The rep mutation VII. Cloning and analysis of the functional rep gene of Escherichia coli K-12

Abstract The rep gene of Escherichia coli was isolated on a 6-kb Pvu II fragment of plasmid pLC44-7 DNA from the Clarke-Carbon collection and cloned into pSC101 (to form pHBH8) and pBR322 (to form pHBH3O). The plasmids pHBH8 and pHBH30 were found to complement all rep mutations tested. The functional rep gene and its promoter were mapped to a 3.2-kb Xho I- Ball fragment on the basis of complementation data with deletion and insertion derivatives of the two plasmids; subcloning of various restriction fragments confirmed the assignment. Eco RI, Hin dIII, and Hpa I restriction sites were found to reside within that region of the DNA required for expression of the rep function. A coupled in vitro transcription-translation system was used to show that only those plasmids containing a functional rep gene encoded a protein of about M r 67 000 (the M r of the rep protein). No plasmids were found that complemented only the A or B classes of rep mutants (which differ in their ability to support the growth of P2 and M13 phages). This result suggests that rep -A and rep -B are alleles of the same structural gene.