Purchase this article with an account.
P.S. Lagali, R. Ambasudhan, J. Liu, R. Ayyagari, P.W. Wong; Use of the Yeast Saccharomyces cerevisiae as a Model Organism to Study Human Retinal Disease Gene Mutations . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5097.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To describe the development of an in vitro yeast expression system for the study of the biochemical and cellular effects of specific mutations in the ELOVL4 macular disease gene encoding a putative fatty acid elongation enzyme. Methods: Library screening was performed to isolate the human wild-type ELOVL4 cDNA which served as a template for site-directed mutagenesis to introduce sequence variations associated with human retinal degenerative phenotypes. Standard molecular cloning techniques enabled the construction of plasmids for expression in the yeast Saccharomyces cerevisiae. Western blotting of extracts from yeast cells expressing the engineered plamids will confirm expression of protein originating from the exogenous cDNAs. Preparation of fatty acid extracts from transformed yeast cells grown on media supplemented with various fatty acid substrates followed by gas chromatography and mass spectroscopy enables characterization of the enzymatic activities of the exogenous wild-type and mutant ELOVL4 cDNA constructs. Results: Wild-type strain SC334 yeast cells grown on rich media in the absence of exogenous fatty acids were found to contain saturated and monounsaturated fatty acid species up to 18 carbon atoms in length but not polyunsaturated fatty acids (PUFAs) or longer chain species, suggesting that the yeast cells lack the biochemical components required to synthesize long-chain and polyunsaturated fatty acids, enabling the characterization of exogenous enzyme activities on the elongation of long-chain PUFA substrates. ELOVL4 cDNA constructs containing either the 797-801delAACTT or 790delT+794delT macular disease-associated mutations were synthesized by site-directed mutagenesis of a human brain-derived wild-type ELOVL4 cDNA clone. The wild-type and mutant cDNAs have been inserted into the yeast expression plasmids pYES2 and pYPGE15 for inducible and constitutive expression respectively. Assessment of substrate specificity of the wild-type ELOVL4 cDNA and defects in enzymatic function of the mutant cDNAs in the yeast functional assay system is currently underway. Conclusions: The yeast Saccharomyces cerevisiae is a valuable model system that can be used to study the effects of exogenously expressed fatty acid biosynthetic enzymes on lipid metabolism. Through its use we aim to gain insight into the biochemical function of the ELOVL4 gene in the normal and degenerating retina.
This PDF is available to Subscribers Only