Non-random distribution of N-methyl-N-nitrosourea sensitive sites in a eukaryotic genome

DNA released from Ehrlich ascites cells by lysis in the presence of 50 microgram X ml-1 of proteinase K contains long alkali-stable strands in the order of 50-100 X 10(6) daltons. In contrast, DNA released in the presence of 6 mg X ml-1 of autodigested pronase is significantly nicked. According to sedimentation rates the number of internal ends liberated during this procedure is 24/200 X 10(6) daltons. The number of alkali-labile sites introduced into DNA by incubation of Ehrlich ascites cells with 1 nM of N-methyl-N-nitrosourea (MNU) followed by cell lysis in the presence of 50 microgram X ml-1 of proteinase K and alkali-denaturation is 16.6/200 X 10(6) daltons. From this one should expect that denatured DNA released from cells pretreated with 1 mM of MNU which are subsequently lysed with 6 mg X ml-1 of pronase would have about 40 single-strand breaks/200 X 10(6) daltons. However, denatured DNA strands released by 6 mg X ml-1 of pronase either from MNU-treated or untreated cells cannot be separated by centrifugation through alkaline sucrose gradients. This phenomenon could be explained by a non-random distribution of MNU-inducible alkali-labile sites of DNA in vivo.