Although recent genetic and molecular evidence has clarified the mechanisms utilized for sex determination in somatic tissues of Drosophila melanogaster, there has been little information relating to the germ line. Recently, however, ovo has been shown to be active in a sex-specific manner as early as the blastoderm stage and that mutations in ovo cause a switch in differentiation into the male germ line pathway. Both molecular and genetic analysis of ovo indicate that it shares sequences with shavenbaby (svb), a polyphasic zygotic lethal. Sequence information has been published for cDNA clones isolated from embryonic libraries which indicates that the gene codes for a putative nuclear protein with four zinc fingers near the C-terminus. We have identified a cDNA from an ovary library which codes for a different isoform containing an additional 177 amino acids inserted prior to the zinc-fingers. We propose to analyze the 5' terminus of the ovo-svb transcript, utilizing PCR methodology. With PCR, we will identify the genetic origin of the different classes of RNAs. We will prepare antibodies both against the zinc-finger region as well as against the 177 amino acid portion specific to one isoform of the protein. In situ hybridization with digoxygenin-labelled probes and immunolocalization will be carried out to identify the cell and tissue basis for ovo and svb expression. We will determine the DNA-binding sites of the ovo protein by staining the nurse cell polytene chromosomes of ovarian tumor mutants. If any of the staining sites correspond to regions where putative interacting genes with ovo are located, we will attempt to identify the specific sites responsible for DNA binding by gel shift assays. From these studies we should gain an understanding of the role of ovo germ cell function. These results will lay the foundation for investigating other genes involved in sex determination in the germ line.
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