A major aim of this work is to produce alterations in known regulatory regions of the genome of the small phage, PHCX174, and then to determine the change in DNA sequence responsible for the altered gene expression. In this way essential coding elements of the regulatory regions can be identified for this phage, whose DNA sequence has been completely determined. Mutants will be isolated by their inability to grow on a cell strain carrying a defective sigma subunit of RNA polymerase. DNA sequence analysis will be carried out by Sanger's method. Another aim is to identify host factors involved in recognition of regulatory signal on PHCX174 DNA and mRNA. Phage-protein ratios will be determined after infection of bacterial strains defective in various transcriptional and translational host factors. These strains include rho mutants, ribosomal protein S12 mutants (strA), 16S RNA mutants (ksgA) and an S1 mutant. An altered phage protein ratio is an indication that the mutant host factor has recognition properties. I plan to see if there are similarities in the DNA replication mechanisms for single-stranded DNA synthesis of PHCX174 and for conjugational replication of the small plasmid Col El. The dependence of Col El conjugational replication on the rep gene will be studied. The ability of the DNA shut-off function of PHCX174 to shut off Col El replication will be tested.