Xenoous 1aerie appears to be an excellent model for. REDACTED studies in that the egg from these amphibians makes a transition into a rapid mitotic cell cycle followlng fertilization. The fertilized egg then undergoes twelve synchronous divisions within six hours. Herein, mitotic events correlate with inhibition of translation initiation which has been shown by decrease.
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January 19, 1994
SUBJECT: "Regulatlon of Translation
Initiation During Xenopus Development"
Application #3076 A
Xenoous 1aerie appears to be an excellent model for. REDACTED
studies in that the egg from these amphibians makes a transition
into a rapid mitotic cell cycle followlng fertilization. The
fertilized egg then undergoes twelve synchronous divisions within
six hours. Herein, mitotic events correlate with inhibition of
translation initiation which has been shown by decrease. Studies
have shown that Xenoous laevis shows almost complete absence of
transcription of the zygotic genome in this six hour window from
1 to 4,000 cells, and during this period, stored maternal mRNAs
provide the sole means to induce new proteins in the cleaving
embryo. The eIF-4 group of translation factors catalyzes the
initial step of translation initiation. It is noteworthy that
phosphorylation of eIF-4E at Ser-53 is crucial to its activity in
delivering mRNA into 48 S initiation complexes. The rate of
phosphorylation is increased in cells stimulated by growth
factors or mitogens which cause cells to proliferate or
differentiate and which fundamentally llnk the regulation of eIF-
4E activity to the cell cycle. The ras signal transduction
pathway mediates eIF-4E phosphorylation and ras induces germinal
vesicle breakdown (GVBD) which is a hallmark of meiotic
maturation when expressed in oocytes.
The PI~s goal is to use the system of Xenopus development to
elucidate mechanisms which link F~-mediated signal transduction,
translation initiation, and the mobilization of mRNAs. He then
hopes to elucidate the roles which eIF-4E and eIF-4T play in
mediating the effects of ras on the cell cycle. He will also
probe the role of eIF-4 factors in the poly(A} elongation and
mobilization of maternal mRNAs, which are a cell cycle-regulated
event. New insights into how the cell coordinates its efforts to
proliferate in concert with the fundamental of protein synthesis
may then ensue.
The ai~s perta£n to the determination of a possible requirement
for eIF-4E and eIF-4T during oocytes mitotic maturation and the
embryonic cell cycle. He will clone XenoDus eIF-4E and eIF-4T
cDNAs, he will determine the pathways of eIF-4E phosphorylation
in ~ oocytes and will then address the roles of eIF-rE and
eIF-4T in polysomal mobilization of stored ~aternal ~RNAs.
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The PI has found a firm llnk between ras and the control of
protein synthesis in cell growth via eIF-4E phosphorylation and
has used one of the two systems from his original grant proposal,
namely CREF cells. He has recruited REDACTED
to work on
the project since R~D~.5A[~D has experience with the mobilization
of maternal mRNAs into the polysomes and their role in XenoDus
embryonic cell cycle and the techniques involved. He thus calls
attention to the fact that this collaboration will involve two
investigators who have expertise in each of the areas to be
A strong case is made for utilizing XenoDus oocytes in embryos
for the study of the mutual dependence of cell cycle progression
and translation initiation, since a strong body of literature is
present on differing facets of cell cycle mechanisms and their
translational regulation in this model.
The supporting data, inclusive of the reprint which acknowledged
CTR, clearly indicate the PI's confidence as well as his
In the experimental design, he will begin by over-expressing
human eIF-4E and eIF-4T separately and together in stage VI
oocytes which are known to be limiting for initiation activity
and he will inject in vitro transcribed human eIF-4E and human
eIF-4T and will assay whether the oocytes undergo GVBD. He will
study the changes in the rate of protein synthesis. He lists
several scenarios which may explain the overexpression. He has
obtained a new XenoDus ovary cDNA library from a colleague in
Vienna (W. Hane).
This is a rather complex proposal, but it has rather compelling
The budget is appropriate._,~C.~.~) will provide 10t of his
time at no cost to CTR. ~ . _. the post-doctoral research
associate, at $34,800. $13,700 is requested for equipment, and
therein is primarily a Zeiss stereomicroscope with attachments.
It appears that the PI is well-funded, but he states that there
is no overlap between his funded proposal entitled "Regulation of
Protein Synthesis Initiation," although this is an interesting
titled when viewed in concert with the present proposal.
The progress report and the appended manuscript clearly indicate
~;I~.DA~~~u is a chemist, obtained his PhD at George Washington
University in Washington D.C., and did post-do=total work in
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page 3 of 3
pharmacology at Stanford.
Two of his publications deal directly with studies on Xenomus
laevis and deal in the general area of REDACTE~ - proposal.
This is an interesting proposal which will extend the work of a
productive investigator and will provide experien=e for a young
investigator who has already begun work on XenoDus laevis.