One hundred and fifty thousand A 260 units of tRNA from C.zeylanoides glycerol (500 µl), and then annealed at room temperature. Magnesium chloride was added to the mixture up to a final concentration of 4 mM cells (3.7 kg) were fractionated on DEAE-Sepharose fast-flow column (3.5ϫ130 cm) with a linear gradient of NaCl from 0.25 to 0.4 M in a and the reaction was carried out at 30°C for 2 h by the addition of 600 units of RNase H (Takara Shuzo). About 60 µg of the cleaved 3Ј-half buffer consisting of 20 mM Tris-HCl (pH 7.5) and 8 mM MgCl 2 . About 300 A 260 units of C.zeylanoides tRNA Ser CAG were finally obtained by fragment was obtained by purification using 10% PAGE containing 7 M urea. Either of two synthetic oligo-RNAs, pCAGAp or pCAGGp, was further column chromatography with Sepharose 4B in a reverse gradient of ammonium sulfate from 1.7 to 0 M with a buffer consisting of ligated with the same 5Ј-half fragment digested by RNase T1 as the variants mutated at position 33 under the conditions described above. 10 mM NaOAc (pH 4.5), 10 mM MgCl 2 , 6 mM β-mercaptoethanol and 1 mM EDTA.