PROTEIN REQUIRED FOR GERM CELL DETERMINATION

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In many organisms early determinative events are established by the differential distribution of informational molecules (cytoplasmic determinants). In Drosophila, the distribution of these determinants is established by genes that make a strictly maternal contribution to the developing embryo. In Drosophila, genetic dissection, micromanipulation, and molecular cloning can be combined to study a specific developmental process. Thus, Drosophila serves as a model system for studying genes that produce these determinants and/or are involved in properly distributing these determinants within the embryo. In particular, we are interested in studying maternal effect genes required both for the determination of the primordial germ cells and establishment of the anteroposterior axis of the embryo. One such locus, tudor (tud), is the basis of this proposal. The tud locus encodes an ~8.0 kb mRNA that is localized within the posterior half of the oocyte during mid-oogenesis. Using antibodies against tud protein we find that the protein is synthesized during the earliest oogenetic stages and fills the perinuclear space of stage 1- oocytes. In early embryogenesis (0-0.25 h) the protein is localized in the posterior pole plasm (germ plasm). During the intravitelline nuclear divisions the protein is sequestered in nuclei and mitochondria along the anteroposterior axis of the embryo. However, the protein accumulates preferentially in posterior pole mitochondria. Experiments outlined below will increase our understanding of the role of tud+ function. First, indirect immunofluorescence and immunoelectron microscopy will be used to determine the subcellular location of tudor protein in oocytes and embryos collected from mutant females. Second, embryos will be fractionated to isolate purified nuclei and mitochondria. Tudor protein will be purified from these fractions and characterized to determine if there are alternate forms of the protein. Third, P element-mediated insertion of various tud genomic and cDNA constructs will be used to identify sequences: (1) complementing subsets of the tud phenotype; and (2) required for targeting the protein. Finally, an antisense construct will be used to determine the requirement for tud during the life cycle.