Berge T, Bergholtz S L, Andersson K B, Gabrielsen O S
Department of Biochemistry, University of Oslo, PO Box 1041, Blindern, N-0316 Oslo 3, Norway.
Nucleic Acids Res. 2001 Oct 15;29(20):E99. doi: 10.1093/nar/29.20.e99.
Yeast (Saccharomyces cerevisiae) has proved to be a highly valuable tool in a range of screening methods. We present in this work the design and use of a novel yeast effector-reporter system for selection of sequences recognised by DNA-binding proteins in vivo. A dual HIS3-lacZ reporter under the control of a single randomised response element facilitates both positive growth selection of binding sequences and subsequent quantification of the strength of the selected sequence. A galactose-inducible effector allows discrimination between reporter activation caused by the protein under study and activation due to endogenous factors. The system mimics the physiological gene dosage relationship between transcription factor and target genes in vivo by using a low copy effector plasmid and a high copy reporter plasmid, favouring sequence selectivity. The utility of the novel yeast screening system was demonstrated by using it to refine the definition of an optimal recognition element for the c-Myb transcription factor (MRE). We present screening data supporting an extended MRE consensus closely mimicking known strong response elements and where a sequence of 11 nt influences activity. Novel features include a more strict sequence requirement in the second half-site of the MRE where a T-rich sequence is preferred in vivo.
酵母（酿酒酵母）已被证明是一系列筛选方法中非常有价值的工具。在这项工作中，我们展示了一种新型酵母效应物-报告基因系统的设计和应用，用于在体内筛选被DNA结合蛋白识别的序列。在单个随机响应元件控制下的双HIS3-lacZ报告基因，既便于对结合序列进行正向生长筛选，又能对所选序列的强度进行后续定量分析。半乳糖诱导型效应物能够区分由所研究蛋白质引起的报告基因激活和内源性因素导致的激活。该系统通过使用低拷贝效应物质粒和高拷贝报告基因质粒，模拟了体内转录因子与靶基因之间的生理基因剂量关系，有利于序列选择性。通过使用新型酵母筛选系统来完善c-Myb转录因子（MRE）最佳识别元件的定义，证明了该系统的实用性。我们展示的筛选数据支持了一个扩展的MRE共有序列，该序列紧密模拟已知的强响应元件，且11个核苷酸的序列影响活性。新特性包括MRE后半位点更严格的序列要求，在体内富含T的序列更受青睐。
