Stringent Mating-Type-Regulated Auxotrophy Increases the Accuracy of Systematic Genetic Interaction Screens with Saccharomyces Cerevisiae Mutant Arrays

By Singh, Indira; Pass, Rebecca et al. | Genetics, January 2009 | Go to article overview

Stringent Mating-Type-Regulated Auxotrophy Increases the Accuracy of Systematic Genetic Interaction Screens with Saccharomyces Cerevisiae Mutant Arrays


Singh, Indira, Pass, Rebecca, Togay, Sine Ozmen, Rodgers, John W., Hartman, John L., IV, Genetics


ABSTRACT

A genomic collection of haploid Saccharomyces cerevisiae deletion strains provides a unique resource for systematic analysis of gene interactions. Double-mutant haploid strains can be constructed by the synthetic genetic array (SGA) method, wherein a query mutation is introduced by mating to mutant arrays, selection of diploid double mutants, induction of meiosis, and selection of recombinant haploid double-mutant progeny. The mechanism of haploid selection is mating-type-regulated auxotrophy (MRA), by which prototrophy is restricted to a particular haploid genotype generated only as a result of meiosis. MRA escape leads to false-negative genetic interaction results because postmeiotic haploids that are supposed to be under negative selection instead proliferate and mate, forming diploids that are heterozygous at interacting loci, masking phenotypes that would be observed in a pure haploid double-mutant culture. This work identified factors that reduce MRA escape, including insertion of terminator and repressor sequences upstream of the MRA cassette, deletion of silent mating-type loci, and utilization of α-type instead of a-type MRA. Modifications engineered to reduce haploid MRA escape reduced false negative results in SGA-type analysis, resulting in >95% sensitivity for detecting gene-gene interactions.

GENE-gene interaction means a phenotype attributable to variation in one gene depends upon the allele status of a second gene (Barton and Keightley 2002). Relatively little is known about the overall contribution of gene interactions to natural genotype-phenotype variation. Systematic analysis of genetic interactions in experimental organisms could help in this regard to infer how naturally occurring genetic variation is expressed phenotypically (Hartman et al. 2001). In Saccharomyces cerevisiae, deletion of all nonessential genes on a single haploid genetic background enables genomewide assessment of gene interactions by parallel analysis of all mutants under various genetic or environmental conditions (Winzeler et al. 1999; Giaever et al. 2002). If performed comprehensively, genetic interaction networks can provide amore global perspective of cellular processes than is possible with studies that focus on only a few or a biased set of genes (Tong et al. 2004). The Boone laboratory developed the synthetic genetic array (SGA) methodology to introduce second mutations into the yeast gene deletion strain array, thereby enabling systematic analysis of gene-gene interactions on a genomewide scale (Tong et al. 2001; Tong and Boone 2006). To construct double mutants by SGA, a ''query'' strain, harboring a second mutation, is mated to the entire array of yeast gene deletion strains, thus creating a new array of heterozygous diploid double mutants. Sporulation and meiotic recombination produce haploid double mutants, which are recovered by media selection. Haploid selection is based on mating-typeregulated auxotrophy (MRA), which results from placing an auxotrophy-complementing gene under control of a mating-type-regulated promoter (referred to here as the ''MRA cassette''). The MRA cassette is activated for prototrophy in the opposite mating type of the query strain; thus only after meiosis are haploids created that obtain the MRA cassette from the query strain and the permissive MAT allele from the deletion array.

The Burke laboratory reported low sensitivity of SGA for detecting known synthetic lethal interactions and described gene conversion between the mating-typeregulated HIS3 auxotrophy and the his3Δ1 allele as a responsible mechanism (Daniel et al. 2006). Replacement of HIS3 with Schizosaccharomyces pombe his5^sup +^ in the MRAcassette eliminated gene conversion and improved sensitivity of SGA analysis.

Similarly, the goal of our study was to improve the purity of haploid double-mutant strains and thus the sensitivity of SGA-type analysis, by reducing formation of heterozygous diploids. …

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Stringent Mating-Type-Regulated Auxotrophy Increases the Accuracy of Systematic Genetic Interaction Screens with Saccharomyces Cerevisiae Mutant Arrays
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