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

Douglas C. Youvan | Zendy; John E. Hearst | Zendy

Open Access

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

Author(s) -

Douglas C. Youvan,

John E. Hearst

Publication year - 1981

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/9.7.1723

Subject(s) - biology , 18s ribosomal rna , ribosomal rna , nucleic acid sequence , rna , complementary dna , rnase p , sequence analysis , microbiology and biotechnology , denaturing high performance liquid chromatography , genetics , dna , gene , polymerase chain reaction

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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