Yokogawa T, Kumazawa Y, Miura K, Watanabe K
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Japan.
Nucleic Acids Res. 1989 Apr 11;17(7):2623-38. doi: 10.1093/nar/17.7.2623.
For large scale preparation of mitochondrial tRNAs, a new hybridization assay method using synthetic oligodeoxyribonucleotide probes (16-17mer) complementary to individual tRNA sequences was developed and applied for the purification of two serine isoacceptor tRNAs (tRNASerAGY and tRNASerUCN) from bovine mitochondria. It is about 100 times more sensitive than the conventional aminoacylation assay method. 2-4 A260 units each of both tRNASer isoacceptors were purified from 17.5 kg of bovine liver, and they were characterized by means of nuclease digestion, melting profiles and aminoacylation activity. It is suggested that tRNASerUCN possesses the D loop/T loop interaction like usual L-shaped tRNAs, and that tRNASerAGY lacking almost an entire D arm is aminoacylated with an efficiency not very much lower than that of tRNASerUCN.
为了大规模制备线粒体tRNA，开发了一种新的杂交检测方法，该方法使用与单个tRNA序列互补的合成寡脱氧核糖核苷酸探针（16 - 17聚体），并将其应用于从牛线粒体中纯化两种丝氨酸同工受体tRNA（tRNASerAGY和tRNASerUCN）。它比传统的氨酰化检测方法灵敏约100倍。从17.5千克牛肝中纯化出了2 - 4个A260单位的两种丝氨酸同工受体tRNA，并通过核酸酶消化、解链曲线和氨酰化活性对它们进行了表征。结果表明，tRNASerUCN像通常的L形tRNA一样具有D环/T环相互作用，而几乎缺失整个D臂的tRNASerAGY的氨酰化效率并不比tRNASerUCN低很多。
