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IN~ER~FFICEMEMO D~TE: January 19, 1994 TO: SUBJECT: "Regulatlon of Translation Initiation During Xenopus Development" Application #3076 A Primary reviewer. 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. 50326084

#3076 ~ page 2 o~ 3 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 studied. 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 productivity. 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 biological significance. 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 progress. ~;I~.DA~~~u is a chemist, obtained his PhD at George Washington University in Washington D.C., and did post-do=total work in 50326085

:~3076 A 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. Merit A. 50326086