Analysis of nuclear reconstitution, nuclear envelope assembly, and nuclear pore assembly using Xenopus in vitro assays.
Analysis of nuclear reconstitution, nuclear envelope assembly, and nuclear pore assembly using Xenopus in vitro assays.
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DOI：
10.1016/b978-0-12-417160-2.00008-4
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发表时间：
2014
影响因子：
--
通讯作者：
Forbes, Douglass J.
中科院分区：
文献类型：
--
作者：
Bernis, Cyril;Forbes, Douglass J.
大而复杂的真核细胞核是DNA复制、RNA转录、剪接和核糖体组装的仲裁者。随着20世纪80年代来自非洲爪蟾卵的体外核重建提取物的出现，在体外围绕添加的DNA或染色质底物组装多个核成为可能。这种重组的细胞核含有核纤层、双核膜、核孔，并且能够进行DNA复制和核输入。体外核重建允许组装“野生型”和“生化突变型”核，其中可以评估单个组分的影响。在这里，我们描述了核重建提取物的制备方案，体外核重建，核膜完整性的评估，和一个更专门的核孔组装成预成型的无孔核中间体的测定。
The large and complex eukaryotic nucleus is the arbiter of DNA replication, RNA transcription, splicing, and ribosome assembly. With the advent of in vitro nuclear reconstitution extracts derived from Xenopus eggs in the 1980s, it became possible to assemble multiple nuclei in vitro around added DNA or chromatin substrates. Such reconstituted nuclei contain a nuclear lamina, double nuclear membranes, nuclear pores, and are competent for DNA replication and nuclear import. In vitro nuclear reconstitution has allowed the assembly of “wild-type” and “biochemically mutant” nuclei in which the impact of individual components can be assessed. Here, we describe protocols for preparation of the nuclear reconstitution extract, nuclear reconstitution in vitro, assessment of nuclear membrane integrity, and a more specialized assay for nuclear pore assembly into preformed pore-free nuclear intermediates.