Figure 3.

GCD10 is dispensable for cell viability in the presence of hcIMT4. (A) Growth of strain YJA146 (gcd10Δ + hcIMT4) and a transformant of its parental GCD10 strain BJ5464 bearing p1775 (GCD10 + hcIMT4) on YPD medium at 26°C for 3 days, and at 30°C or 36°C for 2 days. (B) Northern blot analysis of total RNA (7 μg) isolated from the same two strains described in A (gcd10Δ + hcIMT4 and GCD10 + hcIMT4 lanes) plus isogenic strain YJA143 containing the gcd10Δ chromosomal allele and single-copy GCD10 plasmid p2704 (GCD10). Strains were grown at 26°C or 36°C for 2 and 6 hr as described in Fig. 1. The membrane was probed for tRNA_i^Met and stripped and reprobed for tRNA_e^Met as described in Fig. 1. The different RNA species detected are indicated at left. The various forms of tRNA_i^Met species are labeled at right as in Fig. 2, with the addition of species g, which may be end-trimmed molecules lacking the CCA extension (see text). (C) Northern blot analysis of total RNA (5 μg) isolated from strain Hm296 (gcd14-2) containing wild-type GCD14 on single-copy plasmid pRC62 (GCD14 lanes) or empty vector YEp24 (gcd14 lanes), grown as described in Fig. 1. The membrane was probed for tRNA_i^Met and stripped and reprobed for tRNA_e^Met as described in Fig. 1. Indicated with arrowheads inside the blot and labeled to the right are the various tRNA_i^Met species described in Fig. 2 and above. The indicated ratios of pre-tRNA_i^Met to mature tRNA_i^Met were calculated from the relative intensities of hybridization signals quantitated by PhosphorImager analysis.