Chromatin Structure Analysis of Single Gene Molecules by Psoralen Cross-Linking and Electron Microscopy
Chromatin Structure Analysis of Single Gene Molecules by Psoralen Cross-Linking and Electron Microscopy
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DOI：
10.1007/978-1-4939-1680-1_9
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发表时间：
2015-01-01
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影响因子：
--
通讯作者：
Boeger, Hinrich
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文献类型：
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作者：
Brown, Christopher R.;Eskin, Julian A.;Boeger, Hinrich
核小体通过阻断转录因子进入其染色体DNA上的靶位点而在调节真核基因表达中发挥核心作用。染色质结构和功能的分析主要通过用核酸内切酶探测DNA可及性来进行。这样的实验是对同一基因的大量分子进行平均，最近，是对整个基因组进行平均。然而，消化和平均都消除了指示动态行为的分子之间的结构变化，这对于任何调节理论都必须重建。这个问题的解决需要单基因分子的结构分析。在这一章中，我们描述了一种方法，通过该方法从酵母酿酒酵母中纯化单基因分子，并与pesticen交联，允许通过透射电子显微镜测定核小体构型。我们还提供定制分析软件，可半自动分析显微图像数据。这种单基因技术能够详细检查酵母中任何基因组位点的染色质结构。
Nucleosomes occupy a central role in regulating eukaryotic gene expression by blocking access of transcription factors to their target sites on chromosomal DNA. Analysis of chromatin structure and function has mostly been performed by probing DNA accessibility with endonucleases. Such experiments average over large numbers of molecules of the same gene, and more recently, over entire genomes. However, both digestion and averaging erase the structural variation between molecules indicative of dynamic behavior, which must be reconstructed for any theory of regulation. Solution of this problem requires the structural analysis of single gene molecules. In this chapter, we describe a method by which single gene molecules are purified from the yeast Saccharomyces cerevisiae and cross-linked with psoralen, allowing the determination of nucleosome configurations by transmission electron microscopy. We also provide custom analysis software that semi-automates the analysis of micrograph data. This single-gene technique enables detailed examination of chromatin structure at any genomic locus in yeast.