Stein A, Mitchell M
Department of Biological Sciences Purdue University West Lafayette, IN 47907.
J Mol Biol. 1988 Oct 20;203(4):1029-43. doi: 10.1016/0022-2836(88)90127-1.
We have been able to generate ordered nucleosome arrays that span the physiological range of spacing periodicities, using an in vitro system. Our system (a refinement of the procedure previously developed) uses the synthetic polynucleotide poly[d(A-T)], poly[d(A-T)], core histones, purified H1, and polyglutamic acid, a factor that increases nucleohistone solubility and greatly promotes the formation of ordered nucleosome arrays. This system has three useful features, not found in other chromatin assembly systems. First, it allowed us to examine histones from three different cell types/species (sea urchin sperm, chicken erythrocyte, and HeLa) as homologous or heterologous combinations of core and H1 histones. Second, it allowed us to control the average packing density (core histone to polynucleotide weight ratio) of nucleosomes on the polynucleotide; histone H1 is added in a second distinct step in the procedure to induce nucleosome alignment. Third, it permitted us to study nucleosome array formation in the absence of DNA base sequence effects. We show that the value of the spacing periodicity is controlled by the value of the initial average nucleosome packing density. The full range of physiological periodicities appears to be accessible to arrays generated using chicken erythrocyte (or HeLa) core histones in combination with chicken H5. However, chromatin-like structures cannot be assembled for some nucleosome packing densities in reactions involving some histone types, thus limiting the range of periodicities that can be achieved. For example, H1 histone types differ significantly in their ability to recruit disordered nucleosomes into ordered arrays at low packing densities. Sea urchin sperm H1 is more efficient than chicken H5, which is more efficient than H1 from HeLa or chicken erythrocyte. Sea urchin sperm core histones are more efficient in this respect than the other core histone types used. These findings suggest how different repeat lengths arise in different cell types and species, and provide new insights into the problems of nucleosome linker heterogeneity and how different types of chromatin structures could be generated in the same cell.
我们利用体外系统成功生成了跨越生理间距周期范围的有序核小体阵列。我们的系统（对先前开发的程序进行了改进）使用合成多核苷酸聚[d(A-T)]、聚[d(A-T)]、核心组蛋白、纯化的H1和聚谷氨酸，聚谷氨酸是一种可增加核组蛋白溶解度并极大促进有序核小体阵列形成的因子。该系统具有其他染色质组装系统所没有的三个有用特性。首先，它使我们能够将来自三种不同细胞类型/物种（海胆精子、鸡红细胞和HeLa细胞）的组蛋白作为核心组蛋白和H1组蛋白的同源或异源组合进行研究。其次，它使我们能够控制多核苷酸上核小体的平均堆积密度（核心组蛋白与多核苷酸的重量比）；在该程序的第二个不同步骤中添加组蛋白H1以诱导核小体排列。第三，它使我们能够在不存在DNA碱基序列效应的情况下研究核小体阵列的形成。我们表明，间距周期的值由初始平均核小体堆积密度的值控制。使用鸡红细胞（或HeLa细胞）核心组蛋白与鸡H5组合生成的阵列似乎可以达到整个生理周期范围。然而，在涉及某些组蛋白类型的反应中，对于某些核小体堆积密度无法组装出类似染色质的结构，从而限制了可实现的周期范围。例如，H1组蛋白类型在低堆积密度下将无序核小体募集到有序阵列中的能力存在显著差异。海胆精子H1比鸡H5更有效，而鸡H5又比HeLa细胞或鸡红细胞的H1更有效。在这方面，海胆精子核心组蛋白比其他使用的核心组蛋白类型更有效。这些发现揭示了不同细胞类型和物种中不同重复长度是如何产生的，并为核小体连接体异质性问题以及同一细胞中如何产生不同类型染色质结构提供了新的见解。
