Studies of Bacteriophage P2 DNA Replication: The DNA Sequence of the Cis-acting gene A and ori Region and Construction of a P2 Mini-chromosome

Abstract

A self-replicating plasmid was constructed from the 76·7 to 91·6% region of bacteriophage P2, which contains the P2 origin of replication ( ori) and the genetically defined replication genes B and A. The sequence of the 76·7 to 80·2% has been determined previously, and the sequence of the 80·2 to 91·6% region is now reported. The sequenced region contained gene A, which predicts a 761 amino acid polypeptide known to induce a single-strand cut at or near ori , and ori, which has been located by electron microscopy to about 89% from the left end of the phage genome. Analysis of plasmid-encoded proteins in minicells indicated that the A gene product was about 78 kilodaltons. Five previously unknown open reading frames ( orf-80, orf-81, orf-82, orf-83 and orf-91) were discovered. They have been cloned and their respective products were identified. The products of orf-80, orf-82 and gene A were found to be lethal to the host when overexpressed. The predicted amino acid sequences of the orf-82 and orf-83 gene products were similar to two early gene products of phage 186; the orf-91 product resembled the hypothetical protein of orfd of retron Ec67. Similarities between the products of P2 gene A , 186 gene A and orf2 and orf3 of Ec67 were also found. A P2 mini-chromosome has been constructed that contains only the P2 A gene and the β-lactamase gene as a marker.