_____________________________________ Name BIO320 HW-7 Cell Cycle #1 (due Thursday 04/12/2012)
Problem 1. A common first step in characterizing cell-division-cycle (Cdc) mutants is to define the phase of the cell cycle at which the mutation blocks the cell's progress. Temperaturesensitive Cdc mutants are particularly useful because they grow and divide normally at one temperature (the permissive temperature) but express a mutant phenotype at a higher temperature (the restrictive temperature). One method for characterizing temperature-sensitive Cdc mutants uses the drug hydroxyurea, which blocks DNA synthesis by inhibiting ribonucleotide reductase (which provides deoxyribonucleotide precursors). A useful feature of this method is that the block can be rapidly reversed by changing the incubation medium to one that lacks hydroxyurea. Consider the following results with the hypothetical mutants Cdc101 and Cdc102: You incubate a culture of the yeast Cdc101 mutant at its restrictive temperature (37C) for 2 hours (the approximate length of the cell cycle) so that its mutant phenotype is expressed. Then you change the medium to one containing hydroxyurea, and incubate the culture at the permissive temperature (20C). None of the cells divide. You now reverse the order of treatment. You incubate Cdc101 cells at 20C for 2 hours in medium containing hydroxyurea, and then change to a medium without hydroxyurea, and incubate at 37C. The cells undergo one round of division. You repeat these two experiments with the Cdc102 mutant. The cells do not divide in either case. A) In what phase of the cell cycle is Cdc101 blocked at the restrictive temperature? Explain the results of the two different temperature-shift experiments. B) In what phase of the cell cycle is Cdc102 blocked at the restrictive temperature? Explain the results of the two different temperature-shift experiments. Problem 2. Frog oocytes mature into eggs when incubated with progesterone. Egg maturation is characterized by disappearance of the nucleus (termed germinal vesicle breakdown) and formation of a meiotic spindle. The requirement for progesterone can be bypassed by micro injecting 50 nL of egg cytoplasm directly into a fresh oocyte (1000 nL), which then matures normally (Figure 1). Progesterone-independent maturation is triggered by maturation factor promoting (MPF) activity in the egg cytoplasm (later called mitosis promoting factor and shown to be M-Cdk). At early times after progesterone treatment, inhibition of protein synthesis by cyclohexamide blocks egg maturation. However, a few hours before oocytes become eggs (a time that corresponds to the appearance of MPF activity-progesterone) induced maturation can no longer be blocked by cyclohexamide.
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Is synthesis of MPF itself the cycloheximide-sensitive event? To test this possibility, you transfer MPF serially from egg to oocyte to test whether its activity diminishes with dilution. You first microinject 50 nL of cytoplasm from an activated egg into an immature oocyte as shown in Figure 1; when the oocyte matures into an egg, you transfer 50 nL of its cytoplasm into another immature oocyte; and so on. Surprisingly, you find that you can continue this process for at least 10 transfers, even when the recipient oocytes are bathed in cycloheximide!! Moreover, the apparent MPF activity in the last egg is equal to that in the first egg. A) What dilution factor is achieved by 10 serial transfers of 50 nL into 1000 nL? Do you consider it likely that a molecule might have an undiminished biological effect over this concentration range? B) How do you suppose MPF activity can be absent from immature oocytes, yet appear in activated eggs, even when protein synthesis has been blocked by cycloheximide? C) Propose a means by which MPF activity might be maintained through repeated serial transfers.
Figure 1. Progesterone- and MPF-induced maturation of oocytes. You can induce maturation of oocytes either by giving them Progesterone or by injecting a small amount of cytoplasm from an mature egg. Problem 3. You have isolated two temperature-sensitive strains of yeast (which you've named giant and tiny) that show very different responses to elevated temperature. At high temperature, giant cells grow until they become enormous, but no longer divide. By contrast, tiny cells have a very short cell cycle and divide when they are very much smaller than usual. Based on your understanding of cell-cycle regulation by Wee1, and Cdc25, propose an explanation for how mutations in the genes encoding those proteins might have given rise to the giant and tiny strains.