Annunziato A T, Schindler R K, Riggs M G, Seale R L
J Biol Chem. 1982 Jul 25;257(14):8507-15.
Histone deposition in HeLa cells has been studied by monitoring the fractionation and electrophoresis mobility of pulse-labeled histones under conditions that separate newly replicated from bulk chromatin DNA. The separation efficiency of these two methods is approximately 70%. Following micrococcal nuclease digestion, chromatin was fractionated by salt elution. 50-65% of the newly synthesized histones eluted with bulk chromatin at NaCl concentrations between 0.1 and 0.3 M and were further down to co-electrophorese with bulk chromatin DNA, not with the more extensively digested newly replicated chromatin DNA contained in those fractions. The remaining chromatin fractions, solubilized with 0.4-0.6 M NaCl, were several-fold enriched in nascent DNA (Annunziato, A. T., Schindler, R. K., Thomas, C. A., Jr., and Seale, R. L. (1981) J. Biol. Chem. 256, 11880-11886) and were correspondingly enriched for the balance (35-50%) of newly synthesized core histones. This fraction of newly synthesized core histone may be preferentially deposited onto newly replicated DNA. In contrast, histone H1 showed little tendency toward deposition onto new DNA. Within 15 min all new core histones attained the same solubility and electrophoretic mobility as bulk chromatin. We conclude that newly synthesized histones are deposited onto both replicating and nonreplicating regions of chromatin.
通过在将新复制的染色质DNA与整体染色质DNA分离的条件下监测脉冲标记组蛋白的分级分离和电泳迁移率，对HeLa细胞中的组蛋白沉积进行了研究。这两种方法的分离效率约为70%。在微球菌核酸酶消化后，通过盐洗脱对染色质进行分级分离。50%-65%新合成的组蛋白在NaCl浓度为0.1至0.3M时与整体染色质一起洗脱，并进一步与整体染色质DNA共电泳，而不是与这些级分中消化更充分的新复制染色质DNA共电泳。其余用0.4-0.6M NaCl溶解的染色质级分中，新生DNA富集了几倍（Annunziato，A.T.，Schindler，R.K.，Thomas，C.A.，Jr.和Seale，R.L.（1981）J.Biol.Chem.256，11880-11886），相应地，新合成的核心组蛋白的其余部分（35%-50%）也富集了。新合成的核心组蛋白的这一部分可能优先沉积到新复制的DNA上。相比之下，组蛋白H1几乎没有沉积到新DNA上的趋势。在15分钟内，所有新的核心组蛋白都达到了与整体染色质相同的溶解度和电泳迁移率。我们得出结论，新合成的组蛋白沉积在染色质的复制和非复制区域上。
