CONSIDERATIONS IN THE ISOLATION OF RAT-LIVER NUCLEAR MATRIX, NUCLEAR-ENVELOPE, AND PORE COMPLEX LAMINA
CONSIDERATIONS IN THE ISOLATION OF RAT-LIVER NUCLEAR MATRIX, NUCLEAR-ENVELOPE, AND PORE COMPLEX LAMINA
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DOI：
10.1016/0014-4827(81)90088-4
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发表时间：
1981-01-01
影响因子：
3.7
通讯作者：
SHAPER, JH
中科院分区：
文献类型：
--
作者：
KAUFMANN, SH;COFFEY, DS;SHAPER, JH
最近的一些研究表明，盐，核酸酶和洗涤剂的耐亚核结构称为核蛋白基质，它由一个纤维颗粒核内网络，核仁的残留成分，和周边层。然而，其他工作者已经表明，有些类似的方法导致分离缺乏核内成分的外周板层。在这份报告中，我们表明，看似轻微的变化，在隔离程序中获得的残留结构的形态造成重大变化。当新鲜纯化的大鼠肝细胞核用DNA酶I和RNA酶A消化，然后用低镁离子浓度（LS缓冲液）和高离子强度（HS缓冲液）的缓冲液提取时，在Triton X-100提取之前或之后分离的所得结构缺乏广泛的核内网络和存在于核蛋白基质中的容易识别的残留核仁。系统的修改，这种提取过程中发现，形态学上可识别的残留核仁时，消化与RNA酶A，然后用HS缓冲液提取，但不存在时，这些步骤的顺序是颠倒的。RNase A和HS缓冲液对核仁的去除与相同处理对核RNA的去除相关。这些协调事件无法通过蛋白酶抑制剂处理来预防，但可以通过用RNA酶抑制剂焦碳酸二乙酯处理RNA酶A来预防。广泛的核内网络中看到的核蛋白质基质稀疏或不存在时，残留的结构，从DNA酶和RNA酶处理的核的条件下，最大限度地减少蛋白质巯基的氧化。相比之下，广泛的非染色质的核内网络被视为如果分子间蛋白质二硫键的形成，促进提取细胞核与阳离子去污剂，通过过夜孵育，或通过处理与氧化剂，如连四硫酸钠之前，核酸酶消化和随后的提取。通过改变提取步骤的顺序和二硫化物交联的程度，可以从单批核中分离出具有广泛形态和组成的残留结构。
A number of recent studies have demonstrated a salt-, nuclease, and detergent-resistant subnuclear structure termed the nuclear protein matrix which consists of a fibrogranular intranuclear network, residual components of the nucleolus, and a peripheral lamina. Other workers, however, have shown that somewhat similar methods result in the isolation of the peripheral lamina devoid of the intranuclear components. In this report we demonstrate that seemingly slight changes in the isolation procedure cause major changes in the morphology of the residual structures obtained. When freshly purified rat liver nuclei were digested with DNase I and RNase A and then extracted with buffers of low magnesium ion concentration (LS buffer) and high ionic strength (HS buffer), the resulting structures isolated prior to or after Triton X-100 extraction lacked the extensive intranuclear network and the easily identifiable residual nucleoli present in the nuclear protein matrix. Systematic modification of this extraction procedure revealed that morphologically identifiable residual nucleoli were present when digestion with RNase A followed extraction with HS buffer but were absent when the order of these steps was reversed. The removal of the nucleolus by RNase A and HS buffer correlated with the removal of nuclear RNA by the same treatments. These coordinate events could not be prevented by treatment with protease inhibitors but were prevented by treatment of the RNase A with diethylpyrocarbonate, an RNase inhibitor. The extensive intranuclear network seen in the nuclear protein matrix was sparse or absent when residual structures were prepared from DNase- and RNase-treated nuclei under conditions which minimized the oxidation of protein sulfhydryl groups. In contrast, an extensive non-chromatin intranuclear network was seen if the formation of intermolecular protein disulfide bonds was promoted by extraction of nuclei with cationic detergents, by overnight incubation, or by treatment with oxidizing agents like sodium tetrathionate prior to nuclease digestion and subsequent extraction. By varying the order of extraction steps and the extent of disulfide cross-linking, it is possible to isolate from a single batch of nuclei residual structures with a wide range of morphologies and compositions.