Figure 5. Examination of NRG1 transcript stability.

A. One copy of NRG1 was replaced with lacZ under the control of the tetO promoter (ICY281). In this strain reporter activity, as indicated by the appearance of the blue precipitate, is modulated through the addition of doxycycline to the medium. In the tet-lacZ tet-BRG1 strain (ICY307) simultaneous over-expression of an ectopic copy of BRG1 under the control of the tetO promoter results in a wrinkled morphology as in the tet-BRG1 strain (ICY171), but does not affect expression of the lacZ reporter inserted at NRG1.

B. lacZ expression was measured using quantitative real-time PCR. In agreement with the phenotypic results, over-expression of BRG1 (ICY307) reduced NRG1 expression but not lacZ expression. Levels were normalized by ACT1 and expressed relative to the tet-lacZ strain (ICY281) time zero samples (therefore those results are 1.0). Error bars represent the standard error of the mean.

C. Constitutive expression of NRG1 from an ectopic ACT1 promoter-regulated allele (strain ICY293) is sufficient to block hyphal induction in YPD at 37°C. Addition of a tet-regulated BRG1 allele, to form strain ICY295, overcame this Nrg1p-mediated repression.

D. Northern blot analysis. The abundance of both the endogenous and larger ectopic NRG1 (arrow) transcripts depends on BRG1 over-expression. Strains were grown in YPD at 37°C for 3 h. Ethidium bromide-stained rRNA bands are shown as a loading control. (-) No DOX. (+) Plus DOX