The long term objective of the work described in this application is to understand the genetic mechanisms that regulate the levels of the DNA replication enzymes of T4 phage in infected E. coli hosts. The focus of the proposed studies is the T4DNA polymerase (gene 43) and its accessory proteins, the products of phage genes 45,44, and 62 (proteins that stimulate processivity of the polymerase on its DNA template). These genes map as a cluster on the T4 chromosome and one of our specific goals will be to identify the transcriptional control signals for this cluster. We have developed a large library of recombinant plasmids that harbor defined segments of this T4 genetic segment. We will be using these plasmids to generate and isolate mutants of specific portions of the structural genes for the replication proteins and of subsegments that we suspect play regulatory functions. A major effort will be directed at gene 43, the DNA polymerase gene, with the aim of identifying intragenic segments that determine specific biological functions of this multifunctional protein. By similar approaches, we will generate mutations that affect regions of transcriptional/translational coupling in the gene 45-44-62 cluster and other mutations that affect interactions of the proteins in vivo. Mutations with interesting effects will either be cloned and sequenced at the DNA level or sequenced at the protein level. We will also use S1 mapping techniques to identify regions of initiation, termination, and processing of the transcriptional products encoded by this cluster of replication genes. These analyses will include RNA and DNA blotting techniques and DNA sequence determinations, and will be carried out on wild-type sequences as well as on cis-dominant mutant sequences that we have isolated and shown to affect the levels of expression of genes in the cluster.
