TRANSCRIPTION-INDUCED DELETIONS IN PLASMID VECTORS - M13 DNA-REPLICATION AS A SOURCE OF INSTABILITY

We have previously shown that concurrent progression of pBR322 replica tion and pTac-directed transcription in opposite orientations induces illegitimate recombination events. We tested here the effects of M13 r olling circle replication on the incidence of plasmid deletions. The p rogression of the M13 replication fork leads to an increase of more th an 300-fold in the frequency of transcription-dependent deletion event s. pBR322 derivatives carrying the M13 replication origin and a 511 bp transcribed region under the control of the pTac promoter were used. Up to 12% of the plasmid population has sustained deletions within 4 h following the induction of pTac-directed transcription and M13 DNA re plication, provided that the two proceed in opposite orientations. We observed that induction of transcription of the whole Escherichia coli lacZ gene (3244 bp) in the direction opposite to M13 replication lead s to a fivefold decrease in plasmid copy number within 2 h, which is c onsistent with the proposal that deletions arise because replication f ork progression is impeded. This decrease in parental plasmid copy num ber leads in turn to an enrichment in deleted plasmid forms. Our data confirm and extend the notion that simultaneous transcription and repl ication in opposite directions can efficiently promote deletion format ion. In addition, this instability may be amplified when the rearrange d molecules acquire a replicative advantage.