Validation of glutamine analog hypersensitivity suppressors.

Abstract

(A) 107 cells / mL of the indicated genotype in the S288C background were serially diluted (1:6), spotted on YPD plates with or without 50 μM DON or 1.5 mM HU and grown for 2d (Untreated, HU) or 3d (DON). (B) Exponentially growing cells of the indicated genotypes in the S288c background were treated with 100 μM DON for 2h as indicated. Metabolites were extracted and CTP levels were quantified by nano-LC-MS/MS. Values are ion intensities (peak areas) normalized by the mean of untreated wt samples. Significance was calculated by Kruskal-Wallis rank sum test (p = 0.0012), followed by Wilcoxon rank sum test. (C) 107 cells / mL of the indicated genotype in the S288C background were serially diluted (1:6), spotted on YPD plates with or without 50 μM DON or 1.5 mM HU and grown for 2d (Untreated, HU) or 3d (DON). (D) Cells of the indicated genotypes were arrested in G1 phase with α-factor, released into YPD with or without 50 μM DON for 6h and fixed with sodium azide. Chromosome integrity was analyzed by pulsed-field gel electrophoresis analysis and staining of DNA with ethidium bromide. The left panel shows a representative PFGE result from 2 independent gels. The right panel shows the quantification of DNA signal along the gel lanes containing DON-treated samples. Bars plots with error bars represent mean values and standard deviation. Representative spot assay images are shown. wt = wildtype, NCR = nitrogen catabolite repression, Untr = Untreated.</p