This proposal addresses the question of translational regulation as a fundamental element in the control of gene expression during early development. Specifically, we are asking how protein synthesis is activated following fertilization and how it is regulated during embryogenesis. The system chosen for this study is the sea urchin egg and developing embryo. The general approach proposed is to purify and characterize the key components of translation, and develop tools which can monitor changes in the levels, forms and activities of these components, to ultimately discover the mechanisms which govern their interactions. First, we shall purify and characterize the initiation factors of protein synthesis from sea urchin eggs and embryos and produce antibodies against the purified factors. Using immunochemical and gel electrophoretic techniques, we will determine levels and molecular forms (e.g., phosphorylation) of the factors in unfertilized eggs and embryos. Second, sea urchin egg cell-free lysates active in protein synthesis will be prepared following published procedures. We will also construct an assay system entirely of highly purified sea urchin components, and use these two assays for detailed analysis of the role of the purified translational components of the initiation pathway in activation of protein synthesis. Third, we will examine changes in ribosomal proteins during fertilization and early development, and establish how changes are linked to the translational activation at fertilization. The role of cytoskeletal changes during early development in modulating the rate of protein synthesis will also be studied. Finally, we shall purify and characterize the mRNA and proteins of bulk and specific messenger rebonucleoprotein particles using immunological and gel electrophoretic techniques and try to identify a masking factor. The tools developed and the information obtained from these experiments will then allow us to elucidate the molecular mechanisms of mobilization of mRNP into polysomes. These studies will elucidate not only important controlling mechanisms during early development, but also important general mechanisms of translational control in all eukaryotic cells.
| Lopo, A C; Lashbrook, C C; Hershey, J W (1989) Characterization of translation systems in vitro from three developmental stages of Strongylocentrotus purpuratus. Biochem J 258:553-61 |
| Lopo, A C; MacMillan, S; Hershey, J W (1988) Translational control in early sea urchin embryogenesis: initiation factor eIF4F stimulates protein synthesis in lysates from unfertilized eggs of Strongylocentrotus purpuratus. Biochemistry 27:351-7 |
| Lopo, A C; Lashbrook, C C; Infante, D et al. (1986) Translational initiation factors from sea urchin eggs and embryos: functional properties are highly conserved. Arch Biochem Biophys 250:162-70 |
