FIG. 2.

HPLC of nucleosides from tRNA from strain GRB1108 (tsaA⁺) (A) and strain GRB1109 (tsaA1) deficient in m⁶t⁶A (B). tRNA was degraded to nucleosides and a part (retention time, 0 to 80 min) of the chromatogram is shown. No difference in the patterns of modified nucleosides for the wild type and the mutant than that shown in the figure was observed. The identification of the compound migrating at min 67.538 as m⁶t⁶A was based on the following criteria: (i) the relative retention time was the same as that found for m⁶t⁶A with the HPLC system devised by Gehrke and Kuo (17) and used in the experiment described and also the same as that found by Buck et al. (9), as revised by Limbach et al. (23); (ii) the spectrum of this compound was the same as that reported for m⁶t⁶A (17); and (iii) the molecular weight was that of protonated m⁶t⁶A (427), as determined by the electrospray method. (C) HPLC of nucleosides from in vitro-methylated tRNA from strain GRB1109 (tsaA1). The radioactivity was monitored by a Radiomatic FLO-ONE scintillation counter, and the absorbance at 254 nm was measured by a photodiode array detector (Waters AB). The retention time of the major radioactive compound, which was not observed with tRNA from GRB1108 (tsaA⁺) as the substrate (data not shown), corresponded to that of m⁶t⁶A, as determined by measuring the UV absorbance at 254 nm. Although the retention time for m⁶t⁶A in panel C was several minutes shorter than in panel A, the retention time of m⁶t⁶A relative to m²A was the same for m⁶t⁶A in panel A (0.956) as in panel C (0.957). The small radioactive compound migrating at 31 min coincided with m⁵U, and a similar amount of radioactive m⁵U was also observed with tRNA from strain GRB1108 (tsaA⁺).