ABSTRACT
Despite the scientific and applied interest in the anaerobic metabolism of Saccharomyces cerevisiae, not all genes whose transcription is upregulated under anaerobic conditions have yet been linked to known transcription factors. Experiments with a reporter construct in which the promoter of the anaerobically upregulated TIR1 gene was fused to lacZ revealed a loss of anaerobic upregulation in an snf7Delta mutant. Anaerobic upregulation was restored by expression of a truncated allele of RIM101 that encodes for a constitutively active Rim101p. Analysis of lacZ expression in several deletion mutants confirmed that the effect of Snf7p on anaerobic upregulation of TIR1 involved Rim101p. Further studies with deletion mutants in NRG1, NRG2 and SMP1, which were previously shown to be regulated by Rim101p, could not totally elucidate the TIR1 regulation, suggesting the involvement of a more complex regulation network. However, the aerobic repression mechanism of TIR1 involved the general repressor Ssn6p-Tup1p. Transcriptome analysis in anaerobic chemostat cultures revealed that 26 additional genes exhibited an Snf7p/Rim101p-dependent anaerobic upregulation, among which, besides TIR1, are four other anaerobic genes SML1, MUC1, AAC3 and YBR300C. These results provide new evidence on the implication of the Rim101p cascade in the transcriptional regulation of anaerobic metabolism in S. cerevisiae.
Subject(s)
Endosomal Sorting Complexes Required for Transport/physiology , Gene Expression Regulation, Fungal , Heat-Shock Proteins/biosynthesis , Repressor Proteins/physiology , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/physiology , Transcription, Genetic , Anaerobiosis , Artificial Gene Fusion , Endosomal Sorting Complexes Required for Transport/deficiency , Gene Deletion , Gene Expression Profiling , Genes, Reporter , Saccharomyces cerevisiae/genetics , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
The applicability of transcriptomics for functional genome analysis rests on the assumption that global information on gene function can be inferred from transcriptional regulation patterns. This study investigated whether Saccharomyces cerevisiae genes that show a consistently higher transcript level under anaerobic than aerobic conditions do indeed contribute to fitness in the absence of oxygen. Tagged deletion mutants were constructed in 27 S. cerevisiae genes that showed a strong and consistent transcriptional upregulation under anaerobic conditions, irrespective of the nature of the growth-limiting nutrient (glucose, ammonia, sulfate or phosphate). Competitive anaerobic chemostat cultivation showed that only five out of the 27 mutants (eug1Delta, izh2Delta, plb2Delta, ylr413wDelta and yor012wDelta) conferred a significant disadvantage relative to a tagged reference strain. The implications of this study are that: (i) transcriptome analysis has a very limited predictive value for the contribution of individual genes to fitness under specific environmental conditions, and (ii) competitive chemostat cultivation of tagged deletion strains offers an efficient approach to select relevant leads for functional analysis studies.
