Fig 1. S. pombe trm8Δ mutants lack m⁷G and are temperature sensitive.

(A) trm8Δ mutants have no detectable m⁷G in their tY(GUA) and tF(GAA). S. pombe trm8Δ mutants and WT cells were grown in biological triplicate in YES media at 30°C and tRNAs were purified, digested to nucleosides, and analyzed for modifications by HPLC as described in Materials and Methods. The bar chart depicts average moles/mol values of nucleosides with associated standard deviation; WT, green; S. pombe (Sp) trm8Δ, brown. (B) trm8Δ mutants are temperature sensitive due to lack of trm8⁺. Strains with plasmids as indicated were grown overnight in EMM-Leu media at 30°C, diluted to OD₆₀₀ ~ 0.5, serially diluted 10-fold in EMM-Leu, and 2 μL was spotted onto plates containing EMM-Leu or YES media and incubated at 33°C, 37°C, and 38°C. The two independent trm8Δ mutants were labeled as Sp trm8Δ V1 and V2.