The reverse two-hybrid system: a genetic scheme for selection against specific protein/protein interactions.
The yeast two-hybrid system is a powerful experimental approach for the characterization of protein/ protein interactions. A unique strength of the yeast two-hybrid system is the provision for genetic selection techniques that enable the identification of specific protein/protein interactions. We now report the development of a modified yeast two-hybrid system which enables genetic selection against a specific protein/protein interaction. This reverse two-hybrid system utilizes a yeast strain which is resistant to cycloheximide due to the presence of a mutant cyh2 gene. This strain also contains the wild-type CYH2 allele under the transcriptional control of the Gal1 promoter. Expression of the wild-type Gal4 protein is sufficient to restore growth sensitivity to cycloheximide. Growth sensitivity towards cycloheximide is also restored by the coexpression of the avian c-Rel protein and its I kappa B alpha counterpart, p40, as Gal4 fusion proteins. Restoration of growth sensitivity towards cycloheximide requires the association of c-Rel and p40 at the Gal1 promoter and correlates with the ability of the c-Rel/p40 interaction to activate expression from the Gal1 promoter. A genetic selection scheme against specific protein/protein interactions may be a valuable tool for the analysis of protein/protein interactions.
Leanna
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CA
Biochemistry Department, University of Missouri-Columbia 65212, USA.
Hannink
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D017487:Ankyrins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004352:Drug Resistance, Microbial / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D017930:Genes, Reporter; D020672:I-kappa B Proteins; D016296:Mutagenesis; D000072000:NF-KappaB Inhibitor alpha; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020569:Proto-Oncogene Proteins c-rel; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D012641:Selection, Genetic; D014157:Transcription Factors; D015533:Transcriptional Activation; D014170:Transformation, Genetic
D017487:Ankyrins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D020672:I-kappa B Proteins; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000072000:NF-KappaB Inhibitor alpha; D003513:Cycloheximide