PMID: 7008026Nov 25, 1980Paper

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

Nucleic Acids Research
C TraboniF Salvatore

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.

References

Apr 8, 1977·Science·J S BeckmannJ Abelson
Apr 1, 1979·Proceedings of the National Academy of Sciences of the United States of America·D A Peattie
Oct 1, 1977·Nature·A SimoncsitsH Guilley
Oct 1, 1975·Proceedings of the National Academy of Sciences of the United States of America·M Grunstein, D S Hogness
Sep 15, 1973·Journal of Molecular Biology·S G ClarksonV Serra
Apr 20, 1968·Nature·S K DubeS Cory
Apr 1, 1980·Cell·B HovemannD Söll

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Citations

May 11, 1983·Nucleic Acids Research·J P GoddardJ D Smith
Dec 25, 1991·Nucleic Acids Research·R TanakaA Muto
Jul 17, 1987·Journal of Chromatography·L W McLaughlin, R Bischoff
Dec 1, 1985·Analytical Biochemistry·R Bischoff, L W McLaughlin
Mar 5, 1987·Journal of Molecular Biology·M D MendenhallM R Culbertson
Nov 1, 1984·Journal of Biochemical and Biophysical Methods·G L Igloi

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