ACCURATE AND EFFICIENT RNA POLYMERASE-II TRANSCRIPTION WITH A SOLUBLE NUCLEAR FRACTION DERIVED FROM DROSOPHILA EMBRYOS
ACCURATE AND EFFICIENT RNA POLYMERASE-II TRANSCRIPTION WITH A SOLUBLE NUCLEAR FRACTION DERIVED FROM DROSOPHILA EMBRYOS
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DOI：
10.1073/pnas.88.3.1024
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发表时间：
1991-02-01
影响因子：
11.1
通讯作者：
KADONAGA, JT
中科院分区：
文献类型：
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作者：
KAMAKAKA, RT;TYREE, CM;KADONAGA, JT
我们描述了来自果蝇胚胎的可溶性核组分的制备和生化特性。这种提取物，这可以很容易地在2.5小时内制备，是能够准确和有效的RNA聚合酶II转录的各种不同的基因从果蝇和哺乳动物。使用果蝇Kruppel基因的相对强的启动子，在单轮转录中可以实现20%的模板使用，这大大高于用标准核提取物所看到的几乎等于3%的模板使用。此外，虽然U小核RNA基因难以用HeLa转录提取物转录，但可溶性核级分转录来自果蝇的U1小核RNA基因。此外，通过序列特异性激活剂的转录激活可以在体外用可溶性核级分实现。总体转录效率似乎限于每30分钟每个DNA模板0.45个转录本，但限制机制尚不清楚。当需要高效率的RNA聚合酶II转录时，开发用于重建核内环境的可溶性核级分应该是有用的。
We describe the preparation and biochemical properties of a soluble nuclear fraction derived from Drosophila embryos. This extract, which can be easily prepared in 2.5 hr, is capable of accurate and efficient RNA polymerase II transcription of a variety of diverse genes from both Drosophila and mammals. With the relatively strong promoter of the Drosophila Kruppel gene, it is possible to achieve 20% template usage in a single round of transcription, which is considerably higher than the template usage of almost-equal-to 3% seen with standard nuclear extracts. Further, although U small nuclear RNA genes are refractory to transcription with HeLa transcription extracts, the soluble nuclear fraction transcribes a U1 small nuclear RNA gene from Drosophila. Moreover, transcriptional activation by sequence-specific activators can be attained in vitro with the soluble nuclear fraction. The overall transcriptional efficiency appears limited to 0.45 transcript per template of DNA per 30 min, but the mechanism of limitation is not known. The soluble nuclear fraction, which was developed to recreate the environment within the nucleus, should be useful when high efficiencies of RNA polymerase II transcription are desired.