Figure 6.

Evidence that methionine-accepting and other tRNAs from a gcd10Δ strain are hypomethylated. (A) Total tRNA isolated from YJA146 (gcd10Δ) and the p1775 transformant of Bj5464 (GCD10) was aminoacylated with [³⁵S]methionine and [³H]methionine, respectively, and 500,000 cpm of each were chromatographed on a RPC-5 column. The radioactivity in each fraction (0.6 ml) was measured by liquid scintillation in 7 ml of Ecoscint A (National Diagnostics) and is plotted on different y-axes, as shown, against the fraction number. (█) Results obtained with wild-type tRNA (GCD10); (♦) results with gcd10Δ tRNA. The elution positions of the methionine-accepting tRNAs are indicated at the appropriate positions. (B) Transfer RNAs were digested to nucleosides and chromatographed by HPLC on a Supelcosil LC-18S column. Only the portion of the chromatogram (corresponding to retention times of 2–14.5 min) showing a difference between the GCD10 and gcd10Δ samples is shown here. The peak that is absent in the gcd10Δ sample was identified as m¹A by several means (see text). The identities of other peaks in this portion of the chromatogram are indicated: (Θ) Pseudouridine; (C) cytidine; (U) uridine; (m¹A) 1-methyladenosine; (m⁵C) 5-methylcytidine. (AU₂₅₄) Absorbance units at 254 nm.