Assay for Rifampicin Resistant M13 RF Synthesis

Abstract

We have developed a rapid assay which can detect the function of the origin of 0X174 complementary strand DNA in Ml3 by virtue of its ability to initiate the synthesis of M13 complementary strands in the presence of the antibiotic rifampicin. A segment of DNA carrying the sequence recognized by the replication protein n\u27 has been isolated from bacteriophage ØX174 and cloned into an Ml3 bacteriophage vector. Protein n\u27 is a component of the bacterial primosome, a multi-protein assembly which initiates the synthesis of primers during DNA replication in the bacterium Escherichia coli. The recognition site for n\u27 on the ØXl74 viral strand DNA constitutes the origin of replication for the ØX174 complementary strand. On ØX174 viral single-stranded DNA, synthesis of the complementary strand is initiated by the primosome. RNA polymerase is not involved. By contrast, the formation of primers on M13 viral DNA is accomplished by the direct action of RNA polymerase. Rifampicin inhibits this enzyme and, therefore, prevents the synthesis of M13 complementary strands. M13 clones which include the ØXl74 n\u27 recognition site acquire the rifampicin-insensitive characteristic of the ØX174 replication origin. We have designed an assay in which small cultures of Escherichia coli, incubated with chloramphenicol and rifampicin are infected with M13, ØXl74, or Ml3 phage hybrids which contain a fragment of DNA bearing the ØX174 n\u27 recognition site. Rapid lysates were applied to agarose gels to detect the presence of double-stranded replicative form DNA in the cells. Numerous phage isolates could be tested simultaneously in one day. We will use the assay to screen isolates which have been mutagenized in the region of the ØX174 insert