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7.06_2004_PS5key - 7.06 Spring 2004 PS 5 KEY 1 of 10 1 You...

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7.06 Spring 2004 PS 5 KEY 1 of 10 1) You are an M.D./Ph.D. student investigating a recently discovered genetic disorder, the M IT TA S yndrome (MTAS). This disease was first identified in MIT graduate teaching assistants who complained of ophthalmoplegia after grading several hundred cell biology exams. Ophthalmoplegia is characterized by slowly, progressive, paralysis of the extraoccular muscles. Preliminary studies used pedigree analysis to determine how this disease is inherited. a) Given the following pedigrees, what is your hypothesis for the pattern of inheritance (circle: female, square: male, open: WT, filled: MTAS patient)? Autosomal Recessive b) Linkage analysis suggested that victims of MTAS have a deletion of a region in the long arm of chromosome 13. Using the human genome sequence, you compile a list of genes located in this area. One of the genes in the deleted region encodes a neuron-specific cytochrome c oxidase isoform (cytoc706). As a first step in studying cytoc706, you decide to investigate its subcellular localization. How could you do this? IF/GFP fusion with colocolization (to a known mitochondrial marker such as F1/F0 ATPase). c) Your thesis advisor suggests that you construct a model system to study MTAS and so you decide to examine the loss of function of cytoc706 in yeast. You transform yeast cells with a DNA fragment encoding resistance to the cytotoxic drug G418 that will insert into the cytoc706 gene in yeast, thereby inactivating it (resistance to G418 is a marker for the deletion of cytoc706). You then examine the subcellular localization of cytoc706 in yeast and human cells and find that that protein is localized to mitochondria (you know that the cytoc706 positive green signal is labeling mitochondria because you’ve labeled all mitochondria with fluorescent antibodies against the F1/Fo ATP synthase). NOTE: The schematic diagrams have some mitochondria which are coloured – pay close attention if you are looking at a black and white print-out of this question.
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7.06 Spring 2004 PS 5 KEY 2 of 10 Is there anything odd about the localization pattern? Yeast “knock-outs” have some mitochondria which stain positively for cytoc706 – more than one copy of the gene exists – it is likely mitochondrially encoded in yeast. d) You cross a yeast cell which has a disruption of the cytoc706 (and hence is G418 resistant) to a WT cell and grow the diploids on media containing G418. You then induce sporulation and test the transmission of the G418 resistance (cytoc706 disruption). Three of the four resulting spores are G418 resistant (instead of the expected 2:2 segregation pattern). Confused by this result, you attempt the mating again, but recover 4 G418 resistant spores now. You repeat the mating for a third time and recover two resistant spores and two sensitive spores. When you allow the resistant spores to germinate and divide mitotically (mitosis), you recover some cells that are sensitive to G418! How could you explain this segregation pattern? Why is it different from the human pedigree? Explain.
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