Tsai S Y, Tsai M J, Kops L E, Minghetti P P, O'Malley B W
J Biol Chem. 1981 Dec 25;256(24):13055-9.
Total HeLa cell extract was separated into multiple components using first DEAE-Sephadex and then phosphocellulose column chromatography. Four major fractions, DE175, DE500, P100, and P1000, from HeLa cells are found to be essential for accurate and efficient transcription of cloned ovalbumin DNA fragments in the reconstituted system. DE175 serves as the source for RNA polymerase II and DE500 minimizes the synthesis of small molecular size RNA. P100 enhances the levels of specific transcription, while P1000 is absolutely required for correct initiation. Chick oviduct crude extract was also fractionated into multiple components according to the same procedure. Similar efficiency of specific initiation could be obtained when an individual fraction (DE175, DE500, P100, or P1000) from oviduct cells was exchanged for a fraction from HeLa cells. These results indicate that chick oviduct tissue also contains general transcription factors like that of HeLa cells and these factors can be fractionated according to the same procedure. In this fractionation scheme, we were able to separate the bulk of RNase activity into the P350 fraction which was not required for initiation of ovalbumin RNA transcription. Thus, this reconstituted system is suitable for studying in vitro transcription in a homologous system derived from tissue extracts, even though a substantial amount of cellular ribonuclease may be associated with it.
首先使用二乙氨基乙基葡聚糖（DEAE - Sephadex），然后通过磷酸纤维素柱色谱法，将总的海拉细胞提取物分离成多个组分。发现来自海拉细胞的四个主要组分，即DE175、DE500、P100和P1000，对于重组系统中克隆的卵清蛋白DNA片段的准确高效转录至关重要。DE175作为RNA聚合酶II的来源，而DE500可使小分子RNA的合成降至最低。P100提高特异性转录水平，而P1000是正确起始所绝对必需的。鸡输卵管粗提取物也按照相同程序被分离成多个组分。当用输卵管细胞的单个组分（DE175、DE500、P100或P1000）替换海拉细胞的组分时，可获得相似的特异性起始效率。这些结果表明，鸡输卵管组织也含有像海拉细胞那样的通用转录因子，并且这些因子可以按照相同程序进行分离。在这个分离方案中，我们能够将大部分核糖核酸酶活性分离到P350组分中，而卵清蛋白RNA转录起始并不需要该组分。因此，这个重组系统适用于研究源自组织提取物的同源系统中的体外转录，尽管可能有大量细胞核糖核酸酶与之相关。
