The ctu2-2 mutant is affected in tRNA thiolation. A) Two ctu2 insertion mutants were identified. The T-DNA inserted in the 3′ untranslated region (ctu2-1) and in the third exon (ctu2-2). The insertion sites are indicated with arrowheads. B) RT-PCR experiments were performed on 900 ng of reverse transcribed total RNA from wild-type Columbia (WT), ctu2-1, ctu2-2, and complemented lines using CTU2- and ACTIN2-specific primers. Contaminating genomic DNA in the RNA preparation could be excluded by the length polymorphism to genomic DNA due to intronic sequence. The ACTIN2 PCR was performed to confirm that comparable amounts of RNA were used for the RT-reaction. C) Thiolated tRNAs (arrowhead) show reduced mobility in acrylamide gels in the presence of N-acryloylamino phenyl mercuric chloride (APM; left gel). tRNA of wild-type Columbia (WT) show a retarded band that is absent in the ctu2-2 mutant. The faint bands in the ctu2-2 lane are present also in the gel lacking APM (right gel) and thus do not represent thiolated tRNAs. D) Complementation of ctu2-2 with a wild-type clone of CTU2 reconstituted tRNA thiolation. Three independent transgenic lines are shown which are identical to those shown in B).