BACTERIAL FUNCTION IN PHAGE DEVELOPMENT

拟议研究的总体目标是（a）识别、纯化和 广泛表征大肠杆菌蛋白质， 用于噬菌体λ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 宿主和噬菌体蛋白之间相互作用的确切机制。 各种E.大肠杆菌的功能，最初通过分离 的突变体，阻止繁殖的能力，已被证明是下降 分为三大类：λ DNA复制所需的，λ RNA转录和噬菌体形态发生。 基因和 将采用生化分析以阐明确切的模式 所有这些功能的作用。 遗传实验将包括（a） 具有可辨别表型的突变的分离和表征 (Cs，Ts或无意义）在已经鉴定的基因中以及在任何 （B）分离和表征 这些基因中的基因外抑制因子。 生物化学方法 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）所述证明 纯化的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，包括共沉淀，交联， 和亲和层析，和（c）体外重构的设计 DNA复制、RNA转录和形态发生系统， 能够特异性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.