Chromosome Cohesion and Condensation in Xenopus Egg Extracts.
Chromosome Cohesion and Condensation in Xenopus Egg Extracts.
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非洲爪蟾卵提取物中的染色体凝聚和缩合。
DOI：
10.1101/pdb.prot097121
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发表时间：
2019
影响因子：
--
通讯作者：
Rankin,Susannah
中科院分区：
文献类型：
--
作者：
daSilva,EuláliaML;Rankin,Susannah
间期和M期细胞的染色体结构都受到粘附素和凝集素蛋白复合体作用的强烈影响。在DNA复制后，粘附素复合体将每条染色体的相同拷贝(称为姐妹染色单体)连接在一起，促进正常的间期染色体结构和基因表达。相反，凝聚素主要活跃在M期，并促进单个染色体的紧凑。非洲爪哇的鸡蛋提取系统非常适合分析这两个复合体的功能。可以操纵细胞周期状态的鸡蛋提取物含有足够组装每微升数千个细胞核的核蛋白(包括凝集素和粘附素)。从未受精卵中制备的提取物在具有高水平M期激酶活性的状态下被细胞抑制因子(CSF)抑制，但可以被刺激进入间期，在间期DNA复制自发发生。对于凝聚力分析，去膜精子核在间期提取液中孵育，在那里它们通过从提取液中招募蛋白质和其他因素(如核苷酸)进行快速和同步的DNA复制和凝聚力建立。姐妹染色单体凝聚力的评估是通过添加新鲜的脑脊液抑制提取物将提取物驱动到M期来进行的。相反，由于染色体凝聚在M期提取液中自发发生，精子核被直接添加到脑脊液提取液中以检测凝聚。
Chromosome structure in both interphase and M-phase cells is strongly influenced by the action of the cohesin and condensin protein complexes. The cohesin complex tethers the identical copies of each chromosome, called sister chromatids, together following DNA replication and promotes normal interphase chromosome structure and gene expression. In contrast, condensin is active largely in M phase and promotes the compaction of individual chromosomes. The Xenopus egg extract system is uniquely suited to analyze the functions of both complexes. Egg extracts, in which the cell cycle state can be manipulated, contain stockpiles of nuclear proteins (including condensin and cohesin) sufficient for the assembly of thousands of nuclei per microliter. Extract prepared from unfertilized eggs is arrested by the presence of cytostatic factor (CSF) in a state with high levels of M-phase kinase activity, but can be stimulated to enter interphase, in which DNA replication occurs spontaneously. For cohesion assays, demembranated sperm nuclei are incubated in interphase extract, where they undergo rapid and synchronous DNA replication and cohesion establishment through the recruitment of proteins and other factors (eg, nucleotides) from the extract. Sister chromatid cohesion is assessed by then driving the extract into M phase by the addition of fresh CSF-arrested extract. In contrast, because chromosome condensation occurs spontaneously in M-phase extracts, sperm nuclei are added directly to CSF extracts to assay condensation.