A survey of yeast genomic assays for drug and target discovery

Abstract

Over the past decade, the development and application of chemical genomic assays using the model organism Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of known drugs and novel small molecules in vivo. These assays identify drug target candidates, genes involved in buffering drug target pathways and also help to define the general cellular response to small molecules. In this review, we examine current yeast chemical genomic assays and summarize the potential applications of each approach.

Figure 1

A) Diagram of the HIPHOP assay (1) The yeast deletion collection is pooled and each strain is included at approximately equal abundance. (2) The pool is grown competitively in a compound of choice. (3) Genomic DNA is isolated from the pooled compound treated sample. (4) Up and down barcodes are PCR amplified in 2 separate reactions. (5) The PCR product is hybridized to a TAG4 barcode microarray to assess relative abundance of each strain by hybridization intensity. The intensity on the microarray serves as a proxy for strain abundance, intensities that are significantly reduced compared with the control identify strains sensitive to compound. B) Comparison of genetic interactions and compound-gene interactions (1) A query strain consisting of a mutation in Your Favourite Gene (yfgΔ) is crossed into an ordered array of ~4,000 non-essential deletion strains (designated as 1-4000Δ) of the opposite mating type using the Synthetic Genetic Array (SGA) protocol and (2) the resulting double mutant haploid progeny are selected on plates containing the appropriate media (Tong & Boone, 2006). Colony size is used to identify those strains that are reduced in sized and represent genetic interactions. (3) The same ordered array of ~4,000 non-essential deletion strains, as in (1), is pinned onto plates containing drug targeting Yfg. (4) Colony size is used to identify those colonies that are reduced in sized and therefore identify deletion mutants sensitive to compound. C) Multi-copy suppression profiling (1) An ORFeome library constructed by one of several methods (See Table 1) is transformed en masse, into a wildtype yeast strain. (2) The resulting pool is grown in a compound of choice. (3) Plasmid DNA is isolated. (4) Inserts are amplified using plasmid primers that flank each insert. (5) Amplicons are then labeled using a biotin labeling mix and a Klenow fragment, to generate short strands of labeled DNA molecules (denoted as coloured trapezoids), that are hybridized to a TAG4 microarray carrying the complementary ORF-specific probes. The trapezoid generated from the ORFs are used to distinguish them as ORF probes from the rectangular barcode amplicons in Figure 1A. In this scenario, intensities that are significantly increased on the array compared to the control identify ORFs that confer drug resistance. D) Complementation of compound resistant mutants (1) A haploid drug resistant (resistance designated as the red-hue in the yeast) strain is isolated and confirmed that the resistant phenotype is recessive by crossing to a wildtype haploid strain to verify that drug sensitivity is restored. (1) The original resistant strain is transformed with the MoBY-ORF library. (2) The resulting pool is grown in a high concentration of drug; strains that are sensitive due to complementation by plasmid are depleted from the pool. (3) Plasmid DNA is isolated. (4) Barcodes are amplified using universal primers that flank each barcode. (5) Amplicons are hybridized to a TAG4 microarray carrying the complementary barcode probes. Intensities that are significantly reduced compared with the control identify strains that harbor the ORF carried by plasmid responsible for drug resistance. In a complementary approach, dominant compound-sensitive strains can be transformed with the MoBY-ORF library and those ORFs that render the mutant compound-sensitive can be identified.