PHAGE T4 HEAD ASSEMBLY AND INITIATION OF INFECTION

噬菌体 T4 头部组装和感染起始

• 批准号: 3480617
• 负责人: LINDSAY W BLACK
• 金额: $ 24.24万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-07-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3480617
• 关键词: DNA virus; affinity labeling; bacterial genetics; bacteriophage T4; conformation; crosslink; microorganism growth; microorganism metabolism; molecular pathology; mutant; virus DNA; virus envelope; virus genetics; virus infection mechanism

The research focuses on the mechanism of viral DNA packaging and on the construction of phage T4 in vitro packaging-derived cloning vectors. Our work is especially directed toward 1) understanding the structures and functions of the multifunctional phage T4 packaging (prohead and terminase) proteins. The 3- dimensional basis for enzymatic (DNA translocating) and structural (prohead form-determining) roles of the major capsid protein gp23*), of a minor processed enzymatic product (gp23**), and of capsid gene mutants will be determined. The assembly of the DNA entrance (also the prohead initiation) vertex as an integral membrane protein will be examined using overexpression vectors; the role of a catalytic gene product and host systems in the membrane insertion will be determined. Specific packaging mutations in both these prohead proteins will be selected following gene-directed mutagenesis. The DNA terminase proteins which interact with the DNA entrance vertex protein in the prohead have been overexpressed and purified-their mechanisms of action in DNA translocation and concatemer cutting will be investigated. lambda and lambda-pBR322 derivative DNAs can be packaged into T4 heads in vitro. cos and/or P1 DNAs packaged into T4 and recircularized with the homologous site-specific recombination systems should allow development of T4-hybrid megacosmid vectors. We have proposed a novel "spiral-fold" model for packaged phage DNA. Morphological and (with phage ) chemical work can definitively establish this model. Packaged kinked or non-B form DNA will be detected, and its interaction with the DNA binding site in the major capsid protein and its minor processed enzymatic product (gp23**) will be probed. We will determine what DNA structures can be packaged in vivo and in vitro; e.g. whether nicks and heteroduplex loops are excluded from packaged DNA in vitro, and whether the terminase proteins act to discriminate against such structures in coupled DNA repair processes.

该研究重点是病毒DNA包装的机制 以及噬菌体T4体外包装衍生的 克隆向量。 我们的工作特别针对1） 了解多功能的结构和功能 噬菌体T4包装（Prohead和末端酶）蛋白。 3- 酶促的尺寸基础（DNA易位）和 主要衣帽 蛋白质GP23*），次要加工酶产物（GP23 **）的蛋白质 将确定衣壳基因突变体。 组装 DNA入口（也是启动启动）顶点作为一个 积分膜蛋白将使用过表达检查 向量；催化基因产物和宿主系统在 将确定膜插入。 特定包装 这两种突发蛋白的突变都将被选择 遵循基因定向的诱变。 DNA末端酶 与DNA入口顶点蛋白相互作用的蛋白质 Prohead已过表达和纯化 DNA易位和征服者中的作用机理 切割将进行调查。 Lambda和Lambda-PBR322 衍生物DNA可以在体外包装到T4头部。 cos 和/或P1 DNA包装到T4中，并用 同源特定地点重组系统应允许 T4杂种巨型媒介的开发。 我们提出了 包装噬菌体DNA的一种新颖的“螺旋折”模型。 形态学和（带有噬菌体）化学工作可以确定 建立此模型。 包装的扭结或非B形式DNA将是 检测到，及其与DNA结合位点的相互作用 主要的衣壳蛋白及其次要加工酶促产品 （GP23 **）将被探测。 我们将确定哪些DNA结构 可以在体内和体外包装；例如 是否划伤和 异源环路在体外被排除在包装的DNA之外，并且 末端酶蛋白是否起作用以区分这种 耦合DNA修复过程中的结构。