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. 2013 Dec 11;42(5):3246–3260. doi: 10.1093/nar/gkt1281

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© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 1. — RP-HPLC screening of the mutants for identification of m³U methyltransferase. The 25S rRNA from mutants and isogenic wild type were digested to nucleosides using P1 nuclease and alkaline phosphatase. Nucleosides obtained after digestion was then analyzed by RP-HPLC. For optimum separation of m³U residues, we changed the elution conditions to an isocratic mode using 50% buffer A (2.5% methanol) and 50% buffer B (20% methanol). RP-HPLC chromatogram from the wild type (A), Δyil096c (B), Δylr063w (C) and Δyil096cΔylr063w (D) mutants. The peak corresponding to the m³U with a retention time of ∼9 min reduces to half in both Δyil096c and Δylr063w mutants and disappears in double mutant Δyil096cΔylr063w, highlighting the involvement of these two methyltransferases in m³U methylations of the 25S rRNA.