Purification and characterization of two serine isoacceptor tRNAs from bovine mitochondria by using a hybridization assay method.
Purification and characterization of two serine isoacceptor tRNAs from bovine mitochondria by using a hybridization assay method.
复制标题
使用杂交测定方法纯化和表征来自牛线粒体的两种丝氨酸同工受体 tRNA。
作者：
T. Yokogawa;Y. Kumazawa;K. Miura;K. Watanabe
为了大规模制备线粒体tRNAs，利用人工合成的与tRNA序列互补的寡核苷酸探针(16-17mer)，建立了一种新的杂交分析方法，并用于从牛线粒体中分离纯化两种丝氨酸同工受体tRNAs(tRNASerAGY和tRNASerUCN)。该方法比传统的氨基酰化分析方法灵敏约100倍。从17.5公斤牛肝中分离纯化了2-4A260单位的tRNASer异构体，并用核酸酶消化、熔融图谱和氨基酰化活性等方法对其进行了鉴定。结果表明，tRNASerUCN具有与通常的L型tRNAs相似的D环/T环相互作用，并且tRNASerAGY几乎缺少一整条D臂，其氨基酰化效率并不比tRNASerUCN低太多。
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.