Lockard R E, Connaughton J F, Kumar A
Nucleic Acids Res. 1982 Jun 11;10(11):3445-57. doi: 10.1093/nar/10.11.3445.
By direct RNA sequence analysis we have determined the primary structures of both the 5' and 3' domains for rabbit 18S ribosomal RNA. Purified 18S rRNA was labeled in vitro at either its 5' or 3' terminus with 32P, base-specifically fragmented enzymatically and chemically, and the resulting fragments electrophoretically fractionated by size in adjacent lanes of 140 cm long polyacrylamide sequencing gels run in 90% formamide. A phylogenetic comparison of both the mammalian 5' proximal 400 residues and the 3' distal 301 nucleotides with the previously determined yeast and Xenopus laevis 18S rRNA sequence shows extensive conservation interspersed with tracts having little homology. Clusters of G + C rich sequences are present within the mammalian 5' domain which are entirely absent in both the Xenopus laevis and yeast 18S rRNAs. Most base differences and insertions within the mammalian 18S rRNA when compared with yeast or Xenopus rRNA result in an increase in the G + C content of these regions. We have found nucleotide sequence analysis of the ribosomal RNA directly permits detection of both cistron heterogeneities and mapping of many of the modified bases.
通过直接RNA序列分析,我们确定了兔18S核糖体RNA 5'和3'结构域的一级结构。纯化的18S rRNA在体外分别用32P标记其5'或3'末端,通过酶法和化学法进行碱基特异性切割,然后将所得片段在90%甲酰胺中运行的140厘米长的聚丙烯酰胺测序凝胶的相邻泳道中按大小进行电泳分离。将哺乳动物5'近端400个残基和3'远端301个核苷酸与先前确定的酵母和非洲爪蟾18S rRNA序列进行系统发育比较,结果显示广泛的保守性中穿插着几乎没有同源性的区域。富含G + C的序列簇存在于哺乳动物5'结构域内,而在非洲爪蟾和酵母18S rRNA中则完全不存在。与酵母或非洲爪蟾rRNA相比,哺乳动物18S rRNA中的大多数碱基差异和插入导致这些区域的G + C含量增加。我们发现对核糖体RNA进行核苷酸序列分析直接可以检测顺反子异质性并对许多修饰碱基进行定位。