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. 2015 Jan;21(1):61–74. doi: 10.1261/rna.047639.114

FIGURE 2.

FIGURE 2.

S. pombe trm7Δ mutants are barely viable, and trm7Δ [Pnmt1** Sp trm7+] strains grown in repressive conditions have reduced Cm32, Gm34, and yW37 modification of tRNAPhe. (A) S. pombe trm7▵ mutants are barely viable. S. pombe strains with the indicated plasmids were grown in EMM overnight, diluted to OD600 of ∼0.5 in H2O, and serially diluted 10-fold in H2O, and then 2 µL was spotted onto indicated media, followed by incubation for 4 d at 30°C. (B) HPLC traces of tRNAPhe from an S. pombe trm7Δ [Pnmt1** Sp trm7+] strain. tRNAPhe from indicated strains grown in EMM-Leu with thiamine (+T, repressive conditions) or without thiamine (permissive conditions) was digested to nucleosides and analyzed by HPLC as described in Materials and Methods. (C) Levels of yW on tRNAPhe substantially decrease in an S. pombe trm7Δ [Pnmt1** Sp trm7+] strain grown under repressive conditions. tRNAPhe from indicated strains grown in EMM-Leu with thiamine (+T) was analyzed as described in panel B under conditions optimized for detecting yW as described in Materials and Methods.