THE REP MUTATION .7. CLONING AND ANALYSIS OF THE FUNCTIONAL REP GENE OF ESCHERICHIA-COLI-K-12

The rep gene of E. coli was isolated on a 6-kb (kilobase) PvuII fragment of plasmid pLC44-7 DNA from the Clarke-Carbon collection and cloned into pSC101 (to form pHBH8) and pBR322 (to form pHBH30). The plasmids pHBH8 and pHBH30 complemented all rep mutations tested. The functional rep gene and its promoter were mapped to a 3.2-kb XhoI-BalI fragment on the basis of complementation data with deletion and insertion derivatives of the 2 plasmids; subcloning of various restriction fragments confirmed the assignment. EcoRI, HindIII and HpaI restriction sites resided 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 .apprx. MW 67,000 (the MW of the rep protein). No plasmids were found that complemented that only the A or B classes of rep mutants (which differ in their ability to support the growth of P2 and M13 phages). Evidently, rep-A and rep-B are alleles of the same structural gene.