Gregory P D, Barbaric S, Hörz W
Institut für Physiologische Chemie, Universität München, Germany.
Methods. 1998 Aug;15(4):295-302. doi: 10.1006/meth.1998.0633.
The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell. This article provides two basic procedures for the study of chromatin in vivo. The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cells in vivo to determine the binding of factors to cis elements in the locus of interest. Specific examples illustrating the techniques described are given from our work on the regulation of the yeast PHO8 gene, but have also been successfully and reliably applied to the study of many other yeast loci. These procedures make it possible to correlate the binding of a transactivator with an altered or perturbed chromatin organization at a specific locus.
染色质研究曾被认为是一种纯粹的结构基质，用于将基因组的DNA压缩到细胞核中，而如今对于我们理解DNA在细胞中的功能愈发重要。本文提供了两种在体内研究染色质的基本方法。第一种是基于DNase I的方法，用于处理分离的细胞核以解析特定区域的染色质结构；第二种是在体内对全细胞进行硫酸二甲酯足迹分析，以确定因子与感兴趣位点的顺式元件的结合。文中给出了我们在酵母PHO8基因调控研究中运用这些技术的具体示例，这些技术也已成功且可靠地应用于许多其他酵母基因座的研究。这些方法能够将反式激活因子的结合与特定基因座处改变或受到干扰的染色质组织关联起来。
