title: Replication of ColE2 and ColE3 plasmids: in vitro replication dependent on plasmid-coded proteins.
abstract: We developed an in vitro replication system for ColE2 and ColE3 plasmids using cell extracts prepared from bacteria with or without these plasmids. DNA synthesis depended on host DNA polymerase I and was sensitive to rifampicin and chloramphenicol. Preincubation of the extracts with plasmid DNA, however, allowed replication of template DNA added subsequently in a plasmid-specific manner in the presence of rifampicin and chloramphenicol. The plasmid-specified trans-acting factor(s) was detected in cell extracts from bacteria carrying a recombinant plasmid with the region of ColE2 or ColE3 encoding the Rep protein. The plasmid-specified factor(s) consisted at least in part of protein, probably the Rep protein. In vitro replication started within a region of ColE2 or ColE3 containing the smallest cis-acting segment essential for in vivo replication and proceeded in a fixed direction.
authors.affiliation: Department of Biology, Faculty of Science, Osaka University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Itoh
authors.forename: T
authors.initials: T
authors.lastname: Horii
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D003086:Bacteriocin Plasmids; D002701:Chloramphenicol / Q000494:pharmacology; D004256:DNA Polymerase I / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004269:DNA, Bacterial / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000378:metabolism; D010957:Plasmids; D011402:Pronase / Q000378:metabolism; D015183:Restriction Mapping; D012293:Rifampin / Q000494:pharmacology; D013698:Templates, Genetic
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D002701:Chloramphenicol; D004256:DNA Polymerase I; D008836:Micrococcal Nuclease; D011402:Pronase; D012293:Rifampin