Screen for glutamine analog resistance genes.

Abstract

(A) Schematic of the validation screen for DON resistance genes. The heatmaps represent the difference between DON-treated vs. untreated colony size. All values are log2-transformed, corrected for standard deviation and normalized to the 70-percentile of control clones. Statistical information is provided in S1 Table. (B) 107 cells / mL of the indicated genotype in the S288C background were serially diluted (1:6), spotted on YPD plates with or without 250 μM DON or 200 mM HU and grown for 2d (Untreated), 3d (DON) or 4d (HU). (C, D) Exponentially growing cells of the indicated genotypes in the W303 (C) or S288c (D) background were treated with 300 μM DON for 2h as indicated. Metabolites were extracted and glutamine 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 Student’s t-test (two-sided) over 5 replicate cultures. (E) Exponentially growing cells of the indicated genotypes in the S288c background were cultured in YPD and treated with 300 μM DON for 2h. Samples were collected to quantify IMD2 mRNAs by RT-qPCR using TFC1 as reference (n = 3 independent replicate cultures). Representative spot assay images are shown. wt = wildtype. (TIF)</p