Abstract
Abstract: :
Purpose: Many studies have shown that aldose reductase is etiologically linked to the pathogenesis of diabetic complications in ocular and neuronal tissues. However, little is known about the detailed functions of this protein and of related aldo-reductases in mammals. Gene-knock and functional genomic approaches provide novel tools to study their homologous genes in S. cerevisiae, through which new insights into the general functions of this family of proteins can be obtained. Methods: Three aldo-keto reductase (AKR) genes (YPR1, GRE3, GCY1) from S. cerevisiae were simultaneously deleted by the PCR-mediated gene disruption approach. Transcription profiling of the triple AKR null mutant strain was compared to that of the isogenic wild type strain under normal culture conditions through DNA microarray analysis using Affymetrix chips. Phenotypic rescue experiments were carried out by transforming triple AKR null and control strains with an expression plasmid containing human aldose reductase cDNA. Results: DNA microarray analysis revealed that 91 genes with broad cellular functions were significantly upregulated in the triple AKR null mutant strain as compared with the wild type strain under normal culture conditions. Transcripts from the INO2 gene, which encodes a transcription factor, were the most highly upregulated of all genes in the mutant strain. Experiments with strains transformed with constitutively active Ras mutants revealed that INO2 transcript levels appeared to be negatively regulated at least in the null mutant strain by the activity of Ras-cAMP signaling pathway. Human aldose reductase partially rescued the heat shock phenotype of the triple AKR null strain. Conclusions: Deletion of multiple AKR genes causes a heat shock phenotype in Saccharomyces cerevisiae. Transcription profiling studies suggest that alterations in expression of INO2, a critical transcription factor, may be linked to this phenotype. The heat shock phenotype of the null mutant strain could be rescued by human aldose reductase suggests that the aldo-keto reductase proteins are functionally conserved between yeast and humans.
Keywords: diabetes • gene microarray • cataract