Molecular cloning of bacteriophage PRD1 genomic fragments

Abstract

DNA from bacteriophage PRD1 was extracted and partially digested with restriction endonuclease HaeII. The digest was cloned into the PstI site of plasmid pBR322 by homopolymer tailing with guanidylate tails on the plasmid and cytidylate tails on the phage DNA. Insert bearing plasmids were isolated by transforming E. coli strains for tetracycline resistance and screening for ampicillin sensitivity. These strains were then screened for the ability to accomplish marker rescue of nonsense mutants of bacteriophage PRD1. Additional clones were isolated by screening transformants with radioactively labeled probe PRD1 DNA fragments using colony hybridization. A genetic map was generated by the marker rescue capabilities of overlapping cloned inserts. This map allowed the ordering of fourteen of the known PRD1 complementation groups.