Elledge S J, Sugiono P, Guarente L, Davis R W
Department of Biochemistry, Stanford University School of Medicine, CA 94305.
Proc Natl Acad Sci U S A. 1989 May;86(10):3689-93. doi: 10.1073/pnas.86.10.3689.
We describe a genetic selection method designed to facilitate the cloning of genes encoding sequence-specific DNA-binding proteins. The strategy selects for clones expressing particular sequence-specific DNA-binding activities from a library of clones encoding other, nonspecific proteins. Specific DNA-binding sites have been placed near the start of transcription of the strong synthetic conII promoter to create promoters that can be repressed by the corresponding sequence-specific DNA-binding proteins. Transcription from the conII derivatives in the absence of repression interferes with the phenotypic expression of an adjacent drug-resistance gene, aadA. Sequence-specific DNA-binding proteins are shown to repress these promoters and alleviate transcriptional interference of aadA, resulting in drug resistance in cells expressing the appropriate DNA-binding protein.
我们描述了一种基因筛选方法，该方法旨在促进对编码序列特异性DNA结合蛋白的基因进行克隆。此策略从编码其他非特异性蛋白的克隆文库中筛选出表达特定序列特异性DNA结合活性的克隆。已将特异性DNA结合位点置于强合成conII启动子转录起始位点附近，以创建可被相应序列特异性DNA结合蛋白抑制的启动子。在没有抑制作用的情况下，来自conII衍生物的转录会干扰相邻抗药基因aadA的表型表达。序列特异性DNA结合蛋白可抑制这些启动子并减轻aadA的转录干扰，从而使表达适当DNA结合蛋白的细胞产生抗药性。
