Genetic selection for genes encoding sequence-specific DNA-binding proteins.
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.
Elledge
S J
SJ
Department of Biochemistry, Stanford University School of Medicine, CA 94305.
Sugiono
P
P
Guarente
L
L
Davis
R W
RW
D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012641:Selection, Genetic; D014157:Transcription Factors / Q000235:genetics
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA