The overall objective of the research proposed in this grant is to elucidate the biochemical and regulatory mechanisms of a eukaryotic transposable element in a higher metazoan using an integrated approach that combines biochemistry, genetics and molecular biology. Our efforts will focus on understanding the biochemical mechanism of P element transposition and the mechanisms by which these eukaryotic DNA rearrangement reactions are regulated in the fruit fly, Drosophila melanogaster. We will also investigate the role of the Drosophila Ku and DNA-dependent protein kinase subunits in DNA repair and P element transposition. These studies will provide necessary information for the development of P elements as genetic tools in other organisms as well as provide insights into metazoan DNA repair mechanisms. In order to accomplish our overall objective, we will: 1. Analyze the phenotypes of mutant P element transposase proteins using an in vivo excision assay. 2. Express and purify wild type and mutant transposase proteins using mammalian vaccinia virus vectors. 3. Analyze the biochemical properties of purified recombinant transposase protein; further develop biochemical assays for its activities. 4. Analyze the properties of the 66kD and KP repressor proteins in vitro using purified recombinant E. coli proteins. 5. Identify other proteins that interact with the 66kD P element repressor protein and explore their role in P cytotype repression. 6. Analyze the role of the Drosophila proteins IRBP(Dmku70), DmKu80 and DmDNA-PK(p450) in P element transposition. 7. Develop P element-mediated transposition as a genetic tool in other organisms.
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