No abstract

The PAR1/SNQ3 gene of S. cerevisiae, which increases resistance to iron chelators in multi-copy transformants, is identical to the YAP1 gene, a yeast activator protein isolated as a functional homologue of the human c-jun oncogene by binding specifically to the AP-1 consensus box. The observed H2O2-sensitivity of par1 mutants has been attributed to an increased sensitivity to reduced oxygen intermediates. Accordingly, par1 mutants did not survive an elevated oxygen pressure and were very sensitive to menadione and methylviologene, two chemicals enhancing the deleterious effects of oxygen. The specific activities of enzymes involved in oxygen detoxification, such as superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase, were decreased in par1 mutants and increased after PAR1 over-expression. As in the case of oxygen detoxification enzymes, the cellular levels of glutathione were similarly affected. These observations indicate that PAR1/YAP1/SNQ3 is involved in the gene regulation of certain oxygen detoxification enzymes. The finding that H2O2 promotes DNA-binding of human c-jun is consistent with a similar function for PAR1/YAP1/SNQ3 and c-jun in cellular metabolism.

We have isolated mutants of Saccharomyces cerevisiae with an increased sensitivity to oxidative stress. All pos9 mutants (pos for peroxide sensitivity) were hypersensitive to methylviologene, hyperbaric oxygen or hydrogen peroxide, but grew similarly to the wild-type under all other conditions tested. Isolation and sequencing of the respective POS9 gene revealed that it was identical to SKN7. The predicted Skn7/Pos9 protein possesses a domain with high homology to prokaryotic response regulators. These regulatory proteins are part of a simple signalling cascade termed a "two-component system", where a phosphorylation signal of a histidine kinase is transferred to a conserved aspartate residue of the response regulator. To test the functional role of the respective aspartate residue of Skn7/Pos9 protein in oxidative stress, we mutagenized this residue in vitro to alanine, arginine and glutamate. Only the glutamate allele (D427 to E) was able to rescue the hydrogen peroxide-sensitivity of pos9 mutants. By fusion experiments with the Gal4 DNA-binding domain we identified the isolated response regulator-like domain as a novel eukaryotic domain sufficient for gene activation. Whereas this hybrid protein activated transcription of a lacZ reporter gene under aerobic conditions, no activation was observed under anaerobic conditions, indicating that the response regulator domain is involved in a signalling reaction. Two-hybrid investigations also suggest an oligomerization of the Pos9 protein. Our results indicate that a two-component system is involved in the oxidative-stress response of yeast.

Exposure of Saccharomyces cerevisiae to elevated concentrations of hydrogen peroxide induces transcription of several genes involved in the oxidative stress response. Two major transcription factors are involved in this induction, Pos9/Skn7 and Yap1. Fusions of either Yap1 or Pos9/Skn7 with the Gal4 DNA binding domain are active as transcription factors. Gal4-Yap1-dependent reporter gene activity is only weakly regulated by oxidative stress. In contrast, fusion of the Gal4 DNA binding domain to the Pos9/Skn7 protein results in a transcription factor that is independent of the YAP1 gene and is strictly regulated by oxidative stress, indicating that a signaling cascade impinges on the Pos9/Skn7 protein. We have observed that the Ras/PKA (cAMP-dependent protein kinase A) pathway affects this signaling. When PKA activity was low (in the presence of multicopy PDE2 or a cyr1(D822-->A) mutation) maximum reporter gene activity was observed even in the absence of oxidative stress. In contrast, high PKA activity (in strains mutant for either pde2 or bcy1, or expressing the dominant active Ras2Val19) resulted in a complete loss of activation following oxidative stress. The transcription of Pos9/Skn7 target genes was also affected in Ras/PKA pathway mutants. Furthermore, we demonstrated that activated Pos9/Skn7 is necessary for Yap1-dependent reporter gene induction.

Open reading frames (6116) of the budding yeast Saccharomyces cerevisiae were PCR-amplified from genomic DNA using 12,232 primers specific to the ends of the coding sequences; the success rate of amplification was 97%. PCR-products were made accessible to hybridization by being arrayed at very high density on solid support media using various robotic devices. Probes made from total RNA preparations were hybridized for the analysis of the transcriptional activity of yeast under various growth conditions and of different strains. Experimental factors that proved critical to the performance, such as different RNA isolation procedures and the assessment of hybridization results, for example, were investigated in detail. Various software tools were developed that permit convenient handling and sound analysis of the large data quantities obtained from transcriptional profiling studies. Comprehensive arrays are being distributed within the European Yeast Functional Analysis Network (EUROFAN) and beyond. 1998 John Wiley & Sons, Ltd.