Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences

Abstract

SV40 DNA replication requires interactions between proteins provided by the host cell, the virus-coded A protein and a viral sequence of ∼80 base pairs (bp), which serves as an origin of DNA replication^1,2. The apparent simplicity of this virus has provided a useful means of producing^3,4 SV40 virions that carry specific eukaryotic genes which can infect cells in culture and replicate to a high copy number. One disadvantage to this approach is that the viral DNA sequences necessary for replication and the gene of interest must be precisely tailored to package efficiently into SV40 virus particles. In an attempt to circumvent this limitation, we have studied the replication of SV40–pBR322 plasmids after transfection into simian cells with the ultimate goal of using the viral replicon as a means of amplifying the expression of cloned fragments inserted in these vectors. Such a dual-host replicon also offers the possibility of rescuing and subsequently cloning genes which are properly expressed and stably integrated into the cell genome⁵. We and others (refs 5–8 and Schaffner, personal communication), have noted that such plasmids propagated in Escherichia coli replicate poorly if at all after transfection of simian cells. Further-more, recombinant plasmids isolated from the transfected simian cells subsequently show a reduced ability to retransform E. coli⁵. We now demonstrate that both these phenomena are related to the presence of a cis-acting DNA sequence in the bacterial vector, pBR322. SV40–pBR322 plasmids lacking this sequence replicated as effectively as authentic SV40 DNA in simian cells and were re-established as plasmids in E. coli as effectively as plasmid DNAs extracted from E. coli.