BACTERIOPHAGE P22 ESSENTIAL RECOMBINATION FUNCTION

噬菌体 P22 基本重组功能

• 批准号: 3127777
• 负责人: ANTHONY R. POTEETE
• 金额: $ 13.33万
• 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 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3127777
• 关键词: Escherichia coli; bacteriophage P22; endonuclease; gel electrophoresis; gene expression; genetic manipulation; genetic mapping; genetic recombination; molecular cloning; nucleic acid sequence; operon; plasmids; virus genetics

The mechanism of generalized genetic recombination carried out by the bacteriophage P22 essential recombination function (erf gene) will be studied by biochemical and genetic means. The interaction of purified Erf protein with DNA will be examined by gel electrophoretic techniques and enzymatic probes of the state of the DNA. Fragments of the Erf protein, generated by proteolysis in vitro and by chain terminating mutations in vivo, will be purified. Their structural and DNA binding activities will be examined in parallel with those of intact Erf. The abc gene, a newly discovered P22 recombination-related gene, will be sequenced, and expressed at high levels by genetic engineering techniques. The Abc protein will be purified and its interaction with DNA, Erf protein, and possibly, host cell recombination functions will be examined. Similar studies of Y, another new recombination-related gene of P22, will be carried out. erf-dependent supercoiling of linear, terminally repetitious P22 chromosomes will be examined in order to determine which phage and host cell components are necessary for this process. Attempts will be made to assay this process in vitro, in order to permit reconstruction of the erf recombination system from purified components. Such a reconstruction would greatly facilitate studies of mechanism. 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.

广义遗传重组的机制是由 噬菌体P22的基本重组功能(ERF基因)将 通过生物化学和遗传学手段进行研究。纯化Erf分子间的相互作用 含有DNA的蛋白质将通过凝胶电泳技术和 DNA状态的酶探针。Erf蛋白的片段， 由体外蛋白降解和由链终止突变产生 活体，将被提纯。它们的结构和DNA结合活性将 与完整的ERF进行平行检查。ABC基因，一种新的 发现P22重组相关基因，将进行测序，并进行表达 通过基因工程技术达到很高的水平。ABC蛋白将是 纯化及其与DNA、Erf蛋白、可能与宿主细胞的相互作用 我们将研究重组功能。对Y的类似研究，另一种 将进行P22新的重组相关基因的研究。依赖ERF 线形、末端重复的P22染色体的超级卷曲将是 检查以确定哪些噬菌体和宿主细胞成分是 这是这一过程所必需的。将尝试在以下方面分析这一过程 体外，以允许重建ERF重组系统 从提纯的成分中提取。这样的重建将极大地促进 对机理的研究。 这项研究的长期目标是了解其作用机制 基因重组。重组是一个根本性的过程 在遗传变异的产生中的重要性，在高等生物体中， 免疫反应的多样性。