S'iakste N I, Blokhin D Iu
Biokhimiia. 1989 Jul;54(7):1217-23.
A preparative procedure for obtaining tightly and loosely bound DNA-matrix complexes was developed. To this end, the DNA-matrix complexes were separated from DNA excess by restrictase digestion and solubilized in 0.1% sodium dodecyl sulfate + 8 M urea. To obtain tightly bound complexes, the lysate was transferred to a solution containing 2 M LiCl and 4 mM urea and gel filtered in the same medium. Tightly bound complexes were eluted in the same peak with DNA. Loosely bound complexes were obtained by gel filtration in 0.1% sodium dodecyl sulfate + 8 M urea after preliminary digestion of the original complexes with DNAase I which selectively destroys the tight bonds. Within the composition of loosely bound DNA-matrix complexes, the polypeptides with Mr of 180, 65-75, 58 and 47-50 kDa were identified, whereas tightly bound complexes were shown to contain polypeptides with molecular masses of 180, 65-75, 63, 61, 58 and 52-53 kDa.
开发了一种用于获得紧密结合和松散结合的DNA - 基质复合物的制备方法。为此，通过限制性酶消化将DNA - 基质复合物与过量的DNA分离，并溶解于0.1%十二烷基硫酸钠 + 8M尿素中。为了获得紧密结合的复合物，将裂解物转移至含有2M LiCl和4mM尿素的溶液中，并在相同介质中进行凝胶过滤。紧密结合的复合物与DNA在同一峰中洗脱。松散结合的复合物是在先用DNA酶I对原始复合物进行初步消化（选择性破坏紧密键）后，在0.1%十二烷基硫酸钠 + 8M尿素中通过凝胶过滤获得的。在松散结合的DNA - 基质复合物的组成中，鉴定出分子量为180、65 - 75、58和47 - 50kDa的多肽，而紧密结合的复合物显示含有分子量为180、65 - 75、63、61、58和52 - 53kDa的多肽。
