16 Name Last, First MCB121, Page 2 of 13 Gasser, Burgess Total 200 Name Last, First MCB121, Page 3 of 13 Gasser, Burgess 1. (23 points) You have identified a new member of the human nuclear hormone receptor family. You name it "Kryptonite Receptor" (KR) for its ability to bind the novel small molecule Kryptonite (K). In further work you find that KR appears to be able to regulate genes that include the sequence "ATAGNNCTAT" in their promoter regions. You name this sequence the "kryptonite regulatory element" or KRE. To study how KR regulates gene expression you perform gel shift assays to evaluate interactions between purified KR, a 32P-labeled oligonucleotide including the KRE sequence, and two other purified proteins BAF53 (a component of the SWI/SNF complex) and TAFII250 (a histone acetyltransferase, HAT, that is also a component of TFIID). An autoradiogram of the results of this gel shift assay is shown below. All lanes include the labeled KRE oligonucleotide probe, and the presence or absence of other components is indicated by the "+" and "" signs, respectively. Use the above information to answer the following questions: a) (15) Circle T for "True", F for "False" or N for "no evidence from this experiment" for each of the following. T F N KR can bind to co-repressors in the absence of K. Co-repressors were not tested. T F N KR can only bind KRE when it is also bound to K. Lane 2 shows binding in absence of K. T F N TAF is lower in molecular weight than KR. Migration and molecular weight not well correlated on these gels. T F N BAF binds to KR in the absence of K. BAF does not appear to ever bind to KR. Name Last, First MCB121, Page 4 of 13 Gasser, Burgess T F N KR appears to act in activation of target gene expression in the presence of K. Recruits TAFII250 that would be expected to activate gene expression. b) (8) By which of the following mechanisms does the above experiment indicate that KR can affect transcription of target genes (circle all that apply). Remove methyl groups from cytosine residues in the promoter. Remove acetyl groups from the histones in the promoter region to close the chromatin. TAFII250 would add acetyl groups to histones. Shift the positions of nucleosomes to facilitate factor interactions with the promoter. Recruiting components of the Pol II pre-initiation complex (PIC) to the promoter. TAFII250 is part of the TFIID and the PIC. 2. (24 points) For each of the following circle the "T" if the statement is true and the "F" if the statement is false. (3 points each) T F Transfer of DNA from bacteria to eukaryotic cells is an invention of humans and does not occur in nature without human intervention. Agrobacterium has this ability with no human intervention. F Because many genetic diseases manifest themselves early in development, germ-line gene therapy is the primary course being pursued by medical researchers. Only somatic cell therapy is being pursued. F Two of eleven patients treated with retroviral gene therapy to cure SCID-X1 had insertions near their LMO2 genes leading to the development of leukemia. This indicates that the retroviral vector preferentially inserts near this gene. The insertion was random, overexpression of LMO2 gave cells advantage. F De-ubiquitinating enzymes can hydrolyze either isopeptide or peptide bonds depending on the nature of the substrate. F Lean beef and pork available at US supermarkets most likely comes from genetically engineered animals. Transgenic mammals have not been commercialized for food. F A majority of soybean products produced in the United States derive from genetically engineered plant material. F Random insertion of mobile DNA elements occurs during traditional breeding as well as in production of transgenic (genetically modified) plants and so does not represent a potential hazard unique to genetically engineered plants. Transposons commonly move during breeding programs. T T T T T T Name Last, First T MCB121, Page 5 of 13 Gasser, Burgess F Addition of MG132, a specific inhibitor of the protease activity of the proteosome, causes activation of the the NFB transcription factor in animal cells. This would cause repression because could not degrade I Name Last, First MCB121, Page 6 of 13 Gasser, Burgess 3. (23 points) Huntington's disease is a dominant inherited disease that results in degeneration of neurons (nerve cells) in the brain. The critical difference between the disease-causing and normal alleles is that the disease-causing allele includes a region of repeats of the trinucleotide "CAG" that is expanded (has more repeats) relative to the allele in unaffected individuals. This expanded region is translated in the protein to a polyglutamine repeat not found in the protein of unaffected individuals. Gene therapy is being considered as one route to treat affected individuals. Answer the following questions. a) (8) The expansion of the "CAG" repeat in the genome could result from which of the following: (Circle all that apply). i) ii) iii) iv) Unequal crossing over. Slippage of DNA polymerase. Insertion of a retrotransposon. Alternative splicing. b) (15) Because Huntington's disease results from the presence of a dominant diseasecausing allele, a heterozygous individual with one normal and one disease-causing allele will have the disease. You wish to construct a gene that could be useful in treatment of the disease if it could be introduced into the affected cells of such a heterozygous individual. Place an "X" in the blank next to each of the following that would be an appropriate gene to include in such a vector that would be likely to be effective at curing the disease (mark all correct answers). A full-length copy of the wild-type (normal) gene including all necessary promoter elements. The disease is dominant so this would not help. A gene encoding a CTD kinase that can efficiently phosphorylate the Cterminal domain of RNA Polymerase II even when Pol II is not in contact with general transcription factors. This would stop all transcription and would kill the transformed cell. A gene encoding a factor that would interfere with the ability of U2AF to recognize branch points in splicing reactions. This would stop all splicing and would kill the transformed cell. A gene that would produce a micro-RNA that is complementary to a part of the sequence of the disease-causing allele in a region where the sequence differs from the normal allele. This could shut down the disease-causing allele. A gene encoding a repressor domain and six zinc finger domains designed to bind specifically to the promoter of only the disease-causing allele. This could shut down the disease-causing allele. X X Name Last, First MCB121, Page 7 of 13 Gasser, Burgess 5. (5) Briefly explain why inherited blood diseases are especially good targets for the current technology in use for somatic cell gene therapy in humans. Do not exceed the space provided below. Blood is continually renewed from stem cells. Transformation of the stem cells can thus produce a fully transgenic complement of blood in a patient. Most other cells of the body are not continually renewed and thus must be transformed directly to produce therapeutic effect. _________________________________________________________________ 5. (13 points) A DNA transposon that moves via a "cut and paste" type mechanism may leave a double-strand break at the site of excision. A. (4) What repair mechanism is likely to be used if excision were to occur during G2 of the cell cycle? Homologous recombination B. (4) What if excision were to occur during G1 of the cell cycle? Non-homologous end joining B. (5) In a cell line where non-homologous end joining is blocked by a mutation in Artemis, what outcome(s) of transposon excision might you detect? __x___ formation of a chromosome translocation This can occur by reciprocal homologous recombination between repeated elements located on different chromosomes __x___ expansion of nearby repeated element This can occur by unequal crossing over by homologous recombination involving by repeated elements on sister chromatids. __x___ loss of heterozygosity This can occur of there is a reciprocal crossover mediated by homologous recombination between nonsister chromatids of homologous chromosomes. For the event to create loss of heterozygosity the Name Last, First MCB121, Page 8 of 13 Gasser, Burgess recombination event must occur during G2 at a location between the heterozygous locus and the centromere. ___x__ a fully restored transposon DNA sequence This can occur by reciprocal recombination between sister chromatids or homologous chromosomes. 6. (12 points) For each of the polymerization processes below, fill in the synthesized polymer (e.g. DNA, RNA, etc.) with its template from which information is read (e.g. DNA, RNA, no template, etc.) Synthesized polymer Poly-A polymerase Telomerase RNA polymerase II RNA-dependent RNA polymerase Reverse transcriptase DNA polymerase-alpha (primase) RNA DNA RNA RNA DNA RNA (and some DNA) Template none RNA DNA RNA RNA DNA 1 a a. b c c.. Name Last, First 2 3 MCB121, Page 9 of 13 Gasser, Burgess 7. (8 points) Radiolabeled pre-mRNA has been incubated with nuclear extract and ATP to carry out an in vitro splicing reaction. Three different precursors are used. The first (1) contains a mutation that abolishes interaction of the 5' splice site with U1, the second (2) contains a mutation at the 3' splice site and the third (3) contains all sequences sufficient to promote splicing. d e A. (5) For letters a-e identify the name of the molecule that best describes the structure to the right: __e__ free exon 1 __d__ spliced product __b__ lariat intron __a__ lariat intermediate __c__ unspliced precursor B. (3) Indicate which of the two chemical step(s), if any (i.e. step 1 only; step 1 and step 2; or no splicing) has successfully occurred using the three different precursor substrates. 1. No splicing 2. Step 1 only 3 Step 1 and step 2 Name Last, First MCB121, Page 10 of 13 Gasser, Burgess 8. (16 points) Match the following term with the definition listed below: A. Sliding clamp B. DNA ligase C. Clamp loader D. RNA primer F. Leading strand G. Lagging strand H. Replication fork I. DNA primase J. Single-stranded DNA binding protein (SSB or RPA) K. DNA helicase L. DNA polymerase __B___ Enzyme that joins two adjacent DNA strands together __K___ Enzyme that opens the DNA helix by separating the single strands __A___ A protein complex that encircles the DNA double helix and binds to DNA polymerase, keeping it firmly bound to the DNA while it is moving __H___ Y-shaped region of a replication DNA molecule at which the two daughter strands are formed __F___ One of the two newly made strands of DNA found at a replication fork. It is made by continuous synthesis in the 5' to - 3' direction. __D___ Short length of RNA synthesized on the lagging strand during DNA replication and subsequently removed. __G___ One of the two newly made strands of DNA found at the replication fork. It is made in discontinuous segments that are later joined covalently. __J___ Non enzymatic protein that prevents annealing of unwound DNA strands at the replication fork. Name Last, First MCB121, Page 11 of 11 Burgess, Gasser 9. (6 points) You attended a seminar last week where the speaker presented new data showing that the specialized nucleosome at the centromere comprises a tetramer of subunits (H2a, H2b, CENP-A, and H4). This tetramer is wrapped one time by DNA. List three features of this "centromere nucleosome" that differ from typical nucleosomes. a. tetramer of histones + CENP-A versus octomer b. CENP-A in place of H3 c. DNA wraps one time around tetramer versus ~2 times 10. (14 points) RNAi an important genetic tool for genetic research in C. elegans. Interestingly only a small amount of RNA sequence ingested by the worm is required to elicit gene-specific silencing throughout the animal and its progeny. A. (2) What form is the RNA generally introduced into the animal? [circle one] 1) 2) 3) single stranded DNA double-stranded RNA an RNA/DNA duplex B. (6) Distinguish the enzymatic roles of Dicer and Slicer? Slicer is part of RISC and uses 22nt RNAs to guide cleavage of target sequence. Dicer cleaves all dsRNA in to 22nt fragments. C. (6) RNAi can lead to gene silencing by one of several different mechanisms depending on the origin of the interfering RNA, the organism or cell type. List three different ways that individual Argonaute proteins can silence gene expression. 1) degrade message with slicer 2) transport to P-bodies to inhibit translation 3) modify chromatin structure in nucleus Name Last,5' 5' First MCB121, Page 12 of 11 Burgess, Gasser 5' 5' 11. (12 points) You have cloned a DNA repair gene (DRGL) that is found mutated in some liver 3' cancers. You team up with an oncologist that has a liver cancer patient whose grandmother, 3' 3' have mother and two brothers also 3' liver cancer. The families have agreed to donate healthy tissue and tumor tissue for your studies to test your hypothesis that a mutation in DRGL gives a predisposition to liver cancer. Interestingly all affected family members carry one wild type and one mutated copy of the gene (i.e. DRGL+/-) in a biopsy of healthy tissue. A. (4) Given these data you suspect that DRGL is a(n) [ONCOGENE / TUMOR SUPPRESSOR GENE] circle one word B. (4) The genotype of the tumor cells is very likely a. DRGL+/+ b. DRGL+/c. DRGL -/- or DRGL-/_ C. (4) Next you want to measure whether or not telomerease is active in the tumor cells. You are able to measure telomerase activity in both tumor cells and healthy tissue from the same patient. Your expectation is that telomerease will be (ACTIVE / INACTIVE) in tumor cells and (ACTIVE / INACTIVE) in healthy tissue. (circle one word in each case) 12. (8 points) Shown below is a nucleic acid structure found in cells: A. (4) Where might you find such a structure? (circle one answer) 1. a region of the genome where dsRNAs are formed 2. at the end of a chromosome 3. at the centromere 4. in the spliceosome B. (4) Assuming that the T-loop follows the same rules of polarity as a D-loop, indicate the polarity of both ends of each strand of the nucleic acid. Name Last, First MCB121, Page 13 of 11 Burgess, Gasser 13. (20 points) For each statement determine if it is TRUE (T) or FALSE (F) ____F___ A single event resulting in loss-of-heterozygosity in a Brca2+/- individual is sufficient for malignant tumor formation. Cancer cell progression requires multiple mutations ___T___A proto-oncogene is inherited from one generation to the next. A proto-oncogene encodes a gene with a role in cellular function. So this gene is inherited. Conversion of a proto-oncogene to an activated oncogene occurs spontaneously in the individual that has cancer. ___F___ Oncogenes usually encode DNA repair factors Oncogenes usually are cell growth controlled genes that have been mutated to be inappropriately expressed or activated. Genes involved in DNA repair are generally tumor suppressor genes 14. (16 points) Provide a brief statement of why each of the statements below is false: A. Addition of the 7-me G cap and loading of splicing factors occurs cotranscriptionally. Following transcription termination the transcript is exported from the nucleus and polyadenylated. Poly-adenlyation occurs in the nucleus prior to the transcript being exported B. The spliceosome cycle requires RNA helicase proteins to disrupt RNA base pair interactions that occur between snRNPs, snRNPs and pre-mRNA and even RNA base pair interactions within a single snRNP. The splicing helicases do not require ATP to carry out their function. Splicing helicases require ATP C. The first step of splicing involves the nucleophillic attack of the 2' OH of the branch site nucleotide at the 5' splice site to give two products: a free Name Last, First MCB121, Page 14 of 11 Burgess, Gasser exon 1 terminating with a 3' phosphate and a branched lariat structure containing the unusual 2'-5' phosphodiester linkage involving the first nucleotide of the intron and the branch site nucleotide. The free exon 1 that follows the first step of splicing terminates in a 3'OH D. RNA splicing occurs in regions of the cytosol known as P-bodies, which are enriched for Argonaute-containing complexes RNA splicing takes place in the nucleus ...
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This note was uploaded on 04/06/2011 for the course MCB 121 taught by Professor Gasser during the Winter '09 term at UC Davis.
- Winter '09