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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. Temperature-
sensitive 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 (37°C) 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 (20°C). None of the cells divide.

You now reverse the order of treatment. You incubate Cdc101 cells at 20°C for 2 hours in
medium containing hydroxyurea, and then change to a medium without hydroxyurea, and
incubate at 37°C. 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.
The two different temperature-shift experiments demonstrate that Cdc101 is blocked at 37°C in the G1
phase of the cell cycle. The experiment in which the cells were first incubated at the restrictive
temperature shows that the mutational block precedes or coincides with the hydroxyurea block. If this
were not the case, the cells would have divided when they were shifted to 20°C in the presence of
hydroxyurea. The experiment in which the cells were first blocked with hydroxyurea shows that the
hydroxyurea block occurs after the mutational block, because the cells divided once when they were
shifted to 37°C in the absence of hydroxyurea. Together, the two experiments indicate that the mutational
block in Cdc101 is in the G1 phase of the cell cycle. In the first experiment, the Cdc101 mutants
accumulate in G1 at 37°C; when shifted into hydroxyurea medium at 20°C, they move to S phase, but are
stopped there by the hydroxyurea block, and thus do not divide. In the second experiment, in the
presence of hydroxyurea at 20°C the cells are blocked in S phase; when hydroxyurea is removed and the
cells are shifted to 37°C, they progress normally through G2 and M before they are blocked in G1.
Therefore, they undergo one round of cell division.

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.
The results with Cdc102 indicate that it is blocked at 37°C in S phase. The first experiment shows that
the mutational block precedes or coincides with the hydroxyurea block, or the cells would have divided
when they were shifted to 20°C. The second experiment shows that the hydroxyurea block precedes or
coincides with the mutational block, or the cells would have divided when they were shifted to 37°C.
Together, the two experiments indicate that the mutational block and the hydroxyurea block coincide.
Since hydroxyurea and the Cdc102 mutation both affect the same phase of the cell cycle, the order of
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treatment makes no difference; the cells remain trapped in S phase and therefore do not divide.

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 promoting
factor (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.

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.
ent 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?
Since each transfer accomplishes a 20-fold dilution (50 nL/1000 nL), 10 transfers yield a dilution factor of
2010, which is equal to 1013. It is unreasonable for a molecule to have an undiminished biological effect
over this range of dilution.

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?
The appearance of MPF activity in the absence of protein synthesis suggests that an inactive precursor of
MPF is being activated. In principle, activation could involve one of several kinds of post-translational
modifications such as protease cleavage or changes in protein phosphorylation. From class you know
that MPF is held in an inactive state by phosphorylation of an inhibitory site of Cdk. MPF is activated
when that inhibitory phosphate is removed by the protein phosphatase, Cdc25.

C) Propose a means by which MPF activity might be maintained through repeated serial
transfers.
In order for MPF to propagate its activated state through serial transfers, it must be able to activate itself.
Since MPF is a protein kinase, it is reasonable to suggest that it operates via phosphorylation to trigger
the activation of the inactive pre-MPF. In principle, MPF could activate pre-MPF directly by adding one or
more phosphates. Or it could act indirectly through other kinases or phosphatases to alter the
phosphorylation status of pre-MPF and thereby activate it. As it turns out, MPF triggers the addition of
activating phosphates to Cdc25, which then removes the inhibitory phosphates from pre-MPF, activating
it. This sets up an auto-amplification cycle that rapidly activates all the MPF, thereby stimulating egg
maturation in each serial transfer.
It is also reasonable to postulate that in each transfer you move some phosphorylated (active) Cdc25
from the mature egg to the injected oocyte. This would set off the same auto-amplification cycle by
removing the inhibitory phosphates from pre-MPF, activating it. In reality, you are doing some of both.
Progesterone induces egg maturation by setting up the same auto-amplification cycle, but initiates it in a
different way which we did not cover in class but you can read in the textbook if interested.
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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.

In the normal situation, Wee1 adds phosphates to Cdk1 to inactivate it, and Cdc25 removes those
phosphates, activating Cdk1. In principle, different types of mutation in any one of these genes might
produce the giant and
tiny temperature-sensitive strains of yeast. For example, a mutation in the Cdk1 gene that led to an
inactive protein at high temperature would generate a yeast strain like giant that was unable to proceed
through the cell cycle. A mutation in the same gene that prevented Cdk1 from being inactivated by Wee1
at the restrictive temperature would lead to a constitutively active Cdk1 that would continually push the
cell through the cell cycle, as in the tiny strain.
You could imagine analogous mutations in the Wee1 and Cdc25 genes. An inactive form of Wee1 (or a
hyperactive form of Cdc25) would leave Cdk1 permanently active, generating cells with a short cell cycle,
like the tiny strain. A hyperactive version of Wee1 (or an inactive form of Cdc25) would generated an
inactive Cdk1, would produce cells that were unable to progress through the cell cycle, as in the giant
strain.
In various screens, inactivating mutations in Wee1 and Cdc25 have been found, and they have the
expected phenotypes, but mutations that lead to hyperactive forms of these proteins have not been
isolated. The mutations described in this problem correspond to two alleles of Cdc2 (the original name for
the Cdk1 gene) discovered by Paul Nurse in 1974.