Multiple genes for functional 6 fatty acyl desaturases (Fad) in Atlantic salmon (Salmo salar L.): Gene and cDNA characterization, functional expression, tissue distribution and nutritional regulation

Article abstract of DOI:10.1016/j.bbalip.2010.04.007

Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids, eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs encoding functionally distinct 5 and 6 Fads were isolated, but screening of a genomic DNA library revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes, 6fad_a and 5fad, corresponded to the previously cloned 6 and 5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, 6fad_b and 6fad_c, had only 6 activity, converting 47 % and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of 18:2n-6 to 18:3n-6, for 6Fad_b and 6fad_c, respectively. Both 6fad_a and 6fad_b genes were highly expressed in intestine (pyloric caeca), liver and brain, with 6fad_b also highly expressed in gill, whereas 6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The expression levels of the 6fad_a gene in liver and the 6fad_b gene in intestine were significantly higher in fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for 6fad_a in intestine, 6fad_b in liver, or 6fad_c in liver or intestine of fish fed vegetable oil compared to fish fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes are discussed in relation to gene structures and fish physiology.

Full text of DOI:10.1016/j.bbalip.2010.04.007

Biochimica et Biophysica Acta 1801 (2010) 1072–1081
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbalip
Multiple genes for functional Δ6 fatty acyl desaturases (Fad) in Atlantic salmon
(Salmo salar L.): Gene and cDNA characterization, functional expression, tissue
distribution and nutritional regulation
⁎
Óscar Monroig , Xiaozhong Zheng, Sofia Morais, Michael J. Leaver, John B. Taggart, Douglas R. Tocher
Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids,
eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of
higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions
catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs
encoding functionally distinct Δ5 and Δ6 Fads were isolated, but screening of a genomic DNA library
revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we
show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes,
Δ6fad_a and Δ5fad, corresponded to the previously cloned Δ6 and Δ5 Fad cDNAs. Functional characterization
by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes,
Δ6fad_b and Δ6fad_c, had only Δ6 activity, converting 47 % and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of
18:2n-6 to 18:3n-6, for Δ6Fad_b and Δ6fad_c, respectively. Both Δ6fad_a and Δ6fad_b genes were highly
expressed in intestine (pyloric caeca), liver and brain, with Δ6fad_b also highly expressed in gill, whereas
Δ6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The
expression levels of the Δ6fad_a gene in liver and the Δ6fad_b gene in intestine were significantly higher in
fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to
reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for
Δ6fad_a in intestine, Δ6fad_b in liver, or Δ6fad_c in liver or intestine of fish fed vegetable oil compared to fish
fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes are
discussed in relation to gene structures and fish physiology.
Received 23 January 2010
Received in revised form 25 March 2010
Accepted 7 April 2010
Available online 18 April 2010
Keywords:
Atlantic salmon
cDNA
Fatty acyl desaturase
Long-chain polyunsaturated fatty acids
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
LC-PUFA from C18 fatty acyl precursors [3–5]. It is this fundamental
aspect of human fatty acid metabolism that partly underpins the well-
Biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA)
in vertebrates involves sequential desaturation and elongation of
precursor PUFA, 18:2n-6 and 18:3n-3. Synthesis of arachidonic acid
(ARA; 20:4n-6) is achieved by Δ6 desaturation of 18:2n-6 to produce
18:3n-6 that is elongated to 20:3n-6 followed by Δ5 desaturation [1].
Synthesis of eicosapentaenoic acid (EPA; 20:5n-3) from 18:3n-3
requires the same enzymes and pathway as for ARA, but docosahex-
aenoic acid (DHA; 22:6n-3) synthesis requires two further elongation
steps, a second Δ6 desaturation and a peroxisomal chain shortening
step [2]. However, humans are rather inefficient at the production of
known phenomenon that increasing our dietary intake of the omega-
3 or n-3 LC-PUFA, EPA and DHA, has beneficial effects in a number of
inflammatory and pathological conditions, including cardiovascular
and neurological diseases [6–8], and the much greater efficacy of fish
oil containing EPA and DHA than linseed/flax oil, rich in 18:3n-3, in
intervention studies [9].
As with all vertebrates, PUFA are essential in the diet of fish, but
requirements vary with species [10]. Atlantic salmon (Salmo salar) are
very similar to humans as, although they possess all the enzyme
activities required for the production of LC-PUFA, they are inefficient
in the production of EPA and, especially, DHA from C18 fatty acyl
precursors [11]. In many ways the molecular control and basis of
LC-PUFA synthesis in vertebrates is better understood in fish than in
humans, with key studies in model species such as zebrafish [12–14],
as well as commercial important species such as salmon [15–20]. This
interest has been driven by the crucial role fish play as the primary
source of n-3 LC-PUFA in the human food chain [21]. With declining
fisheries worldwide [22], farmed fish constitute an ever-increasing
Abbreviations: DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FO, fish oil;
LC-PUFA, long-chain polyunsaturated fatty acids; ORF, open reading frame; qPCR,
quantitative (real-time) polymerase chain reaction; RACE, rapid amplification of cDNA
ends; UTR, untranslated region; VO, vegetable oil
⁎
Corresponding author. Institute of Aquaculture, University of Stirling, Stirling FK9
4LA, Scotland, UK. Tel.: +44 1786 467993; fax: +44 1786 472133.
E-mail address: oscar.monroig@stir.ac.uk (Ó. Monroig).
1388-1981/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbalip.2010.04.007

Ó. Monroig et al. / Biochimica et Biophysica Acta 1801 (2010) 1072–1081
1073
proportion of the fish in the human food basket amounting to almost
50 % in 2008 [23]. Until now, high n-3 LC-PUFA levels in farmed fish
have been obtained by the use in the feeds of fish oils (FOs),
paradoxically themselves derived from industrial (reduction) marine
fisheries, but this is not sustainable and will constrain continuing
growth of aquaculture activities [24,25]. In consequence, alternatives
to FO are urgently required, with vegetable oils (VOs) as the prime
candidates [26]. However, VOs are rich in C18 PUFA, but devoid of the
n-3 LC-PUFA abundant in FO [27]. Feeding fish on VO can have
important consequences for the human consumer as it lowers the n-3
LC-PUFA content of the flesh compromising the nutritional value [28].
Our overarching hypothesis is that understanding the molecular
basis of LC-PUFA biosynthesis and regulation will allow us optimise
the activity of the pathway to enable efficient and effective use of
sustainable plant-based alternatives in aquaculture while maintaining
the nutritional quality, in particular the n-3 LC-PUFA content, of
farmed fish for the human consumer. The extent to which any species
can convert C18 PUFA to LC-PUFA varies, associated with their
complement of fatty acyl desaturase (Fad) and very long-chain fatty
acyl elongase (Elovl) enzymes. Elovl enzymes account for the
condensation reaction of activated fatty acids with malonyl-CoA in
the elongation multi-enzyme complex, and three members of the
family, Elovl2, Elovl5 and Elovl4, are involved in LC-PUFA biosynthesis
in mammals [29]. Molecular studies have revealed that salmon
express several Elovl enzymes, and cDNAs for Elovl2 and two distinct
Elovl5s have been isolated and functionally characterized [19,20]. The
Fad enzymes introduce double bonds in the fatty acyl chain at carbon
6 (Δ6 Fad) or carbon 5 (Δ5 Fad) from the carboxyl group [1], and two
distinct cDNAs encoding functional Δ5 and Δ6 Fads have been isolated
from salmon [16,19]. However, in a recent study characterizing fad
gene promoters [18], screening performed on a genomic DNA library
revealed the existence of more putative fad genes in the Atlantic
salmon genome.
uncharacterized fad genes. When intronic sequences were removed,
alignment of the predicted cDNAs for the four fad genes showed the
entire open reading frame (ORF) was obtained for one of the new
desaturases (subsequently termed Δ6fad_b/Δ6 Fad_b), whereas only
partial ORF sequence from the start codon (∼1000 bp, cf. 1373 full-
length ORF) was available for the other (subsequently named
Δ6fad_c/Δ6 Fad_c). To obtain the complete ORF sequence of Δ6
Fad_c and the 3′ untranslated regions (UTR) of both uncharacterized
fad genes, specific PCR primers were designed, and 3′ rapid
amplification of cDNA ends (RACE) PCR (FirstChoice® RLM-RACE
kit, Ambion, Applied Biosystems, Warrington, UK) was performed on
cDNA synthesized from 5 μg of total brain RNA. Details of all PCR
primers used in this study are given in Table 1. For Δ6fad_b, a positive
3'RACE PCR fragment was obtained by two-round PCR. The first round
PCR was performed using the gene specific sense primer SSD6bF1 and
the adapter specific 3'RACE OUTER primer, with an initial denaturing
step at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C
for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 2 min,
followed by a final extension at 72 °C for 5 min (GoTaq® Colorless
Master Mix, Promega, Southampton, UK). First round PCR products
were used as template for nested PCR with primers SSD6bF2 and
3'RACE INNER in a 30-cycle reaction under the same thermal conditions
as above. A similar approach was followed for Δ6fad_c, with first round
PCR performed with primers SSD6cF1 and 3'RACE OUTER, with an initial
denaturating step at 95 °C for 1 min, followed by 32 cycles of
denaturation at 95 °C for 30 s, annealing at 65 °C for 30 s, extension at
68 °C for 3 min, followed by a final extension at 68 °C for 3 min
(Advantage 2 Polymerase Mix®, Clontech, CA, USA). First round PCR
product wasthen used as template for nested PCR with primers SSD6cF2
and 3'RACE INNER, with thermal conditions as above except initial
denaturating was for 1 min, and annealing was at 60 °C (Advantage 2
Polymerase Mix). 3'RACE PCR products were cloned into pBluescript,
sequenced, assembled with their corresponding ORF sequences, and
deposited in the GenBank database under accession numbers
GU207400 (Δ6fad_b) and GU207401 (Δ6fad_c).
In the present study, we present evidence that there are at least
four genes encoding putative Fad proteins in the Atlantic salmon
genome, and that these do not represent alleles as each has distinct
sizes and sequences of introns. Two genes corresponded to the
previously cloned Δ6 and Δ5 Fad cDNAs. Here, we thus describe the
cloning of the cDNAs for the two further putative fad genes and their
phylogenetic analyses. Functional characterization by heterologous
expression in yeast showed both these cDNAs have Δ6 activities.
Tissue expression and nutritional regulation of the newly cloned
salmon Fad cDNAs were determined by quantitative real-time PCR
(qPCR).
2.3. Sequence and phylogenetic analysis
Deduced amino acid sequences of the two newly cloned fad cDNAs
were aligned with those of human Δ6 Fad (gb|AF126799|) and fish
Fads including zebrafish (Danio rerio) dual Δ6Δ5 Fad (gb|AF309556|),
salmon Δ5 Fad (gb|AF478472|) and Δ6 Fad_a (gb|AY458652|) using
ClustalW and Bioedit (BioEdit version 5.0.6, Tom Hall, Department of
Microbiology, North Carolina State University, USA). Pairwise se-
quence comparisons were performed using the EMBOSS Pairwise
Alignment Algorithms tool (http://www.ebi.a.uk/Tools/Wise2/index.
html) was used. Phylogenetic analysis of the amino acid sequences of
the newly cloned salmon Fads and those from other organisms obtained
from GenBank database using BLASTX was performed by constructing a
tree using the Neighbour Joining method [30], with confidence in the
resulting tree branch topology measured by bootstrapping through
1000 iterations.
2. Materials and methods
2.1. Genomic screening for fatty acyl desaturases
An Atlantic salmon genomic DNA library was constructed with the
lambda FIX II/Xho I partial fill-in vector kit (Stratagene, La Jolla, USA)
and probed with a full-length cDNA of salmon Δ5 Fad (gb|AF478472|).
Inserts of positive recombinant phages were isolated and subcloned
into the pBluescript KS II vector (Stratagene, La Jolla, USA) for
sequencing (CEQ-8800 Beckman Coulter Inc., Fullerton, USA). The
positive phage sequences were then assembled (BioEdit version 5.0.6,
Tom Hall, Department of Microbiology, North Carolina State Univer-
sity, USA) to obtain the sequences and structures of all putative fad
genes found in the Atlantic salmon genome.
2.4. Heterologous expression of desaturase ORFs in Saccharomyces cerevisiae
Comparison of cDNA nucleotide sequences of Δ6fad_b and Δ6fad_c
enabled design of sequence specific primers for PCR amplification of
the respective ORFs. For Δ6fad_b, sense primer SSD6bU5F and
antisense primer SSD6bU3R were designed for first round PCR,
whereas sense primer SSD6cU5F and antisense primer SSD6cU3R
were used for Δ6fad_c. The isolation of both Δ6fad_b and Δ6fad_c ORFs
was achieved in a second round (nested) PCR using first round PCR
products primed with SSD6VF and SSD6VR containing HindIII and
XhoI restriction sites, respectively (Table 1). PCR was performed using
the high fidelity PfuTurbo® DNA polymerase (Stratagene, Agilent
Technologies, Cheshire, UK), with an initial denaturing step at 95 °C
2.2. Fatty acyl desaturase cDNA cloning
Preliminary results from the genomic library screening revealed
the existence of four putative fad genes in the Atlantic salmon
genome, including the genes for the previously cloned Δ5 Fad [16] and
Δ6 Fad [18] cDNAs, termed Δ5fad and Δ6fad_a, and two further

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Ó. Monroig et al. / Biochimica et Biophysica Acta 1801 (2010) 1072–1081
Table 1
Details of PCR primer pairs used in this study for Δ6fad_b and Δ6fad_c ORF and 3'UTR cloning and for real-time quantitative PCR (qPCR) determinations of the transcript levels of
Atlantic salmon Δ6 fad genes. The data include sequences and annealing temperatures (Ta) for primer pairs, amplicon sizes and accession number of the sequence used as reference
for primer design. Primers SSD6VF and SSD6VR (ORF cloning) contained restriction sites (underlined) for HindIII and XhoI, respectively.
Aim
Transcript
Primer
Primer sequence
Amplicon size
Ta
Accession No.
1
3'RACE
Δ6fad_b
SSD6bF1
3'RACE OUTER
SSD6bF2
3'RACE INNER
SSD6cF1
3'RACE OUTER
SSD6cF2
3'RACE INNER
SSD6bU5F
SSD6bU3R
SSD6VF
5′-TCCGCATTCCATCCTGATC-3′
1841 bp
62 °C
GU207400
5'-GCGAGCACAGAATTAATACGACT-3′
5′-GATGTCAACTCACTGCATGTC-3′
5'-CGCGGATCCGAATTAATACGACTCACTATAGG-3'
5′-ACTTCCAGGCCTTGCGCG-3′
5'-GCGAGCACAGAATTAATACGACT-3'
5′-CAAGACTTTGAAGAAAGGCATCAT-3′
5'-CGCGGATCCGAATTAATACGACTCACTATAGG-3'
5′-TTGGACAGACTGGACAGAGC -3′
5′-GCGATGGATTTAAAATCCCG-3′
5′-CCCAAGCTTAGGATGGGGGGCGGAGGCC-3′
5′-CCGCTCGAGTTATTTATGGAGATATGCAT-3′
5′-GTGAGTGGAGCAGAGAGAACC-3′
5′-GCAGCGATCATTATCAACACA-3′
5′-CCCAAGCTTAGGATGGGGGGCGGAGGCC-3′
5′-CCGCTCGAGTTATTTATGGAGATATGCAT-3′
5′-CCCCAGACGTTTGTGTCAG-3′
1319 bp
2442 bp
1529 bp
1481 bp
1395 bp
1514 bp
1395 bp
180 bp
204 bp
171 bp
175 bp
141 bp
224 bp
189 bp
57 °C
68 °C
61 °C
60 °C
60 °C
59 °C
60 °C
60 °C
58 °C
60 °C
60 °C
56 °C
60 °C
65 °C
Δ6fad_c
Δ6fad_b
Δ6fad_c
GU207401 1
GU207400 1
GU207401 1
ORF cloning
SSD6VR
SSD6cU5F
SSD6cU3R
SSD6VF
SSD6VR
SSD6aF
SSD6aR
SSD6bF
SSD6bR
SSD6cF
1
qPCR
Δ6fad_a
Δ6fad_b
Δ6fad_c
elf-1α
AY458652
5′-CCTGGATTGTTGCTTTGGAT-3′
1
5′-ATAGAGGGTTTATATAGTAGGGCC-3′
5′-GGTGGGACGCTAGAAGTTAA-3′
5′-CCCACCCCCATCTTAAAACT-3′
5′-CTGGGGTCCAAACAAGGTTA-3′
5′-CTGCCCCTCCAGGACGTTTACAA-3′
5′-CACCGGGCATAGCCGATTCC-3′
5′-ACATCAAGGAGAAGCTGTGC-3′
5′-GACAACGGAACCTCTCGTTA-3′
5′-AGCCTATGACCAACCCACTG-3′
GU207400
1
GU207401
SSD6cR
1
ELF-1α jbt2
ELF-1α jbt2
BACT-F
BACT-R
B2F
B2R
ss18S-2F
ss18S-2R
AF321836
1
β-actin
AF012125
2
cofilin-2
18 S rRNA
TC63899
5′-TGTTCACAGCTCGTTTACCG-3′
5′-GGCGCCCCCTCGATGCTCTTA-3′
5′-CCCCCGGCCGTCCCTCTTAAT-3′
1
AJ427629
1GenBank (http://www.ncbi.nlm.nih.gov/).
2Atlantic salmon Gene Index (http://www.compbio.dfci.harvard.edu/tgi/).
for 2 min, followed by 30 cycles of denaturation at 95 °C for 30 s,
annealing at 58 °C for 30 s, extension at 72 °C for 1 min 30 s, followed
by a final extension at 72 °C for 5 min. The DNA fragments were
digested with the corresponding restriction endonucleases (New
England BioLabs, Herts, UK) and ligated into a similarly restricted
pYES2 yeast expression vector (Invitrogen, Paisley, UK). The purified
plasmids (GenElute™ Plasmid Miniprep Kit, Sigma) containing
Δ6fad_b and Δ6fad_c ORFs were used to transform Saccharomyces
cerevisiae competent cells (S.c. EasyComp Transformation Kit, Invitro-
gen). Transformation and selection of yeast with recombinant pYES2
plasmids, yeast culture and fatty acid (FA) analyses were performed
as described in detail previously [12,13,16]. Briefly, cultures of
recombinant yeast were grown in S. cerevisiae minimal medium-uracil
supplemented with one of the following FA: 18:3n-3, 18:2n-6,
20:4n-3, 20:3n-6, 22:5n-3 and 22:4n-6. The FA were added to the
yeast cultures at final concentrations of 0.5 (C18), 0.75 (C20) and 1.0
(C22) mM. After 2-days culture at 30 °C, yeast were harvested and
washed, and lipid extracted by homogenization in chloroform/
methanol (2:1, v/v) containing 0.01% butylated hydroxy toluene
(BHT) as antioxidant. Methyl esters of FA were prepared, extracted,
purified, and analyzed by GC in order to calculate the proportion of
substrate FA converted to desaturated FA product as [product area/
(product area+substrate area)]×100. Identities of FA peaks were
based on GC retention times and confirmed by GC-MS as described
previously [12,13].
tanks at ambient temperature, salinity and photoperiod in the Marine
Environment Research Laboratory, Machrihanish, Scotland, UK, and
fed a commercial salmon feed. Intestine, liver, white muscle, red
muscle, kidney, spleen, heart, brain, gill and visceral adipose tissue
were dissected from three salmon and frozen at -70 °C. Total RNA was
extracted using Tri Reagent (Sigma, Poole, UK) according to the
manufacturer's protocol, and 1 µg of total RNA reverse transcribed
into cDNA using random hexamers (VersoTM cDNA kit, ABgene,
Surrey, UK). A second experiment investigating the effects of diet on
fad expression was performed with salmon post-smolts (∼132 g) fed
four diets with the same basal composition (47% protein and 33% oil)
but formulated with different oils including FO, soybean oil (SO),
linseed oil (LO) or rapeseed oil (RO), as described previously [31].
After 16 weeks (∼430 g), 0.5 g of liver and intestine of five fish per
dietary treatment were dissected and rapidly disrupted in 5 ml of TRI
Reagent (Ambion, Applied Biosystems) using an Ultra-Turrax ho-
mogenizer (Fisher Scientific, Loughborough, UK), and immediately
frozen in liquid nitrogen and stored at –70 °C prior to RNA extraction.
All samples for qPCR analyses were obtained from fish starved for 24 h
prior to sample collection. The details of the qPCR analysis were as
described previously [20].
2.6. Statistical analysis
For tissue distribution, results were expressed as mean ( SE)
normalized ratios between the copy numbers of the putative
desaturase transcripts and the copy numbers of the reference gene,
18s rRNA [20]. Differences in the expression of each fad gene among
tissues were analyzed by one-way analysis of variance (ANOVA)
followed by Tukey's multiple comparison test at a significance level of
P≤0.01 (SPSS Inc., Chicago, IL, USA). The effects of diet on fad
expression, expressed as the relative expression ratio of each gene in
2.5. Tissue distribution and nutritional regulation
Expression of the new fad genes was measured by quantitative
real-time PCR (qPCR; see Table 1 for primer details). In a first
experiment, tissue distribution of fad expression was determined in
samples collected from ∼150 g post-smolts held in 7 m3 seawater

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1075
fish fed one of the VOs in relation to those fed FO (control) and
normalized by two reference genes (elf-1α and cofilin-2, which were
previously shown to be unresponsive under the nutritional conditions
tested [31]), were analyzed for statistical significance using the
relative expression software tool (REST-2008©, http://www.gene-
quantification.info/), which employs a pair-wise fixed reallocation
randomization test (10,000 randomizations) with efficiency correc-
tion [32].
Δ6fad_a (gb|AY736067|). The complete gene sequence for Δ6fad_a
was obtained, but only the sequence corresponding to the last five
introns and exons was obtained for Δ5fad. The other two distinct DNA
sequences suggested additional putative fad genes for which the
cDNA had not yet been cloned or characterized. After cloning and
functional characterization of the cDNAs (see below), these genes
were termed Δ6fad_b (compiled from gb|GU294486-8|) and Δ6fad_c
(gb|GU294489|). All four fad genes had identical coding exon
structures, being comprised of 12 coding exons, with a high degree
of sequence identity (Fig. 1). Identification of these as separate genes
was confirmed as each had introns of distinct size and sequence
composition. Clearly, they were not allelic variants of a single gene
(Table 2). Intron regions that could not be aligned across all four genes
had high identity to multiple sequences in diverse salmon database
entries and included degenerate salmon Class I transposons [35], or
could be aligned with sequences of RepBase (http://www.girinst.
org/repbase/index.html) entries for various transposons [34]. The
first coding exons of Δ6fad_b and Δ6fad_c contain a nine base pair
insertion compared to Δ6fad_a and Δ5fad, resulting in a three amino
acid insertion in the predicted polypeptide sequences.
Comparison of the cDNA and gene sequences for Δ6fad_a enabled
the identification of an upstream 5′ non-coding exon, whilst
comparison of the cDNAs for Δ6fad_b showed the presence of a
further two 5′ non-coding exons for Δ6fad_b. The first of these non-
coding exons mapped to the first 5′ non-coding exon of Δ6fad_a. Two
cDNAs were obtained for Δ6fad_c, (see section below) and compar-
ison of these with the gene sequence indicated that these cDNAs
represented alternatively spliced products, both derived from the
Δ6fad_c gene. These exons did not show similarity to the exons for
Δ6fad_a and Δ6fad_b, although they represented sequences which
could be mapped elsewhere in the 5′ regions of Δ6fad_a and Δ6fad_b.
One of these alternative exons was similar to the previously
characterized promoter region of Δ6fad_a [18], whilst the other
2.7. Materials
Eicosatetraenoic (20:4n-3), docosapentaenoic (22:5n-3) and doc-
osatetraenoic (22:4n-6) acids (all N 98-99% pure) were purchased from
Cayman Chemical Co., Ann Arbor, USA. Linoleic (18:2n-6), α-linolenic
(18:3n-3), eicosatrienoic (20:3n-6) acids (all N99% pure), BHT, fatty
acid-free BSA, galactose, 3-(hydroxymethyl) pyridine, nitrogen base,
raffinose, tergitol NP-40 and uracil dropout medium were obtained
from Sigma Chemical Co. Ltd., Dorset, UK. Thin-later chromatography
(TLC) (20×20 cm×0.25 mm) plates pre-coated with silica gel 60
(without fluorescent indicator) were purchased from Merck, Darm-
stadt, Germany. All solvents were HPLC grade and were from Fisher
Scientific, Loughborough, UK.
3. Results
3.1. Salmon fatty acyl desaturase gene structure
Six lambda phage salmon genomic inserts with homologous
regions to the previously described salmon desaturase cDNAs were
obtained after screening the library. These six phage inserts resolved
into four distinct fad genes. Two of these corresponded to the
previously cloned and functionally characterized Δ5 and Δ6 Fad
cDNAs [16,19], and so were termed Δ5fad (gb|GU294485|) and
Fig. 1. Structures of Atlantic salmon Δ5 and Δ6 fatty acyl desaturase (Δ5/Δ6fad) genes. Gene structures were determined from genomic sequences from a library constructed from a
single fish. Sequenced regions (not to scale) showing high levels of identity across all genes (using DIALIGN, http://www.bibiserv.techfak.uni-bielefeld.de/dialign/) [33] are
indicated by a black line, and sequenced coding exons predicted from alignment with cDNAs are indicated by black boxes. Non-coding exon regions are indicated by unfilled boxes.
Hatched lines indicate regions of the genes that have not been sequenced and grey boxes indicate exons predicted from cDNAs, but not sequenced from genomic DNA. Asterisks
indicate an alternatively utilised exon predicted from cDNA sequences. Vertical arrows indicate the position of a nine base pair insertion in Δ6fad_b and Δ6fad_c compared to Δ6fad_a
and Δ5fad. Horizontal arrows indicate the position and orientation of an inverted repeat sequence in Δ6fad_c. The black triangle indicates the position of the promoter region for
Δ6fad_a [18]. The positions, sizes (base pairs) and similarity hit to RepBase (http://www.girinst.org/repbase/index.html) [34] of intron regions which could not be aligned across all
four genes are indicated. These sequences all match multiple sequences in diverse salmon database entries. DTSsa1, Tss, pTSsa1 and SSTN are degenerate salmon Class I transposons
[35]. Rex1, Tol2, PiggyBac, and FuguRep2 denote sequences with extensive similarity to RepBase entries for various transposons. RPT is an uncharacterized sequence with N100 high
similarity hits to diverse salmonid sequences in Genbank.

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Table 2
Asian sea bass or barramundi (Lates calcarifer), zebrafish and common
carp (Cyprinus carpio). The salmon Fads clustered more distantly from
Fads of other organisms including mammalian (mouse and human),
fungus (Mortierella alpina) and worm (Caenorhabditis elegans).
Exon and intron sizes (bp) of Atlantic salmon fatty acyl desaturase (fad) genes.
Exon size (bp)
Intron size (bp)
Δ6fad-a Δ6fad_b Δ6fad_c Δ5fad Δ6fad-a Δ6fad_b
Δ6fad_c Δ5fad
1
2
3
4
5
6
7
8
60a
103a
320d
111
198
102
126
61
77
98
97
80
532
358
912
4525
203
152
3.3. Functional characterization
22a
496b
111
198
102
126
61
77
98
97
80
698
1127
744
228
345
515
128
303
161
1011
383
216
321f
111
198
102
126
61
77
98
97
1172
987
The salmon Fad proteins were functionally characterized by
determining the fatty acid profiles of transformed S. cerevisiae contain-
ing either the pYES2 vector alone or the vector with the salmon fad
cDNA inserts, grown in the presence of a variety of potential fatty acid
substrates, including Δ6 substrates (18:2n-6 and 18:3n-3), Δ5 sub-
strates (20:3n-6 and 20:4n-3) and Δ4 substrates (22:4n-6 and 22:5n-3).
The fatty acid composition of the yeast transformed with the vector
alone showed the main fatty acids normally found in S. cerevisiae,
namely 16:0, 16:1 isomers, 18:0 and 18:1n-9, together with the
exogenously derived fatty acids (data not shown). This is consistent
with S. cerevisiae not possessing Δ4, Δ5 or Δ6 desaturase activities [12].
Based on GC retention time and confirmed by GC-MS, salmon Δ6 Fad_b
and Δ6 Fad_c showed activity towards Δ6 substrates 18:3n-3 (Fig. 4A
and B) and 18:2n-6 (Fig. 4C and D), which were converted to 18:4n-3
and 18:3n-6, respectively. No Δ5 or Δ4 desaturase activity was detected
for any of the cDNA constructs, as evidenced by the lack of additional
peaks representing desaturated products of 20:4n-3, 20:3n-6, 22:5n-3
or 22:4n-6 (Fig. 4E-H; Table 3). Conversion rates indicated that Δ6Fad_b
protein was more active than Δ6Fad_c in the recombinant yeast system,
with Δ6 Fad_b desaturating 47 % of 18:3n-3 to 18:4n-3, and 25 % of
18:2n-6 to 18:3n-6, whereas Δ6 Fad_c showed conversions of 12 and
7 %, respectively (Table 3). As reported for other teleost Fads, salmon Δ6
Fad_b and Δ6 Fad_c displayed more activity towards the n-3 fatty acids
than n-6 (Table 3).
1935
342
3208/3222h 1265
128/124h
3283/3348h
158
236
1883
128
206
1292
9
10
11
12
98
97
80
161
237
410
130
13 126
126
126
132
14 509c
400e
1124g
a
Exons are 5′ untranslated regions (UTR).
Includes a 5′-UTR of 259 bp.
Includes a 3′-UTR of 457 bp.
Includes a 5′-UTR of 74 bp.
Includes a 3′-UTR of 348 bp (not end of gene).
Includes a 5′-UTR of 75 bp.
b
c
d
e
f
g
h
Includes a 3′-UTR of 1072 bp.
Δ6fad_b was compiled from different clones, which differered in intron size due to
variable lengths of simple sequence repeats in the conserved regions, indicating allelic
variance.
appeared to be an inversion of a sequence immediately 5′ of the first
coding exon. Significantly, these exons were not located in sequenced
portions of Δ6fad_c. Although the 5′ regions of Δ6fad_a and Δ6fad_b
were very similar, including the sequence and position of the promoter
region, the equivalent region in Δ6fad_c indicated the presence of an
inserted repeat sequence (Fig. 1).
3.4. Fatty acyl desaturase gene expression: tissue distribution and
nutritional regulation
3.2. Salmon fatty acyl desaturase cDNAs
Tissue distributions of the three Δ6fad genes including Δ6fad_a
(gb|AY458652|) and the newly cloned Δ6fad_b (gb|GU207400|) and
Δ6fad_c (gb|GU207401|), were determined by qPCR performed on
cDNA obtained from different tissues. The results showed that
Δ6fad_a and Δ6fad_b genes were expressed at significantly higher
levels (P≤0.01) in intestine (pyloric caeca) and brain compared to
other tissues, with liver also having high transcript copy number for
Δ6fad_a (Fig. 5). Interestingly, Δ6fad_c transcript showed a different
tissue distribution pattern, with transcripts being predominantly
found in brain (P≤0.01), with an expression level exceeding that
quantified in all other tissues by over 30-fold (Fig. 5). The regulation of
Δ6fad_a, Δ6fad_b and Δ6fad_c in response to dietary FA composition
was determined in intestine (pyloric caeca) and liver of salmon fed
diets containing either FO rich in n-3 LC-PUFA (EPA and DHA) or VOs
rich in C18 fatty acids, SO (18:2n-6), LO (18:3n-3) or RO (18:1n-9). In
intestine, there was significantly higher expression of Δ6fad_b
transcripts in SO- and RO-fed salmon compared to the fish fed FO,
with normalized expression ratios of 3.8 and 2.9, respectively
(Fig. 6A). No significant differences were found between transcript
levels for Δ6fad_a and Δ6fad_c in intestine of VO-fed fish compared to
FO-fed fish. In liver, expression of the Δ6fad_a gene was up-regulated
by VO treatments, with significantly higher transcript levels in fish fed
SO (2.7-fold), LO (2.2-fold) and RO (3.0-fold) (Fig. 6B). The levels of
Δ6fad_b mRNA were also higher in liver of fish fed SO and RO
treatments and, although not statistically significant (REST-2008),
this trend was consistent with the results observed in intestine.
The new putative fad cDNAs isolated from Atlantic salmon were both
shown to have ORFs of 1373 bp encoding proteins of 457 amino acids,
with 3'UTRs of 642 bp for Δ6fad_b and 1408 bp for Δ6fad_c. The protein
sequences included all the characteristic features of microsomal fatty
acyl desaturases, including three histidine boxes and an N-terminal
cytochrome b5 domain containingtheheme-bindingmotif, H-P-G-G and
two transmembrane regions (Fig. 2). These features are similar to those
of other Fad proteins including salmon Δ5 Fad (gb|AF478472|) and Δ6
Fad_a (gb|AY458652|), the zebrafish Δ6/Δ5 Fad (gb|AF309556|), and the
human Δ5 (gb|AF199596|) and Δ6 (gb|AF126799|) Fads. A pair-wise
comparison was made between the newly cloned Atlantic salmon Fads
with each other, and with other fish and human Fad sequences. The
amino acid sequences predicted by the salmon putative Δ6 Fad_b and Δ6
Fad_c ORFs showed 96.7% identity to each other. When compared to
other fish Fads, Δ6 Fad_b and Δ6 Fad_c amino acid sequences were
90.4 - 92.8 % identical to salmon Δ6 Fad_a and Δ5 Fad, 76.6 - 78.1 %
identical to marine fish Δ6 Fads including those from cobia (Rachycen-
tron canadum) (gb|FJ440238|) and Atlantic cod (Gadus morhua) (gb|
DQ054840|), and 65.6 & 65.0 %, respectively, to the dual zebrafish Δ6/Δ5
Fad. Additionally, Δ6 Fad_b and Δ6 Fad_c proteins showed, respectively,
63.9 & 63.7 % identity to the human Δ6 and 56.6 & 57.3 % to human Δ5
Fad. Phylogenetic analysis clustered the new Atlantic salmon putative
Fad sequences with other salmonid Fads, including the formerly studied
Atlantic salmon Δ6 and Δ5 Fads, Δ6 Fad from rainbow trout
(Oncorhynchus mykiss), and uncharacterized putative Fads of masou
(cherry) salmon (Oncorhynchus masou) (Fig. 3). The salmonid Fads also
clustered with other fish Fads including those of Atlantic cod, gilthead
sea bream (Sparus aurata), turbot (Psetta maxima), cobia, tilapia
(Oreochromis niloticus), European sea bass (Dicentrarchus labrax),
4. Discussion
Prior to this study cDNAs for two fads (with Δ6 and Δ5 activities)
had been identified and characterized in Atlantic salmon [16,19].

Ó. Monroig et al. / Biochimica et Biophysica Acta 1801 (2010) 1072–1081
1077
Fig. 2. Alignment of the deduced amino acid sequence of the newly cloned Δ6 fatty acyl desaturases from Atlantic salmon with formerly cloned Salmo salar Δ6 (Ss Δ6Fad_a) and
Δ5 (Ss Δ5Fad) desaturases, the Δ6/Δ5 bifunctional desaturase from Danio rerio (Dr Δ6/Δ5Fad) and human Δ6 desaturase (Hs Δ6FAD). Deduced amino acid sequences were aligned
using ClustalW. Identical residues are shaded black and similar residues are shaded grey. Identity/similarity shading was based on the BLOSUM62 matrix, and the cut-off for shading
was 70%. The cytochrome b5-like domain is dot-underlined, the two transmembrane regions are dash-underlined, and the three histidine-rich domains are solid-underlined. The
asterisks on the top mark the heme-binding motif, HPGG.
However, despite having distinct enzymatic activities, these two
proteins showed a remarkable level of amino acid sequence similarity,
indicating that they had arisen within the evolutionary line leading to
salmonids, and after divergence from other fish groups. This finding
raised questions regarding the nature of the genes encoding these
enzymes, including the possibility that the identified cDNA sequences
may have been allelic variants rather than distinct genes. In this study,
following sequencing of genomic clones, it is clear that salmon possess
at least four fad genes. Although all of the products of these genes
show the same remarkable levels of amino acid sequence similarity
(up to 97 % for Δ6fad_b and Δ6fad_c, for example) the evidence that
these sequences represent distinct loci is most clear from the
differential and highly distinctive distribution of transposable (and
other repeat) elements within the introns of each gene. In addition,
while the salmon fad genes all have identical genomic coding exon
structures consisting of 12 coding exons, they differ in the identity and
positions of 5′ non-coding exons, and in the size and sequence of 3′
non-coding regions. The genomic coding organization is identical to
that previously reported for the human FADS1 (Δ5 desaturase),
FADS2 (Δ6 desaturase) and FADS3 genes, which also consist of 12
coding exons [36].
Although the salmon fad genes show several interesting features,
particularly in the intron structures, which may eventually reveal
important information on the evolutionary history of fad genes in

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Ó. Monroig et al. / Biochimica et Biophysica Acta 1801 (2010) 1072–1081
Fig. 3. Phylogenetic tree comparing putative amino acid sequences of Atlantic salmon, S. salar, fatty acyl desaturases Δ6Fad_b and Δ6Fad_c, together with the formerly cloned salmon
Δ5 Fad, Δ6 Fad_a, and other desaturase proteins from fish and other organisms. Desaturases that have not been functionally characterized are marked with an asterisk. The tree was
constructed using the Neighbour Joining method [30] with MEGA4. The horizontal branch length is proportional to amino acid substitution rate per site. The numbers represent the
frequencies (%) with which the tree topology presented was replicated after 1000 iterations.
salmon and fish generally, it is the heterogeneity in the 5′ end of the
genes that is of particular interest for the present study. The
differential splicing of the non-coding exons in this region of the
Δ6fad_a, Δ6fad_b and Δ5fad genes and the contrasting quite different
structure of the region in the Δ6fad_c gene may have possible
functional consequences. Comparison of the upstream sequences of
Δ6fad_a and Δ6fad_b revealed a high degree of similarity, and
although Δ6fad_b possesses an extra non-coding exon, both of these
genes share a similar first exon, which in both genes is preceded by a
conserved sequence previously shown to act as a functional and PUFA-
responsive promoter in Δ6fad_a [18]. In contrast the Δ6fad_c gene has a
repeat element insertion in the equivalent region. This appears to have
resulted in a very different 5′non-coding exon structure, including the
utilization of alternative splice sites. Although we were unable to map
these exons to the available Δ6fad_c genomic sequence, one of these
alternative exons is an inverted repeat of a region immediately
following the 5′ end of the repeat element and before the first coding
exon. It is possible that the insertion of the repeat element has caused
the repetition and inversion of the host DNA at the insertion site, and
the inverted sequence has been utilized as an alternative exon. The
other alternative exon comprises a sequence that matches a major
portion of the promoter region of Δ6fad_a. Based on these observations
it is plausible to hypothesize that Δ6fad_b may be regulated similarly to
Δ6fad_a, and that Δ6fad_c is regulated differently. This prediction is
supported by the gene expression data which showed substantial
differences between Δ6fad_c and the other Δ6fad genes, with Δ6fad_c
expression being very largely confined to brain whereas Δ6fad_a was
expressed highly in intestineNliverNbrain and Δ6fad_b was expressed
in brainNintestineNgillNliver. Furthermore, Δ6fad_c expression
showed no nutritional regulation whereas expression of Δ6fad_a and
Δ6fad_b were significantly increased in liver and intestine, respectively,
in fish fed diets with reduced levels of LC-PUFA.
In humans, the desaturase gene cluster (FADS1, FADS2 and FADS3)
has most likely arisen by tandem gene duplication. This is on the basis
that the genes are in a head to head, or head to tail arrangement, and
the exon organization is nearly identical in the three family members.
Each gene consisting of 12 exons, with splice and acceptor sites
interrupted at identical nucleotide positions within highly conserved
codons [36]. The first coding exons in the salmon Δ5fad and Δ6fad_a
genes are 30 bp longer (as a single block) than the corresponding
exons in the human FADS1 (Δ5 FAD) and FADS 2 (Δ6 FAD) genes,
corresponding to the additional 10 amino acids found in several
salmonid desaturases [16,17,19,37]. The additional two salmon fad
genes isolated here have a further segment comprising nine additional
bases (three amino acids) in their first coding exon. Therefore,
excluding the UTR, the first coding exons contain 246 coding bp in
Δ6fad_b and Δ6fad_c, and 237 bp in Δ6fad_a and Δ5fad. In contrast, the
remaining exons are exactly the same size in all the salmon fad genes
as the human Δ6 desaturase gene (FADS2), with splice and acceptor
sites interrupted at similar nucleotide positions, despite the lengths of
the introns being very different [16,36]. Interestingly, the human FADS
genes vary by 3 bp in coding exon 5, FADS1/Δ5 comprising 129 bp,
FADS2/Δ6 comprising 126 and FADS3 comprising 123 bp [36], but this
exon in all the salmon fad genes, including Δ5fad, is 126 bp in length.

Ó. Monroig et al. / Biochimica et Biophysica Acta 1801 (2010) 1072–1081
1079
Fig. 4. Functional characterization of the newly cloned salmon fatty acyl desaturases Δ6fad_b (panels A, C, E and G) and Δ6fad_c (panels B, D, F and H) in transgenic yeast
(Saccharomyces cerevisiae) grown in the presence of Δ6 substrates 18:3n-3 (A and B) and 18:2n-6 (C and D), and Δ5 substrates 20:4n-3 (E and F) and 20:3n-6 (G and H). Fatty acids
were extracted from yeast transformed with pYES2 vector containing the ORF of the putative fatty acyl desaturase cDNA as an insert. The first five peaks in all panels are the main
endogenous fatty acids of S. cerevisiae, namely 16:0 (1), 16:1n-9 (2), 16:1n-7 (3), 18:0 (4), and 18:1n-9 (5). Substrates (“*”) and their corresponding desaturated products are
indicated accordingly in panels A-H. Vertical axis, FID response; horizontal axis, retention time.
While the different enzymatic activities of Δ6 Fad_a and Δ5 Fad
clearly discriminate their functional roles, however the three different
Δ6 Fads may also have distinct biological/physiological roles in salmon.
The activityofΔ6 Fad_ain theheterologous yeast systemwaspreviously
reported as 60 % and 14 % for desaturation of 18:3n-3 and 18:2n-6,
respectively [16]. With the additional data reported in the current study,
an activity rank order of aNbNc for three Δ6 isoenzymes is apparent. All
three were found to be more active towards the n-3 fatty acid substrate,
as observed previously for all other fish Fad enzymes [12,16,19,38–40],
with the rank order for n-3/n-6 activity ratio being a (4.2)Nb (1.9)Nc
(1.7). As mentioned above, tissue and nutritional regulation also
showed functional differences, with Δ6fad_a being the main fad gene
expressed in liver, and also being under nutritional regulation in that
tissue. In contrast, Δ6fad_b was highly expressed and nutritionally
regulated in intestine. Although Δ6fad_a was also highly expressed in
intestine it showed no nutritional regulation in that tissue, and Δ6fad_b
was not significantly regulated in liver. In contrast, Δ6fad_c was
expressed predominantly in brain, as has been observed with the Δ6
Fads in marine fish [39,40]. Unfortunately, brain was not sampled in the
earlier nutritional experiment and so we were unable to determine if
Δ6fad_c was nutritionally regulated in that tissue. However, the data are
consistent with the different Fads having different functional roles in
different tissues. In this respect, the high levels of expression of two of
the Δ6fad genes in both intestine (pyloric caeca) and brain perhaps
reflect the importance of fatty acid metabolism in these tissues, with
Table 3
Functional characterization of salmon Δ6 Fad_b and Δ6 Fad_c proteins in Saccharomyces
cerevisiae. Results are expressed as a percentage of total fatty acid substrate converted
to desaturated product.
FA substrate
Product
Conversion (%)
Activity
Δ6 Fad_b
Δ6 Fad_c
18:3n-3
18:2n-6
20:4n-3
20:3n-6
22:5n-3
22:4n-6
18:4n-3
18:3n-6
20:5n-3
20:4n-6
22:6n-3
22:5n-6
47
25
0
0
0
12
7
0
0
0
Δ6
Δ6
Δ5
Δ5
Δ4
Δ4
0
0

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Ó. Monroig et al. / Biochimica et Biophysica Acta 1801 (2010) 1072–1081
Fig. 5. Tissue distribution profile of Δ6fad_a, Δ6fad_b and Δ6fad_c transcripts in Atlantic
salmon, determined by qPCR. Absolute copy numbers were quantified for each
transcript and normalized by absolute levels of ribosomal 18s. Mean values are shown
on top of each column and represented diagrammatically in logarithmic scale. Error
bars are standard errors (n=3). The columns marked with asterisks indicate that the
expression of these fad genes in these tissues is significantly higher than the expression
in any of the other tissues (P≤0.01). A, adipose tissue; B, brain; G, gill; H, heart;
K, kidney; L, liver; PC, pyloric caeca (intestine); RM, red muscle; S, spleen; WM, white
muscle.
intestine being the tissue first exposed to dietary fatty acids, and the
brain having strict LC-PUFA requirements including high DHA and ARA
[11]. Thus high expression of both Δ6fad_a and b in intestine, with one
being regulated by dietary LC-PUFA composition is consistent with it
encountering and responding to dietary lipid, and high expression of
both Δ6fad_b and c in brain is consistent with the critical importance of
high LC-PUFA levels in neural tissue [39,40]. The fact that Δ6fad_a, the
highest activity enzyme, is the gene with highest expression in liver is
consistent with liver being the major tissue for regulating overall body
lipid metabolism. Determining how the same genes are differentially
regulated in different tissues will be an interesting challenge, and
presumably related to the expression, activities and regulation of
transcription factors that could include sterol regulatory element
binding protein-1c, liver X receptor and, possibly, peroxisome pro-
liferator activated receptors and retinoid X receptors [18,41,42].
Fig. 6. Nutritional regulation of desaturases Δ6fad_a, Δ6fad_b and Δ6fad_c in Atlantic
salmon fed diets containing fish oil (FO), soybean oil (SO), linseed oil (LO) or rapeseed
oil (RO), in the intestine (A) and liver (B), determined by qPCR. The results show
normalized expression ratios (reference genes: elf-1α and cofilin-2, an unresponsive
gene identified in prior Atlantic salmon dietary microarray studies [31]) of the target
transcripts in one of the VO treatments (SO, LO or RO), in relation to the FO control
treatment. Values are means with S.E. (n=5) and asterisks represent significant
differences (P≤0.05) between the column's dietary treatment and the FO treatment,
for the respective transcript (REST-2008©).
The evolutionary context of the presence of four fad genes in
Atlantic salmon is interesting. Salmonids, unlike the Actinopterygian
fish species studied to date, have relatively higher activities of
LC-PUFA biosynthesis and contain genes for both Δ5 and Δ6 Fads. In
these other fish, such as sea bass and sea bream, there appears to be
no detectable Δ5 Fad activity or gene expression [10] and sequenced
genomes have numbers of fad genes varying from three in the case of
stickleback (Gasterosteus aculeatus) to one in the case of medaka
(Oryzias latipes), or even zero, as in the pufferfish (Takifugu rubripes,
Tetraodon nigroviridis) although this may change as the depth of
sequencing increases [41]. It is also clear that all four genes have been
duplicated relatively recently, and within the salmonid evolutionary
line. Preliminary evidence from limited pedigree analyses in our
laboratory (not shown) suggests that all four salmon desaturase genes
are located in the same linkage group, indicating that may have arisen
as a result of segmental duplications and not as a result of the
salmonid tetraploidization event hypothesized to have occurred
between 25 and 100 million years ago [43]. Regardless of the
mechanism of duplication, the separate evolution by sub- or neo-
functionalization of Δ5 and Δ6 desaturase activities, together with the
tissue specific regulation of these genes, is likely to be responsible for
the high biosynthetic LC-PUFA capacity in salmon compared to other
fish. It has been suggested that the relatively high LC-PUFA
biosynthesis in salmonids is an evolutionary adaptation to a nutrient
poor, freshwater habitat, enabling Atlantic salmon and their relatives
to exist on a diet with a high input of terrestrially-derived foodstuffs
that are relatively deficient in EPA and DHA. The presence of four very
similar, sub-functionalized desaturase genes which group together in
phylogenetic plots supports this contention. The three mammalian
PUFA desaturase genes [36] are also the result of segmental
duplications, followed by a sub-functionalization, giving rise to
separate genes for Δ5, Δ6, and a third uncharacterized desaturase
(FADS3). However, considering the phylogenetic relationships
amongst vertebrate desaturases, these mammalian genes are clearly
the result of a separate, earlier duplication which must have occurred
in the evolutionary line leading to mammals after its split from fish.
Thus, both the salmon and mammalian desaturase duplications and
subsequent sub-functionalizations might represent separate evolu-
tionary adaptations to diets containing terrestrial foodstuffs, ulti-
mately derived from plants which are devoid of LC-PUFA.
In summary, we have demonstrated that there are at least four
functional genes encoding putative Fad proteins in Atlantic salmon. Two
genes, Δ6fad_a and Δ5fad, corresponded to the previously cloned Δ6 and
Δ5 Fad cDNAs, and cDNAs for the two further genes, Δ6fad_b and

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Acknowledgements
OM was supported by the postdoctoral research programme of the
Fundación Española para la Ciencia y la Tecnología and a EU Marie
Curie Intra-European Fellowship (PIEF-GA-2008-220929, COBIA-
GENE), and SM by “Fundação para a Ciência e a Tecnologia”, Portugal
(grant SFRH/BPD/34247/2006). This work and XZ were supported by
the Biotechnology and Biological Sciences Research Council (BBSRC)
Responsive Mode Grant, BB/C51237X/1 (Transcriptional control of
polyunsaturated fatty acid synthesis in fish).
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