Analysis of DNA attached to the chromosome scaffold.
Analysis of DNA attached to the chromosome scaffold.
复制标题
DOI：
10.1083/jcb.93.2.278
复制
发表时间：
1982-05
期刊：
影响因子：
--
通讯作者：
Kuo MT
中科院分区：
文献类型：
--
作者：
Kuo MT
已经描述了两种不同的方法来研究是否有任何特定的DNA序列与中期染色体支架密切相关。通过用2 M NaCl使染色体去组织化制备的染色体支架是一种非组蛋白蛋白复合物，许多环状DNA分子附着于其上（Laemmli et al.，1977年，冷泉港会议。定量42：351- 360）。以鸡MSB染色体为材料，经限制性内切酶EcoRI酶切，蔗糖梯度沉降去除环状DNA，制备染色体支架DNA。或者，使用含有2 M NaCl的蔗糖梯度从微球菌核酸酶消化的完整染色体制备支架DNA。放射性标记的支架DNA与大量过量的总核DNA的溶液杂交揭示，在任一情况下，支架DNA不是基因组DNA的独特序列类。Southern印迹杂交还显示，从EcoRI消化的去组蛋白染色体制备的支架DNA在卵清蛋白基因序列中没有富集（或耗尽）。蛋白质和DNA在染色体支架的动态相互作用的可能性和支架是一个准备工件的可能性进行了讨论。
Two different methods have been described to investigate whether any specific DNA sequences are intimately associated with the metaphase chromosome scaffold. The chromosome scaffold, prepared by dehistonization of chromosomes with 2 M NaCl, is a nonhistone protein complex to which many looped DNA molecules are attached (Laemmli et al., 1977, Cold Spring Harbor Symp. Quant. Biol. 42:351--360). Chromosome scaffold DNA was prepared from dehistonized chicken MSB chromosomes by restriction endonuclease EcoRI digestion followed by removal of the looped DNA by sucrose gradient sedimentation. Alternatively, the scaffold DNA was prepared from micrococcal nuclease- digested intact chromosomes using sucrose gradients containing 2M NaCl. Solution hybridization of the radioactively labeled scaffold DNA with a large excess of total nuclear DNA revealed that, in either case, the scaffold DNA is not a unique sequence class of genomic DNA. Southern- blotting hybridization also showed that the scaffold DNA prepared from EcoRI-digested dehistonized chromosomes was not enriched (or depleted) in the ovalbumin gene sequences. The possibility of a dynamic interaction of protein and DNA in the chromosome scaffold and the possibility that the scaffold is a preparative artifact are discussed.