Figure 1

Location of novel 2'-O-methylated nucleotides in human 28S rRNA using reverse transcription of partially hydrolyzed rRNA and reverse transcription of rRNA at low dNTP concentrations. The positions of novel 2'-O-methylated nucleotides are shown in bold together with the putative snoRNA guide in parenthesis. Three previously detected methylations Cm2838, Am3794 and Am3799 are also indicated. Lanes 0', 30', 45' and 60' show primer extension products of partially hydrolyzed 28S rRNA, where the hydrolysis time is 0, 30, 45 and 60 min, respectively. Lanes, labeled 1 mM, 0.02 mM and 0.002 mM represent reverse transcription products of 28S rRNA at the indicated dNTP concentrations. Lanes A, C, G and T contain dideoxy sequencing ladders. Note that primer extension products generated with 5'-end-phosphorylated oligodeoxynucleotide primers migrate about 1 nucleotide farther than the respective sequencing reaction products produced with oligodeoxynucleotide primers bearing a hydroxyl group at the 5' end and terminating with dideoxynucleotides.