M F Law

Bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector.

A novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. The foreign deoxyribonucleic acid (DNA) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. We have constructed a DNA hybrid molecule, BPV69T-rI1, containing the transforming region of bovine papilloma virus DNA and the rat preproinsulin gene I (rI1), and used it to transform susceptible mouse cells. DNA hybridization analysis has demonstrated the presence of multiple unintegrated copies of hybrid DNA molecules, with the bovine papilloma virus 1 DNA segment and the rI1 gene covalently linked in selected transformed cell lines. S1 nuclease analysis revealed the presence of a correctly spliced coding segment of the preproinsulin transcript similar or identical in its electrophoretic mobility to that of messenger ribonucleic acid produced in rat insulinoma cells. Significant levels of a protein immunoreactive with anti-insulin serum were detected by radioimmunoassay in the culture medium of transformed cells. Immunoprecipitation analysis in conjunction with competitive binding to bovine proinsulin established the identity of the protein as that of rat proinsulin.

A stable bovine papillomavirus hybrid plasmid that expresses a dominant selective trait.

We describe a bovine papillomavirus hybrid plasmid containing the neomycin resistance gene from Tn5 inserted into a mammalian cell transcriptional unit. This plasmid is maintained as a stable extrachromosomal element (20 to 100 copies per diploid genome) in mouse cells selected either for the transformed phenotype or for resistance to the aminoglycoside G418. Cells selected for G418 resistance initially display a flat, nontransformed phenotype before exhibiting the gross morphological characteristics of transformation. The delay in the appearance of the transformed phenotype indicated that some intracellular event or series of events subsequent to the establishment of transcriptionally active bovine papillomavirus 1 hybrid plasmid is required for the manifestation of the transformed phenotype.

The colinear alignment of the genomes of papovaviruses JC, BK, and SV40

Abstract The DNAs of polyomaviruses JC, BK, and SV40 were analyzed, under a range of nonstringent hybridization conditions, for nucleotide sequence homology. When the hybridizations were performed at T m - 36°, conditions which would detect regions of homology with as much as 26% base mismatch, extensive homology was detected in all gene regions between the JC genome and both BK and SV40 DNAs. The regions of strongest sequence homology between these genomes formed stable duplexes at T m - 21°, indicating at least 85% base match. By two-dimensional cross-hybridization of restriction endonuclease cleavage fragments of JC to both those of BK and SV40 under nonstringent conditions, it was possible to map the homologous DNA fragments of each pair of viruses with respect to each other. The physical maps of these polyomaviruses could be colinearly aligned using the conserved single Eco RI site in each genome as the 0 map position. The region of strongest homology among these three genomes was localized in a narrow segment (0.76 to 0.85 map unit) in the late region, which in the SV40 genome contains the codons for the N-terminal half of the minor viral protein VP2.