Activity in Vitro of Three Replication Origins of the Antibiotic Resistance Plasmid RSF1040

Replicating molecules of plasmid RSF1040, a deletion mutant of R6K, were synthesized in vitro and analyzed by electron microscopy. Initiation of replication occurs at three unique sites, ori alpha, ori beta, and ori gamma, within a 3900-base pair segment of the R6K genome. These sites are indistinguishable from the origins that are active in vivo. Frequencies of initiation at these three origins, however, are different from those observed in vivo. Replication proceeds unidirectionally in either direction from ori beta and ori gamma and in one direction from ori alpha. The replication terminus of the R6K genome is inactive in the in vitro system.