Cloning and mapping of the replication origin of Escherichia coli.

Abstract

The replication origin of E. coli was cloned on a nonreplicating DNA fragment for ampicillin resistance. This recombinant DNA, named pSY211, replicates depending on the presence of the replication origin and can be recovered as a closed circular plasmid DNA of 10.7 megadaltons (Mdal). A restriction map was constructed. EcoRI cleaves pSY211 into 2 fragments: one is the ampicillin fragment of 4.5 Mdal and the other is a chromosomal fragment of 6 Mdal and contains the origin. The 6 Mdal EcoRI fragment has 4 BamHI sites, 3 HindIII sites and 1 Xho I site. A mutant of pSY211 was isolated which lacks 2 BamHI fragments of the chromosomal fragment. In recA hosts, pSY211 is lost at a high frequency. In recA+ hosts, pSY211 is integrated into the chromosome due to nucleotide sequence homology between pSY211 and the replication origin of the E. coli chromosome. The integration site was mapped. The replication origin apparently located at a site between uncA and rbsK, at about 83 min on the genetic map of E. coli.