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Fig. 5. scn1 and scn2 are both Ala-tRNA anticodon mutants. (A) The genomic DNAs were purified from wild-type, scn2-7 and scn2-27 cells. The 2 kb long genomic DNA fragments corresponding to the 2425-4520 region of the chromosome I cosmid c4C5 were amplified by PCR, followed by DNA sequencing. Amplified DNAs from the mutant strains scn2-7 and scn2-27 showed a single mutation (indicated in red) in alanine-tRNA gene (SpaTRNAala.02). Otherwise the sequences were identical to that obtained from the wild-type. (B) The predicted cloverleaf structure of the tRNA-Ala (UGC) in wild-type and scn2. The mutated scn2 tRNA is presumed to recognize the ACA codon in mRNA that encodes threonine. (C) The plasmid containing the mutant tRNA gene of scn2 conferred cold-sensitivity on wild-type (WT) cells and suppressed the temperature sensitivity of cut9-665. Wild-type and cut9-665 (cut9) carrying plasmid of the wild-type tRNA gene (pTRNAala.02) and plasmid of scn2 mutant tRNA gene (pTRNAala.02(UGC>UGU)) were incubated on the minimal EMM2 plate at 22, 26, 33 and 36°C. (D) Wild-type cells carrying multicopy plasmid containing the mutant Ala-tRNA(UGU) gene showed mitotic phenotypes sim ilar to those of scn1 and scn2 mutants. Wild-type cells carrying pTRNAala.02 or pTRNAala.02(UGC>UGU) were cultured in the EMM2 medium at 20°C for 9 hours and fixed with glutaraldehyde. DNA was stained with DAPI. The phenotype of scn1-17 was also shown. Brightly stained cells were dead and accumulated the dye. Bar, 10 µm. (E) The scn1 mutant contained a substitution mutation in the second copy of alanine-tRNA gene, but not in the scn3+ gene. The genomic DNA of wild-type and scn1-17 was purified, and DNA sequencing of the region containing the second tRNA gene (SpaTRNAala.03) was done. The SpaTRNAala.03 near to the scn3+ gene was mutated in scn1-17 in the same position in anticodon as in scn2.
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