Highly unsaturated fatty acid synthesis in marine fish: cloning, functional characterization, and nutritional regulation of fatty acyl delta 6 desaturase of Atlantic cod (Gadus morhua L.).

This study reports the cloning, functional characterization, tissue expression, and nutritional regulation of a delta6 fatty acyl desaturase of Atlantic cod (Gadus morhua). PCR primers were designed based on the sequences of conserved motifs in available fish desaturases and used to isolate a cDNA fragment from cod liver, with full-length cDNA obtained by rapid amplification of cDNA ends. The cDNA for the putative desaturase was shown to comprise 1980 bp, including a 261-bp 5'-UTR, a 375-bp 3'-UTR, and an ORF of 1344 bp that specified a protein of 447 amino acids. The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all characteristic of microsomal fatty acyl desaturases. The cDNA displayed delta6 desaturase activity in a yeast expression system. Quantitative real-time PCR assay of gene expression in cod showed that the delta6 desaturase gene was expressed highly in brain, to a slightly lesser extent in liver, kidney, intestine, red muscle, and gill, and at much lower levels in white muscle, spleen, and heart. The expression of the delta6 desaturase gene did not appear to be under significant nutritional regulation, with levels in liver and intestine being barely altered in fish fed a vegetable oil blend, in comparison with levels in fish fed fish oil. This was reflected in enzyme activity, as hepatocytes or enterocytes showed very little highly unsaturated FA biosynthesis activity irrespective of diet. Further studies are required to determine why the delta6 desaturase appears to be barely functional in cod under the conditions tested.

Cloning and functional characterisation of polyunsaturated fatty acid elongases of marine and freshwater teleost fish.

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids are key to the biosynthesis of the highly unsaturated fatty acids, arachidonic, eicosapentaenoic and docosahexaenoic acids from linoleic and alpha-linolenic acids. A Mortierella alpina cDNA polyunsaturated fatty acid elongase sequence identified mammalian, amphibian, zebrafish and insect expressed sequence tags (ESTs) in GenBank. Consensus primers were designed in conserved motifs and used to isolate full length cDNA from livers of several fish species by Rapid Amplification of cDNA Ends (RACE). The amplified cDNAs encoded putative open reading frames (ORFs) of 288-294 amino acids that were highly conserved among the fish species. Heterologous expression in yeast, Saccharomyces cerevisiae, demonstrated that all of the ORFs encoded elongases with the ability to lengthen polyunsaturated fatty acid substrates with chain lengths from C18 to C22 and also monounsaturated fatty acids, but not saturated fatty acids. There were differences in the functional competence of the elongases from different fish species. Most of the fish elongases showed a pattern of activity towards different fatty acid substrates in the rank order C18>C20>C22, although the tilapia and turbot elongases had similar activity towards 18:4n-3 and 20:5n-3. The fish elongases generally showed greater activity or similar activities with n-3 than with n-6 homologues, with the exception of the cod enzyme which was more active towards n-6 fatty acids.

Highly unsaturated fatty acid synthesis in vertebrates: new insights with the cloning and characterization of a delta6 desaturase of Atlantic salmon.

Fish are an important source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a delta5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterized previously. Here we report cloning and functional characterization of a delta6 fatty acyl desaturase of Atlantic salmon and describe its genomic structure, tissue expression, and nutritional regulation. A salmon genomic library was screened with a salmon delta5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106 bp containing an open reading frame of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly delta6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the delta6 desaturase gene constituted 13 exons in 7965 bp of genomic DNA. Quantitative real-time PCR assay of gene expression in Atlantic salmon showed that both delta6 and delta5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver, and brain, and less so in kidney, heart, gill, adipose tissue, muscle, and spleen. Furthermore, expression of both delta6 and delta5 fatty acyl desaturase genes in intestine, liver, red muscle, and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil.

Zebrafish cDNA encoding multifunctional Fatty Acid elongase involved in production of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids.

Enzymes that increase the chain length of fatty acids are essential for biosynthesis of highly unsaturated fatty acids. The gLELO gene encodes a protein involved in the elongation of polyunsaturated fatty acids in the fungus Mortierella alpina. A search of the GenBank database identified several expressed sequence tag sequences, including one obtained from zebrafish (Danio rerio), with high similarity to gLELO. The full-length transcript ZfELO, encoding a polypeptide of 291 amino acid residues, was isolated from zebrafish liver cDNA. The predicted amino acid sequence of the open reading frame shared high similarity with the elongases of Caenorhabditis elegans and human. When expressed in Saccharomyces cerevisiae, the zebrafish open reading frame conferred the ability to lengthen the chain of a range of C18, C20, and C22 polyunsaturated fatty acids, indicating not only that biosynthesis of 22:6n-3 from 18:3n-3 via a 24-carbon intermediate is feasible, but also that one elongase enzyme can perform all three elongation steps required. The zebrafish enzyme was also able to elongate monounsaturated and saturated fatty acids, and thus demonstrates a greater level of promiscuity in terms of substrate use than any elongase enzyme described previously.

Isolation and physical mapping of sex-linked AFLP markers in nile tilapia (Oreochromis niloticus L.).

Gynogenetically produced XX and YY Nile tilapia (Oreochromis niloticus) and diploid control groups were screened for amplified fragment length polymorphisms (AFLPs) to search for sex-linked or sex-specific markers. Family-level bulked segregant analysis (XX and YY gynogenetic family pools) and individual screening (XX and YY gynogenetics and XX and XY control individuals) identified 3 Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420) AFLP markers. OniX420 and OniY425 were shown to be allelic. Single locus polymerase chain reaction assays were developed for these markers. Tight linkage was demonstrated between the AFLP markers and the sex locus within the source families. However, these markers failed to consistently identify sex in unrelated individuals, indicating recombination between the markers and the sex-determining loci. O. niloticus bacterial artificial chromosome clones, containing the AFLP markers, hybridized to the long arm of chromosome 1. This confirmed previous evidence, based on meiotic chromosome pairing and fluorescence in situ hybridization probes obtained through chromosome microdissection, that chromosome pair 1 is the sex chromosomes.

Molecular cloning and functional characterization of fatty acyl desaturase and elongase cDNAs involved in the production of eicosapentaenoic and docosahexaenoic acids from alpha-linolenic acid in Atlantic salmon (Salmo salar).

Fish are the only major dietary source for humans of omega-3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities. Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids. The protein sequence includes all the characteristics of microsomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b(5) domain containing a heme-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an omega-3 delta5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from alpha-linolenic acid (18:3n-3). The desaturase showed only low levels of delta6 activity toward C(18) polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C(18) > C(20) > C(22). Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3.

Responses of bovine chimaeras combining trypanosomosis resistant and susceptible genotypes to experimental infection with Trypanosoma congolense.

West African N'Dama cattle have developed a genetic capacity to survive, reproduce and remain productive under trypanosomosis risk. The cellular and molecular bases of this so-called trypanotolerance are not known, but the trait is manifested by the N'Dama's greater capacity to control parasitaemia and anaemia development during an infection. In order to examine the role of the haematopoietic system in trypanotolerance, we have exploited the tendency for the placentas of bovine twin embryos to fuse. Placental fusion in cattle results in bone marrow chimaerism in twins. By comparison with the N'Dama, cattle of the East African Boran breed are relatively susceptible. We evaluated the role of the haemopoietic system in trypanotolerance by comparing the performance of five Chimaeric Boran/N'Dama twin calves with that of singletons of the two breeds. Chimaeric Boran/N'Dama pairs of twins were produced in recipient Boran cows by embryo transfer, and the majority of haemopoietic cells in all twinned individuals were of Boran origin. Thus, N'Dama chimaeras differed from N'Dama singletons in that the bulk of their haemopoietic system was derived from their susceptible Boran twins, while Boran chimaeras differed little from Boran control animals. All cattle became parasitaemic and developed anaemia. The N'Dama chimaeras did not manage their anaemia and white blood cell counts effectively. However, they were able to limit parasitaemia development. These results suggest that trypanotolerance is the result of two mechanisms, one that improves parasite control and is independent of the genetic origin of the haemopoietic tissue, and another that is influenced by haemopoietic tissue genotype and which improves control over anaemia. The capacity to maintain growth during infection was similarly dependent on the genetic origin of the haemopoietic tissue.

Chromosomal regions controlling resistance to gastro-intestinal nematode infections in mice.

Quantitative trait loci (QTL) associated with resistance to an intestinal worm in a well-defined murine model are described. These have been identified in an F(2) population derived from resistant (SWR) and susceptible (CBA) parental mouse strains infected with the gastro-intestinal nematode parasite Heligmosomoides polygyrus. Seven QTL located on six chromosomes are described, associated with components of the complex host response and the differential regulation of parasite survival and reproduction. The combined additive effects of the five significant QTL associated with worm survival (total worm count at necropsy) account for about 60% of the difference in worm count between the parental lines. The dominance effect for these five QTL are all in the direction of resistance, supporting the heterosis for resistance established from the mean worm count for the F(2) line relative to the parental lines. It is now possible to identify the comparative chromosomal regions of these QTL in livestock and humans and to consider the possibility of future improved control strategies. These may include breeding of resistant or tolerant livestock, development of vaccines, or identification of new anthelmintic drugs.