Open AccessConstruction of a novel plasmid-phage hybrid: use of the hybrid to demonstrate ColE1 DNA replication in vivo in the absence of a ColE1-specified protein.
Author(s) -
Michael L. Kahn,
Donald R. Helinski
Publication year - 1978
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.75.5.2200
Subject(s) - cole1 , replicon , plasmid , bacteriophage , biology , dna replication , plasmid preparation , microbiology and biotechnology , origin of replication , t dna binary system , dna polymerase i , dna , escherichia coli , genetics , rna , recombinant dna , gene , pbr322 , reverse transcriptase , vector (molecular biology)
A hybrid bacteriophage, P420, was constructed in vitro; it contains part of bacteriophage P4 and a 3.6-kilobase derivative of plasmid ColE1. In Escherichia coli, the plasmid-phage hybrid can exist as a stable plasmid or can be packaged into infective bacteriophage particles. Replication of P420, directed by the ColE1 replicon, was found to occur after P420 phage infection of E. coli cells that had been incubated in the presence of chloramphenicol. Replication began without a lag period and resulted in the synthesis of covalently closed circles of P420 DNA. Like ColE1 DNA replication but unlike that of P4, replication was dependent on DNA polymerase I and was sensitive to rifampicin. The presence of a resident ColE1 plasmid in the infected cells resulted in an inhibition of the replication of the incoming P420 DNA. These results indicate that ColE1 does not require a plasmid-coded protein to replicate its DNA in vivo and demonstrate the utility of P4 bacteriophage for coupling bacteriophage properties to a plasmid replicon.