Figure 7. Effect of CUG reassignment on yeast fitness. (A) Partial CUG reassignment had a strong negative impact on yeast growth rate. Yeast cell cultures transformed with WT and mutant C. albicans tRNACGASer were inoculated at an initial OD600 of 0.02 and were grown in selective medium at 30°C, 180 rpm, until they reached stationary phase. (B) The relative growth rate of cells transformed with WT and mutant tRNACGASer was determined using exponential growth phase values, relative to the control cells. (C) To determine the effect of Ser misincorporation on protein structure E. coli β-gal was co-expressed with WT tRNACGASer and mutant tRNACGASer in yeast cells. Thermoinactivation profiles were determined by measuring β-gal activity after a denaturation step at 47°C. The β-gal activity that remained functional after thermal inactivation was measured by incubating cells at 30°C for 2 min with ONPG. β-gal activity at each time point corresponds to the % of activity relative to total activity measured in cells prior to denaturation. (D) β-gal activity was quantified after incubating total cell protein extracts at 30°C with ONPG until a pale yellow color appeared. Reactions were stopped with addition of 1M sodium carbonate. Data represent the mean ± s.e.m. of triplicates of three independent clones. (***p < 0.001 for one-way Anova post Bonferroni’s multiple comparison test with CI 95%, relative to pRS315).