Cloning of the replication gene P of bacteriophage lambda: Effects of increased P-protein synthesis on cellular and phage DNA replication

Summary

A restriction fragment of λDNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/λE complement λPam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/glE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of λDNA proceeds normally under these conditions.

Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type λ (λwt), acquire the ability to replicate λPam80 phage but not λwt when they are transformed with a plasmid carrying the λP gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of λwt phage when infected at a high multiplicity. λPam80 phage does not multiply in these cells.