BACTERIAL FUNCTION IN PHAGE DEVELOPMENT

拟议研究的总体目标是：(A)识别、提纯和 广泛鉴定必需的大肠杆菌蛋白 用于噬菌体Lambda的裂解生长，(B)鉴定和纯化 与它们相互作用的噬菌体基因产物，以及(C)阐明 宿主与噬菌体蛋白相互作用的确切机制。 各种大肠杆菌功能，最初是通过分离鉴定的 阻止病毒繁殖能力的突变体的数量已经显示出下降 分为三大类：Lambda DNA复制所需的Lambda RNA转录与噬菌体形态发生。基因和基因的结合 将使用生化分析来阐明确切的模式 所有这些功能的作用。基因实验将包括：(A) 具有明显表型的突变的分离和鉴定 (CS，TS或胡说八道)在已经识别的基因中以及在任何 有待发现，以及(B)分离和表征 这些不同基因中的基因外抑制因子。生化方法 将包括：(A)野生型的克隆、过量生产和纯化 以及突变细菌和噬菌体基因产物，(B)演示 纯化基因产物之间潜在的蛋白质-蛋白质相互作用 使用各种方法，包括共编辑、交叉链接 和亲和层析，以及(C)体外重组的设计 DNA复制、RNA转录和形态发生的系统 能够特异性地检测这些基因产物的活性。

The overall object of the proposed research is (a) to identify, purify and extensively characterize the Escherichia coli proteins that are necessary for the lytic growth of bacteriophage Lambda, (b) to identify and purify the phage gene products that interact with them, and (c) to elucidate the exact mechanism of the interactions between the host and phage proteins. The various E. coli functions, originally identified through the isolation of mutants that block the ability of to propagate, have been shown to fall into three major groups: those required for Lambda DNA replication, Lambda RNA transcription and phage morphogenesis. A combination of genetic and biochemical analysis will be employed in order to elucidate the exact mode of action of all these functions. The genetic experiments will include (a) the isolation and characterization of mutations with discernible phenotypes (Cs, Ts or Nonsense) in the genes already identified as well as in any remaining to be discovered, and (b) the isolation and characterization of extragenic suppressors among these various genes. The biochemical approach will include (a) the cloning, overproduction and purification of wild type as well as mutant bacterial and phage gene products, (b) the demonstration of potential protein-protein interactions among the purified gene products using a variety of approaches that include cosedimentation, cross-linking and affinity chromatography, and (c) the design of in vitro reconstituted systems for DNA replication, RNA transcription and morphogenesis that are capable of specifically detecting the activities of these gene products.