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.
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