FIGURE 4.

Trm8p protein, but not mRNA, is severely reduced in a trm82-Δ strain. (A) Comparison of Trm8p expression in a trm82-Δ and a TRM82⁺ strain. Otherwise isogenic haploid strains containing wild-type TRM8 or chromosomally epitope-tagged TRM8 (tagged with His⁶-HA-3C^{protease site}-ZZ^{Protein A}, abbreviated ZZ), and TRM82⁺ or trm82-Δ as indicated, were grown, harvested, and lysed with SDS and glass beads as described in Materials and Methods. Lysates were serially diluted fourfold, starting with 2.7 OD₆₀₀ units of cells, resolved by SDS-PAGE, and analyzed for Trm8-ZZ expression by Western blot analysis, as described in Materials and Methods. Lanes a–c, TRM8-ZZ TRM82⁺ strain; d–f, TRM8-ZZ trm82-Δ strain; g–i, TRM8-ZZ trm82-Δ strain transformed with plasmid containing CEN LEU2 P_TRM82TRM82; j–l, TRM8 TRM82⁺ strain, with no epitope tag. (B) SDS-PAGE analysis of lysates described in (A) and stained with Coomassie. (C) Comparison of TRM8 mRNA in a trm82-Δ and a TRM82⁺ strain. RNA from otherwise isogenic wild-type (lanes a–c), trm82-Δ (d–f), and trm8-Δ (g–i) haploid strains was resolved on a 1% agarose gel, transferred to membrane, and hybridized with ³²P-labeled TRM8 or ACT1-specific probes, as described in Materials and Methods. Successive lanes contain 15, 7.5, and 3.8 μg RNA.