Replication of pSV2-gpt in COS-1 cells: stability of plasmid DNA in the presence and absence of biochemical selection.
We have previously demonstrated that COS-1 cell lines transformed by pSV2-gpt and maintained under biochemical selection replicate multiple copies of extrachromosomal plasmid DNA (1). We have now examined the replication and stability of this DNA in a representative cell line. In situ hybridization analyses revealed that intense replication of pSV2-gpt occurs in only a small subpopulation of cells and results from bursts of plasmid replication that occur periodically and spontaneously in the cell population. This suggests that COS-1 cells are only semipermissive for pSV2-gpt replication. No correlation was observed between levels of pSV2-gpt replication and the presence or absence of biochemical selection for the Gpt marker. However, growth of cells under nonselective conditions led to a rapid and progressive loss of pSV2-gpt DNA. This loss correlated with segregation of Gpt- revertants that lacked detectable plasmid sequences. Hence, maintenance of pSV2-gpt in the cell line was dependent on continuous biochemical selection. Stable replication of pSV2-gpt could be observed as late as four months after transfection, suggesting that this system might be useful for propagation of cloned DNA in COS-1 cells for extended periods of time. However, by nine months, extensive rearrangements of pSV2-gpt sequences were detected, indicating ultimate instability of the plasmid in the host cells.
Tsui
L C
LC
Breitman
M L
ML
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003470:Culture Media; D004261:DNA Replication; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics*; D000882:Haplorhini; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D003470:Culture Media; D004274:DNA, Recombinant; D004279:DNA, Viral