A sister chromatid cohesion assay using Xenopus egg extracts
A sister chromatid cohesion assay using Xenopus egg extracts
复制标题
使用非洲爪蟾卵提取物进行姐妹染色单体凝聚力测定
DOI：
10.1007/978-1-4939-6545-8_1
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发表时间：
2017
影响因子：
--
通讯作者：
Tatsuya Hirano
中科院分区：
文献类型：
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作者：
Keishi Shintomi;Tatsuya Hirano
从非洲爪蟾提取的无细胞提取物使我们能够在试管中重现许多与细胞周期进程相关的染色体事件。当精子染色质与这些提取物一起孵育时，它首先在组装的细胞核内复制，然后转化为有丝分裂染色体，在每个染色体中，姐妹染色单体以粘着蛋白依赖的方式彼此并列。在这里，我们描述了我们的协议，组装重复的染色体使用卵子提取物，沿着细胞学和生化分析，以解决姐妹染色单体凝聚的分子机制。还包括一个强大的方法，涉及免疫耗竭的粘连蛋白及其监管机构。
Cell-free extracts made fromXenopus laeviseggs enable us to recapitulate many chromosomal events associated with cell cycle progression in a test tube. When sperm chromatin is incubated with these extracts, it is first duplicated within an assembled nucleus, and is then transformed into mitotic chromosomes, in each of which sister chromatids are juxtaposed with each other in a cohesin-dependent manner. Here we describe our protocols for assembling duplicated chromosomes using egg extracts, along with cytological and biochemical assays for addressing the molecular mechanisms of sister chromatid cohesion. A powerful approach involving immunodepletion of cohesin and its regulators is also included.