Janet C. Byrne

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.

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

Abstract The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The 32 P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R I restriction endouclease from Escherichia coli ( Eco RI‡ restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.

Characterization of supercoiled oligomeric SV40 DNA molecules in productively infected cells.

Abstract We have characterized oligomeric supercoiled forms of SV40 DNA which appear during the latter stages of productive infection and account for more than 10% of the intracellular viral DNA 70 hr after infection. These DNA molecules form a series of oligomers that are integral multiples (dimers through hexamers) of supercoiled SV40 DNA. We have determined the sedimentation constants of oligomeric DNA under neutral and alkaline conditions and ascertained electrophoretic mobilities of the different supercoiled forms. Nucleic acid hybridization studies as well as cleavage with bacterial restriction endonucleases both indicate that oligmeric supercoiled DNA molecules contain only SV40 DNA sequences. These unintegrated viral DNA forms appear predominantly in the Hirt pellet and sediment in regions of alkaline sucrose gradients characteristic of high molecular weight cellular DNA.

Biologic activity of oligomeric forms of SV40 DNA.

Abstract We have analyzed the biologic activity of supercoiled oligomeric forms of SV40 DNA during productive and transforming infection. These DNA molecules had equal infectivity as assayed by plaque formation; however, the ability of such DNA molecules to transform nonpermissive cells increased linearly with their size. Oligomeric linear and circular forms of SV40 DNA produced by in vitro ligation of linear SV40 monomeric DNA had similarly enhanced transforming activity. Nucleic acid hybridization studies suggest that the amount of viral DNA in rat cells transformed by the variously sized oligomeric DNAs is similar.

INTERFERON INHIBITS BOVINE PAPILLOMAVIRUS TRANSFORMATION OF MOUSE CELLS AND INDUCES REVERSION OF ESTABLISHED TRANSFORMANTS

ABSTRACT Mouse L cell interferon reduced the level of bovine papillomavirus (BPV-1) induced transformation of mouse C127 cells approximately 20-fold. Continued treatment of established BPV-1 transformed mouse cells with mouse L cell interferon lead to a reduction in the copy number of the plasmid viral genomes present in these transformed cells. Flat revertants were selected from two independent transformed lines carried 60 generations in the presence of 200 units/ml of interferon. These flat revertants had the biologic characteristics of their untransformed parent C127 cells which included low cell saturation density and the inability to form colonies in soft agar. The revertants could be retransformed by BPV and each of 8 different revertant lines examined was found to be “cured” of their viral DNA sequences as determined by Southern blot hybridization.