Jackson V, Chalkley R
Cell. 1981 Jan;23(1):121-34. doi: 10.1016/0092-8674(81)90277-4.
We have developed a new method for isolating subcellular components after fixation of whole cells with formaldehyde. By a number of criteria we establish that the fixation does not alter or cause rearrangement of nucleosomal structure of either newly replicated or old chromatin. Using this approach we can almost completely resolve newly replicated chromatin from preexisting material on the basis of the difference in density. Newly replicated chromatin (even from cycloheximide-treated cells) appears to contain nucleosomes on both daughter strands. Exploiting the ability to separate newly synthesized chromatin, we have reexamined the question of the deposition of new histone at the replication fork. We find that newly synthesized histones H3 and H4 are deposited onto new DNA and stay in place for a significant time. In contrast new H1 is deposited on old DNA and new H2A-H2B, while they may be transiently bound to new DNA, are largely associated with preexisting chromatin.
我们开发了一种新方法，用于在用甲醛固定全细胞后分离亚细胞成分。通过多项标准，我们确定这种固定不会改变或导致新复制或旧染色质的核小体结构重排。使用这种方法，我们几乎可以根据密度差异将新复制的染色质与先前存在的物质完全分离。新复制的染色质（即使来自环己酰亚胺处理的细胞）似乎在两条子链上都含有核小体。利用分离新合成染色质的能力，我们重新审视了新组蛋白在复制叉处沉积的问题。我们发现新合成的组蛋白H3和H4沉积在新DNA上，并在相当长的时间内保持原位。相比之下，新的H1沉积在旧DNA和新的H2A-H2B上，虽然它们可能会短暂地与新DNA结合，但主要与先前存在的染色质相关。
