BACTERIOPHAGE P22 ESSENTIAL RECOMBINATION FUNCTION

噬菌体 P22 基本重组功能

• 批准号: 3127775
• 负责人: ANTHONY R. POTEETE
• 金额: $ 19.16万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3127775
• 关键词: DNA binding protein; Escherichia coli; bacteriophage P22; endonuclease; enzyme mechanism; gel electrophoresis; gene expression; genetic manipulation; genetic mapping; genetic recombination; host organism interaction; molecular cloning; nucleic acid sequence; plasmids; protein structure function; virus genetics

The long-term goal of this research is an understanding of the mechanisms of genetic recombination. Recombination is a process of fundamental importance in the generation of genetic variation, and, in higher organisms, of diversity in the immune response. The system to be studied is the homologous recombination system of bacteriophage P22, which consists of the protein products of four phage genes: Erf, the key component, a strand-exchanging protein; Arf (accessory recombination function), which enhances Erf activity in vivo; and Abc1 and Abc2 (anti-RecBCD), which modulate the activity of the host cell RecBCD protein, diverting it into the phage recombination pathway. The mechanism of homologous recombination carried out by the P22 system will be studied by biochemical and genetic means. In vivo assays of recombination between phages will pe employed to examine the role of double-chain breaks in the Erf system, and to test the activity of Arf in promoting homologous recombination by other systems. Individual components of the recombination system will be purified and characterized biochemically: structural and functional alteration of RecBCD by Abc (ana by the Gam protein of phage lambda), modulation of Erf activity by Arf, and the function of the carboxy-terminal domain of Erf will be examined in particular. Attempts will be made to assemble, from purified components in vitro, systems and sub-systems that faithfully mimic the activity of the P22 homologous recombination in vivo. The actions of these systems on substrate DNA will be studied by gel electrophoretic and electron microscopic techniques, with the aim of establishing the individual steps of the process and characterizing the intermediates formed.

这项研究的长期目标是了解 基因重组机制。 净化是一个过程 对遗传变异的产生至关重要， 以及高等生物免疫反应的多样性。 的 要研究的系统是同源重组系统， 噬菌体P22，它由四种噬菌体的蛋白质产物组成， 噬菌体基因：Erf，关键成分，链交换蛋白; Arf（辅助重组功能），增强Erf活性 体内;和Abc 1和Abc 2（抗RecBCD），其调节 宿主细胞RecBCD蛋白的活性，将其转移到 噬菌体重组途径 P22进行同源重组的机制 系统将通过生物化学和遗传学手段进行研究。 体内 采用重组测定法来检测 双链断裂在ERF系统中的作用，并测试 Arf在促进其他基因同源重组中的活性 系统. 重组系统的各个组成部分将 进行纯化和生物化学表征：结构和 RecBCD的功能改变由Abc（ana由Gam蛋白的 噬菌体λ），Arf对Erf活性的调节，以及 Erf的羧基末端结构域的研究将在 特别的。 将尝试组装，从纯化 体外组件、系统和子系统， 体内P22同源重组的活性。 的 这些体系对底物DNA的作用将通过凝胶电泳进行研究 电泳和电子显微镜技术，目的是 建立过程的各个步骤， 表征所形成的中间体。