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706_F06exam2_key - Answer Key Name If you do not follow...

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Name: ___________________________ 1 Answer Key 7.06 Cell Biology EXAM #2 October 24, 2006 This is an open book exam, and you are allowed access to books and notes but not computers or any other types of electronic devices including cell phones. Please write your answers in pen (not pencil) to the questions in the space allotted. Please write only on the FRONT SIDE of each sheet. And be sure to put your name on each page in case they become separated! Remember that we will Xerox all of the exams. Good luck! Question 1. 25 pts __________ Question 2. 30 pts __________ Question 3. 25 pts __________ Question 4. 20 pts __________ If you do not follow instructions, we WILL take off points!
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Name: ___________________________ 2 Answer Key Question 1 (25 points) You are interested in studying the enzyme Air1 responsible for Histone H3 phosphorylation in mam- malian tissue culture cells. Your friend raised antibodies against the enzyme and is kind enough to send you an aliquot. A. (6 points) Describe how you could use this antibody to examine whether the protein that is recog- nized by the antibody is indeed responsible for Histone H3 phosphorylation. Be sure to include details on the experimental process and controls. I would perform a kinase assay. I would use the Air1 antibody to immunoprecipitate Air1. Using the precipitate I would perform an invitro kinase reaction. I would add substrate (Histone H3), Magnesium (to stabilize the protein kinase), and radioactive (p32)ATP. To ensure that the phosphorylation of Histone H3 is due to the precipitated protein and not pro- tein kinases sticking to the beads and antibody in an unspecific manner, I could use an unrelated antibody or pre-immune serum in the immunoprecipitation experiment. The positive control would be incubation of Histone H3 and radioactive ATP with a known protein that phosphorylates Histone H3. You would run these samples on a gel and phosphoimage it. If Air1 is responsible for Histone H3 phosphorylation, you should see a radioactively labeled Histone H3 in your experimental sample as well as the positive control but no band for the negative control. You decide to characterize the regulation of Air1 in more detail by first examining the levels of the protein during the cell cycle in synchronized wild type cell cultures. You arrest the cells in G1 by se- rum starvation and release them into the cell cycle synchronously by re-adding serum. To monitor cell cycle progression you measure DNA content. This is the result that you obtain. B. (3 points) Which stages of the cell cycle is Air1 absent from? When is the protein present at high levels and when at low levels? The protein is absent during G1, is present at low levels during S phase and present at high levels during G2 and/or mitosis.
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Name: ___________________________ 3 Answer Key C. (6 points) How could you test the hypothesis that ubiquitin-proteasome dependent protein degra- dation is responsible for the loss of Air1 in certain stages of the cell cycle? Include controls. Draw the expected result if the hypothesis is true and explain your reasoning.
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