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用β-32P-焦磷酸基团对RNA进行3'末端标记及其在灰色链霉菌5S RNA序列分析中的应用。

3' Terminal labelling of RNA of RNA with beta-32P-pyrophosphate group and its application to the sequence analysis of 5S RNA from Streptomyces griseus.

作者信息

Simoncsits A

出版信息

Nucleic Acids Res. 1980 Sep 25;8(18):4111-24. doi: 10.1093/nar/8.18.4111.

DOI:10.1093/nar/8.18.4111
PMID:6159585
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC324222/
Abstract

Nucleotide pyrophosphate transferase isolated from Streptomyces griseus is used to transfer pyrophosphate group from gamma-32P-ATP to the 3'-OH of tRNA, generating a strictly terminal label at its 3' end. Using yeast tRNAPhe as model compound, it is demonstrated that the labelled molecule is suitable for rapid gel sequencing by both enzymatic and chemical methods. RNA molecules terminated by pyrimidine nucleoside are poor pyrophosphate acceptors. To label RNAs of this kind, first guanosine 5'-phosphate 3'-(beta-32P)-pyrophosphate (pGpp) is prepared from gamma-32P-ATP and GMP by nucleotide pyrophosphate transferase. pGpp is then ligated to the 3' end of RNA by T4 RNA ligase. The complete nucleotide sequence of 5S RNA from Streptomyces griseus is established by rapid gel sequencing methods performed on 3'-(beta-32P)-pyrophosphate labelled molecule.

摘要

从灰色链霉菌中分离出的核苷酸焦磷酸转移酶用于将焦磷酸基团从γ-32P-ATP转移至tRNA的3'-OH，在其3'端产生一个严格的末端标记。以酵母苯丙氨酸tRNA作为模型化合物，证明标记后的分子适用于通过酶法和化学法进行快速凝胶测序。由嘧啶核苷终止的RNA分子是较差的焦磷酸受体。为标记这类RNA，首先通过核苷酸焦磷酸转移酶由γ-32P-ATP和GMP制备鸟苷5'-磷酸3'-(β-32P)-焦磷酸（pGpp）。然后通过T4 RNA连接酶将pGpp连接至RNA的3'端。通过对3'-(β-32P)-焦磷酸标记的分子进行快速凝胶测序方法确定了灰色链霉菌5S RNA的完整核苷酸序列。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7a7b/324222/49bdc5383831/nar00435-0072-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7a7b/324222/f22b872db844/nar00435-0067-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7a7b/324222/dd7909d032d4/nar00435-0068-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7a7b/324222/7317eab9b080/nar00435-0071-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7a7b/324222/49bdc5383831/nar00435-0072-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7a7b/324222/f22b872db844/nar00435-0067-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7a7b/324222/dd7909d032d4/nar00435-0068-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7a7b/324222/7317eab9b080/nar00435-0071-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7a7b/324222/49bdc5383831/nar00435-0072-a.jpg

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