Glucosylated nucleotide sequences from T2-bacteriophage deoxyribonucleic acid

Phages T 2, T 4 and T 6 contain glucose attached as a-glucosyl, fl-glucosyl and a-gentiobiosyl groups to the hydroxymethylcytosine of the DNA (Lehman & Pratt, 1960; Kuno & Lehman, 1962).Although each type of phage DNA contains virtually the same amount of hydroxymethylcytosine, there are large differences in the total amounts of glucose and in the amounts of each of the three types of glucosylated hydroxymethylcytosine.In T 2-phage DNA, 24-28 % of the total hydroxy- methylcytosine is not glucosylated; 5-6 % is substituted with oc-gentiobiosyl residues and the remainder bears oc-glucosyl groups (Lehman & Pratt, 1960).It has been shown (Kornberg, Zimmerman & Kornberg, 1961) that the hydroxymethylcytosine nucleotides are glucosylated after they have been incorporated into DNA, but it is not known whether the glucose is randomly distributed among these nucleotides or whether its attachment to a hydroxymethylcytosine residue is affected by the nature of the neighbouring nucleo- tides.We have investigated these possibilities by studying the nucleotide products obtained by digesting T 2-phage DNA with diphenylamine in aqueous formic acid (Burton & Petersen, 1960) and with pancreatic deoxyribonuclease.A preliminary note describing part of this work has been published (Lunt & Burton, 1962). MATERIALS AND METHODSBacteriophage.Two wild-type strains of T2 phage were used: T2(K) phage was from Dr D. Kay, Department of Pathology, Oxford, and T2(H) phage originated from Dr Hershey and was obtained via Dr G. L. Brown, King's College, London.The phages were prepared by lysis of Escherichia coli strain B grown in 1 1. batches of medium [composition (per 1.): KH2PO4, 2-1 g.; Na2HPO4, 12-5 g.; NH4Cl, 2-0 g.; MgSO4, 0-8 g.; and glucose, 1-8 g., added after separate autoclaving] in a rotating-flask apparatus of the type described by Mitchell (1949).The bacteria were grown to a cell density of 2 x 108/ml.and sufficient phages were added to give a multiplicity of infection of 2-3.After