Combining the Dihydrofolate Reductase Protein-Fragment Complementation Assay with Gene Deletions to Establish Genotype-to-Phenotype Maps of Protein Complexes and Interaction Networks.
Systematically measuring the impact of gene deletion on protein-protein interactions is a promising approach to reveal the structural bases of protein interaction networks and to allow a better understanding of how genotypes translate into phenotypes. Genetic and protein-interaction tools in yeast now allow us to explore this third dimension of protein-protein interaction networks. Because it is scalable and quantitative, the protein-fragment complementation assay (PCA) using dihydrofolate reductase (DHFR) as the reporter protein provides an exceptionally powerful tool for such a purpose. Here, we describe a fully automated protocol that combines DHFR PCA for protein-protein interaction measurement and synthetic genetic array (SGA) technology for introducing mutant and other alleles into PCA strains using genetic crosses. In this, PCA strains are crossed with strains carrying a gene deletion and SGA markers, and the recombinant haploid progeny are selected by SGA. The resulting haploid strains, each expressing a DHFR-fragment fusion protein in a gene-specific haploid deletion background, are crossed to measure the interaction between the two recombinant proteins by PCA in a diploid homozygous deletion background. This approach can be used to measure a single protein interaction in a large array of genetic backgrounds or a large number of protein interactions in a small number of genetic backgrounds.
Département de Biologie, Institut de Biologie Intégrative et des Systèmes, PROTEO-Québec Research Network on Protein Function, Structure and Engineering, Université Laval, Québec, Québec G1V 0A6, Canada.
Guillaume
G
Diss
Département de Biologie, Institut de Biologie Intégrative et des Systèmes, PROTEO-Québec Research Network on Protein Function, Structure and Engineering, Université Laval, Québec, Québec G1V 0A6, Canada.
Christian R
CR
Landry
D017353:Gene Deletion; D017930:Genes, Reporter; D005820:Genetic Testing / Q000379:methods*; D050296:Microbial Viability; D025941:Protein Interaction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development*; D012641:Selection, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
D013762:Tetrahydrofolate Dehydrogenase