The overall objective of the research proposed in this grant is to elucidate mechanisms used to regulate gene expression during development of a higher metazoan using an integrated approach combining biochemistry, molecular biology, and genetics. The major portion of our efforts will be focused on understanding the biochemical mechanisms governing transposition and expression of P transposable elements in Drosophila melanogaster as a model for genetic and tissue-specific mechanisms controlling gene expression. It is anticipated that the results obtained from these studies will be useful in understanding mechanisms governing gene expression during human as well as Drosophila development. In order to accomplish our overall objectives we will: (1) isolate and characterize a series of monoclonal antibodies directed against different determinants on the two P element proteins. (2) Express the P element proteins to high levels using expression host-vector systems to facilitate their biochemical isolation. (3) Purify and characterize the P element proteins using conventional biochemical techniques. (4) Analyze the biochemical activities and structure-function relationships of the P element proteins and relate these activities to their biological role in P element transposition and its regulation. (5) Define the DNA sequences necessary to initiate P element transcription by in vitro mutagenesis using both in vitro and in vivo assays. (6) Develop in vitro assays to identify transcription factors that recognize the P element promoter; study the possible regulatory interactions between these factors and the P element proteins. (7) Study the regulation of P element transposition and gene expression in vivo using classical genetics in combination with gene fusions and P element-mediated transformation. (8) Develop an in vitro system to study the processing of Drosophila mRNA in somatic cells; attempt to develop assays to study P element germline-specific splicing in vitro. (9) Attempt to develop an in vitro system to study P element transposition and its regulation.. (10) Investigate the possible use of P elements as transformation vectors in other organisms.

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Whitehead Institute for Biomedical Research
United States
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