Nawaz Z, Tsai M J, McDonnell D P, O'Malley B W
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Gene Expr. 1992;2(1):39-47.
A rapid method for defining novel steroid-responsive elements has been developed. Large libraries of degenerate oligonucleotides were analyzed using a yeast-based screen to identify estrogen-responsive DNA sequences. From a library of 40,000 recombinants, seven estrogen-responsive clones were identified. When sequenced, these elements showed remarkable diversity and were different from the consensus vitellogenin A2 ERE. One surprising result was the presence of the two half sites as direct repeats in some of the clones. This implies that in vivo estrogen receptor can bind and transactivate yeast genes through response elements in which the two half sites align as direct repeats. This protocol requires no purified protein and specifically selects for functional response elements. It has a wide application in the study of any transcription factor/DNA interaction.
已开发出一种定义新型类固醇反应元件的快速方法。使用基于酵母的筛选方法分析了大量简并寡核苷酸文库，以鉴定雌激素反应性DNA序列。从一个包含40,000个重组体的文库中，鉴定出了七个雌激素反应性克隆。测序时，这些元件显示出显著的多样性，并且与卵黄蛋白原A2雌激素反应元件的共有序列不同。一个令人惊讶的结果是，在一些克隆中，两个半位点以直接重复的形式存在。这意味着在体内，雌激素受体可以通过两个半位点以直接重复形式排列的反应元件来结合并激活酵母基因。该方案无需纯化的蛋白质，可特异性选择功能性反应元件。它在任何转录因子/DNA相互作用的研究中都有广泛应用。
