Distribution of O6-ethylguanine in DNA exposed to ethylnitrosourea in vitro as visualized by electron microscopy using a monoclonal antibody.
Application of a monoclonal antibody (Mab ER-6; Rajewsky et al., 1980) specific for O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), in conjunction with a protein-free spreading procedure for double-stranded DNA molecules and transmission electron microscopy, permits the visualization of antibody molecules complexed to O6-EtdGuo residues formed in DNA upon reaction with the carcinogen N-ethyl-N-nitrosourea (EtNU) (Nehls et al., 1984). To obtain information on the distribution of O6-EtdGuo in native DNA exposed to EtNU in vitro, samples of purified rat brain DNA were briefly incubated with EtNU at concentrations differing by a factor of 8 (0.5 and 4 mg of EtNU/ml, respectively). As determined in DNA hydrolysates by competitive radioimmunoassay, the resulting DNA preparations contained O6-EtdGuo at O6-EtdGuo/2'-deoxyguanosine molar ratios of 15.1 X 10(-5) and 116 X 10(-5), respectively. Interspace distances between Mab-binding sites in both sets of ethylated DNA were determined by electron microscopy both in individual DNA fragments of different size, and in computer-generated, long-thread DNA constructs. Comparative statistical analyses by a newly developed MOLRANDO computer program show a non-random distribution pattern of Mab-binding O6-EtdGuo residues.
Nehls
P
P
Institut für Zellbiologie (Tumorforschung), Universität Essen GH, F.R.G.
Spiess
E
E
Weber
E
E
Berger
J
J
Rajewsky
M F
MF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D004247:DNA / Q000187:drug effects* / Q000648:ultrastructure; D004249:DNA Damage; D005038:Ethylnitrosourea / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology; D008854:Microscopy, Electron; D051381:Rats; D012984:Software
D000911:Antibodies, Monoclonal; C007673:6-ethylguanine; D006147:Guanine; D004247:DNA; D005038:Ethylnitrosourea