Figure 1.

Inactivation of DRN causes alteration of the UPR activity in a reciprocal manner. Northern blot (A) and histogram obtained from the qPCR analysis (B) revealing the levels of HAC1 pre-mRNA in a wild type (yBD-117), cbc1-Δ (yBD-131), rrp6-Δ (yBD-129) strains in the absence of ER stress. The northern blot was hybridized with a DNA fragment encompassing both the intronic and exonic sequences of the HAC1 gene. For qRT-PCR analysis, the amplicon used to quantify the HAC1 species encompasses the intronic sequence of HAC1. SCR1 RNA in these strains was used as internal loading control. Three independent cDNA preparations (biological replicates, n = 3) were used to determine the levels of HAC1 mRNA. Normalized value of HAC1 mRNA from normal samples was set to 1. (C) Relative steady state level of Hac1p protein in wild type (yBD-117), cbc1-Δ (yBD-131), rrp6-Δ (yBD-129) strains in presence of 1 μg/ml tunicamycin. α-tubulin (lower panel) was used as the loading control. Cells of indicated strains were grown in absence and in the presence of tunicamycin, harvested and total protein extract were prepared as described in Materials and Methods. 50 μg of total cellular protein was loaded in each lane, resolved in 12% SDS-PAGE and probed with anti-Hac1p and anti-tubulin antibodies to detect these proteins as described in Materials and Methods. (D) Relative growth of wild type (yBD-117), cbc1-Δ (yBD-131), rrp6-Δ (yBD-129) strains in YPD solid growth media in the absence (–Tm) and presence of 1 μg/ml tunicamycin (+Tm). (E) Relative growth of Wild type (yBD-117) (Black Bar), cbc1-Δ (yBD-131) (Red Bar), rrp6-Δ (yBD-129) (Blue Bar) strains in YPD liquid growth media in the absence (–Tm) and the presence of 1 μg/ml tunicamycin (+Tm). The histogram represents the mean colony numbers obtained from three independent experiments (n = 3) (biological replicates), and the standard error of means was plotted as error bars. (F) Relative fitness of sustenance of Wild type (yBD-117) (Black), cbc1-Δ (yBD-131) (red), rrp6-Δ (yBD-129) (blue) strains in presence tunicamycin. Relative index values were estimated from the experiment described in panel E, which is defined as the ratio of no. colonies of a given strain in the presence of tunicamycin to the number of colonies in the absence of tunicamycin. (G) Histogram showing the relative growth efficiency of wild type (yBD-117), cbc1-Δ (yBD-131), rrp6-Δ (yBD-129) strains in liquid YPD medium in absence and presence of 5 mM DTT. Cells of indicated strains were grown in YPD liquid medium till the A₆₀₀ of the cultures reach 0.6 as mentioned in the Materials and Methods. Either water (–DTT) or 5 mM DTT (+DTT) was then added to them, and they were continued to grow for another 240 min. The relative viability count was determined as described in the Materials and Method section.