Skip to main content
. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: Mol Microbiol. 2012 Jul 5;85(3):557–573. doi: 10.1111/j.1365-2958.2012.08127.x

Figure 4. Analysis of BRG1 and NRG1 expression during hyphal induction.

Figure 4

BRG1 and NRG1 transcription was examined during hyphal induction (YPD+10% FBS, 37°C) by northern blot analyses. RNA was isolated, transferred to a Nytran membrane and probed with DNA fragments specific to the BRG1 or NRG1 coding sequence. Ethidium bromide-stained rRNA bands are shown as a loading control. Numbers represent time post-induction.

A. In a wild-type strain (SC5314), BRG1 transcript is undetectable in yeast cells (Time 0), is abundant 30 minutes after induction and subsequently declines. Expression of NRG1 is high in yeast cells (Time 0), falls during the first hour of hyphal induction and gradually returns to pre-induction levels.

B. In the brg1Δ strain (ICY326), no BRG1 transcript is detectable and during hyphal induction, NRG1 levels remain high.

C. In the tet-BRG1 strain (ICY171), when BRG1 expression is induced from a tet-regulated allele, BRG1 levels rise steadily over time, whilst NRG1 levels fall. When BRG1 is not over-expressed, through the addition of DOX to the medium, NRG1 levels remain stable (YPD 28°C).

D. When BRG1 is over-expressed in yeast conditions (YPD 28°C) in the tet-BRG1 strains ICY171 and ICY175, NRG1 levels fall. Note that in ICY175, transcription from both the endogenous and tet-regulated (arrow) NRG1 alleles is affected. (-) No DOX, (+) Plus DOX.

E. Quantitative real-time PCR analysis confirmed that NRG1 mRNA destabilization depends on BRG1. The tet-NRG1 (SSY50-B) and brg1Δ (ICY277) strains were grown overnight (YPD 28°C No DOX) then diluted intro fresh medium and grown under hypha inducing conditions (YPD+10% FBS 37°C Plus DOX). In the tet-NRG1 strain, NRG1 levels fall substantially by 30 minutes after induction whilst in the brg1Δ strain NRG1 levels remain stable. Levels were normalized by ACT1 and expressed relative to the respective time zero samples (therefore those results are 1.0). Error bars represent the standard error of the mean.

F. During hyphal induction (YPD+10% FBS 37°C 2 h) NRG1 transcript is most abundant in the brg1Δ (ICY326) strain. Levels are returned to those observed in the BRG1/brg1Δ heterozygote (ICY318) by reintegrating a copy of BRG1 at its native locus (ICY330).