A sequence from Drosophila melanogaster 18S rRNA bearing the conserved hypermodified nucleoside am psi: analysis by reverse transcription and high-performance liquid chromatography.

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RESUMO

The naturally occurring modified nucleoside 3-[3-amino-3-carboxypropyl]-1-methylpseudouridine (abbreviated am psi) is found in eukaryotic 18S rRNA. We localized am psi to sequence resolution in D. melanogaster 18S rRNA. This hypermodified base causes an absolute stop in cDNA elongation. The RNA sequence bearing am psi was determined by dideoxy-sequencing with reverse transcriptase. The rDNA coding for this part of the 18S rRNA was sequenced by the Maxam-Gilbert method. Together these two sequencing methods can be used to position the cDNA stop (am psi) in the rRNA sequence. Chemical evidence for the existence of am psi in this RNA sequence was obtained by high-performance liquid chromatography (HPLC) of 18S rRNA nucleosides from radioactive-labeled cells. L-[2-14C] methionine will selectively label am psi in eukaryotic 18S rRNA. Using HPLC, we found a single 14C-labeled nucleotide in digests of 18S rRNA. This nucleotide is in the RNA sequence bearing the cDNA stop since a restriction fragment which hybridizes to this sequence protects the modified base from RNase T1 digestion.

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