Isolation and characterization of cDNA sequences of L-gulono-gamma-lactone oxidase, a key enzyme for biosynthesis of ascorbic acid, from extant primitive fish groups.

Most advanced teleosts lack L-gulono-gamma-lactone oxidase (GULO), a key enzyme required for the biosynthesis of ascorbic acid. However, extant representatives of primitive species including sturgeon and many cartilaginous fishes, are exceptional in their ability to synthesize ascorbic acid de novo. In the present study, full-length GULO cDNAs were isolated from white sturgeon (Acipenser transmontanus) and two shark species belonging to the Triakidae (Triakis scyllium and Mustelus manazo). The open reading frames from all three species contained 440 amino acids and the deduced polypeptides had similar hydropathy profiles, predicted molecular masses and theoretical pI values. These GULO sequences exhibited high amino acid identity (67-97%) with each other, and also shared 61-71% identity with mammalian GULOs. Based on the GULO sequences obtained from these species, we developed degenerate primers for the isolation of partial GULO sequences by RT-PCR from other primitive species including another shark (Mustelus griseus, Triakidae), a spiny dogfish (Squalus acanthias, Squalidae), two ray species (Raja kenojei, Rajidae and Dasyatis akajei, Dasyatidae) and four sturgeons (Acipenser baeri, A. gueldenstaedtii, A. naccarii and A. ruthenus, Acipenseridae). Overall, sequence identities of these amplified GULO segments among primitive species were 63-99% at the nucleotide level and 67-100% at the amino acid level. Considerable numbers of amino acid residues were unique to either fish or mammals, and Acipenseriform species occupied an intermediate position, sharing several residues with either fish or mammalian GULOs. Phylogenetic analyses based on parsimony, distance and likelihood methods of both nucleotide and amino acid sequences resulted in trees that were in agreement with known taxonomy. The transcription and enzyme activity of GULO were kidney-specific when measured by biochemical assay and reverse transcription-PCR.

Dietary rapeseed oil affects the expression of genes involved in hepatic lipid metabolism in Atlantic salmon (Salmo salar L.).

Supplies of marine fish oils (FO) are limited, and sustainable production in aquaculture dictates that alternatives that do not compromise fish health and product quality, such as vegetable oils, must be found. Nutrigenomics will increase our understanding of how nutrition influences metabolic pathways and homeostatic control, and may be used to measure and validate subtle changes in organ-specific, metabolic gene expression signatures. We compared 2 groups of Atlantic salmon fed diets containing 100% FO or 75% rapeseed oil (RO) for 42 wk. A small-scale cDNA microarray was constructed to screen for changes in the expression of lipid metabolism genes in the liver resulting from this partial substitution of RO for FO. Delta5 fatty acid desaturase gene expression was significantly greater in fish fed 75% RO than in fish fed the control diet; this was confirmed by quantitative real time PCR analysis. In addition, several genes, among these mitochondrial proteins, peroxisome proliferator-activated receptor gamma, as well as other transcription factors, coactivators, and signal transducers, showed significant differential regulation. This partially validated microarray may be used for further gene expression profiling using other dietary comparisons, and for further characterization of selected genes.