da Silva Eulália M L, Rankin Susannah
Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104.
Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104;
Cold Spring Harb Protoc. 2019 Feb 1;2019(2):pdb.prot097121. doi: 10.1101/pdb.prot097121.
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 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 (e.g., 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.
在间期细胞和M期细胞中，染色体结构都受到黏连蛋白和凝聚蛋白复合体作用的强烈影响。黏连蛋白复合体在DNA复制后将每条染色体的相同拷贝（即姐妹染色单体）拴在一起，并促进正常的间期染色体结构和基因表达。相比之下，凝聚蛋白主要在M期发挥作用，并促进单个染色体的压缩。卵提取物系统特别适合分析这两种复合体的功能。卵提取物中细胞周期状态可被操控，其中含有核蛋白储备（包括凝聚蛋白和黏连蛋白），每微升足以组装数千个细胞核。从未受精卵制备的提取物因细胞静止因子（CSF）的存在而停滞在具有高水平M期激酶活性的状态，但可被刺激进入间期，在此期间DNA会自发复制。对于黏连测定，将去膜的精子核在间期提取物中孵育，在那里它们通过从提取物中募集蛋白质和其他因子（如核苷酸）进行快速同步的DNA复制和黏连建立。然后通过添加新鲜的CSF停滞提取物将提取物驱动到M期，以此评估姐妹染色单体的黏连。相比之下，由于染色体凝聚在M期提取物中会自发发生，因此将精子核直接添加到CSF提取物中以测定凝聚。
