Hancock R
Proc Natl Acad Sci U S A. 1978 May;75(5):2130-4. doi: 10.1073/pnas.75.5.2130.
The assembly of chromatin from newly synthesized nucleosomal histones (labeled with [3H]arginine) and new DNA (density-labeled with [125I]iododeoxyuridine)was studied in growing cultured mouse cells. The nucleosomal histones were specifically examined by dissociating histone H1 and nonhistone proteins from unsheared chromatin either by incubation in 0.6 M NaCl or by digestion with micrococcal nuclease to release nucleosomes. In both cases, the four nucleosomal histones (H2A, H2B, H3, and H4) are essentially the only proteins that remain bound to DNA and that are labeled by [3H]arginine. After formaldehyde fixation, H1-depleted chromatin containing dense DNA can be completely resolved in CsCl buoyant density gradients from that containing unreplicated DNA; separation of nucleosomes is satisfactory although less complete. New DNA and new histones are already assembled into chromatin possessing characteristic nucleosomal structure after 3 min of synthesis (the shortest time studied), as shown by the kinetics of digestion of new DNA by micrococcal nuclease, by the distribution of new DNA and new histones in nucleosomes. However, after 3-30 min of synthesis most new nucleosomal histones are associated with unreplicated DNA rather than with new DNA. It is concluded that new nucleosomes are assembled on DNA at some distance from DNA replication sites, with concomitant migration of preexisting nucleosomes onto new DNA.
在生长的培养小鼠细胞中研究了由新合成的核小体组蛋白（用[3H]精氨酸标记）和新DNA（用[125I]碘脱氧尿苷进行密度标记）组装染色质的过程。通过在0.6M NaCl中孵育或用微球菌核酸酶消化以释放核小体，从未剪切的染色质中解离组蛋白H1和非组蛋白来特异性检测核小体组蛋白。在这两种情况下，四种核小体组蛋白（H2A、H2B、H3和H4）基本上是仅有的与DNA结合并被[3H]精氨酸标记的蛋白质。甲醛固定后，含有致密DNA的H1缺失染色质可以在CsCl浮力密度梯度中与含有未复制DNA的染色质完全分离；核小体的分离虽然不太完全，但令人满意。如微球菌核酸酶对新DNA的消化动力学、新DNA和新组蛋白在核小体中的分布所示，合成3分钟（研究的最短时间）后，新DNA和新组蛋白已组装成具有特征性核小体结构的染色质。然而，合成3 - 30分钟后，大多数新的核小体组蛋白与未复制的DNA相关，而不是与新DNA相关。得出的结论是，新核小体在距DNA复制位点一定距离处组装在DNA上，同时先前存在的核小体迁移到新DNA上。
