Academic journal article Genetics

Eliminating Gene Conversion Improves High-Throughput Genetics in Saccharomyces Cerevisiae

Academic journal article Genetics

Eliminating Gene Conversion Improves High-Throughput Genetics in Saccharomyces Cerevisiae

Article excerpt


Synthetic genetic analysis was improved by eliminating leaky expression of the HIS3 reporter and gene conversion between the HIS3 reporter and hisΔ1. Leaky expression was eliminated using 3-aminotriazole and gene conversion was eliminated by using the Schizosaccharomyces pombe his5^sup +^ gene, resulting in a 5- to 10-fold improvement in the efficiency of SGA.

SYNTHETIC genetic array analysis (SGA) detects genetic interactions using the collection of deletion mutants of nonessential genes in yeast (ToNG et al. 2001, 2004). We adopted SGA for use on a small scale in laboratories that do not have access to robotics. SGA performed manually had a low success rate as judged by a number of criteria. We developed a "gold standard" set of genes that were synthetic lethal with mad2 and showed that a typical SGA screen, using manual manipulations, identified <10% of the bona fide interactions. SGA efficiency improved after eliminating leaky expression of the MFAl promoter-driven HIS3 reporter. Gene conversion between the HIS3 reporter gene and the his3α. 1 allele in strains isogenic with BY4741, BY4742, and BY4743 increased false negatives and conversion was eliminated by using the Schizosaccharomyces pombe his5+ gene.

The success of SGA depends on being able to select haploid spores after meiosis. SGA utilizes a MATa query strain that contains the mutant of interest marked by a deletion with a nourseothricin resistance marker (NAT), a fusion gene with HIS3 under the control of the MFA^ promoter and canl (TONG et al. 2001 ). MFA^ is a haploid-specific gene that is expressed only in MATa. cells (MiCHAELis and HERSKOWITZ 1988). The query strain is mated to the collection of deletion mutants where every mutation is a deletion replaced by G418 resistance (WACH et al. 1994). Diploids are isolated and sporulated, and haploids are selected on SC-his medium containing canavanine. After meiosis, populations of spores from many méioses are transferred to the haploid selection plate; the exact number depends on the sporulation efficiency of the individual cross. The haploids are then tested on medium containing NAT and G418 to determine if double mutants are inviable or compromised for growth. We constructed a query strain with mad2::NAT, performed SGA, and assayed for synthetic lethal interactions using manual manipulations (see supplemental material at http:/7 supplemental/ ).

Three evaluation criteria indicated that our SGA screens were yielding limited success. The first was that three successive screens using the identical mad2::NAT query strain identified <10% overlap between the screens. The second indicator was that we failed to identify mutations in the full set of genes encoding proteins of multisubunit complexes. The third indicator was that we found little linkage to MAD2 in three separate screens (ToNG et al. 2001, 2004). To determine the overall efficiency of the mad2 screens, we chose 170 mutants that are annotated as affecting functions relating to microtubules, cytoskeleton, kinetochore, chromosome segregation, and cell cycle (supplemental Table 1 at We reasoned that a subset of these mutants should compromise chromosome segregation and show a synthetic phenotype in combination with mad2. The mutants were mated to mad2::NATand each cross was evaluated directly using random spore analysis. We identified 22 mutants that showed synthetic lethal interactions with mad2::NAT and served as a gold standard tojudge the efficiency of a given SGA screen (supplemental Table 2 at http://wvm.

Two independent mad2 screens, performed with the entire arrays of ^47OO mutants by manual pinning, identified only one and three of the known synthetic lethal interactions, respectively, and therefore detected 4 and 9% of the interactions. We picked 10 of the false negative colonies and in every case there were mixtures of colonies that were MATa and colonies that were sterile, suggesting they were a mixture of haploids and MATa/ MATa diploids. …

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