The genomic instability associated with integrated simian virus 40 DNA is dependent on the origin of replication and early control region

D J Hunter, E G Gurney
1994 Journal of Virology  
DNA rearrangements in the form of deletions and duplications are found within and near integrated simian virus 40 (SV40) DNA in nonpermissive cell lines. We have found that rearrangements also occur frequently with integrated pSV2neo plasmid DNA. pSV2neo contains the entire SV40 control region, including the origin of replication, both promoters, and the enhancer sequences. Linearized plasmid DNA was electroporated into Xl, an SV40-transformed mouse cell line that expresses SV40 large T antigen
more » ... (T Ag) and shows very frequent rearrangements at the SV40 locus, and into LMtk-, a spontaneously transformed mouse cell line that contains no SV40 DNA. Stability was analyzed by subcloning G-418-resistant clones and examining specific DNA fragments for alterations in size. Five independent Xl clones containing pSV2neo DNA were unstable at both the neo locus and the T Ag locus. By contrast, four Xl clones containing mutants of pSV2neo with small deletions in the SV40 core origin and three Xl clones containing a different neo plasmid lacking SV40 sequences were stable at the neo locus, although they were still unstable at the T Ag locus. Surprisingly, five independent LMtkclones containing pSV2neo DNA were unstable at the neo locus. LMtk-clones containing origin deletion mutants were more stable but were not as stable as the Xl clones containing the same plasmid DNA. We conclude that the SV40 origin of replication and early control region are sufficient viral components for the genomic instability at sites of SV40 integration and that SV40 T Ag is not required. . untranslated TnS neo sequences, which are not found in pLTRneo, are present in pSV2neo, accounting for the difference in size of the neo region found in the two plasmids. The replication activity of the SV40 origin in pSV2neo was inhibited by making small deletions in T Ag binding domain II, 787 on May 9, 2020 by guest
doi:10.1128/jvi.68.2.787-796.1994 fatcat:ojpbbscb5jfipfczmdruv3t7zi