Aspects structuraux et biochimiques des effets de quelques milieux utilises pour l'isolement et le fractionnement du noyau de foie de souris
Aspects structuraux et biochimiques des effets de quelques milieux utilises pour l'isolement et le fractionnement du noyau de foie de souris
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环境影响的结构和生物化学方面利用了酸菜汤的隔离和分离
DOI：
10.1016/0014-4827(73)90238-3
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发表时间：
1973
影响因子：
3.7
通讯作者：
M. Philippe
中科院分区：
文献类型：
--
作者：
P. Chevaillier;M. Philippe
通过电子显微镜检查和生化评估回顾了通常用于分离和分级小鼠肝细胞核的各种方法。先前使用的所有方法产生的核都不够稳定，无法进行正确的细分。在分离和分级分离过程中，核成分经历结构修饰，导致常染色质和异染色质的错误分离。我们开发了一种方法来真正分离这两个染色质部分。根据 Chauveau 程序，在含有 Tris 1 mM pH 7.5、25 mM KCl、CaCl2 和 MgCl20.9 mM 以及 0.14 mM 亚精胺的 2.2 M 蔗糖中制备小鼠肝核。超声处理后，根据弗伦斯特技术的改进，通过差速离心回收核成分。获得三组级分：重级分（Frenster的级分I和II），主要由核仁、核仁相关染色质和异染色质组成； 78000g离心1小时获得的中间级分（级分III），由核膜和颗粒组成；轻组分（组分 IV）对应于常染色质，可通过 105 000 g 离心 18 小时收集。目前正在研究这些核亚级分的进一步纯化。
Various methods generally used for isolation and fractionation of mouse liver nuclei are reviewed by electron microscopic examination and biochemical estimations. All methods previously employed yield nuclei which are not sufficiently stabilized to enable correct subfractionation. During isolation and fractionation, nuclear components undergo structural modifications which lead to false separation of euchromatin and heterochromatin. A method was developed to enable true separation of these two fractions of chromatin. Mouse liver nuclei are prepared according to Chauveau's procedure in 2.2 M sucrose containing Tris 1 mM pH 7.5, 25 mM KCl, CaCl2and MgCl20.9 mM each and 0.14 mM spermidine. After sonication, nuclear components are then recovered by differential centrifugation according to a modification of Frenster's technique. Three groups of fractions are obtained: a heavy fraction (fractions I and II of Frenster) which is mainly composed of nucleoli, nucleolus-associated chromatin and heterochromatin; an intermediate fraction (fraction III) obtained by centrifugation at 78 000 g for 1 h and composed of nuclear membranes and particles; a light fraction (fraction IV) which corresponds to euchromatin and which can be collected by centrifugation at 105 000 g for 18 h. Further purification of these nuclear subfractions is now being investigated.