Holmberg L, Melander Y, Nygård O
Department of Zoological Cell Biology, Arrhenius Laboratories E5, Stockholm University, Sweden.
Nucleic Acids Res. 1994 Apr 25;22(8):1374-82. doi: 10.1093/nar/22.8.1374.
The secondary structure of mouse Ehrlich ascites 18S, 5.8S and 28S ribosomal RNA in situ was investigated by chemical modification using dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulphonate. These reagents specifically modify unpaired bases in the RNA. The reactive bases were localized by primer extension followed by gel electrophoresis. The three rRNA species were equally accessible for modification i.e. approximately 10% of the nucleotides were reactive. The experimental data support the theoretical secondary structure models proposed for 18S and 5.8/28S rRNA as almost all modified bases were located in putative single-strand regions of the rRNAs or in helical regions that could be expected to undergo dynamic breathing. However, deviations from the suggested models were found in both 18S and 28S rRNA. In 18S rRNA some putative helices in the 5'-domain were extensively modified by the single-strand specific reagents as was one of the suggested helices in domain III of 28S rRNA. Of the four eukaryote specific expansion segments present in mouse Ehrlich ascites cell 28S rRNA, segments I and III were only partly available for modification while segments II and IV showed average to high modification.
利用硫酸二甲酯和1-环己基-3-(吗啉代乙基)碳二亚胺对甲苯磺酸盐进行化学修饰，对小鼠艾氏腹水瘤18S、5.8S和28S核糖体RNA的二级结构进行了原位研究。这些试剂特异性修饰RNA中的未配对碱基。通过引物延伸和凝胶电泳确定反应性碱基的位置。三种rRNA对修饰的可及性相同，即约10%的核苷酸具有反应性。实验数据支持为18S和5.8/28S rRNA提出的理论二级结构模型，因为几乎所有修饰碱基都位于rRNA的推定单链区域或预期会发生动态呼吸的螺旋区域。然而，在18S和28S rRNA中均发现了与建议模型的偏差。在18S rRNA中，5'-结构域中的一些推定螺旋被单链特异性试剂广泛修饰，28S rRNA结构域III中的一个建议螺旋也是如此。在小鼠艾氏腹水瘤细胞28S rRNA中存在的四个真核生物特异性扩展片段中，片段I和III仅部分可用于修饰，而片段II和IV显示出中等至高修饰水平。
