CONTROLLING THE ACTIVITY OF A DROSOPHILA TGF-B HOMOLOG

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Dr. Michael B. O'Connor is currently an assistant professor in the Department of Molecular Biology and Biochemistry at the University of California - Irvine. The candidates long term research interest is to establish how, at the molecular level, positional information is specified and interpreted during development. The fruit fly Drosophila Melanogaster is to be used as the experimental system. The immediate aim is to investigate the roles played by the tolloid (tld), screw (scw), and tld related (tld-r) gene products (TLD, SCW, TLD-R) in the process that directs dorsal/ventral (D/V) pattern formation in the developing 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 or organisms and appear to control a wide range of biological processes including cell growth and differentiation. To clarify the nature of tld-dpp and scw-dpp interactions, the structure, expression, and biochemical activities of these gene products will be examined. For tld a baculovirus expression system will be used to over-express the protein. DNA sequence analysis has revealed that tld is 45% identical to human bone morphogenetic protein-1 (BMP-1), a putative metalloprotease. Purified TLD protein will be tested for proteolytic activity using radiolabeled casein and coumarin derivatized peptides as substrates. To search for TLD-DPP complexes, antibodies will be raised against different parts of TLD and used in co-immunoprecipitation experiments. The ability of TLD to cleave DPP will be monitored by SDS-PAGE electrophoresis of co-expressed proteins. To correlate specific tld domains with function, the lesions associated with 11 different tld mutations, which cause abnormal interactions with dpp, will be identified by DNA sequencing. this work will necessitate that Dr. O'Connor acquire several new experimental skills including the ability to purify proteins, make antibodies, and develop biochemical assays for protein function. The department has several established investigators including Dr. Krishna Tewari, Dr. Agnes Henschen, Dr. Charles Glabe and Dr. Barbara Burgess, whose research utilizes various techniques in protein chemistry on a daily basis. Under their tutelage, Dr. O'Connor will develop the necessary skills to competently address problems in protein structure and function. In the long term, the ability to apply a full complement of genetic, molecular and biochemical techniques to experimental problems will significantly enhance Dr. O'Connor's research capabilities. The department will relieve the candidate from teaching and committee responsibilities during the award period so that he can devote his full efforts towards establishing himself as an independent investigator.