Molecular cloning of a functional dam+ gene coding for phage T4 DNA adenine methylase

Abstract

Phages T2 and T4 induce synthesis of a DNA-adenine methylase which is coded for by a phage gene, dam +. These enzymes methylate adenine residues in specific sequences which include G-A-T-C, the methylation site of the host Escherichia coli dam + methylase. Methylation of G-A-T-C to G-m 6A-T-C protects the site against cleavage by the MboI restriction nuclease. We have taken advantage of this property to enrich and screen for transformants which contain a cloned, functional T4 dam + gene. These recombinant molecules consist of a 1.85-kb HindIII fragment inserted into the plasmid pBR322; both orientations of the fragment express the methylase gene, suggesting that transcription is from a T4 promoter. We have tested the 1.85-kb insert for sensitivity to a variety of restriction nucleases and have found single sites for EcoRI, BalI, XbaI, and at least two sites for Bst/NI ( EcoRII). The relative positions of these restriction sites have also been determined. Physical mapping was carried out by Southern blot hybridization with 32P-labeled (nick-translated clone) probe. These experiments showed that the insert corresponds to a HindIII fragment located on the physical map of T4 between positions 16.2 and 18.1 kb from the T4 r-IIA- rIIB junction. E. coli dam − possesses several phenotypic differences from the wild-type dam + parent, including an increased sensitivity to 2-aminopurine (2-AP). We found that T4 dam + clones could relieve dam − cells of their increased sensitivity to 2-AP.