Salas C E, Dirheimer G
Nucleic Acids Res. 1979 Mar;6(3):1123-33. doi: 10.1093/nar/6.3.1123.
Rat brain cortices from young animals contain large amounts of tRNA (adenine-1)methyltransferase(s). The enzyme(s) can methylate E. coli tRNA and to a lower degree yeast tRNA. Among yeast tRNA species which can be methylated we have selected tRNAAsp as a substrate for the brain enzyme. The digestions of in vitro methylated [Me-3H]-tRNAAsp with pancreatic and/or T1 ribonucleases followed by chromatographies on DEAE-cellulose, 7 M urea, suggested that the methylation of tRNAAsp occurred at a single position within the D-loop. Further digestion of the radioactive oligonucleotide recovered after DEAE-cellulose chromatography by phosphomonoesterase and snake venom phosphodiesterase enzymes followed by bidimensional thin layer chromatography enabled us to determine the location of the adenine residue which becomes methylated by the brain enzyme. This one resulted to be the adenine 14 in the D-loop of yeast tRNAAsp.
幼龄动物的大鼠脑皮层含有大量的tRNA（腺嘌呤-1）甲基转移酶。该酶能使大肠杆菌tRNA甲基化，对酵母tRNA的甲基化程度较低。在可被甲基化的酵母tRNA种类中，我们选择了tRNAAsp作为脑酶的底物。用胰核糖核酸酶和/或T1核糖核酸酶对体外甲基化的[Me-3H]-tRNAAsp进行消化，然后在DEAE-纤维素、7M尿素上进行色谱分析，结果表明tRNAAsp的甲基化发生在D环内的一个单一位置。通过磷酸单酯酶和蛇毒磷酸二酯酶对DEAE-纤维素色谱后回收的放射性寡核苷酸进行进一步消化，然后进行双向薄层色谱分析，使我们能够确定被脑酶甲基化的腺嘌呤残基的位置。结果发现，该腺嘌呤位于酵母tRNAAsp的D环中的第14位腺嘌呤。
