Bloom K S, Carbon J
Cell. 1982 Jun;29(2):305-17. doi: 10.1016/0092-8674(82)90147-7.
We have examined the chromatin structure of the centromere regions of chromosomes III and XI in yeast by using cloned functional centromere DNAs (CEN3 and CEN11) as labeled probes. When chromatin from isolated nuclei is digested with micrococcal nuclease and the resulting DNA fragments separated electrophoretically and blotted to nitrocellulose filters, the centromeric nucleosomal subunits are resolved into significantly more distinct ladders than are those from the bulk of the chromatin. A discrete protected region of 220-250 bp of CEN sequence flanked by highly nuclease-sensitive sites was revealed by mapping the exact nuclease cleavage sites within the centromeric chromatin. On both sides of this protected region, highly phased and specific nuclease cutting sites exist at nucleosomal intervals (160 bp) for a total length of 12-15 nucleosomal subunits. The central protected region in the chromatin of both centromeres spans the 130 bp segment that exhibits the highest degree of sequence homology (71%) between functional CEN3 and CEN11 DNAs. This unique chromatin structure is maintained on CEN sequences introduced into yeast on autonomously replicating plasmids, but is not propagated through foreign DNA sequences flanking the inserted yeast DNA.
我们使用克隆的功能性着丝粒DNA（CEN3和CEN11）作为标记探针，研究了酵母中III号和XI号染色体着丝粒区域的染色质结构。当用微球菌核酸酶消化分离细胞核中的染色质，然后将所得DNA片段进行电泳分离并印迹到硝酸纤维素滤膜上时，着丝粒核小体亚基比大部分染色质中的核小体亚基能分辨出明显更多不同的梯状条带。通过绘制着丝粒染色质内确切的核酸酶切割位点，揭示了一个220 - 250 bp的CEN序列离散保护区域，其两侧为高度核酸酶敏感位点。在该保护区域两侧，以核小体间隔（160 bp）存在高度有序且特定的核酸酶切割位点，总长度为12 - 15个核小体亚基。两个着丝粒染色质中的中央保护区域跨越了130 bp片段，该片段在功能性CEN3和CEN11 DNA之间表现出最高程度的序列同源性（71%）。这种独特的染色质结构在自主复制质粒上引入酵母的CEN序列上得以维持，但不会通过插入的酵母DNA侧翼的外源DNA序列进行传播。
