FIG. 3.

Aminoacylation status of yeast tRNA^Phe expressed in mammalian cells. Northern blot analyses of cytoplasmic yeast tRNA^Phe and tRNA^Lys from cells transfected with either pU6^PheWt or pU6^PheUUA are shown. All lanes contain 5 μg of cytoplasmic RNA isolated under acidic conditions; the RNAs loaded in lanes 2 and 4 were adjusted to pH 9.0 and incubated at 37°C for 1 h to serve as the deacylated controls. The migration of aminoacylated tRNA and deacylated control tRNA are denoted AA and DA, respectively. Lanes 1 and 2 were probed for tRNA^Lys, and lanes 3 and 4 were probed for yeast tRNA^Phe. Phosphorimager analysis of the aminoacylated versus deacylated tRNA in lanes 1 and 3 revealed a ratio of greater than 99 to 1, indicating that the vast majority of the yeast tRNA^Phe and tRNA^Lys isolated from the cells was aminoacylated. However, for RNA collected from cells transfected with pU6^PheUUA, no aminoacylation of tRNA^PheUUA was detected, while the majority of mammalian tRNA^Lys from the same sample was aminoacylated. Note that the blots were not performed simultaneously and autorad exposure times varied.