Okada N, Sakamoto K, Itoh Y, Ohshima Y
J Biochem. 1982 Apr;91(4):1281-91. doi: 10.1093/oxfordjournals.jbchem.a133813.
Using post-labeling techniques, the nucleotide sequence of a major species of U5 RNA isolated from rat liver was determined to be: XpppAmUmACUCUGGUUUCUCUUCAGAUCGUAUAAAUCUUUCGmCCUUmUpsiACmNAAAGAUpsiUCCGUGGAGAGGA ACAACUCUGAGUCUUAAACCAAUUUUUUGAGGCCUUGUCUUGA(G)CAAGGCUOH. The 5'-end of the RNA is blocked with a cap structure. In addition to the modified nucleotides around the 5'-end (XpppAmUmA), U5 RNA contains Gm at position 38, Um at position 42, psi at position 44, Cm 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 second structure, which causes sequence compression on electrophoresis 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.
采用后标记技术，测定了从大鼠肝脏中分离出的一种主要U5 RNA的核苷酸序列为：XpppAmUmACUCUGGUUUCUCUUCAGAUCGUAUAAAUCUUUCGmCCUUmUpsiACmNAAAGAUpsiUCCGUGGAGAGGA ACAACUCUGAGUCUUAAACCAAUUUUUUGAGGCCUUGUCUUGA(G)CAAGGCUOH。该RNA的5′端被帽结构封闭。除了5′端周围的修饰核苷酸（XpppAmUmA）外，U5 RNA在第38位含有Gm，第42位含有Um，第44位含有psi，第46位含有Cm，第47位含有N，第54位含有psi作为修饰核苷酸。U5 RNA以几种具有微异质性的物种混合物形式存在，其长度为117、118或119个核苷酸。主要物种有117个核苷酸，约占U5 RNA总量的60%。3′端附近的一个区域形成了一个稳定的二级结构，这导致在聚丙烯酰胺凝胶电泳时出现序列压缩。为了克服这一障碍，我们在甲酰胺中进行部分水解之前，开发了一种用亚硫酸氢钠进行化学修饰的方法，该方法可使含有7 M尿素的聚丙烯酰胺凝胶中的二级结构变性。该方法为检查在聚丙烯酰胺凝胶电泳中测定的RNA序列提供了一个良好的系统，这些序列可能由于序列压缩而出现明显的缺失。
