Antibodies specifically directed to 5-methylcytidine were raised in rabbits and purified by affinity chromatography. The accessibility of 5-methyldeoxycytidine (m'dcyd) to such antibodies was studied with DNAs from various origins. The reaction was followed by measuring the retention of radiolabelled DNA by antibodies on nitrocellulose filters, by immunoprecipitation, by gel filtration and was visualized with the electron microscope. Antibodies did not bind to Escherichiu coli B DNA, which is deficient in m'dCyd. Denatured and native DNA from calf thymus, which contains m'dCyd as a minor nucleoside, was weakly retaincd on the filters whereas DNA extracted from Xanthomonus oryzae XP12 bacteriophage, which is rich in m'dCyd, was well recognized even in the native form.Several lines of evidence suggest that methylation of cytosine residues at the 5 position in DNA plays an important role in the mechanisms which control the expression of eukaryotic genes [l -71. The amount of 5-methylcytosine (m'Cyt) in total DNA or DNA fragments can be quantified by thin-layer chromatography or high-performance liquid chromatography [8 -121. These methods allow the detection of small amounts of the modified nucleoside but require chemical or enzymatic hydrolysis of the polynucleotide and do not provide information on the location of m'Cyt on individual molecules. Digestion of DNA with restriction endonucleases sensitive to methylation of CpG sequences, the main site of m'Cyt in vertebrate DNA, and visualization of specific gene regions by blot hybridization with radiolabelled cloned DNA probes [I, 13, have revealed that tissue-specific differences in methylation of cytosine residues are common. However, restriction enzymes sensitive to methylation will only recognize m'Cyt occurring in d(CpG) sequences at certain oligonucleotide sites. Another approach for analysing the distribution of m'Cyt in DNA utilises iinmunochemical methods. Specific antibodies were widely used to demonstrate the presence of methylated bases in RNA, DNA and chromosomes [18]. For DNA, specific antibodies were used mostly to detect m'Cyt either on DNA fragments imniobilised on nitrocellulose paper by Southern transfer [19] or on fixed metaphase chromosomes [20]. Available antisera to 5-methylcytidine (m'Cyd) were shown to react with chromosomes only after ultraviolet irradiation or photooxidation. It had been concluded that the accessibility of m5Cyt residues to antibodies was limited to denatured DNA [21]. Abbreviations. m'Cyt, 5-methylcytosine; m'Cyd, 5-methylcytidine; m'dCyd, 5-methyldcoxycytidine; dCyd, deoxycytidine; HPLC, high-performance liquid chromatography; kb, lo3 base pairs.Enzynfe. Nuclease S1 (EC 3.1.30.1).Here we report results obtained with antibodies directed to m5Cyd and reacted with native or denatured DNAs from various origins. The very sensitive filter-binding method [22] was used to measure the reactivity of these antibodies with bacteriophage XP12 DNA, in which virtually all cytosine residues are methylated [23], with calf thymus DNA in...
