MECHANISMS & REGULATION OF DROSOPHILA P ELEMENT TRANSPST

机制

• 批准号: 2734742
• 负责人: DONALD C RIO
• 金额: $ 29.41万
• 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 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734742
• 关键词: DNA binding protein; Drosophilidae; Escherichia coli; gene mutation; genetic techniques; immunoaffinity chromatography; laboratory mouse; laboratory rabbit; molecular cloning; molecular genetics; protein kinase; recombinant proteins; transposon /insertion element; vaccinia virus

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.

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 将重点了解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。我们还将研究果蝇 Ku 和 DNA 修复和 P 元件中的 DNA 依赖性蛋白激酶亚基 换位。这些研究将为 P元素作为其他生物体遗传工具的发展以及 提供对后生动物 DNA 修复机制的见解。为了 实现我们的总体目标，我们将： 1. 使用分析突变 P 元件转座酶蛋白的表型 体内切除测定。 2. 使用表达和纯化野生型和突变型转座酶蛋白 哺乳动物痘苗病毒载体。 3. 分析纯化的重组转座酶的生化特性 蛋白质;进一步开发其活性的生化测定。 4. 体外分析66kD和KP阻遏蛋白的特性 使用纯化的重组大肠杆菌蛋白。 5. 识别与 66kD P 元件相互作用的其他蛋白质 阻遏蛋白并探讨其在 P 细胞型抑制中的作用。 6. 分析果蝇蛋白 IRBP(Dmku70)、DmKu80 和 P 元件转座中的 DmDNA-PK(p450)。 7. 开发P元件介导的转座作为其他领域的遗传工具 有机体。