Replication of ColE2 and ColE3 plasmids In vitro replication dependent on plasmid-coded proteins |
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Authors: | Tateo Itoh and Toshihiro Horii |
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Affiliation: | (1) Laboratory of Genetics, Department of Biology, Faculty of Science, Osaka University, 560 Toyonaka, Osaka, Japan |
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Abstract: | Summary 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. |
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Keywords: | ColE2 and ColE3 In vitro DNA replication Rep protein Replication origin Plasmid specificity |
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