Figure 2. Mutations in the coding and promoter sequences of P_TDH3-YFP as well as changes in its copy number were found among the 231 mutant genotypes.

(A) The schematic depicts the P_TDH3-YFP reporter gene and is drawn to scale. Annotations indicate the location of each mutation relative to the transcription start site (+1) of TDH3 [68], the nucleotide substitution observed, and the change in amino acid sequence if applicable. Asterisks indicate mutations observed in two mutant genotypes. Mutations at positions −255, −240 and −140 are located within the promoter, and the mutation at position 348 is a synonymous change. (B) In 221 of the 231 mutants, the relative copy number of P_TDH3-YFP and P_TDH3-CFP was determined by pyrosequencing. Clusters of points representing genotypes with one copy of P_TDH3-YFP (CFP and YFP≈1/2) and two copies of P_TDH3-YFP (CFP≈1/3 and YFP≈2/3) are indicated with arrows. (C) Median YFP fluorescence is plotted for each of the replicate populations analyzed for eight distinct genotypes: unmutagenized cells containing the “wildtype” P_TDH3-YFP sequence (diamonds); each of the four regulatory mutant genotypes (8Q1D4, 4Q4E4, 3Q3C11, and 1Q4D11 in Table S3) that contained a promoter mutation (filled circles); and genotypes in which one of the promoter mutations (−255, −240, or −140) was introduced into same genetic background as the wildtype P_TDH3-YFP gene (open circles). Populations of cells containing any one of the promoter mutations showed a significant change in YFP fluorescence relative to the wildtype genotype (P₋₂₅₅ = 0.002, P₋₂₄₀ = 0.002, P₋₁₄₀ = 0.015, MWW test). The mutation at −255 had an effect on YFP fluorescence equivalent to that of mutant genotype 8Q1D4 (P = 0.261, MWW), but not to that of mutant genotype 4Q4E4 (P = 0.0002, MWW). The effects on YFP fluorescence of mutations at −240 and −140 were equivalent to those of the mutant genotypes (3Q3C11 and 1Q4D11, respectively) that harbored them (P₋₂₄₀ = 0.262 and P₋₁₄₀ = 0.262, MWW).