Tener G M, Delaney A D, Grigliatti T A, Cowling G J, Gillam I C
Biochemistry. 1978 Feb 21;17(4):741-5. doi: 10.1021/bi00597a029.
A method for the isolation and labeling to high specific radioactivity of individual isoaccepting tRNAs is described. After blocking reactive minor bases by acetylation and iodination of the crude tRNA, a single family of isoacceptors was aminoacylated. Individual isoacceptors were separated by chromatography on RPC-5 and then acylated with the 3-(4-hydroxyphenyl)propionyl ester of N-hydroxysuccinimide. The product was purified by chromatography on BD-cellulose and RPC-5. This derivatized tRNA was then iodinated with 125I- and Chloramine-T to give a product containing between 5 X 10(7) and 3 X 10(8) dpm/microgram. The suitability of such labeled tRNAs for hybridization to homologous DNA in solution and cytological preparations of chromosomes is discussed with particular reference to Drosophila melanogaster.
本文描述了一种分离并标记具有高比放射性的单个同功tRNA的方法。通过对粗制tRNA进行乙酰化和碘化来封闭反应性次要碱基后,单个同功受体家族被氨酰化。单个同功受体通过RPC-5柱色谱法分离,然后用N-羟基琥珀酰亚胺的3-(4-羟苯基)丙酰酯进行酰化。产物通过BD-纤维素和RPC-5柱色谱法纯化。然后用125I和氯胺-T对这种衍生化的tRNA进行碘化,得到每微克含5×10(7)至3×10(8) 每分钟衰变数(dpm)的产物。特别参照黑腹果蝇,讨论了这种标记的tRNA在溶液中与同源DNA杂交以及染色体细胞学制备中的适用性。