Figure 2

Experimental refinement procedure. (a) Overview of procedure and error detection. Beginning with 77 of the 87 deletion mutants whose experimentally measured growth phenotype differed from the model prediction under at least one condition, we tested the presence of the correct deletion mutation by PCR and whether the phenotypes were linked to the gene of interest (Materials and methods). Strains that were incorrect by PCR (12), failed to form haploid progeny in the high throughput linkage method (6), or had phenotypes unlinked to the deletion mutation (3) were excluded from further analysis. (b) High-throughput linkage analysis method. MATa haploids containing the gene deletions of interest and gridded in 96-well format are mated to a lawn of the MATα strain containing a HIS3 reporter gene under the control of the MFA1 promoter. This construct only expresses HIS3 in MATa haploid strains, and in this scheme is used to select haploid progeny that have undergone meiosis (half of which will also contain the G418-marked deletion of interest). Following mating and sporulation in 96-well format, tetrads are disrupted by digestion with zymolyase and MATa haploid progeny are selected by plating for single His+, G418r colonies. For each deletion mutant, 10 of these progeny colonies were assayed for the phenotypes of interest. Mutants in which all 10 progeny exhibited the phenotype were considered linked and candidates for further analysis.