Accurate initiation of rat ribosomal RNA gene transcription using a fractionated nuclear extract from normal liver and a hepatoma.
Accurate initiation of rat ribosomal RNA gene transcription using a fractionated nuclear extract from normal liver and a hepatoma.
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使用正常肝脏和肝癌的分级核提取物准确启动大鼠核糖体 RNA 基因转录。
DOI：
10.1073/pnas.82.4.1059
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发表时间：
1985
影响因子：
11.1
通讯作者：
Jacob,ST
中科院分区：
文献类型：
--
作者：
Kurl,RN;Jacob,ST
用DEAE-Sephadex柱层析法分离了正常大鼠肝和Morris肝癌3924 A细胞核提取物。用175 mM（NH 4）2SO 4（DE-B）洗脱的馏分，其中含有大于90%的RNA聚合酶I活性，支持克隆大鼠rDNA的准确转录。从胞质溶胶中获得的类似组分具有rDNA转录所需的所有因子。然而，它的转录活性最多是相应核组分的六分之一，这取决于产生类似数量的转录物所需的蛋白质量。未分级的细胞核或细胞质制备物不能产生准确的转录本。最佳KCl和镁浓度分别为60 mM和5-7.5 mM。转录活性的程度按以下顺序：肝癌细胞核组分DE-B大于来自大鼠乳腺癌细胞的全细胞提取物，远大于正常肝组分DE-B。肝癌制备物产生的转录物的量是相应肝核制备物产生的转录物的量的至少10倍。转录活性与RNA聚合酶I的水平和这些组织中rRNA合成的速率成正比。
Nuclear extracts obtained from normal rat liver and from Morris hepatoma 3924A were fractionated by DEAE-Sephadex chromatography. The fraction eluted with 175 mM (NH4)2SO4 (DE-B), which contains greater than 90% of RNA polymerase I activity, supported accurate transcription of cloned rat rDNA. A similar fraction obtained from the cytosol had all of the factors required for rDNA transcription. However, its transcriptional activity was at most one-sixth that of the corresponding nuclear fraction, as determined by the amount of protein needed to produce a similar quantity of the transcript. Unfractionated nuclear or cytosol preparations did not yield an accurate transcript. Optimal KCl and magnesium concentrations for rDNA transcription were 60 mM and 5-7.5 mM, respectively. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-B greater than whole cell extract derived from rat mammary adenocarcinoma cells much greater than normal liver fraction DE-B. The hepatoma preparation produced at least 10 times the amount of transcript produced by the corresponding liver nuclear preparation. Transcriptional activity was proportional to the levels of RNA polymerase I and to the rate of rRNA synthesis in these tissues.