CLASSICAL AND NOVEL APPROACHES TO THE DETECTION AND LOCALIZATION OF THE NUMEROUS MODIFIED NUCLEOTIDES IN EUKARYOTIC RIBOSOMAL-RNA

Human ribosomes contain more than 200 modified nucleotides. These are made up as follows: more than 100 2'-O-methyl groups, 10 methylated ba ses, about 95 pseudouridines and at least one other modification. Othe r mammalian sources that have been examined, as well as the lower vert ebrate Xenopus laevis, show very similar patterns of nucleotide modifi cations, especially as revealed by oligonucleotide fingerprinting for methyl groups. Most of the methyl groups have been located along the r RNA primary structure by matching oligonucleotide sequence data to the complete sequences derived from rDNA. Nearly all of the methyls are i n conserved core regions. Saccharomyces carlsbergensis ribosomes conta in about 55% as many methyls as vertebrate ribosomes. The locations of most of the S carlsbergensis methyls are also known. However, of the numerous other eukaryotes whose rRNA sequences have been determined in directly from rDNA, few have yielded detailed data on modified nucleot ides. This is in part because the methods applied to vertebrate and ye ast ribosomes are highly laborious and are not universally applicable. Therefore in the final part of this paper we briefly review other met hods that have been applied to the detection and localization of modif ied nucleotides in rRNA. In particular, we outline progress towards de veloping a method whereby reverse transcription shows characteristic p ausing at most of the 2'-O-methylation sites in human and Xenopus 18S rRNA. 2'-O-Methylation pauses are distinguishable from most other inte rruptions; the 2'-O-methyl pauses occur more strongly at low than at h igh dNTP concentration, whereas most other interruptions are independe nt of dNTP concentration.