Liew C C, Chan P K
Proc Natl Acad Sci U S A. 1976 Oct;73(10):3458-62. doi: 10.1073/pnas.73.10.3458.
Rat liver chromatin was digested by micrococcal nuclease. More than 80% of the enzyme-digested chromatin could be recovered after centrifugation. Treatment with sodium deoxycholate and Triton X-100 at concentrations of 0.5% in the final chromatin suspension gave a higher recovery. Chromatin subunits were fractionated on a 5-30% linear sucrose density gradient. Approximately 35% of the chromatin subunits could be recovered from the gradient. Chromatin subunits and their DNA fragments were identified by gel electrophoresis and ultracentrifugation. The presence of nonhistone chromatin proteins (NHCP) in chromatin subunits was demonstrated by the following criteria: (i) Quantitative analysis showed that the mass ratio of histone to NHCP, in the presence or absence of detergents, was 1:0,25 or 1:0.1, respectively. (ii) After the removal of acid-soluble protein from the subunits, it was found that most of the phenol-soluble NHCP were similar to total chromatin NHCP. However, four major fractions of these phenol-soluble NHCP were found to be enriched in the subunits as identified by two-dimensional polyacrylamide gel electrophoresis. (iii) Experiments using an exchange of isotope-labeled and nonlabeled chromatin showed that NHCP were tightly bound to the chromatin subunits.
用微球菌核酸酶消化大鼠肝脏染色质。经离心后，超过80%的酶消化染色质能够回收。在最终染色质悬浮液中用浓度为0.5%的脱氧胆酸钠和 Triton X - 100处理可获得更高的回收率。染色质亚基在5 - 30%的线性蔗糖密度梯度上进行分级分离。约35%的染色质亚基可从梯度中回收。通过凝胶电泳和超速离心鉴定染色质亚基及其DNA片段。通过以下标准证明染色质亚基中存在非组蛋白染色质蛋白（NHCP）：（i）定量分析表明，在有或没有去污剂的情况下，组蛋白与NHCP的质量比分别为1:0.25或1:0.1。（ii）从亚基中去除酸溶性蛋白后，发现大部分酚溶性NHCP与总染色质NHCP相似。然而，通过二维聚丙烯酰胺凝胶电泳鉴定发现，这些酚溶性NHCP的四个主要组分在亚基中富集。（iii）使用同位素标记和未标记染色质交换的实验表明，NHCP与染色质亚基紧密结合。
