Genome sequencing projects have revealed thousands of genes of unknown function. For budding yeast, large-scale gene deletion analysis has shown that over 80% of the ~6,200 predicted or known yeast genes are not required for viability - this remarkable result means that many genes and pathways in eukaryotic cells must be functionally redundant or buffered from phenotypic consequences following genetic perturbation. To address this problem, our collaborative yeast group in Toronto - led by Charlie Boone's group - has developed an automated method for systematic construction of double mutants. We call this approach synthetic genetic array or SGA analysis - the inviability or compromised growth of double mutant meiotic progeny identifies functional relationships between genes.

The basic method is diagrammed below and our proof of principle study with screens using several genes with roles in cell polarity or DNA repair generated a network of 291 interactions involving 204 genes (see network figure below). The Boone, Andrews and Tyers' labs are now systematically applying this approach to produce a gene interaction map in which each gene is assigned a function based on its position in the global network. We are also working with colleagues in Toronto to integrate information derived from DNA microarray experiments and systematic proteomics approaches with our genetic interaction map. We are currently expanding our SGA platform to other types of genetic interactions. For example, we can now efficiently introduce any plasmid of interest into the yeast deletion array enabling us to extend the application of SGA analysis to examine synthetic dosage lethality (SDL). Functional genomics approaches are currently being applied to specific projects in the lab (see people of details) and also to a larger effort to map genetic networks controlling cell cycle progression and cell polarity in yeast.

 

Synthetic Genetic Array methodology for the construction of double mutant meiotic progeny and synthetic lethal analysis.

First, a MATa strain carrying a query mutation (bni1D in this example) linked to a dominant selectable marker, such as the nourseothricin-resistance marker natMX that confers resistance to the antibiotic nourseothricin, and an MFA1pr-HIS3 reporter, is crossed to an ordered array of MATa viable yeast deletion mutants, each carrying a gene deletion mutation linked to a kanamycin-resistant marker (kanMX). Growth of resultant heterozygous diploids is selected for on medium containing nourseothricin and kanamycin. Second, the heterozygous diploids are transferred to medium with reduced levels of carbon and nitrogen to induce sporulation and the formation of haploid meiotic spore progeny. Third, spores are transferred to synthetic medium lacking histidine, which allows for selective germination of MATa meiotic progeny because these cells express the MFA1pr-HIS3 reporter specifically. Finally, the MATa meiotic progeny are transferred to medium that contains both nourseothricin and kanamycin, which then selects for growth of double mutant meiotic progeny. Within budding yeast cells or spores, the gene deletions are represented as filled circles, whereas the wild-type gene is represented as an open circle.