Chromosomal integration of a binding domain:bait gene into yeast enhances detection in the two-hybrid system.
We have developed a modified yeast two-hybrid system using the GAL4 transcription activator by integrating a BD:bait gene (GAL4 binding domain:bait gene) into the host chromosome. Locus-specific integration by homologous recombination and use of a strong transcription promoter enabled uniform expression of the integrated BD:bait gene in all host cells. Moreover, after introduction of an AD:prey (GAL4 activation domain:prey) plasmid, false positives were nearly undetectable and false negatives were decreased significantly. Using this improved method a cDNA library could be efficiently screened using roughly 1/10 of the materials required for conventional sequential two-hybrid screen.
Miyashita
Shuhei
S
Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Shirako
Yukio
Y
D015825:Chromosomes, Fungal; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins; D015723:Gene Library; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics; D020798:Two-Hybrid System Techniques
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors