NAME: _____________________________________ STUDENT ID #: ______________________________ Page 1 of 5 Questions on Prof. Urnov’s section Question 4 (25 points) – genetics of inducible gene control A. E. coli and S. cerevisiae activate transcription of lacZ and GAL1-10, respectively, when lactose and galactose, respectively, are added to the medium. Explain the biological rationale behind this evolutionary conservation. (5 points) B. In a further analogy, the addition of glucose to the medium represses transcription both of lacZ and GAL1-10 even if lactose or galactose are also present. What is the biological rationale behind this fact? (5 points) C. The rendition of the PaJaMo experiment in your textbook (shown on the right) is woefully incomplete. Write out (or draw out) the missing part of this experiment – the part that convinced Pardee and Jacob/Monod that the “inducer” theory was wrong (10 points) D. The PaJaMo experiment worked because of two separate features of E. coli biology. The first is that E. coli mate. The second? … (5 points)NAME: _____________________________________ STUDENT ID #: ______________________________ Page 2 of 5 Question 5 (30 points) Epigenetic silencing – such as X chromosome inactivation – is used in many different organisms, and for different reasons. In class, we discussed work from Jasper Rine and Michael Grunstein that led to our present understanding of molecular mechanisms of budding yeast mating type loci silencing. In addition to HML and HMR, yeast epigenetically silence regions adjacent to their telomeres (see schematic below). A. The RED1 gene makes a product that turns yeast colonies a vivid bright red (normal yeast colonies are cream-colored). Using the drawing above as a template, sketch out a chromosome you would engineer to do a screen – with RED1 as the “reporter” gene – for genes required for telomeric silencing in yeast (5 points). B. Explain how you would do the screen using the strain you have just engineered. Use a numbered list format for your answer. (8 points) C. Assuming complete conservation of molecular pathways between mating type loci and telomeres, in the screen that you are doing, what would be the phenotype of a colony that lacks the histone H4 tail? A colony lacking the SIR2 gene? A colony carrying 2 extra copies of SIR3 and SIR4? (9 points) ∆H4? ______________ ∆SIR2 _______________ 2× SIR3/SIR4 _______________ D. In your screen, you isolate a mutation that is not linked to any of the histone or SIR loci. In this mutant strain the silencing is much stronger than normal. Write out the experiment could you do to find out whether the product of this new gene (say, DOA1) functions upstream or downstream of SIR2 at the telomere. (8 points) centromeretelomere telomereactive activesilent silentcentromeretelomere telomereactive activesilent silentNAME: _____________________________________ STUDENT ID #: ______________________________ Page 3 of 5 Question 6 (40 points) – genetics of cancer A. As discovered by Harold Varmus and J. Michael Bishop, retroviruses that cause cancer frequently carry mutated version of normal cellular genes called “protooncogenes.” An interesting feature of such “stolen” protooncogenes is they do not belong to the same functional category: src is a receptor tyrosine kinase, ras is a G-protein, myc is a transcription factor. How can different viruses accomplish the same goal – oncogenic transformation of the cell – by using completely different proteins? (10 points) B. A mouse knockout for p53 has a remarkable phenotype: the animal goes through embryonic development and its “childhood” with no seeming abnormalities, but by 6 months of age, succumbs to multiple tumors and dies. Explain the reason for the delayed onset of cancer in this animal. (10 points) C. Humans who carry germline mutations in pRB are predisposed to cancer of the retina. This is just one of many known examples of inherited cancer in humans. A common and interesting aspect of many “hereditary cancer” syndromes is they exhibit a dominant mode of inheritance. What three different types of mutation could act in such a dominant fashion? For full credit, illustrate your answer with examples of cellular genes affected by such mutations in cancer, clearly explaining why these mutations are dominant (12 points) 1. _________________________________________________________________________ 2. _________________________________________________________________________ 3. _________________________________________________________________________ D. Gleevec is highly efficient in the treatment of chronic myelogenous leukemia. Most significantly, over 80% of CML patients respond to Gleevec even when it is used in a single-agent regimen (i.e., by itself). This is puzzling, because Gleevec inhibits the activity of the oncogene bcr-abl, but the cancer cell still has mutations both in p53 and pRB, and Gleevec has does not affect either one of those. How can Gleevec be effective if it doesn’t address all the mutations in the cancer cell? (8 points)NAME: _____________________________________ STUDENT ID #: ______________________________ Page 4 of 5 Question 7 (40 points). A. Leland Hartwell received a Nobel prize for his studies on cell cycle control in budding yeast, S. cerevisiae. His forward genetic screen identified a large number of cdc genes in the yeast genome, products of which are required for the cell to correctly execute the G1–S–G2–M sequence. Given that inability to divide leads to cell death (a phenotype that can be invoked by mutating many different genes), Hartwell used an elegant trick to ensure that the mutants he identified were specifically in the cell-cycle control machinery, as opposed to some other essential cell system (e.g., glycolysis, or RNA polymerase). What was that trick? (10 points) B. The most important gene identified by Hartwell, CDC28, codes for a cyclin-dependent kinase that is required for yeast to traverse the “start” point of the cell cycle. Cdc28p functions in a complex with a “cyclin” – a protein that directs the kinase to act on specific substrates; the interaction with the cyclin is absolutely required for Cdc28p function in driving the cell cycle. Describe in reasonable detail a set of genetic experiments that would prove (or at least strongly suggest) that Cdc28p physically interacts
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