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.

研究的重点是病毒DNA包装的机制 并对噬菌体T4体外包装衍生的构建进行了研究 克隆载体。 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（前头部和末端酶）蛋白。 第三个- 酶促（DNA易位）和 主要衣壳的结构（前头部形状决定）作用 蛋白质gp 23 *），少量加工的酶产物（gp 23 **）， 和衣壳基因突变体的数量将被确定。 大会 DNA入口（也是头端起始）顶点作为 将使用过表达来检查整合膜蛋白 载体;催化基因产物和宿主系统在 将确定膜插入。 特定包装 将选择这两种前头蛋白中的突变 在基因定向诱变之后。 DNA末端酶 与DNA入口顶点蛋白相互作用的蛋白质， 该探针已经过表达和纯化--它们 DNA易位和多联体的作用机制 切割将被调查。 λ和λ-pBR 322 衍生DNA可以在体外包装到T4头部中。 cos 和/或P1 DNA包装到T4中，并与 同源位点特异性重组系统应该允许 T4-杂合巨噬细胞载体的开发。 我们提出 一种新的噬菌体DNA螺旋折叠模型。 形态学和（与噬菌体）化学工作可以明确地 建立这个模型。 包装的扭结或非B型DNA将 检测到，其与DNA结合位点的相互作用， 主要衣壳蛋白及其次要加工酶产物 (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， 异源双链体环在体外从包装的DNA中排除，并且 终止酶蛋白是否起区别这种 在DNA修复过程中的作用。