猴病毒40 DNA在哺乳动物细胞提取物中的核小体组装。

Nucleosome assembly of simian virus 40 DNA in a mammalian cell extract.

作者信息

Banerjee S, Cantor C R

机构信息

Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032.

出版信息

Mol Cell Biol. 1990 Jun;10(6):2863-73. doi: 10.1128/mcb.10.6.2863-2873.1990.

We report here a mammalian cell-free system that can support chromatin assembly. Effective nucleosome assembly in HeLa cell extracts occurred at 125 to 200 mM KCl or potassium glutamate. At this physiological K+ ion concentration, two types of chromatin assembly were observed. The first was interfered with by Mg2+. Other cations such as Mn2+, Ca2+, Fe3+, and spermidine also inhibited this type of nucleosome assembly. The second type of assembly occurred in the presence of Mg2+ and at least equimolar ATP. However, even in the presence of ATP, excess Mg2+ inhibited assembly and promoted catenation of DNA; these effects could be circumvented by excess ATP, GTP, EDTA, or polyglutamic acid. The critical DNA concentration for optimum assembly in both pathways suggested a stoichiometric association of histones with DNA. The spacing of nucleosomes formed by both types of assembly on linear and circular DNA was reasonably regular, but chromatin assembled in the presence of ATP and Mg2+ was more stable.

我们在此报告一种能够支持染色质组装的哺乳动物无细胞系统。在HeLa细胞提取物中，有效的核小体组装发生在125至200 mM的氯化钾或谷氨酸钾浓度下。在此生理钾离子浓度下，观察到两种类型的染色质组装。第一种受到镁离子的干扰。其他阳离子如锰离子、钙离子、铁离子和亚精胺也抑制这种类型的核小体组装。第二种组装类型发生在镁离子存在且至少等摩尔ATP存在的情况下。然而，即使在ATP存在的情况下，过量的镁离子也会抑制组装并促进DNA的连环化；这些影响可以通过过量的ATP、GTP、EDTA或聚谷氨酸来规避。两种途径中最佳组装的关键DNA浓度表明组蛋白与DNA存在化学计量关联。两种组装类型在线性和环状DNA上形成的核小体间距相当规则，但在ATP和镁离子存在下组装的染色质更稳定。