Abstract: : Purpose: To describe the development of an in vitro yeast expressionsystem for the study of the biochemical and cellular effectsof specific mutations in the ELOVL4 macular disease gene encodinga putative fatty acid elongation enzyme.Methods: Library screeningwas performed to isolate the human wild-type ELOVL4 cDNA whichserved as a template for site-directed mutagenesis to introducesequence variations associated with human retinal degenerativephenotypes. Standard molecular cloning techniques enabled theconstruction of plasmids for expression in the yeast Saccharomycescerevisiae. Western blotting of extracts from yeast cells expressingthe engineered plamids will confirm expression of protein originatingfrom the exogenous cDNAs. Preparation of fatty acid extractsfrom transformed yeast cells grown on media supplemented withvarious fatty acid substrates followed by gas chromatographyand mass spectroscopy enables characterization of the enzymaticactivities of the exogenous wild-type and mutant ELOVL4 cDNAconstructs.Results: Wild-type strain SC334 yeast cells grownon rich media in the absence of exogenous fatty acids were foundto contain saturated and monounsaturated fatty acid speciesup to 18 carbon atoms in length but not polyunsaturated fattyacids (PUFAs) or longer chain species, suggesting that the yeastcells lack the biochemical components required to synthesizelong-chain and polyunsaturated fatty acids, enabling the characterizationof exogenous enzyme activities on the elongation of long-chainPUFA substrates. ELOVL4 cDNA constructs containing either the797-801delAACTT or 790delT+794delT macular disease-associatedmutations were synthesized by site-directed mutagenesis of ahuman brain-derived wild-type ELOVL4 cDNA clone. The wild-typeand mutant cDNAs have been inserted into the yeast expressionplasmids pYES2 and pYPGE15 for inducible and constitutive expressionrespectively. Assessment of substrate specificity of the wild-typeELOVL4 cDNA and defects in enzymatic function of the mutantcDNAs in the yeast functional assay system is currently underway.Conclusions: The yeast Saccharomyces cerevisiae is a valuablemodel system that can be used to study the effects of exogenouslyexpressed fatty acid biosynthetic enzymes on lipid metabolism.Through its use we aim to gain insight into the biochemicalfunction of the ELOVL4 gene in the normal and degenerating retina.