Construction and phenotype of the trmY-deleted strain of H. volcanii. (A) Deletion of the trmY gene was confirmed by PCR; (left) PCR products using primers designed to anneal outside the gene (HvO_1989_Ext_F and HvO_1989_Ext_R) confirm a genomic rearrangement of correctly predicted sizes in WT and mutant strains; (middle) PCR products using primers designed to anneal within the target gene (HvO_1989_Int_F and HvO_1989_Int_R) show the absence of the trmY internal segment in the mutant and its presence in the WT strain; (right). To confirm the presence of VNG_1980c in trans, primers were designed to anneal to the complementing gene (HsaI_COG1901_Fwd and HsaI_COG1901_Rev). The predicted size of the fragment is observed in the rescued strain. (B) LC-MS/MS analysis of tRNA extracted from H26 (wild-type) and VDC2376 (ΔtrmY) showing the UV trace at 254nm (top). The 259 m/z ion that corresponds to the protonated molecular weight (MH+) of m1Ψ was detected by MS at 13.36 min in the WT background, while no 259 m/z ion was detected in the ΔtrmY strain (bottom).
