Cell Cycle Control 
James L. Mailer, Ph.D. — Investigator 
Dr. Mailer is also Professor of Pharmacology at the University of Colorado School of Medicine. He received 
his B.S. degree in biochemistry from Cornell University and his Ph.D. degree in molecular biology from 
the University of California, Berkeley, where he worked with John Gerhart. He then carried out 
postdoctoral studies with Edwin Krebs at both the University of California, Davis, and the University 
of Washington before moving to Colorado. 
TWO events mark the reproductive life of a 
cell: replication of the DNA, and its distribu- 
tion to daughter cells at mitosis. Because of the 
central importance of cell reproduction to or- 
dered cell growth and to birth of the next genera- 
tion, cells have evolved rigorous controls to en- 
sure that both events are carried out with high 
fidelity and at the appropriate time. My labora- 
tory is interested in understanding the nature and 
regulation of these controls with respect to how a 
cell commits itself to replicate its DNA and how it 
knows when to divide. 
The cell cycle has four main phases: Gi, S (syn- 
thesis), G2, and mitosis. The decision to synthe- 
size DNA (to enter the S phase) is made in Gj , and 
the decision to begin cell division (to enter the M 
phase of mitosis) is made in G2. There is abun- 
dant evidence that these decisions are made at 
checkpoints, or restriction points, in the cycle. 
The nature of these Gj and G2 decision-making 
periods in the cell cycle underlies fundamental 
processes operative in early embryonic develop- 
ment and in malignant cells. 
G2 ^ M Regulation 
Our laboratory developed a G2-phase extract 
from frog eggs in which synthetic nuclei entered 
mitosis at the addition of mitotic signals. We then 
purified the mitosis-signaling enzyme (called 
maturation-promoting factor, or MPF) and found 
that it was composed of a protein kinase com- 
plexed to a G2 cyclin. Kinases have the ability to 
attach a phosphate group to many different cellu- 
lar proteins, modifying their function and caus- 
ing profound changes in cellular biochemistry. 
The protein kinase was identified as a vertebrate 
homologue of the cdc2 gene, which had been 
genetically implicated in the control of mitosis 
by the study of certain mutants in yeast. 
G2 cyclins are proteins that accumulate during 
interphase, reach high levels in late G2, and are 
then degraded near the metaphase anaphase 
transition in mitosis. This degradation is required 
in order for cells to complete mitosis successfully 
and enter Gj . In most cells there are two classes of 
G2 cyclins, termed A and B cyclins, that differ in 
sequence similarity and have different kinetics of 
accumulation and degradation. Both bind cdc2 
kinase, but A- type complexes are activated much 
earlier in the cell cycle than B-type complexes, 
and only B-rype cyclins are found in puri- 
fied MPF. 
To investigate the role of cyclin A, we utilized 
extracts from metaphase-arrested eggs that are 
able to exit mitosis in vitro, to undergo DNA syn- 
thesis, and then to reenter mitosis. These extracts 
retain the characteristic dependence of mitosis 
upon completion of DNA synthesis — that is, will 
not enter mitosis if DNA synthesis has not been 
completed, which can result from an excess of 
DNA in the system or the presence of aphidicolin, 
an inhibitor of DNA polymerase. By using anti- 
sense oligodeoxynucleotides to ablate cyclin A 
mRNA from the system, we were able to show that 
activation of MPF (cyclin B/cdc2) occurred even 
when DNA synthesis had not been completed. 
Readdition of recombinant cyclin A protein to the 
antisense-ablated extracts restored the depen- 
dence of mitosis on DNA synthesis by causing a 
lengthening of S phase until DNA synthesis was 
complete. 
This provides evidence that one of the func- 
tions of cyclin A is in the crucial checkpoint that 
prevents the activation of cyclin B/cdc2 (MPF) 
until DNA synthesis has been completed. It also 
explains why cyclin A/cdc2 complexes are acti- 
vated earlier in the cell cycle than cyclin B/cdc2 
complexes and why cyclin A is degraded before 
cyclin B. The latter phenomenon, long known, is 
clearly appropriate, since cyclin A exerts an inhib- 
itory function on cyclin B. A question that now 
merits attention concerns what substrates exist 
for phosphorylation by cyclin A/cdc2 that are in- 
volved in this feedback control mechanism. 
We are interested in the mechanism of activa- 
tion of MPF in oocytes during the cell cycles of 
meiosis I and II. In these cycles the synthesis of 
proteins other than cyclin are required for MPF 
activation. One protein required for meiosis I and 
II is the product of the mos proto-oncogene. 
Proto-oncogenes are the normal cellular counter- 
part of mutated oncogenes found in cancer cells, 
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