Repeat sequence expression congruent with genome annotation. a) All RNA-seq data is shown as plots of read coverage against the annotation of the represented genomic locus. Along the bottom of these panels, CDSs and non-coding RNAs, coloured according to function (see ref. ), are represented as blocks above or below the scale line, depending on their orientation. BOX repeats are shown as red blocks on the scale line. Primer binding sites are indicated by blue blocks labelled using dashed lines. Above the annotation, as part of the coverage plots, blue lines indicate transcription of the upper strand of the genome, while red lines show transcription of the reverse strand. Solid lines represent the result of fully redundant mapping, where reads mapping to multiple sites on the chromosome are randomly distributed between them. Dashed lines represent locally redundant mapping, where reads that might map to regions outside the displayed locus are excluded from the graph (see Methods). i) The region upstream of the trp operon. The trpE gene is adjacent to two T box riboswitch motifs separated by an intervening BOX element, represented as four adjacent red boxes representing the A1B2C1 structure of the repeat. The RNA-seq data suggests the T box motifs and BOX element are cotranscribed as a composite element, repressing the transcription of the downstream biosynthetic operon. ii) Locus surrounding SPN23F16220. This small CDS is annotated as being encompassed by a BOX element. RNA-seq data suggested it was cotranscribed with SPN23F16230, present on the other side of the repeat relative to the more highly expressed galE gene. b) RT-PCR to confirm transcription of these BOX elements. The positions of the primers used in these reactions are indicated by the blue boxes labelled PL (left primer) and PR (right primer) in a) i) and ii). In each case, the three lanes correspond to a positive control reaction using a genomic DNA (+), a test using cDNA produced through reverse transcription of an RNA sample (RTase) and a negative control using a non-reverse transcribed RNA sample (No RTase). The bands indicate that these BOX elements are expressed, as suggested by the RNA-seq data.
Croucher et al. BMC Genomics 2011 12:120 doi:10.1186/1471-2164-12-120