Zaboĭkin M M, Alekhina R P, Likhtenshteĭn A V
Mol Biol (Mosk). 1989 May-Jun;23(3):851-61.
Nuclei isolated from Djungarian hamster fibroblasts transformed by SV40 were treated with restriction endonuclease Bsp RI, fixed on Celite columns and underwent successive gradients of dissociating agents, such as NaCl, LiCl-urea, and temperature. This procedure leads to fractionation of DNA fragments in accordance with the tightness of DNA-protein bonds in situ. The fractions obtained were analysed by agarose gel electrophoresis and dot-hybridization technique with the use of various DNA probes. The results received are as follows: a) a DNA fragment size is not a factor determining the chromatographic position, the latter is probably stipulated by DNA-protein interactions; b) an analysis of cells synchronized at the G1/S border shows that the distribution of specific DNA sequences, such as actin, histone, hsp 70, and c-Ha-ras genes as well as reiterated DNA sequences, does not coincide with that of total genomic DNA; the nuclear matrix-attached fragments of those sequences are enriched to various extents. By nick-translation labeling in situ, DNase I-sensitive and hypersensitive regions were tentatively identified among subdomain chromatin fragments.
从经SV40转化的黑线仓鼠成纤维细胞中分离出的细胞核，用限制性内切酶Bsp RI处理，固定在硅藻土柱上，并经历了诸如NaCl、LiCl-尿素和温度等解离剂的连续梯度处理。该程序导致DNA片段根据原位DNA-蛋白质键的紧密程度进行分级分离。对所得级分进行琼脂糖凝胶电泳分析，并使用各种DNA探针进行点杂交技术分析。得到的结果如下：a）DNA片段大小不是决定色谱位置的因素，后者可能由DNA-蛋白质相互作用决定；b）对在G1/S边界同步化的细胞进行分析表明，特定DNA序列（如肌动蛋白、组蛋白、hsp 70和c-Ha-ras基因以及重复DNA序列）的分布与总基因组DNA的分布不一致；这些序列的核基质附着片段在不同程度上富集。通过原位缺口平移标记，在亚结构域染色质片段中初步鉴定出DNase I敏感和超敏感区域。
