Molecular cloning of a functional dam+ gene coding for phage T4 DNA adenine methylase.
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-m6A-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 BstNI (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 T4rIIA-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.
D003001:Cloning, Molecular; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005796:Genes; D005814:Genes, Viral; D008780:Methyltransferases / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D013604:T-Phages / Q000201:enzymology / Q000235:genetics*
D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes