Characterization of nuclear matrices prepared without salt extraction.
Characterization of nuclear matrices prepared without salt extraction.
复制标题
未经盐提取制备的核基质的表征。
DOI：
10.1016/0003-2697(91)90507-p
复制
发表时间：
1991
影响因子：
2.9
通讯作者：
Wheeler,KT
中科院分区：
文献类型：
--
作者：
Dwarakanath,BS;Wallen,CA;StClair,DK;Wheeler,KT
用蛋白质和DNA凝胶电泳法、流式细胞仪和扫描电子显微镜对经DNase I和几种蛋白酶(PRT-基质)消化的小鼠乳腺癌细胞(66系)制备的核基质的结构和组成进行了表征。将这些PRT-矩阵的特性与采用高盐提取步骤的常规制备的核基质(HS-矩阵)的特性进行比较，以便选择一种可用于生化和/或生物物理研究的制剂，其中盐提取影响数据的分析或解释。在特征PRT-基质中，只有用XIV酶(链霉酶)制备的PRT-基质具有HS-基质的大部分特性。它们(I)保持其结构的完整性，(Ii)其核⩽的5%与基质相关，(Iii)没有证据表明高阶染色质结构，(Iv)dna大小分布在400-1100bp范围内。PRT-矩阵和HS-矩阵之间的主要区别是PRT-矩阵的蛋白质含量降低。虽然PRT-矩阵可能不适合于研究参与复制、转录和分化等功能的独特的核基质相关蛋白，但它们显然适合于研究核基质相关DNA的性质。
The structure and composition of the nuclear matrices prepared from a mouse mammary adenocarcinoma cell (line 66) by digestion with DNase I and several proteases (PRT-matrices) were characterized by protein and DNA gel electrophoresis, flow cytometry, and scanning electron microscopy. The characteristics of these PRT-matrices were compared with the characteristics of conventionally prepared nuclear matrices that employ a high salt extraction step (HS-matrices) in order to select a preparation that can be used in biochemical and/or biophysical studies where salt extraction compromises either the analysis or the interpretation of the data. Of the characterized PRT-matrices, only those prepared with Type XIV protease (pronase) had most of the characteristics of HS-matrices. They, (i) maintained their structural integrity, (ii) had ⩽5% of their nuclear DNA associated with the matrix, (iii) had no evidence of higher-order chromatin structure, and (iv) had a DNA size distribution in the range of 400–1100 bp. The major difference between the PRT-matrices and the HS-matrices was a decrease in the protein content of the PRT-matrices. Although the PRT-matrices may not be appropriate for studying the unique nuclear matrix associated proteins that are involved in functions such as replication, transcription, and differentiation, they are clearly suitable for studying the properties of the nuclear matrix associated DNA.