There are 4 projects in this proposal. The first concerns the mechanisms which regulate the translation of maternal mRNA in Xenopus oocytes/embryos. We will measure the capacity of oocytes to form pre-initiation complexes after injection of radioactive met-tRNAf and we will develop a Xenopus oocyte in vitro translation system to test the efficacy of adding components of the translational machinery on protein synthesis. Also, we will fractionate cytosol from maturing oocytes to isolate proteins which remove """"""""masking"""""""" proteins from maternal mRNA as well as to identify proteins which recruit mRNA into polysomes. Finally, we determine whether or not 3' poly (A) on mRNA increases the efficiency of translation by measuring ribosome transit times and number of ribosomes on mRNAs with and without poly(A). The second project is concerned with the hypothesis that cytoplasmic interspersed poly(A) RNA results from inefficient processing of nuclear RNAs. We will inject components, such as snRNA, known to be involved in nuclear processing into oocytes coupled with assays to determine if transport of unprocessed transcripts to the cytoplasm is reduced. We also will test the capacity of fertilized eggs to process pre-mRNAs or interspersed transcripts injected into the cytoplasm. The third project will test the hypothesis that agonists which induce oocyte maturation act by inhibiting kinase C activity by reducing the level of the kinase activator diacylglycerol (DAG). This will involve thin layer chromatography to measure changes in the level of various phospholipids in the pathways to DAG production as well as DAG itself. To test the possible role of kinase C in later events of maturation, the effects of kinase C on nuclear envelope breakdown will be assayed cytologically using an in vitro Xenopus egg lysate. The final project concerns observations that injection of antibodies to cyto-skeletal proteins into the germ plasm region of 2-cell embryos leads to a reduction in germ cell numbers. We will assess antibody effects on: intracellular location of germ plasm, number of cells containing germ plasm, and ultrastructural integrity of germ plasm using light and electron microscopy.
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