Figure 2

The Sfi I-site in the 5′ UTR near the AUG start codon increases the levels of PDR5 mRNA. Northern blot analysis was performed on late-logarithmically grown AD and AD/sec6-4 cells that expressed PDR5 with differently modified 5′ UTRs (WT = unmodified; strains labeled SP, S or P contained the SfiI/PacI, the SfiI or the PacI site, respectively). AD strains contained the PGK1 terminator and the URA3 marker at the 3′ end of PDR5 while AD/sec6-4 strains contained the 3′ UTR of PDR5. Three control strains were included: two wild-type PDR5 expressing strains (AH22 and SY1) and AD/pABC3 as the negative (∆PDR5) control. A Upper panel - 10 μg total RNA extracts separated on a 1.2% denaturing agarose gel and stained with EtBr (top), lower panels - autoradiographs of blots probed with PDR5 and ACT1. The band intensities for the top two PDR5 and ACT1 panels can be directly compared as they experienced the same treatment (PDR5 and ACT1 probes were combined for the hybridization with the Northern blot) while the autoradiograph at the bottom was overexposed so that PDR5 bands of weaker intensities could be measured accurately. B, C, and D show the intensities of bands in panel A quantified with the ImageJ software program [30]. B shows the expression of PDR5 relative to the expression of the housekeeping gene ACT1 that was used as an internal standard (the intensities for ACT1 in AD/SP-PDR5 were ~10-times higher than in AD/pABC3 while ACT1 varied no more than +/− 50% in the remaining samples). C and D show the -fold differences in normalized PDR5 mRNA levels relative to AD/sec6-4/PDR5 (C) and AD/P-PDR5 (D), respectively (the results for the Sfi I-site containing strains are shown with black bars, for wt-PDR5 with dark grey and Pac I-site containing strains with light grey bars). The numbers above individual bars in B, C, and D give the actual values represented by the bars.