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Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K

1978; Cell Press; Volume: 15; Issue: 4 Linguagem: Inglês

10.1016/0092-8674(78)90046-6

1097-4172

Roberto Kolter, Manabu Inuzuka, Donald R. Helinski,

DNA and Nucleic Acid Chemistry

A non-self-replicating segment (1370 base pairs) of plasmid R6K was cloned in E. coli and shown to trans-complement temperature-sensitive replication mutants of this plasmid. This segment contains the gene which codes for a protein required for initiation of replication of the plasmid, and was used as a helper in a functional assay for an origin of replication in R6K derivatives. A 420 bp fragment, derived from R6K DNA, was shown to carry a functional origin since it was capable of replicating as a plasmid in E. coli cells carrying the helper segment either on the host chromosome or on a plasmid Col E1 derivative. The copy number of the origin fragment in cells carrying the helper segment on the chromosome is essentially the same as the copy number of R6K. A model for the positive regulation of plasmid R6K replication is presented.