Grosschedl R, Birnstiel M L
Proc Natl Acad Sci U S A. 1982 Jan;79(2):297-301. doi: 10.1073/pnas.79.2.297.
Sea urchin (psammechinus miliaris) H2A histone genes shown to be promoter mutants from oocyte injection experiments were tested for their ability to initiate transcription in vitro. Circular templates were transcribed with HeLa cell extracts, and the transcripts were assayed by mung bean or S1 nuclease mapping of the 5' ends. The transcripts of the H2A mutants produced in vitro were qualitatively similar and, in most cases, identical to those seen in oocyte injection experiments, but quite large quantitative differences were observed for some H2A mutant genes. Both the T-A-T-A box and far upstream sequences residing in the modulator segment E [Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 7102--7106] were found to be essential for maximal transcription in vitro. Deletion of either of these sequence elements reduced transcription to 20%. A similar reduction in the amount of H2a transcripts was found when a T-A-T-A-to-T-A-G-A point mutant was tested in vitro. Essential far upstream sequences were mapped between nucleotides -139 and -111, 5' to the initiation site of transcription. In the standard run-off transcription test using restriction fragments, the effects of these sequences could be mimicked by free DNA ends, suggesting that the function of this in vitro upstream sequence might be to provide an entry side for RNA polymerase II.
通过卵母细胞注射实验已证明海胆（球海胆）H2A组蛋白基因是启动子突变体，对其体外启动转录的能力进行了测试。用HeLa细胞提取物转录环状模板，并通过绿豆核酸酶或S1核酸酶对5'端进行图谱分析来检测转录本。体外产生的H2A突变体转录本在质量上相似，并且在大多数情况下与卵母细胞注射实验中观察到的转录本相同，但对于一些H2A突变基因观察到了相当大的数量差异。发现位于调节区段E [格罗斯切德尔，R.和伯恩施泰尔，M. L.（1980年）美国国家科学院院刊77，7102 - 7106]中的T - A - T - A框和远上游序列对于体外最大转录是必不可少的。删除这些序列元件中的任何一个都会使转录降低到20%。当在体外测试T - A - T - A到T - A - G - A点突变体时，发现H2a转录本的量有类似的减少。必需的远上游序列被定位在转录起始位点5'端的核苷酸-139和-111之间。在使用限制性片段的标准径流转录测试中，这些序列的作用可以被游离DNA末端模拟，这表明这种体外上游序列的功能可能是为RNA聚合酶II提供一个进入位点。
