Isolation and visualisation of alkali stable protein/DNA complexes

Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo.

“…Fragments were collected by centrifugation, and dissolved at a concentration of 25 Ag of DNA/mi (0.5 A260 units) in 1/10 EDTA buffer containing I M NaCl. The enrichment of protein-associated DNA fragments was performed as described elsewhere (Spiess et al, 1982). Briefly, DNA samples were filtered through Whatman GF/C filters ( <0.5 A 2w units/filter).…”

“…DNA-containing fractions were collected and the DNA was directly mounted onto mica according to the procedure of Koller et al (1974) modified as described (Spiess et al, 1982). As determined in independent experiments (P. Nehls, E. Spiess, E. Weber and M.F.…”

“…DNA fragments containing such DNA-protein complexes may be essential constituents of higher order chromatin structures and/or be involved in the anchorage of DNA in nuclear structures (Bodnar et al, 1983;Werner et al, 1980Werner et al, , 1981a. Since DNAprotein complexes of this type can be selectively enriched on glass fiber filters (Bodnar et al, 1983;Spiess et al, 1982), the frequency of a specific carcinogen-induced structural modification in the DNA of these complexes can be studied in comparison with the remaining DNA. Using a high-affinity anti-(06-EtdGuo) monoclonal antibody (ER-6;Rajewsky et al, 1980;Adamkiewicz et al, 1982) (Bodnar et al, 1983;Spiess et al, 1982).…”

“…Since DNAprotein complexes of this type can be selectively enriched on glass fiber filters (Bodnar et al, 1983;Spiess et al, 1982), the frequency of a specific carcinogen-induced structural modification in the DNA of these complexes can be studied in comparison with the remaining DNA. Using a high-affinity anti-(06-EtdGuo) monoclonal antibody (ER-6;Rajewsky et al, 1980;Adamkiewicz et al, 1982) (Bodnar et al, 1983;Spiess et al, 1982). The material specifically adsorbed to the filters was recovered and, in parallel with equal amounts of the material that had passed through the filters, was radiolabeled with 1251I exclusively on tyrosines (Neuer et al, 1983).…”

“…The concentration of antibody chosen for the binding reaction in all experiments was such that 99.9%o of the 06-EtdGuo residues in DNA would theoretically bind an antibody molecule. After separation of the DNA from unreacted antibody molecules by gel filtration, samples were processed for electron microscopy by a protein-free preparation technique, allowing visualization of ABS without labeling by a second electron-dense marker (Koller et al, 1974;Spiess et aL, 1982). Although a large number of AIuI-DNA fragments were screened in the electron microscope, no DNA molecule was found in the (06-EtdGuo)-containing bulk DNA with ABS appearing at a frequency significantly above the background seen in nonethylated control DNA (i.e., -1 ABS/100 Am of DNA).…”