Screening for termination sequences of a rolling-circle plasmid: a novel scheme using genomic DNA.

Abstract

The Escherichia coli genome was searched for potential terminators of the rolling-circle replication of staphylococcal plasmid pC194. The replication origin of pC194 was randomly inserted into the E. coli chromosome and rolling-circle replication was initiated by producing pC194's replication protein from a plasmid. Circular DNA resulting from termination in the chromosome was recovered from 42 of the 100 insertion clones screened. The nucleotide sequences at the ends of the chromosomal segment in the recovered DNA were determined and used to identify the locus of integration and the point of termination. The sequence beyond the termination point was retrieved from the database. This information would have been unrecoverable if synthetic random sequences had been used for screening. The consensus sequence based on the discovered potential terminators was consistent with the results of previous and new experiments. The recovered circular DNAs contain a hybrid origin consisting of a 5' part derived from the chromosomal DNA and a 3' part of the integrated origin. Two such hybrid origins were examined for initiation function and shown to be as effective as the authentic pC194 origin. These results suggest a possible evolutionary mechanism in which a rolling-circle plasmid may acquire genes from the host organism.