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对总tRNA群体中单个物种进行选择性32P标记。

Selective 32P-labelling of individual species in a total tRNA population.

作者信息

Traboni C, Cortese R, Salvatore F

出版信息

Nucleic Acids Res. 1980 Nov 25;8(22):5223-32. doi: 10.1093/nar/8.22.5223.

DOI:10.1093/nar/8.22.5223
PMID:7008026
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC324297/
Abstract

A simple procedure to label individual tRNA species in a total tRNA preparation has been developed. The principle of the method is as follows: total crude tRNA (from E. coli) is incubated in the presence of a crude aminoacyl-tRNA synthetase preparation, containing most aminoacyl-tRNA synthetases and only one specific amino acid corresponding to the tRNA species which is intended to be labelled. This achieves the purpose of charging the desired tRNA species thereby protecting its 3'OH-terminus; obviously all the other tRNA species will have a free 3'OH group. Periodate oxidation, followed by beta-elimination, destroys any free 3'OH. After deacylation of the specific aminoacylated tRNA at pH 8.8 the only free 3'OH group will be the one of the desired tRNA species. High specific activity (32P)-pCp is ligated to this 3'OH by means of T4-RNA ligase. Two-dimensional polyacrylamide gel electrophoresis (2D-PGE) and sequence analysis of the isolated tRNA show that the method is very specific. Individually labelled tRNA species can be used as probes for cloning tRNA genes.

摘要

已开发出一种在总tRNA制剂中标记单个tRNA种类的简单方法。该方法的原理如下：将来自大肠杆菌的总粗制tRNA在含有大多数氨酰-tRNA合成酶且仅有一种与打算标记的tRNA种类相对应的特定氨基酸的粗制氨酰-tRNA合成酶制剂存在下孵育。这实现了给所需tRNA种类充电从而保护其3'-OH末端的目的；显然所有其他tRNA种类将具有游离的3'-OH基团。高碘酸盐氧化，随后进行β-消除，会破坏任何游离的3'-OH。在pH 8.8下对特定氨酰化tRNA进行脱酰化后，唯一的游离3'-OH基团将是所需tRNA种类的基团。通过T4-RNA连接酶将高比活性的（32P）-pCp连接到该3'-OH上。对分离的tRNA进行二维聚丙烯酰胺凝胶电泳（2D-PGE）和序列分析表明该方法具有很高的特异性。单独标记的tRNA种类可作为克隆tRNA基因的探针。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/c8430310ee88/nar00439-0099-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/3b17e0a60485/nar00439-0095-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/7ef77c9ba6b8/nar00439-0096-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/b969d424bc59/nar00439-0096-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/90cbf9f2fc02/nar00439-0097-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/43287ead8a9c/nar00439-0098-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/c8430310ee88/nar00439-0099-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/3b17e0a60485/nar00439-0095-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/7ef77c9ba6b8/nar00439-0096-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/b969d424bc59/nar00439-0096-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/90cbf9f2fc02/nar00439-0097-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/43287ead8a9c/nar00439-0098-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75e7/324297/c8430310ee88/nar00439-0099-a.jpg

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