DEPOSITION OF NEWLY SYNTHESIZED HISTONES - NEW HISTONES H2A AND H2B DO NOT DEPOSIT IN THE SAME NUCLEOSOME WITH NEW HISTONES H-3 AND H-4
DEPOSITION OF NEWLY SYNTHESIZED HISTONES - NEW HISTONES H2A AND H2B DO NOT DEPOSIT IN THE SAME NUCLEOSOME WITH NEW HISTONES H-3 AND H-4
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DOI：
10.1021/bi00382a037
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发表时间：
1987-04-21
期刊：
影响因子：
2.9
通讯作者：
JACKSON, V
中科院分区：
文献类型：
--
作者：
JACKSON, V
我们开发了研究复制细胞中组蛋白沉积过程中组蛋白-组蛋白相互作用的程序。用致密氨基酸标记细胞 60 分钟，随后核小体内的组蛋白与甲醛交联成八聚体复合物。这些复合物在密度梯度中沉淀至平衡，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离八聚体和双八聚体复合物。随着交联的逆转，八聚体中各个密度标记的组蛋白的分布被确定。新合成的 H3 和 H4 以四聚体形式沉积，并与旧的 H2A 和 H2B 相关。新合成的 H2A 和 H2B 沉积为与旧 H2A、H2B、H3 和 H4 相关的二聚体。讨论了这些结果对于细胞核中组蛋白相互作用动力学的意义。提出了对照实验来测试这些复合物在制备过程中的人为形成。此外，进行重构实验以证明这些八聚体复合物的组成可以根据它们在密度梯度上的分布来确定。
We have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposit as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution on density gradients.