The Laboratory of Chromatin Biology, The Rockefeller University, New York, NY 10065, USA.
Methods. 2010 May;51(1):3-10. doi: 10.1016/j.ymeth.2009.12.014. Epub 2010 Jan 4.
Histones are the major protein components of chromatin, the physiological form of the genome in all eukaryotic cells. Chromatin is the substrate of information-directed biological processes, such as gene regulation and transcription, replication, and mitosis. A long-standing experimental model system to study many of these processes is the extract made from the eggs of the anuran Xenopus laevis. Since work in recent years has solidified the importance of post-translational modification of histones in directing biological processes, the study of histones in a biochemically dissectible model such as Xenopus is crucial for the understanding of their biological significance. Here we present a rationale and methods for isolating and studying histones and chromatin in different Xenopus egg and oocyte extracts. In particular, we present protocols for the preparation of: cell-free egg and oocyte extract; nucleoplasmic extract ("NPE"); biochemical purification of maternally-deposited, stored histones in the oocyte and the egg; assembly of pronuclei in egg extract and the isolation of pronuclear chromatin and histones; and an extract chromatin assembly assay. We also demonstrate aspects of the variability of the system to be mindful of when working with extract and the importance of proper laboratory temperature in preparing quality extracts. We expect that these methods will be of use in promoting further understanding of embryonic chromatin in a unique experimental system.
组蛋白是染色质的主要蛋白质成分，染色质是所有真核细胞中基因组的生理形式。染色质是信息指导的生物过程（如基因调控和转录、复制和有丝分裂）的底物。长期以来，研究这些过程的实验模型系统是来自非洲爪蟾（Xenopus laevis）的卵提取物。由于近年来的工作巩固了组蛋白翻译后修饰在指导生物过程中的重要性，因此在类似于非洲爪蟾的生化可分析模型中研究组蛋白对于理解其生物学意义至关重要。在这里，我们提出了一种从不同的非洲爪蟾卵和卵母细胞提取物中分离和研究组蛋白和染色质的原理和方法。特别是，我们提出了以下方案：无细胞卵和卵母细胞提取物的制备；核质提取物（“NPE”）；卵母细胞中母源沉积、储存组蛋白的生化纯化；卵提取物中原核的组装以及原核染色质和组蛋白的分离；以及提取染色质组装测定。我们还展示了该系统的可变性方面，以提醒在使用提取物时要注意的问题，以及在制备优质提取物时适当的实验室温度的重要性。我们期望这些方法将有助于在独特的实验系统中进一步了解胚胎染色质。
