Figure 6. The mutants tRNACAGSer are expressed at low level in yeast. (A) To check the in vivo expression of the WT tRNACGASer and mutant tRNACGASer in yeast, 50 µg of total tRNA were extracted and purified under acidic conditions and were fractionated on 15% polyacrylamide gels containing 8M urea at room temperature. tRNACGASer and tRNACCCGly were detected using ɣ-32P-ATP-tDNACGASer and ɣ-32P-ATP-tDNACCCGly probes. Cai4 corresponds to total tRNA purified from C. albicans. Membranes were exposed for 24 h to a K-screen and were visualized using a Bio-Rad Molecular Imager FX. (B) Quantification of expression of the mutant tRNAs relative to the WT tRNA. Data represent the mean ± s.e.m. of three independent experiments (***p < 0.001, one-way Anova post Bonferroni’s test with CI 95% relative to Wt tRNACGASer). (C) In vivo aminoacylation of the WT and mutant tRNACGASer in yeast. In vivo aminoacylation of WT and mutant tRNACGASer was evaluated using acidic northern blot analysis. In vitro deacylated and in vivo acylated tRNAs were fractionated on 6.5% polyacrylamide gels containing 8M urea at 4°C, using 10 mM sodium acetate (pH = 4.5) buffer. The tRNACGASer was detected using ɣ-32P-ATP-tRNACGASer probe. CaI4 corresponds to tRNA extracted from C. albicans. Membranes were exposed for 24 h to a K-screen and were visualized using the Bio-Rad Molecular Imager FX.