Disruption and phenotypic analysis of six open reading frames from chromosome VII of Saccharomyces cerevisiae reveals one essential gene

Six open reading frames (ORFs) located on chromosome VII of Saccharomyces cerevisiae (YGR205w, YGR210c, YGR211w, YGR241c, YGR243w and YGR244c) were disrupted in two different genetic backgrounds using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YGR211w, recently identified as the yeast ZPR1 gene, is an essential gene. The other five genes are non-essential, and no phenotypes could be associated to their inactivation. Two of these genes have recently been further characterized: YGR241c (YAP1802) encodes a yeast adaptor protein and YGR244c (LSC2) encodes the b-subunit of the succinyl-CoA ligase. For each ORF, a replacement cassette with long flanking regions homologous to the target locus was cloned in pUG7, and the cognate wild-type gene was cloned in pRS416.

Hydrogen peroxide sensing, signaling and regulation of transcription factors

The regulatory mechanisms by which hydrogen peroxide (H2O2) modulates the activity of transcription factors in bacteria (OxyR and PerR), lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4) and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1) are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1) synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii) stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii) cytoplasm-nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and, (iv) DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M-1s−1 and ≥ 1.3 × 103 M-1s−1 were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for highly specific effects on gene regulation that depend on the cell type and on signals received from the cellular microenvironment.