Nightingale K P, Becker P B
Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Methods. 1998 Aug;15(4):343-53. doi: 10.1006/meth.1998.0638.
In this review we describe how the extract-mediated chromatin assembly system derived from preblastoderm Drosophila embryos can be modified to assemble chromatin from defined histones. This approach combines the advantages of assembling (i) chromatin templates from homogeneous histones with (ii) an assembly system that generates chromatin with physiological nucleosome spacing and density and that contains the biological complexity of in vivo chromatin. We have used this technique to assemble nonacetylated and hyperacetylated histones into chromatin (K. P. Nightingale, R. Wellinger, J. Sogo, and P. B. Becker, 1998, EMBO J. 17, 2865-2876; W. A. Krajewski and P. B. Becker, 1998, Proc. Natl. Acad. Sci. USA 95, 1540-1545), and use this as an example to detail the structural and transcriptional assays used to compare and characterize these chromatin templates. The application of this procedure to assemble chromatin from recombinant histones should facilitate a wide variety of studies on the role(s) of histone mutants and variants.
在本综述中，我们描述了如何对源自果蝇胚盘前胚胎的提取物介导的染色质组装系统进行改造，以便从特定组蛋白组装染色质。这种方法结合了以下两方面的优势：（i）用同质组蛋白组装染色质模板，以及（ii）一种能产生具有生理核小体间距和密度且包含体内染色质生物学复杂性的染色质的组装系统。我们已使用该技术将未乙酰化和高度乙酰化的组蛋白组装到染色质中（K.P. 南丁格尔、R. 韦林格、J. 索戈和P.B. 贝克尔，1998年，《欧洲分子生物学组织杂志》17卷，2865 - 2876页；W.A. 克拉耶夫斯基和P.B. 贝克尔，1998年，《美国国家科学院院刊》95卷，1540 - 1545页），并以此为例详细介绍用于比较和表征这些染色质模板的结构和转录分析方法。将该程序应用于从重组组蛋白组装染色质，应有助于开展关于组蛋白突变体和变体作用的各种研究。
