pset2(2) - 7.06 Spring 2007 PROBLEM SET 2 Question 1. You...

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7.06 Spring 2007 PROBLEM SET 2 Question 1. You are studying the cell division cycle of a mammalian cell line grown in culture. Your goal is to determine how long cells spend in each part of the cell cycle. The culture is not synchronized: each cell in the culture is going through a cell cycle independent of the other cells. Therefore, at any point in time, a range of cell cycle states is present. You pulse the cells for 30 minutes with BrdU, wash it out, and then add an excess of cold thymidine (in what is called a pulse-chase experiment). Following the Pulse-Chase treatment, you remove an aliquot of cells every hour and perform the following types of analysis. Analysis 1 : First you count the number of cells in each aliquot and calculate the total number cells in the culture flask at various times. Your results are shown in figure 1. Analysis 2: Next, you stain the cells with fluorescent anti-tubulin antibodies and (using a microscope) determine the percentage of cells that are undergoing mitosis. At each time point, you consistently find that approximately 5% of the cells are undergoing mitosis.
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Analysis 3 : From the hourly aliquots you isolate the mitotic cells. You use autoradiography to determine what fraction have been labeled with BrdU. Your results are shown in Figure 2. Figure 2: Fraction of mitotic cells labeled with Brd U 1a. Draw a simple representation of the cell cycle showing the relationship of G1, mitosis, S-phase and G2. Indicate the position of START (the restriction point). 1b. What is the division time for this cell line? What data lead you to this conclusion?
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1c. What is the duration of mitosis in the cell line used in analyses 1-3? What data lead you to this conclusion? 1d. What is the duration of S phase, G1 and G2 in the cell line in analyses 1-3? What data lead you to this conclusion? Question 2. You are a graduate student in the laboratory of Paul Nurse and you have obtained several S. pombe strains that are mutant in various aspects of cdc2 regulation. You would like to determine the effect that each of the mutations has on cdc2 activity. (Note S. pombe cdc2 is the same cdk as cdk1) 2a. How will you assay the activity of cdc2/cdk1 in the various mutants?
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2b. Indicate the activity of cdc2/cdk1 protein in the following mutants by comparing to cdc2/cdk1 activity that would be observed in a wild-type strain. Would the mutant:wild type activity ratio be equal to 1, less than 1, or greater than 1? Why? 1)
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This note was uploaded on 01/07/2011 for the course BIOLOGY 7.06 taught by Professor Nitro during the Spring '10 term at MIT.

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pset2(2) - 7.06 Spring 2007 PROBLEM SET 2 Question 1. You...

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