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Problem Set 3: The Eukaryotic Cell Cycle

Bio 102

1.What cellular mechanism(s) ensure that passage through the cell cycle is
unidirectional and irreversible? What molecular machinery underlies these

2. What is the functional definition of START? Cancer cells typically lose START
control. Explain how the following mutations, which are found in some cancer cells,
lead to a bypass of START controls: (a) overexpression of cyclin D, (b) loss of Rb
function, (c) loss of p16 function, (d) hyperactive E2F.

3. The Rb protein has been called the master brake of the cell cycle. Describe how
the Rb protein acts as a cell cycle brake. How is the brake released in mid- to late
G1 to allow the cell to proceed to the S phase?

4. A common feature of cell cycle regulation is that the events of one phase ensure
progression into a subsequent phase. In S. cerevisiae, G1 and G1/S phase CDKs

promote S-phase entry. Name two ways in which they promote the activation of S

5. Many of the proteins that regulate transit through the cell cycle have been
characterized. Xnf7, identified in extracts of Xenopus eggs, binds to the anaphasepromoting complex/ cyclosome (APC/C). To elucidate the function of this protein,
studies have been undertaken in which Xnf7 either has been depleted from extracts
using an antibody raised against it or has been augmented in the extracts through
addition of extra Xnf7. The consequences on transit through mitosis were then
assessed (see J. B. Casaletto et al., 2005,/. Cell Biol. 1 6 9:61-71).
a. Xenopus egg extracts, arrested in metaphase, were either depleted of Xnf7 or
were mock depleted (subjected to the same treatment as the first sample but
without Xnf7 antibody), then released from metaphase arrest by addition of Ca 2+.
Aliquots of the extract were then sampled at various times after Ca2+ addition and
the amounts of mitotic cyclin determined, as shown on the Western blot below.
What information do these data provide about a possible function for Xnf7?

b. In additional studies, exogenous Xnf7 was added to Xenopus egg extracts,

arrested at metaphase, so that the total amount of this protein in the extracts was

higher than normal. The extracts, released from arrest by Ca2+ addition, were then
assessed at various times after release for mitotic cyclin ubiquitinylation (cyclin-Ub
conjugates). What is the rationale for examining ubiquitinylation? Using the
following figure, determine what information these studies add beyond that
obtained from part (a).

c. The spindle checkpoint pathway prevents cells with unattached kinetochores from
proceeding into anaphase. Thus cells in which this checkpoint has been activated do
not enter anaphase and do not degrade mitotic cyclin. Nocodazole, a drug that
prevents microtubule assembly, can be used to activate the spindle checkpoint.
Cells in nocodazole become arrested in early mitosis because they cannot form a
spindle, and so all kinetochores remain unattached. To determine if Xnf7 is required
for a functional spindle checkpoint, Xenopus egg extracts, arrested in metaphase,
were subjected to various protocols (see the following figure): untreated (no
nocodazole) or treated with nocodazole and either mock depleted (preimmune) or
immunodepleted of Xnf7 (alpha-Xnf7), The extracts were then treated with Ca 2+ to
overcome arrest, and aliquots of the extracts were assessed at various times for
mitotic cyclin, as shown on the Western blot below. What can you conclude about
Xnf7 from these data?