Figure 4.
In vivo aminoacylation assays. A, 2 μg of total mitochondrial RNA purified from six cell lines under acid conditions were electrotrophoresed at 4 °C through an acid (pH 4.5) 10% polyacrylamide, 8 m urea gel, electroblotted, and hybridized with DIG-labeled oligonucleotide probes specific for tRNALeu(UUR), tRNAIle, tRNAThr, and tRNALeu(CUN), respectively. B, the samples from one control (C47–1) and one mutant (II-5–10) cell lines were deacylated (DA) by heating for 10 min at 60 °C at pH 8.3, electrophoresed, and hybridized with DIG-labeled oligonucleotide probes specific for the tRNALeu(UUR) and tRNALeu(CUN). C, quantification of aminoacylated proportions of tRNALeu(UUR) in the mutant and controls. The calculations were based on three independent experiments. Graph details and symbols are explained in the legend and key to Fig. 3.