Hayes J J, Lee K M
Department of Biochemistry, University of Rochester Medical Center, New York, 14642, USA.
Methods. 1997 May;12(1):2-9. doi: 10.1006/meth.1997.0441.
Increasingly, biochemical analyses of processes that occur within eukaryotic nuclei such as transcription and replication require the construction of specific chromatin substrates. Nucleosome complexes reconstituted in vitro have been key elements in a variety of recent studies of polymerase progression and trans-acting factor binding activities. Reconstituted complexes can be easily constructed from purified components in quantities suitable for biochemical and biophysical studies. In addition, reconstituted mononucleosome complexes exhibit native biochemical and biophysical properties but necessarily contain much less heterogeneity with regard to both protein and DNA components than bulk complexes isolated from natural sources. This review details the protocols for reconstitution of model mononucleosome complexes that contain unique DNA sequences and specifically tailored core histone proteins and describes common pitfalls associated with these procedures.
越来越多的针对真核细胞核内发生的过程（如转录和复制）的生化分析需要构建特定的染色质底物。体外重建的核小体复合物已成为近期各种关于聚合酶进程和反式作用因子结合活性研究的关键要素。重组复合物可以很容易地从纯化的组分中构建出来，其数量适合进行生化和生物物理研究。此外，重建的单核小体复合物具有天然的生化和生物物理特性，但与从天然来源分离的整体复合物相比，其蛋白质和DNA组分的异质性必然要小得多。本综述详细介绍了含有独特DNA序列和经过特殊定制的核心组蛋白的模型单核小体复合物的重建方案，并描述了与这些操作相关的常见问题。
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