MECHANISMS & REGULATION OF DROSOPHILA P ELMNT TRANSPSTN

机制

• 批准号: 3308326
• 负责人: DONALD C RIO
• 金额: $ 23.83万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3308326
• 关键词: DNA; DNA binding protein; Drosophilidae; Escherichia coli; developmental genetics; enzyme mechanism; eukaryote; gene rearrangement; guanine nucleotide binding protein; immunoaffinity chromatography; laboratory mouse; laboratory rabbit; protein sequence; tissue /cell culture; transcription factor; transfection; transposon /insertion element

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 combined biochemistry, genetics and molecular biology. Our efforts will be focused 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. DNA rearrangements play a variety of roles in the normal development and genomic flux of eukaryotic cells. These studies will also 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.Express the Drosophila P element 87kD transposase and 66kD repressor proteins and shorter peptides in mammalian or E. coli host-vector systems. 2.Develop rapid immunoaffinity or affinity chromatography methods to facilitate the biochemical analysis of the P element proteins. 3.Analyze the biochemical activities and properties of the P element proteins as they relate to their biological role in transposition and its regulation. 4.Define the domain structures of transposase and the 66kD proteins; determine the location of the DNA and nucleotide ligand binding sites. 5.Analyze the biochemical and genetic properties of the DNA binding region(s), the nucleotide binding domain and leucine zipper regions of the P element proteins. 6.Develop an in vitro assay for P element transposition and analyze the mechanism and regulation of transposition. 7.Identify other proteins that interact with the P element proteins using genetic and biochemical methods and explore their role in transposition. 8.Develop P element-mediated transposition as a genetic tool in other organisms.

这笔赠款中提出的研究的总体目标是 阐明真核生物的生化和调节机制 使用综合方法在高等后生动物中转座元件 结合了生物化学、遗传学和分子生物学。 我们的努力将是 专注于了解P元素的生化机制 转座以及这些真核DNA的转座机制 重排反应在果蝇、果蝇中受到调节 黑腹果蝇。 DNA重排在正常情况下发挥着多种作用 真核细胞的发育和基因组通量。 这些研究也将 为P元素作为遗传物质的发育提供必要的信息 其他生物体中的工具。 为了实现我们的总体目标，我们 将要： 1.表达果蝇P元件87kD转座酶和66kD阻遏蛋白 哺乳动物或大肠杆菌宿主载体系统中的蛋白质和较短的肽。 2.开发快速免疫亲和或亲和层析方法 促进P元素蛋白的生化分析。 3.分析P元素的生化活性和性质 蛋白质，因为它们与转座中的生物学作用及其相关 规定。 4.定义转座酶和66kD蛋白的结构域； 确定 DNA 和核苷酸配体结合位点的位置。 5.分析DNA结合的生化和遗传特性 区域、核苷酸结合结构域和亮氨酸拉链区域 P元素蛋白质。 6.开发P元件转座的体外测定并分析 转位机制和调控。 7.使用以下方法识别与 P 元素蛋白相互作用的其他蛋白 遗传和生化方法并探讨它们在转座中的作用。 8.开发P元件介导的转座作为其他遗传工具 有机体。