Corresponding chapter(s) in the textbook: Chapter 19 (and 17)
Review the following terms before working on the problem:
human papilloma virus, [35S]methionine labeling, [32P]phosphate
labeling, phorbol ester, retinoblastoma protein,
immunoprecipitation, SDS-polyacrylamide gel electrophoresis,
autoradiography, pull-down assay, Western blotting
Read the paper and answer the questions below that refers to
the data described in Figures 4 and 5 of the paper.
Be prepared to discuss the other experiments described, in
class.
Experiment
Human papilloma virus strain 16 (HPV-16) is one of the
causative agents of cervical cancer in women. The purpose of the
research presented in the figure was to study the mechanism of
action of the E7 oncoprotein of HPV-16.
Graph A shows the results of a preliminary experiment. Cells
of a human leukemia cell line were cultured without (samples 1 and
3) or in the presence of phorbol ester (samples 2 and 4) and
concurrently labeled with [35S]methionine (samples 1 and 2) or
[32P]phosphate (samples 3 and 4). (Note: Phorbol ester stimulates
protein kinase C.) Cell extracts were immunoprecipitated with an
antibody specific for the retinoblastoma protein (anti-RB).
SDS-polyacrylamide gel electrophoresis was performed, followed by
autoradiography.
Graph B shows the results of the experiment designed to
analyze the function of the E7 protein. Human leukemia cells were
cultured in the absence (samples 1 to 3) or presence of phorbol
ester (samples 4 to 6). Cell extracts were prepared, and aliquots
were incubated with agarose beads to which E7 protein molecules had
been covalently attached. After incubation, the beads were
sedimented by centrifugation, and Western blot analysis was
performed using the anti-RB antibody on the supernatants (S;
samples 2 and 5), pellets (P; samples 3 and 6), or total cell
extracts (T; samples 1 and 4).
Figure
A) Analysis of RB-immunoprecipitates from human leukemia cell
extracts
B) Analysis of RB and E7 protein interactions by pull-down
assay (T: total cell extract; S: supernatant; P: pellet)
Questions
1. What was the goal of [35S]methionine and [32P]phosphate
labeling?
2. What do bands a and b represent?
3. What does the state of RB protein (graph A) tell you about
the cell cycle conditions of the leukemia cells?
4. How did phorbol ester treatment affect the synthesis and
phosphorylation of the RB protein?
5. How would the effect of phorbol ester treatment affect the
leukemia cell culture?
6. What phenomenon was studied in the experiment that
produced graph B?
7. Which form of the RB protein is affected by the E7
oncoprotein?
8. What effect would expression of the E7 protein in the
leukemia cells have?
9. Summarize the mechanism of action of the E7 oncoprotein.
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