Expression of Cloned Vaccinia Virus DNA Sequences Introduced into Animal Cells

Abstract

SUMMARYIndividual cloned HindlII fragments of vaccinia virus DNA were introduced into cells by DNA-mediated gene transfer. The presence of individual fragments in the different transformants was confirmed by Southern blot hybridization analysis. Several of the transformants were found to express viral sequences at various levels. The sizes of the transcripts containing vaccinia virus sequences were highly heterogeneous, with no discrete species of RNA. Positive clones contained vaccinia virus sequences in both the poly(A) ÷ and poly(A)-RNA fractions, although the prevalence of these sequences was variable in the two fractions. The S1 nuclease map of the 5' end of the transcripts from transformants containing the HindlII-J fragment revealed a unique 5' end, similar to RNA from virus-infected cells• In contrast, analysis of the 3' end of RNAs from these transformants showed a high degree of heterogeneity, which might explain the heterogeneity found in Northern blot patterns. In this report, it is shown for the first time that in cells transformed with vaccinia virus DNA there is proper initiation for, at least, the viral thymidine kinase gene.

Generation of a dominant 8-MDa deletion at the left terminus of vaccinia virus DNA.

“…Conditions for extraction of high molecular weight DNA from whole cell lysates, isolation of WR-DNA from purified virus, digestion of DNA with restriction endonucleases, gel electrophoresis, Southern blotting, and hybridization with specific nick-translated DNA probes have been previously described (5,6). Virus from persistently infected cells was plaque purified by selection of well-isolated plaques on monolayers of BSC-40 cells.…”