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Additional Suggested Problems for Exam II
Note: All of the following are drawn from the study materials (old exams and
problems sets on the web). However, I didn’t completely agree with all of the
answers, so I made a few minor adjustments. - Emily
Exam 4, 2000: Question 1!!!! (I didn’t retype this one, but it’s a great problem
and definitely worth working through.)
From Problem Set 8, 2001:
Question 1
You have just identified three proteins you think are important for progression
through the cell cycle. Specifically, you know that they affect the activity of Cdc2p
by altering its phosphorylation state.
A) Design an experiment to determine if each protein is a kinase or a
phosphatase for Cdc2p.
B) From the above experiment you know that you have one phosphatase (P1)
and two kinases (K1 and K2). You add these enzymes in different
combinations and find that they have different effects on Cdc2p activity. Here
are part of your results:
Cdc2p
Cdc2p
Cdc2p
Cdc2p
Cdc2p
Cdc2p
Cdc2p
Cdc2p
Æ inactive
+ K1 Æ active
+ K2 Æ inactive
+ K1 + K2 Æ inactive
+ P1 Æ inactive
+ K2 + P1 Æ inactive
+ K1 + P1 Æ active
+ K1 + K2 + P1 Æ active
What does this tell you about K1, K2, and P1? If these proteins regulate the G1/S
transition, what might their identities be?
Similar to Question 3…
You’ve purified a protein involved in cell cycle regulation called cdc500. While
analyzing this protein, you’ve learned that residue 246 (a Threonine) is sometimes
phosphorylated. Design an experiment to determine if phosphorylation of this
residue is important for its function or regulation.
Question 4
You are a researcher interested in microtubule dynamics during mitosis. Mitotic
spindle assembly requires the separation of the duplicated centrosomes. The current
model is that centrosome separation requires kinesin-like (or kinesin-related)
proteins (KLPs). You have at you disposal a candidate KLP, klp999, from the frog
Xenopus laevis. You would like to address whether the protein klp999 is indeed
required for centrosome separation and consequent spindle assembly.
A) First you would like to know whether klp999 is actually present near the
centrosomes and the mitotic spindles of Xenopus eggs undergoing mitosis.
What experiment will provide you with an answer?
B) You find the klp999 is indeed localized at the centrosomes. Encouraged, you
want to address whether klp999 plays a critical role in mitotic spindle
assembly. What kind of experiments would you do?
C) In your spare time, you’ve generated various deletion mutants of klp999.
You are particularly impressed with one deletion construct in which the klp999
“tail” region is intact but the “motor” region is completely deleted (and you
call it klp999-noM). You introduce klp999-noM into wildtype Xenopus eggs,
and you discover that the centrosome separation and spindle formation is
inhibited. What do you think is happening and how can you test your
hypothesis?
Question 5
You are interested in studying the cell cycle in budding yeast.
A) How would you go about creating, identifying, and studying yeast with
mutations in genes that regulate the cell cycle?
B) You find on ts (temperature-sensitive) mutant that at the restrictive
temperature gives an arrested phenotype that looks like a large cell with the
beginnings of a small bud formed. At what stage are these cells arrested?
What genes do you think could possibly be mutated? (In other words, genes
involved in what cellular processes could lead to arrest at this point?)
C) You are interested in cloning the gene responsible for this arrest. How would
you go about doing this?
D) You isolate the gene whose absence is responsible for the arrest and call it
CCM1, for Cell Cycle Mutant 1. How would you go about determining if it is
differentially expressed during the cell cycle?
From Problem Set 7, 2000:
Question 1. What is a kinase? Give an example of a kinase mentioned in lecture.
Question 2. All of the following are true about destruction of cyclins except:
i. Destruction is carried out by proteases.
ii. Destruction is preceded by polyubiquitination.
iii. Ubiquitination occurs at specific sites on target molecules.
iv. Ubiquitin targets cdc2 to destroy cyclins.
v. Destruction of cyclins inactivates cdc2’s kinase activity.
Question 3. M-cyclin levels are regulated by:
vi. phosphorylation
vii. synthesis and degradation of its mRNAs
viii. ubiquitination by APC
ix. ubiquitination by MPF
Question 4. Proteosome is another term for which of the following:
x. MPF
xi. APC
xii. Ubiquitin
xiii. SCF
xiv. HU
xv. None of the above
Question 5. How does the addn of hydroxyurea invoke the S-phase checkpoint?
Question 6
One approach to the study of cell cycle regulation has been to fuse cultured cells (the
membranes fuse and their cytoplasms mix-there are now 2 nuclei in one but cell)
that are at different stages of the cell cycle and observe the effect of the fusion on
the nuclei. When cells in G1 were fused with cells in S, the nuclei from the G1 cells
were observed to begin DNA replication earlier than they would have if they had not
been fused. In fusions of G2 and S cells, however, both nuclei continued their
previous activities, apparently uninfluenced by the fusion. Fusions between M and
G1 cells always led to immediate chromatin condensation of the non-mitotic DNA.
Based on these results, identify each of the following statements as probably true
(T), probably false (F) or not sufficient for evaluation based on the given data (NS).
xvi.
xvii.
xviii.
xix.
xx.
xxi.
The activation of DNA synthesis may result form the positive action of one
or more cytoplasmic factors.
The transition from S to G2 may result from the presence of a cytoplasmic
factor that inhibits DNA synthesis.
The transition from G2 to M (mitosis) may result from the presence in the
G2 cytoplasm of one or more factors that induce chromatin condensation.
G1 is not an obligatory phase of all cell cycles.
Like the transition from G1 to S, the transition from G2 to M appears to be
under positive control.
The transition from mitosis to G1 appears to be under negative control.
Question 7
A normal diploid cell from the insect Creapus crawlus contains ten chromosomes and
0.02ng (nanograms) of DNA in its nucleus. Elevated amounts of DNA are found in
some Creapus cells:
Cell Type
Muscle
Liver
Salivary Gland
DNA/cell
20ng
20ng
20ng
Nuclei/cell
1000
1
1
Chromosomes/nucleus
10
10,000
10
i.
Explain how the elevated DNA content of each of these cell types
might have arisen due to altered cell cycles.
ii.
Which of these cell types would you expect to have the shortest
generation time? Why?
iii.
Would you expect the salivary gland cells to be undergoing mitosis and
dividing actively? Explain.
iv.
How might you induce the liver cells to begin dividing again?
Question 8
You have discovered a protein that is secreted by tulips to kill off parasitic fungi.
This protein, UCK, binds to the fungi S-cyclin when you mix them together in a test
tube. You believe that UCK is preventing fungus growth by either stabilizing the Scyclin or cause it to be degraded in the cell.
i.
At what stage of the cell cycle would a fungal cell stop if UCK stabilizes the
S-cyclin?
ii.
At what stage of the cell cycle would a fungal cell stop if UCK promotes Scyclin degradation?
From Problem Set 6, 2000:
Question 8c
Both taxol and vinblastin cause cell cycle arrest in mitosis. Why? How do taxol and
vinblastin affect the microtubules? What does this tell you about the behavior of
microtubules during mitosis?
Question 12
What happens to a microtubule (MT) that loses its “GTP cap”?
Question 13
You have discovered a new cell type that has long protrusions coming from its cell
body.
i.
How would you test if these structures contain actin and/or tubulin?
ii.
You discover that both MTs and microfilaments are present in these
structures. Design an experiment to test if the stability of these
protrusions is maintained solely by MTs.
iii.
How would you determine the polarity of the MTs in these protrusions?