Cloning of DNA segments of phage 2C, which allows autonomous plasmid replication in Bacillus subtilis

Abstract

The chromosome of Bacillus subtilis phage 2C is a 100-MDa double-stranded DNA molecule, containing hydroxymethyluracil in place of thymine and carrying redundant ends each encompassing 10% of the genome. 2C DNA was cleaved with EcoRI and HindIII, and cloned in the shuttle plasmids pSC 540 and pCP 115, both containing segments originating from B. subtilis and Escherichia coli plasmids. These chimaerical plasmids, carrying the chloramphenicol resistance gene, were unable to replicate in B. subtilis; this ability was restored, however, after the insertion of viral DNA segments. Physical maps of the recombinant plasmids were made; a large deletion of the E. coli-derived segment of pSC 540 was observed (which paralleled a loss of replication in this host), whereas addition of 2C DNA segments in pCP 115 was not accompanied by deletion (replication in E. coli was conserved in this case). Cloned viral segments mapped mostly, but not exclusively, within the redundant ends of 2C DNA. It is suggested that the thirteen recombinant clones carried the replication origin region of phage 2C DNA, and that these sequences originated within or close to the redundant extremities of the viral chromosome.