During late oogenesis in Drosophila, ovarian follicle cells synthesize and secrete several proteins that become incorporated into the highly organized vitelline membrane and chorion sublayers that comprise the eggshell. Our research is directed toward understanding the molecular mechanisms that underlie developmental regulation of egg-shell protein synthesis and morphogenesis. Towards this end we have been studying the molecular basis of mutations that effect eggshell production and assembly. This proposal is directed towards extending our analysis of recently isolated eggshell mutants to the nucleic acid level and identifying vitelline membrane structural gene sites by in situ hybridization of vitelline membrane cDNA clones. cDNA clones complementary to egg chamber poly A containing RNA synthesized during the stages of chorion deposition will be isolated from an existing cDNA plasmid library. Following this initial round of hybridization, prospective cDNA clones of interest will be identified by appropriate counterscreens. Selected clones will be characterized by in situ hybridization to salivary gland chromosomes, in vitro translation of hybrid selected RNA, in situ hybridization to tissue sections, and a quantitative analysis of complementary genomic sequences. cDNA clones homologous to region 12E of the X chromosome will be used to initiate a """"""""chromosome walk"""""""" to obtain cloned genomic DNA sequences that cover the entire 12E region. The 12E region is of a particular interest since flies carrying a homozygous deletion for this region are viable, female sterile and produce mature eggs with several phenotypic abnormalities. Following isolation of the appropriate cloned probes, the molecular basis of existing and newly induced female sterile mutations in this region will be studied. Vitelline membrane cDNA clones will be selected from a cDNA library that we propose to construct containing sequences complementary to poly A containing RNA isolated from egg chambers in the period of vitelline membrane deposition. Following identification of vitelline membrane structural gene loci on salivary gland chromosomes, vitelline membrane gene regulation will be approached at the molecular level. Since vitelline membrane deposition represents the initial phase of eggshell formation in Drosophila, activation of vitelline membrane genes may be important in regulation expression of the better studied chorion genes.
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