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Fig. 1 | BMC Molecular and Cell Biology

Fig. 1

From: Exonuclease resistant 18S and 25S ribosomal RNA components in yeast are possibly newly transcribed by RNA polymerase II

Fig. 1

5′-end analysis of Terminator resistant 18S and 25S molecules. a Schematic representation of oligo ligation to indicate presence of more than one phosphate at the 5′-end of terminator resistant molecules. Ribosomal RNA is first treated with alkaline phosphatase, resulting in complete phosphate removal, if there is nothing protecting the triphosphate (ii and iii) or an intact cap-protected rRNA molecule (i). (iv) Use of the decapping enzyme CAP-Clip after CIP treatment makes a phosphate available for RNA oligo ligation (v) and indicates protection of the triphosphate molecules from CIP digestion. Reverse transcription using rDNA primers is performed followed by PCR and the amplicons of predicted size are sequenced. b SYBR-gold stained gel and sequences of PCR products amplified with oligo and internal specific primers (see Table S1) for 18S and 25S. Multiple amplifications and amplicons (at least 5) for each 18S and 25S generated same sequences. No amplifications were obtained for samples treated with CIP alone (lane 2). CIP followed by decapping (Lane 3) shows an amplification product of the predicted size. Untreated RNA (Lane 4) also resulted in the predicted amplicon size. Sequences of lane 3 products shown in detail. Red enclosed letters indicate oligo sequence. Green arrows indicate the 5′-end of these terminator resistant molecules being the same as the 5′-end of processed 18S and 25S [18]. RNA was extracted from stationary phase C. albicans

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