As exemplified in Figure 5A KPT-8602 solubility dmso Δphx1 mutant became sensitive to oxidants such as H2O2 (peroxidation
agent), paraquat and menadione (superoxide-generating agent), diamide (thiol-specific oxidant) and also to heat at 42°C. These results indicate clearly that Phx1 confers fitness to cells not only during nutrient starvation but also under oxidative and heat stress conditions. We analyzed whether these stress conditions induce the expression of the phx1 + gene by analyzing its RNA by qRT-PCR. The results in Figure 5B demonstrate that these acute stresses indeed elevated the level of phx1 + mRNA. Figure 5 Stress-sensitivity of Δphx1 mutant and the inducibility of phx1 + gene by various stresses. (A) Stress-sensitivity of Δphx1 mutant. To examine INK1197 manufacturer sensitivity of the wild-type (JH43) and Δphx1 mutant to various oxidants and heat, exponentially growing cells in liquid EMM at 30°C were treated with 10 mM of H2O2, 20 mM of paraquat, 20 mM of diamide, or 2 mM menadione for 40 min each, or transferred to 42°C incubator for 30 min. Following stress treatment, equal number of cells were serially diluted, spotted onto EMM plates, and incubated at 30°C for 4 to 5 days. (B) Inducibility of phx1 + gene by various stresses. The wild-type (JH43) cells were grown to mid-exponential phase (OD600 of 0.5-1) in liquid EMM at 30°C, and treated
with 10 mM hydrogen peroxide, 20 mM paraquat (PQ), 20 mM diamide (DA), or 2 mM menadione (MD) for 40 min each, or heat-shocked at 50°C for 30 min. RNA samples were analyzed for the level of phx1 + transcript
in comparison with act1 + , an internal control, by qRT-PCR. The average induction folds with standard deviations (error bars) from three independent experiments were presented. The Δphx1/Δphx1 diploid is defective in sporulation When cells are starved of nutrients such as nitrogen or carbon sources, haploid yeast cells find other mating-type partners, conjugate to form diploids, which subsequently undergo meiotic division and sporulation. All of these sexual development processes are controlled by an extensive gene expression program [28, 29]. A genome-wide analysis of S. pombe transcriptome has revealed that phx1 + (SPAC32A11.03 c) is one of the genes that are highly induced during meiotic spore formation . This led us to examine Tryptophan synthase whether Phx1 plays any role in meiosis. We first examined the mating efficiency of Δphx1 mutant cells. Crossing h – and h + haploid Δphx1 strains showed similar mating efficiency (54.2 ± 0.5%) to that of the wild type (56.7 ± 0.9%). Crossing between the wild type and Δphx1 was similarly effective (53.1 ± 2.9%). This suggests that Δphx1 mutation does not significantly impair conjugation and diploid formation. Therefore we obtained homozygous diploid strain Δphx1/Δphx1 and examined the formation of tetrad meiotic spores by incubating in EMM.