The purpose of this research is to understand at the molecular level, how positional information is specified and interpreted during development. The roles played by the tolloid (tld) and screw (scw) gene products (TLD), SCW) in the process that directs dorsal/ventral (D/V) pattern formation in the D. melanogaster embryo will be investigated. Mutations in tld and scw cause a dorsal shift in the blastoderm fate map resulting in a partial ventralization of the embryo. Genetic analysis suggests that both tld and scw act to boost the activity of a third D/V patterning gene decapentaplegic (dpp), whose product (DPP) is a member of the TGF-beta superfamily of growth factors. The TGF-beta family of secretory polypeptides mediate intercellular communication in a multitude of organisms and appear to control a wide range of biological processes including cell growth and differentiation.
The specific aims of this proposal are: 1) to biochemically characterize the TLD protein. 2) to examine the nature of the TLD-DPP interaction, and 3) to molecularly characterize the scw locus. DNA sequence analysis has revealed that tld is 45% identical to human bone morphogenetic protein-1 (BMP-1), a putative metalloprotease. A baculovirus expression system will be use to produce large quantities of TLD protein. Purified TLD will be assayed for protease activity, metal ion requirements, and self association properties. Site-directed mutations and available tld alleles will be assayed for effects on these properties. To search for TLD-DPP complexes, antibodies will be raised against different parts of TLD and used to coprecipitate TLD and associated proteins from perivitelline fluid and from tissue culture cells expressing both TLD and DPP. The ability of TLD to process DPP will be monitored by SDS-PAGE electrophoresis of co-expressed proteins. For scw the transcription unit will be identified by P element mediated transformation and by injection of screw mRNA into mutant embryos. A representative cDNA will be sequenced and analyzed for the presence of homologies which may give a clue to the biological function of the scw gene product. Antibodies will be generated and used to examine the tissue distribution and subcellular localization of the gene product. These studies will contribute to our understanding of how spatial information is specified during development and may provide a paradigm for how the activity of TGF-beta family members is regulated in other organisms and processes.
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