GENETIC AND MOLECULAR ANALYSIS OF GERMLINE DETERMINANTS

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The long term goal of this project is to understand the biochemical nature and function of the germ plasm. The posterior pole plasm of Drosophila plays a critical role for the development of somatic and germ line tissues. These functions depend on the normal activity of the maternal effect gene oskar. Mature oocytes derived from homozygous mutant oskar females lack the morphological specializations found at the posterior pole of the normal oocyte. Embryos developing from mutant oocytes do not form pole cells, the germ line precursor cells, and do not develop abdominal segments. Oskar RNA is localized at the posterior pole of the embryo and the oskar protein may thus be a component of the pole plasm. Genetic evidence indicates that the oskar gene product is required for the localization of additional gene products critical for germ cell determination and that it is also required for the localization of the nanos gene product which controls segmentation in the abdominal region. Initially, the molecular nature of the oskar gene product and its distribution during oogenesis and embryogenesis will be characterized. The oskar transcription unit will be defined, the gene will be cloned and the coding region sequenced. The distribution of oskar RNA and protein will be studied during oogenesis and embryogenesis using nonradioactively labelled oskar DNA and/or antisense RNA probes and antibodies directed against the oskar protein. Subsequently, the mechanisms by which oskar products become localized to the posterior pole plasm will be analyzed. The ability of mutants in other pole plasm components to localize oskar RNA and/or protein will be tested. Cis acting sequences in the oskar RNA and protein necessary for proper localization will be determined by analysis of mutant oskar alleles and deletion assays. The sufficiency of these sequences to localize oskar RNA and/or protein will be tested using reporter gene fusions. Finally, to understand the role of oskar for the localization of germ cell and somatic determinants, the effect of oskar mutations on the localization of other components of the posterior pole plasm, e.g. the nanos RNA and the vasa protein, will be studied. Direct interactions between the oskar RNA and protein with other components of the pole plasm will be studied by copurification assays using antibodies directed against pole plasm products.