Regulated replication of an episomal simian virus 40 origin plasmid in COS7 cells.
AUTOR(ES)
Chittenden, T
RESUMO
The replication of a simian virus 40 (SV40) origin-containing plasmid, pSLneo, stably transfected COS7 cells has been studied. pSLneo contains the SV40 origin of replication and encodes the positive selectable marker for G418 resistance. In transient replication assays, pSLneo replicates to a high copy number in COS7 cells. Uncontrolled SV40 plasmid replication has been reported to be lethal to such transfected cells. Thus, it was anticipated that extensive plasmid replication would preclude isolation of permanent cell lines containing pSLneo. However, significant number of G418-resistant colonies arose after transfection of COS7 cells with pSLneo. Cell lines established from these drug-resistant colonies contained between 100 and 1,000 extrachromosomal pSLneo copies per cell. Episomal plasmid DNA in pSLneo/COS7 lines was stably maintained after 2 months of continuous culture in selective medium. Bromodeoxyuridine labeling and density shift experiments demonstrated that replication of pSLneo closely paralleled that of cellular DNA. On average, plasmid DNA did not replicate more than once during a single cell generation period. Regulation of pSLneo replication appeared to be negatively controlled by a cis-acting mechanism. Endogenous copies of episomal pSLneo remained at a stable low copy number during the simultaneous, high-level replication of a newly transfected plasmid encoding SV40 large T antigen in the same cells. These results indicate that regulated replication of an SV40 origin plasmid can be acquired in a cell and does not require the presence of additional genetic elements. The molecular mechanism by which cells enforce this regulation on extrachromosomal SV40 plasmids remains to be defined.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=250258Documentos Relacionados
- The adeno-associated virus rep gene inhibits replication of an adeno-associated virus/simian virus 40 hybrid genome in cos-7 cells.
- Archetype JC Virus Efficiently Replicates in COS-7 Cells, Simian Cells Constitutively Expressing Simian Virus 40 T Antigen
- Replication of a plasmid bearing a human Alu-family repeat in monkey COS-7 cells.
- Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells.
- Origin of replication in episomal bovine papilloma virus type 1 DNA isolated from transformed cells.