Rat embryo nonhistone chromosomal proteins: interaction in vitro with normal and bromodeoxyuridine-substituted DNA

Nonrandom Correlation between Selective Distribution of Virogenic 5-Bromodeoxyuridine and DNA-Binding Nonhistone Proteins in Rat DNA

“…In this particular system, minimal replacement (<7%) of bromouracil for thymine into finite nucleotide sequence elements was sufficient for substantial virogenic induction to occur. A related study from our laboratory concerned the nature of rat embryo DNA-binding nonhistone proteins as well as their increased affinity for BrdU-containing DNA (23). This communication further documents the localization and distribution of the nonhistone protein binding regions of rat chromosomal DNA.…”

“…Thermal stability determinations were performed by denaturation over heated hydroxy apatite columns (22,23).…”

“…Rat embryo cell nonhistone proteins were isolated, extracted and characterized from purified nuclei as described (23,31). In order to generate reconstituted DNA-protein complexes, each purified reactant was brought up individually in 2M NaCl, 5 M urea, 0.01 M Tris-HCl (pH 8.0) and 1 mM EDTA as reported earlier (23,31) after the method of Bekhor et al (4).…”

“…In order to generate reconstituted DNA-protein complexes, each purified reactant was brought up individually in 2M NaCl, 5 M urea, 0.01 M Tris-HCl (pH 8.0) and 1 mM EDTA as reported earlier (23,31) after the method of Bekhor et al (4). The DNA and protein samples were then respectively mixed together at a ratio of 1:2 and successively dialyzed for 2 h against 1,0.8, 0.6, and 0.4M NaCl at 5 °C in the presence of 5 M urea.…”

“…The solutions were finally brought to 0.01 M Tris-HCl (pH 8.0), 0.01 M NaCl and 1 mM KDTA after an additional 16 h of dialysis. In order to confirm the extent of reconstitution, 0.1-ml aliquots were layered onto linear 5 25% sucrose gradients and sedimented at 50,000 rpm for 210 min in a Spinco SW-50.1 rotor (23). Fractions of 0.25 ml were subsequently assayed for | 3H| in a Packard Model 3380 scintillation spectrom eter, and protein according to Lowry et al (16).…”