We will continue our studies on control of plasmid DNA replication in E. coli using P1, a bacteriophage whose prophage is a plasmid, as a model system. From P1 carrying multiple antibiotic resistance determinants, we have isolated mutants that are maintained at multiple copies per cell. We plan to analyze these in detail to determine the molecular mechanisms involved in copy number regulation. We will also isolate mutants that overproduce a factor we believe to be required for P1 plasmid replication and characterize the product they define. We will study the interaction of the different regulatory products by suppressor analysis. We plan to identify and characterize the RNAs and proteins involved in the regulation of replication of this model plasmid and to begin work on an in vitro replication system. In addition, we will complete our work on the generation of a free resistance determinant from P1ApCm.