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J. Biochem, 1982, Vol. 91, No. 4 1281-1291
© 1982 Japanese Biochemical Society

research-article

Sequence Determination of Rat U5 RNA Using a Chemical Modification Procedure for Counteracting Sequence Compression1

Norihiro OKADA2, Kazuichi SAKAMOTO, Yasuaki ITOH and Yasumi OHSHIMA

Institute of Biological Sciences, The University of Tsukuba Sakura-mura, Ibaraki 305

Using post-labeling techniques, the nucleotide sequence of a major species of U5 RNA isolated from rat liver was determined to be: XpppAmUmACUCUGGUUU-CUCUUCAGAUCGUAUAAAUCUUUCGmCCUUmU{Psi}ACmNAAAGAU{Psi}UC-CGUGGAGAGGAACAACUCUGAGUCUUAAACCAAUUUUUUGAGGCCU-UGUCUUGA(G)CAAGGCUOH. The 5'-end of the RNA is blocked with a cap structure. In addition to the modified nucleotides around the 5'-end (XpppAm-UmA), U5 RNA contains Gm at position 38, Um at position 42, {Psi} at position 44, '-end (XpppAm-UmA), U5 RNA contains Gm at position 38, Um at position 42, {Psi} at position 44, at position 46, N at position 47, and {Psi} at position 54 as modified nucleotides. U5 RNA is present as a mixture of several species with microheterogeneity, whose lengths are 117, 118, or 119 nucleotides. The major species, with 117 nucleotides, comprised approximately 60% of the total U5 RNA. A region near the 3'-end forms a stable secondary structure, which causes sequence compression on electro-phoresis in polyacrylamide gel. To surmount with this obstacle, we developed a chemical modification procedure with sodium bisulfite prior to partial hydrolysis in formamide, which allows denaturation of the secondary structure in polyacrylamide gel containing 7 M urea. The procedure provides a good system for checking RNA sequences determined by electrophoresis in polyacrylamide gel which might have apparent deletions on account of sequence compression.

1This work was supported in part by Grant-in-Aid for Cancer Research and Scientific Research Funds to N.O. and Y.O. from the Ministry of Education, Science and Culture of Japan.

2To whom reprint should be requested.


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