Genetic selection for genes encoding sequence-specific DNA-binding proteins.

Abstract

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.