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Q Chang, L Rikimaru, J Petrash; A Functional Genomics Model for Diabetic Eye Disease . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1331.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Aldose reductase has been implicated as a factor in the pathogenesis of diabetic eye disease. However, little is known about the physiological role of this enzyme and related aldo-keto reductases in the eye. The purpose of the present study is to utilize the budding yeast Saccharomyces cerevisiae for a functional genomics study of the physiological function of aldose reductase. Methods: Yeast open reading frames (ORF) with sequence similarity to human aldose reductase (HAR) were identified by BLAST analysis. Targeted genes were ablated by a PCR-mediated recombination method, utilizing a variety of auxotrophic markers to select for deletion mutants. Mutant strains containing deletions of up to three of the yeast ORFs with highest similarity to HAR were subjected to a range of phenotypic screens. These included morphology and viability, utilization of alternate carbon sources, growth sensititivity to PKC and MAPK-linked pathways, and stress response. Phenotypic suppressor genes were identified by screening a yeast expression library for ORFs that rescued the heat shock phenotype in the AR triple-null strain. Results: BLAST and functional studies identified six yeast ORFs whose encoded proteins were structurally and/or functionally similar to HAR. A triple-null deletion strain involving genes of the three ORFs with the highest similarity to HAR (YDR368W, YHR104W, YOR120W) showed no detectable change in morphology or selective auxotrophies for carbon sources. Compared with wild type strains, the triple-null mutant showed a modest increase in doubling time as well as a marked increase in glucose-dependent sensitivity to oxidative stress and heat shock. Suppression of the heat stress phenotype was observed when the triple null strain was transformed with genomic fragments containing ORFs encoding negative regulators of the RAS-cAMP pathway. Conclusion: The budding yeast Saccharomyces cerevisiae represents a useful genetic model in which to probe the physiological role of aldose reductase. The phenotypes of mutant strains containing selective deletions of multiple aldo-keto reductases suggest a role in stress response for this family of genes.
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