A recombination based method to rapidly assess specificity of two-hybrid clones in yeast.
The yeast two-hybrid system is frequently used to identify protein-protein interactions. Confirming the specificity of candidate clones requires separation and isolation of yeast plasmids, propagation in bacteria and testing combinations of DNA-binding and activation domain hybrids in yeast. In order to simplify this procedure, we developed a rapid method based on PCR amplification of library insert DNAs and in vivo cloning into the activation domain hybrid vector. Reporter gene activity is assayed in parallel for combinations with different DNA-binding domain hybrids. Further characterization of inserts does not require plasmid isolation and intermediate hosts.
Petermann
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R
Children's Cancer Research Institute, St. Anna Kinderspital, Kinderspitalgasse 6, A-1090 Vienna, Austria.
Mossier
B M
BM
Aryee
D N
DN
Kovar
H
H
D000957:Antigens, Viral, Tumor / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins; D005069:Evaluation Studies as Topic; D005189:False Positive Reactions; D005822:Genetic Vectors; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000235:genetics*; D011995:Recombination, Genetic; D012261:Ribonucleoproteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013539:Simian virus 40 / Q000235:genetics; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation
D000957:Antigens, Viral, Tumor; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors