RT-PCR localization of transcriptional start site of <i>bzpR</i> gene and determination of relative mRNA levels.

<p><b>A</b>. RNA and genomic DNA were isolated from growing Ax4 cells and used as templates in RT-PCR (r) or PCR (g) reactions. A 3′ primer (bzr-3) corresponding to sequences at the beginning of the coding region was used with three different 5′ primers corresponding to sequences progressively further upstream of the coding region (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032500#pone-0032500-g001" target="_blank">Fig. 1A</a>). Bzr-9 lies within the first uORF, bzr-10 lies just upstream of uORF 1, and bzr-5 is 41 residues further upstream of -10. <b>B</b>. RNA was isolated from Ax4 growing cells (0) and cells plated for development for the indicated times (in hours) and used in RT-PCR reactions with bzr-3 and -10. <i>H7</i> specific primers were used as an internal control as <i>H7</i> is expressed constitutively during growth and development. Conditions were optimized to reveal differences in RNA levels of two to ten-fold.