MECHANISMS & REGULATION OF DROSOPHILA P ELEMENT TRANSPST

机制

基本信息

批准号：
6636071
负责人：
DONALD C RIO
金额：
$ 34.23万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-07-01 至 2006-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6636071
关键词：
DNA binding protein DNA repair Drosophilidae Escherichia coli gene mutation genetic techniques laboratory mouse laboratory rabbit molecular cloning molecular genetics protein kinase recombinant proteins transposon /insertion element vaccinia virus

项目摘要

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 how these eukaryotic transposable DNA elements tie into the DNA repair pathways of the fruit fly, Drosophila melanogaster. The experiments in this proposal will provide necessary information for the development of P elements as genetic tools in other organisms. 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. Analyze the biochemical properties of wild type and mutant transposase proteins; further develop biochemical assays for their activities. 3. Analyze the biochemical effects of phosphorylation of the transposase protein; identify the kinases involved in these phosphorylation events. 4. Determine what role GTP binding and/or hydrolysis play in P element transposition. 5. Analyze the role of the Drosophila proteins IRBP, DmKup70, DmKup80 and DmDNA-PKcs (p470) in P element transposition. 6. Facilitate the development of P element-mediated transposition as a genetic tool in other organisms.

该资助提出的研究的总体目标是利用结合生物化学、遗传学和分子生物学的综合方法来阐明高等后生动物中真核转座元件的生化和调控机制。我们的工作重点是了解 P 元件转座的生化机制，以及这些真核转座 DNA 元件如何与果蝇 (Drosophila melanogaster) 的 DNA 修复途径结合起来。本提案中的实验将为P元素作为其他生物体遗传工具的开发提供必要的信息。为了实现我们的总体目标，我们将： 1. 使用体内切除测定分析突变 P 元件转座酶蛋白的表型。 2. 分析野生型和突变型转座酶蛋白的生化特性；进一步开发其活性的生化测定。 3.分析转座酶蛋白磷酸化的生化效应；鉴定参与这些磷酸化事件的激酶。 4. 确定 GTP 结合和/或水解在 P 元件转座中起什么作用。 5. 分析果蝇蛋白 IRBP、DmKup70、DmKup80 和 DmDNA-PKcs (p470) 在 P 元件转座中的作用。 6. 促进 P 元件介导的转座作为其他生物体遗传工具的发展。