REPLICATION OF CIRCULAR AND LINEAR SV40-BASED PLASMIDS IN MONKEY CELLS

Three plasmids were derived from a common SV40-based parent. A circula r plasmid (pYACneoC) contained the SV40 ori and two sets of 50 bp of h uman telomeric sequences. By differential enzyme digestion, two linear plasmids were generated from the circular form, one (pYACneoL) termin ating with, and the other (pYACneoN) free of telomeric sequences. The replicative features of the circular and of both linear plasmids were assayed by transfecting COS-7 cells. At various times after transfecti on, the low-molecular-weight DNA was extracted, and the fraction of mo lecules that had replicated was determined by Dpnl digestion. We demon strate that about half of the circular plasmid molecules replicate, bu t only during a short time interval immediately following transfection . No replication was observed in the case of the two linear plasmids. However, the function of the SV40 origin is potentially present in the molecules, since circular forms that do replicate can be recovered fr om both linear plasmids. The extent of replication of circularized pYA CneoL is similar to that of pYACneoC, whereas a lower fraction of circ ularized pYACneoN molecules replicate. These results are discussed in terms of the possible influence of the DNA structure on the viral ori, and of the influence of the host cell functions on viral replication.