Open AccessTransformation and replication in mouse cells of a bovine papillomavirus--pML2 plasmid vector that can be rescued in bacteria.
Author(s) -
Nava Sarver,
Janet C. Byrne,
Peter M. Howley
Publication year - 1982
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.79.23.7147
Subject(s) - plasmid , replicon , biology , transformation (genetics) , pbr322 , dna , bovine papillomavirus , microbiology and biotechnology , in vitro recombination , transformation efficiency , t dna binary system , plasmid preparation , dna replication , molecular cloning , transfer dna , recombinant dna , cloning vector , genetics , vector (molecular biology) , gene , genome , agrobacterium , peptide sequence
The unique ability of bovine papillomavirus (BPV-1) DNA to replicate as a stable, multicopy plasmid in transformed mouse cells has led to its utilization as a eukaryotic cloning vector. One limitation of the system has been the marked reduction in transformation efficiency when BPV-1 DNA or the subgenomic transforming segment of BPV-1 DNA (BPV69T) is covalently linked to pBR322 sequences. A dual host replicon consisting of BPV-1 DNA and pML2d, a deletion variant of pBR322, was constructed and shown to be highly efficient for transformation of mouse cells in vitro. The hybrid molecule replicates as a stable, unintegrated, multicopy plasmid in transformed mouse cells. The resident BPV-1-pML2d plasmid DNA was rescued in bacteria and the recovered plasmids were shown to be identical in structure and to have the same transformation efficiency as the original transforming DNA. In contrast, the transforming efficiency of BPV69T DNA is less than 1/100th that of BPV-1 DNA when the DNA is left covalently linked to pML2d. These observations indicate that, although the nontransforming region of BPV-1 (BPV31NT) DNA is not essential for transformation, it has a facilitative role in the transformation process.