The long-term goal of this research is to understand how cells interact in the development of the Drosophila oocyte. The work will focus on the mechanism and regulation of cytoplasm transport from the nurse cells to the oocyte through the intercellular bridges that connect these cells. Aspects of the regulation of cytoplasm transport are likely to be common to many other cell types in invertebrate as well as vertebrate systems. In addition, the mechanism of communication between germ line cells connected by intercellular cytoplasmic bridges may be relevant to the cause of mammalian germ line tumors.
The specific aims of the proposal are: (1) to determine the nucleotide sequences of two genes, chickadee and kelch, mutations in which disrupt the transport of cytoplasm to the oocyte; (2) to determine the expression pattern of chickadee and kelch at both the mRNA and protein levels; (3) to test models for the function of chickadee and kelch; (4) to search for additional mutants in cytoplasm transport using single P element insertional mutagenesis; and (5) to investigate the regulation of transport specificity. Using controlled single P element insertional mutagenesis, insertional alleles were recovered of two second chromosome genes, chickadee and kelch, that are clearly associated with nurse cell cytoplasm transport; the nurse cells of mutant egg chambers fail to empty their cytoplasm into the oocyte late in oogenesis. The genes were cloned by plasmid rescue of DNA flanking the inserted P elements and putative protein sequence will be examined for any similarities to known proteins or protein motifs. The expression pattern of chickadee and kelch will be examined by Northern hybridization and in situ hybridization. Additional mutants in nurse cell cytoplasm transport will be isolated by insertional mutagenesis of the autosomes with an """"""""enhancer trap"""""""" P element construct. The mutants will be mapped cytogenetically and used in appropriate complementation tests to determine if other alleles of the genes exist. Phenotypic analysis will include staining nurse cell cytoplasmic actin filaments (required for cytoplasm transport) to determine whether the contractile apparatus is intact and determining whether the cytoplasmic bridges are """"""""open"""""""" to cytoplasm flow by studying the distribution of maternal mRNA by in situ hybridization. Two classes of mRNA are transported into the oocyte: those that appear in the oocyte early in oogenesis and those that are sequestered in the nurse cells until the bulk of cytoplasm transport late in oogenesis. Mutant egg chambers will be examined by in situ hybridization for any effect on this transport specificity. The model that the sequestered mRNAs are actively translated will be tested by fractionating egg chamber extracts on sucrose gradients and examining polysome fractions for specific mRNAs.
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