During Drosophila egg development, ring canals provide the pathway for intercellular cytoplasm transport from nurse cells to the growing oocyte. These cells are mitotic descendants of a single germline cell. Development of germline cells occurs in interconnected clusters in many other animal species, including humans. However, no directed nutritive role has been described in mammalian germline development and the significance of syncytial organization in higher eukaryotes is unclear. There is evidence that human germline dysgerminomas may be related to faulty intercellular connections between germline cells in development, leading to unchecked mitotic proliferation. The study of insect ring canals and the regulation of syncytial development should provide the basis for studies on germline development in mammals. This work proposed in this application will focus on three genes that are involved with ring canal morphogenesis in Drosophila-kelch, hu-li tai shao (hts) and cheerio. Two of them, kelch and hits, have already been cloned and we have previously shown that kelch and hits proteins are specifically located at ring canals. The kelch mRNA encodes two tandem open reading frames separately by an-frame stop codon. Germline transformation with fragments of the kelch gene will be used to determine whether the products of both open reading frames are required for kelch function. Conserved domains in kelch will be tested for function by assaying rescue of kelch mutants with germline transformation constructs. Kelch proteins produced in E. coli or in a baculovirus expression system will be assayed for actin binding activity and for interaction with other ring canal proteins. The sequence of the mRNA encoding the hts ring canal protein predicts a protein of much higher molecular weight than the protein found in ring canals. The hypothesis that the hts protein is proteolytically released from a precursor will be tested using germline transformation experiments. A full length cDNA will be transformed into flies to test if it produces the short ring canal protein. Truncated cDNA constructs will be made to determine the shortest rescuing fragment. Based on these results, a hts ring canal protein will be produced either in E. coli or baculovirus and tested for binding to actin and kelch. The mutation phenotype of the third gene, cheerio, suggests that it also encodes a ring canal protein. In cheerio egg chambers, ring canals fail to accumulate actin, kelch and hts proteins and the ring canals do not grow to normal diameter. Experiments are proposed to clone and characterize this gene.
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