This study has been undertaken in order to elucidate the mechanisms of incorporation of Se into glutathione peroxidase (GSHPx), in which seleaocysteine corresponds to the opal termination codon UGA on the mRNA. We studied the above mechanisms using an opal suppressor tRNA, prepared from bovine liver, and caseia as a model protein for the GSHPx apo-enzyme which might contain phosphoserine. The results showed that opal suppressor tRNA did not accept seleaocysteine (lower than 0.1 mmol/mol) under the standard conditions. A trace amount of phosphoseryl-tRNA was converted to selenocysteyl-tRNA by incubation with &Se and some enzymes. Meanwhile, a number of phosphoserine residues in casein were converted to seleaocysteiae residues by incubation with H,Se and enzymes. These results suggest that opal suppressor tRNA plays a role in synthesizing GSHPx via co-and/or post-translational mechanisms.

In order to gain an insight into the pathogenesis of mouse muscular dystrophy, we investigated the natural suppressor serine tRNA. The natural suppressor seryl-tRNA was distinguished from the other seryl-tRNAs on the basis of its specific property of being converted into phosphoseryl-tRNA by a tRNA kinase. On a wet-weight basis, the content of total tRNA in dystrophic muscles was 47% of that in normal muscles. Although the serine-accepting activities of tRNA were similar in muscles of 3-month-old dystrophic and normal mice, the ratio of [32P]phosphoseryl-tRNA (suppressor tRNA) to the total serine tRNA was significantly enhanced in dystrophic muscles compared with that in normal muscles. This high content of suppressor tRNA in dystrophic muscles was further confirmed by dot-blot hybridization experiments with the DNA probes CGTAGTCGGCAGGAT and CGCCCGAAAGGTGGAA for major tRNA(IGASer) and suppressor tRNA respectively. At the early postnatal age of 3 weeks, when only a week had elapsed since the first manifestation of the dystrophic symptom (hindleg dragging), the ratio of suppressor tRNA to major tRNAs in dystrophic hindleg muscles was abnormally increased. Thereafter it decreased with age in normal mice but remained almost unchanged in dystrophic mice. Consequently, at 3 months old, it was 1.7 times higher in dystrophic than in normal mice. The suppressor tRNA is now accepted to play a role in the synthesis of glutathione peroxidase. The present study showed that the content of this enzyme was abnormally elevated in dystrophic mice. Previously we had demonstrated that the docosahexaenoic (C22:6) acid content in phospholipids was decreased, possibly resulting from the enhanced oxidative milieu caused by the dystrophic condition. Thus far, the findings suggest that an increase in the contents of suppressor tRNA and glutathione peroxidase in dystrophic muscle may have been secondarily induced by such a highly oxidative state in the dystrophic condition. However, it is difficult to exclude the possibility that the natural suppressor tRNA plays a primary role in the pathogenesis of muscular dystrophies.

It was believed that there was no natural suppressor tRNA in Escherichiu coli, however, it has been suggested that selC', relating to the synthesis of formate dehydrogenase of a selenoprotein [(1988) Nature 331, 723-7251, codes for tRNA, even though the presence of tRNA has not yet been demonstrated. We detected the product of se/C in the tRNA preparation of the E. cofi MC 4100 strain by the dot blot hybridization method with a DNA probe (ACCGCTGGCGGC) corresponding to the extra arm of sefC tRNA. Two hybridization peaks were found in the chromatographic pattern from Sephadex ASO. The amount of tRNA was estimated to be about 0.03% of the total tRNA. The suppressor PH]seryl-tRNA was phosphorylated by a tRNA kinase in E. co/i B. These results suggest that the opal suppressor seryl-tRNA in E. coli should be converted to selenocysteyl-tRNA through phosphoseryl-tRNA, and occurs in vertebrates as a general phenomenon Suppressor tRNA; Formate dehydrogenase; Selenocysteine; Opal termination codon; tRNA, phosphoseryl-

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