The ligated and dephosphorylated 5Ј-half fragments were annealed and ligated with the 3Ј-half fragment digested by RNase H. About 50 µg of each of the two variants from C.zeylanoides mutated at position 37-Construction of tRNA variants with mutation at position 33 The microsurgery procedures were basically carried out according to the m 1 G37A and m 1 G37G-was obtained by the phosphorylation of the 5Јend and purification by 12% PAGE containing 7 M urea. literature (Ohyama et al., 1985 (Ohyama et al., , 1986 . Limited digestion of 4 mg purified tRNA Ser CAG from C.zeylanoides with RNase T1 was performed at 0°C for 30 min in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), Identification of amino acids attached to tRNA Ser CAGs in 100 mM MgCl 2 , 0.5 mg/ml of the tRNA and 25 000 units/ml RNase the cells T1 (Sigma). After phenol extraction, the resulting fragments were treated Identification of aminoacyl-tRNA Ser CAG from Candida cells was carried with 0.1 N HCl at 0°C for 12 h in order to cleave the 2Ј, 3Ј cyclic out by a new method developed recently by us (Suzuki et al., 1996) . phosphate of the 3Ј-end of the fragments formed in the limited digestion,