Tutter Antonin V, Walter Johannes C
Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
Methods Mol Biol. 2006;322:121-37. doi: 10.1007/978-1-59745-000-3_9.
Cytoplasmic egg extracts from the frog Xenopus laevis represent a powerful cell-free system to study eukaryotic chromosomal DNA replication. In the classical approach, sperm chromatin is added to unfractionated egg cytoplasm, leading to the assembly of transport-competent nuclei that undergo a single, complete round of DNA replication. The need for nuclei in this system has been circumvented. Sperm chromatin or plasmid DNA is first incubated with clarified egg cytoplasm to form chromatin-bound prereplication complexes. Subsequently, a highly concentrated nucleoplasmic extract is added that stimulates initiation from these prereplication complexes, and a single complete round of chromosomal DNA replication ensues. This review describes the preparation of the cytosolic and nucleoplasmic extracts, as well as their use in DNA replication, origin unwinding, and chromatin isolation assays.
非洲爪蟾（Xenopus laevis）的细胞质卵提取物是研究真核染色体DNA复制的强大无细胞系统。在经典方法中，将精子染色质添加到未分级的卵细胞质中，导致形成具有运输能力的细胞核，这些细胞核会经历一轮完整的DNA复制。该系统对细胞核的需求已被规避。首先将精子染色质或质粒DNA与澄清的卵细胞质孵育，以形成与染色质结合的预复制复合物。随后，加入高浓度的核质提取物，刺激这些预复制复合物的起始，接着会发生一轮完整的染色体DNA复制。本综述描述了细胞质和核质提取物的制备方法，以及它们在DNA复制、起始点解旋和染色质分离分析中的应用。
