Selective cloning of Co1E1 DNA initiation sequences using the cloning vector M13 delta E101

Abstract

DNA sequences required to direct single-strand to double-strand conversion by a phi X174-type mechanism have been detected in Co1E1 plasmid DNA. The sites responsible for this DNA strand initiation, named rriA and rriB, have been localized to the L strand of the HaeII-E fragment and to the H strand of the HaeII-C fragment of Co1E1. To define rriA and rriB more precisely, random fragments of Co1E1 DNA have been cloned into the single-stranded phage cloning vector M13 delta E101. This phage contains a viable deletion of the M13 complementary strand origin and makes small turbid plaques. Cloning of DNA initiation determinants into the unique EcoRI site of M13 delta E101 rescues the mutant phage and restores the ability to form clear plaques. DNA sequence analysis of the random, 100-400 bp inserts in clear plaque isolates has localized the rriA and rriB determinants within DNA fragments of about 100 bp. These initiation determinants promote assembly of a functional, multiprotein priming complex, "primosome", identical to that utilized in the in vitro conversion of phi X174 single-stranded DNA to the duplex replicative form.