Random insertion mutations in genes were induced in a large population of yeast cells; the mutagenizing agent was a marked Ty1 transposon carried on a plasmid and driven by the GAL1 promoter. After mutagenesis, each cell within the population contained, on average, one ORF with an inserted transposon, presumably inactivating the ORF. A sample of the initial mutagenized population, which had not undergone any growth under selective conditions, was retained to provide a time-zero reference. Portions of the remaining mutagenized population were further subjected to five alternative (non-mutagenizing) growth situations: 20 doublings in YPD (YPD20), 60 doublings in YPD (YPD60); 20 doublings in minimal medium (YNB), 20 doublings in YP plus 2% Lactate (LAC), and 20 doublings in YP plus 0.9M NaCl (NACL). After growth in these conditions, we measured the proportion of cells carrying Ty insertions in a given gene that remained in the population; this proportion is a measure of that gene's contribution to fitness in each growth environment. We determined this proportion by performing PCR reactions using a gene-specific primer and a reverse primer unique to the marked mutagenizing Ty1 element; the template DNAs used were from the zero-time reference population and from the selected populations. We then derived a ratio of the number of bands (i.e., independent insertion mutations) seen in the selected population sample to the number of bands seen in the zero-time sample. Bayesian analysis was employed, using the ratios from a test set containing several hundred known "essential" genes and proposed "dubious" genes, to generate a "probability of essentiality" curve for each growth condition. Each gene assayed was then assigned a specific "probability of essentiality" for each growth condition, based on where its raw ratio score fell on the curve. These curves are shown in the SGD database, on the Phenotype Details page for each assayed gene. We took the second derivative of each curve to determine the bend points at both the top and bottom of each curve and used those points as cut-off thresholds: genes with ratios falling either below or above the bend points are determined to have either a "severe effect on growth" or "no effect on growth" respectively. Genes with ratios falling in the intermediate region between the two bend points are determined to have a "moderate growth defect" for that growth condition. Note that this study, being a genome-wide screen, relied in most cases on only one experiment per gene. Therefore, appropriate caution must be employed when interpreting the results.

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Personal Communication to SGD

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Dunn B, Ferea T, Spellman P, Schwarz J, Terraciano J, Troyanovich J, Walker S, Greene J, Shaw K, DiDomenico B, ... Show all

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