Axelrod N J, Carmichael G G, Farabaugh P J
Department of Microbiology, University of Connecticut Health Center, Farmington 06032.
Mol Cell Biol. 1990 Mar;10(3):947-57. doi: 10.1128/mcb.10.3.947-957.1990.
Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synthesis, and three fragments from the polyomavirus enhancer region stimulated moderate levels. Three of the four controls of random DNA sequences failed to stimulate significant levels, and the fourth stimulated moderate levels. The stimulation in all cases was independent of the orientation of the inserted fragment. Two series of clones were examined in which between one and six tandemly arranged copies of a fragment were inserted into the XhoI site of the vector. Very interestingly, we detected an apparent exponential relationship between the number of copies of a fragment and the amount of beta-galactosidase produced. Southern analysis showed that increases in enzyme activity were not a result of increased plasmid copy number. Rather, quantitative S1 nuclease analysis demonstrated that the increases were correlated with steady-state levels of lacZ-specific mRNA. We suggest that there may be an evolutionary relationship between some transcriptional activation sequences in yeast cells and the higher eucaryotic regulatory elements that we tested.
通过将十个高等真核生物DNA片段插入缺乏上游激活序列的CYC1::lacZ启动子上游，在酿酒酵母中测试了它们的上游激活序列活性。含有21个碱基对重复区域、猿猴病毒40增强子或两者的片段强烈刺激β-半乳糖苷酶的合成，来自多瘤病毒增强子区域的三个片段刺激水平中等。四个随机DNA序列对照中有三个未能刺激到显著水平，第四个刺激水平中等。所有情况下的刺激都与插入片段的方向无关。检查了两个系列的克隆，其中一个片段的一到六个串联排列的拷贝被插入载体的XhoI位点。非常有趣的是，我们检测到片段拷贝数与产生的β-半乳糖苷酶量之间存在明显的指数关系。Southern分析表明酶活性的增加不是质粒拷贝数增加的结果。相反，定量S1核酸酶分析表明这些增加与lacZ特异性mRNA的稳态水平相关。我们认为酵母细胞中的一些转录激活序列与我们测试的高等真核生物调控元件之间可能存在进化关系。
