Bożenna Rempoła

Functional analysis of RRD1 (YIL153w) and RRD2 (YPL152w), which encode two putative activators of the phosphotyrosyl phosphatase activity of PP2A in Saccharomyces cerevisiae.

Abstract In the context of the cooperative project for functional analysis of novel genes uncovered during the systematic sequencing of the Saccharomyces cerevisiae genome, we deleted two paralogous ORFs: YIL153w and YPL152w. Based on the resulting phenotypes, the corresponding genes were named RRD1 and RRD2, respectively. Rrd proteins show significant similarity to the human phosphotyrosyl phosphatase activator (PTPA). Both single mutants, rrd1Δ and rrd2Δ, were viable. Deletion of RRD1 caused pleiotropic phenotypes under a wide range of conditions, including sensitivity to Ca2+, vanadate, ketoconazole, cycloheximide and Calcofluor white, and resistance to caffeine and rapamycin. The only phenotypes found for rrd2Δ– resistance to caffeine and rapamycin – were weaker than the corresponding phenotypes of rrd1Δ. The double mutant rrd1,2Δ was inviable on rich glucose medium, but could grow in the presence of an osmotic stabilizer. The rrd1,2Δ mutant was partially rescued by inactivation of HOG1 or PBS2, suggesting an interaction between the RRD genes and the Hog1p signal transduction pathway. Introduction of slt2Δ into the rrd1,2Δ background improved the growth of rrd1,2Δ on sorbitol-containing medium, indicating that the Rrd proteins also interact with the Slt2p/Mpk1p signaling pathway. Suppression of the lethal phenotype of the rrd1,2Δ mutant by overexpression of PPH22 suggested that the products of the RRD genes function positively with catalytic subunits of PP2A. The synthetic lethality was also suppressed by the “viable” allele (SSD1-v1) of the SSD1 gene.

Mutants of the Saccharomyces cerevisiae VPS genes CCZ1 and YPT7 are blocked in different stages of sporulation

The CCZ1 gene is a member of the class B VPS (vacuolar protein sorting) genes and it is engaged in the last stage of delivery of multiple kinds of cargo to the yeast vacuole. In the process of fusion of the multivesicular body (MVB) with the vacuole, Ccz1p forms a complex with Ypt7p. Both genes are non-essential for vegetative growth, but their deletions cause a complete block in spore formation. The results of this study indicate that ccz1Δ cells initiate the meiotic program, properly proceed through premeiotic DNA replication and through the pairing of homologous chromosomes, but fail to progress through the first meiotic divisions and arrest in prophase I with a single nucleus. The mutant cells are defective in spindle formation as well as in duplication and/or separation of the SPBs. ypt7Δ cells, on the other hand, cannot execute DNA synthesis. We also show that expression of a mutated variant of the YPT7 gene suppresses the sporulation and autophagy defects of ccz1Δ cells to a quantitatively similar level, suggesting that restoration of autophagy in the ccz1Δ strain is sufficient to enable its sporulation.

Genotoxicity assessment of low-molecular weight interferon inducers by the SOS Chromotest

Tilorone and its aza-analogs, as well as CMA and its butyric analog (CNPA) were investigated as potential genotoxic agents by the SOS Chromotest. The SOS-inducing potency values (SOSIP) were 0.0033 and 0.0009 for SAF and vivakorfen, respectively, after activation with S9 fraction of mouse liver only. In contrast, an SOSIP value for tilorone of 0.0011 was observed in a non-activated assay. The SOSIPs of investigated compounds were low and comparable to the lowest values determined for other genotoxins. CMA and CNPA were not SOS inducers in any test system.