Vol. 284, Issue 1, R41-R50, January 2003
Differential expression of cold- and diet-specific
genes encoding two carp liver 
9-acyl-CoA desaturase
isoforms
S. D.
Polley,
P. E.
Tiku,
R. T.
Trueman,
M. X.
Caddick,
I. Y.
Morozov, and
A. R.
Cossins
School of Biological Sciences, University of Liverpool,
Liverpool L69 7ZB, United Kingdom
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ABSTRACT |
Carp respond to cold
by the upregulated expression of 
9-acyl-CoA desaturase. Here we
report the cloning and characterization of Cds2, a second
9-acyl CoA-desaturase expressed in carp liver. Both Cds1
and Cds2 complemented the ole1 mutation in
Saccharomyces cerevisiae, permitting the synthesis of
9-monounsaturates, confirming their identity as 9-desaturases. We
demonstrate that under a standard feeding regime it is the
Cds2, and not Cds1, transcript that is
transiently upregulated during the first few days of cooling from
30°C to 10°C, the period when cold-induced membrane restructuring occurs. Cds2 exists as two differentially spliced
transcripts, differing by a small segment from the 3'-untranslated
region, the ratio of which varies with temperature. Feeding a diet
enriched in saturated fats produced a fourfold increase in
Cds1 transcript levels, which was blocked by cooling to
15°C. Cds2 transcript levels, however, showed no
substantial response to the saturated diet. Thus carp liver uniquely
expresses two isoforms of 9-acyl CoA desaturase, possibly formed by
a recent duplication event, that are differentially regulated by
cooling and dietary treatment.
temperature adaptation; lipid adaptation; membrane adaptation; homeoviscous adaptation
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INTRODUCTION |
THE PHYSICAL PROPERTIES
of the phospholipid membranes are heavily dependent on the
saturation of their constituent fatty acids (11).
Maintaining an appropriate balance between saturated and unsaturated
fatty acids, in the face of a variable dietary supply, is therefore an
essential compositional requirement for all living organisms. This
situation is further complicated by changes in cell temperature. This
is because membrane physical properties are highly temperature
dependent, and fluctuations in cellular temperature may disturb the
normal function of membrane systems. Organisms that regularly
experience variations in body temperature (i.e., poikilotherms), and
therefore cell temperature, mitigate these effects by activating a
series of corrective mechanisms to preserve function over the normal
range of temperatures and to prevent breakdown at thermal extremes
(7). In the case of cellular membranes, this is evident as
a cold-induced increase in fatty acid unsaturation that provides a
disordering influence to offset the direct ordering effect of cooling.
Warm acclimation induces the reverse response. The resulting
homeostatic regulation of membrane physical structure is termed
homeoviscous (28) or homeophasic adaptation
(14) and is a highly conserved process observed widely in
microorganisms, plants, and animals.
Recent progress using molecular genetic techniques in a wide range of
organisms has identified a central role of acyl-desaturases in this
environmental response (19). For example, in the
cyanobacterium Synechocystis, cold causes the rapid
transcriptional upregulation of the acyl-CoA 9-desaturase
(18), and a similar response has been recorded in higher
plants (22). This enzyme inserts the first double bond
typically at the 9-10 position of a saturated carbon chain, a
position that maximizes the change in physical properties
(3).
In the common carp Cyprinus carpio, a hepatic desaturase is
transiently upregulated in the few days after a slow progressive cooling treatment (27, 36), and this correlates
particularly with an increase in monoenoic fatty acids in the
sn-1 position of ethanolamine phosphoglycerides
(32). We have previously cloned a carp homolog of a rat
stearoyl-CoA 9-desaturase (SCD1) and have shown that
transcript amounts increase 8- to 10-fold in the few days after cold
treatment, due at least in part to enhanced transcription
(32). The induction of desaturase activity was also
brought about by the activation of preexisting but latent desaturase
protein, perhaps posttranslationally. The transcriptional response
occurs with more extreme cooling treatments and with a slower time
course than the activation response, the two offering a graded response
of desaturase activity to the magnitude and speed of the change in
temperature (33). In mammals the expression of the hepatic
9-desaturase is subject to dietary control (29), although little is known about dietary influences on the cold-induced carp 9-desaturase.
We now report the cloning and characterization of a second carp
desaturase, termed Cds2, which is also expressed mainly in the liver. We have developed probes to distinguish between the two
coexpressed transcripts and demonstrate that expression of Cds2 is upregulated by cooling from 30°C to 15°C,
instead of Cds1 as previously reported (32). We
demonstrate that both genes code for 9-desaturases by heterologous
complementation analysis of a Saccharomyces cerevisiae
mutant strain deficient in this enzyme. Finally, we establish that
Cds1 is strongly induced by feeding a saturated diet,
indicating a quite different physiological regulation compared with
Cds2. This situation appears to have arisen by promoter
divergence of duplicated carp desaturases after a genome duplication event.
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EXPERIMENTAL PROCEDURES |
Carp maintenance and cooling treatment.
Carp (Cyprinus carpio L., 0.2-0.5 kg) were obtained
from a local fish farm (Clearwater, Fiddlers Ferry Power
Station, Widnes, UK) and held for at least 2 mo at 30 ± 0.5°C
in large 2,000-liter tanks provided with recirculation filters.
The carp were routinely fed twice daily on trout pellets (Trouw UK,
Preston, UK) containing 21% (wt/vol) crude oils and 49% (wt/vol)
crude protein. For cooling treatment, fish were transferred to
1,000-liter tanks and cooled at 1°C/h to a maximum of
7°C/day, reaching a temperature of 10°C on day 3 of the
cooling (27) at which temperature they were held for up to
69 days.
Dietary treatment.
Fish were transferred to 1,000-liter tanks and fed at 0.5% of
their body weight twice daily, two groups being fed the trout pellet
diet and another two groups a specially formulated and pelleted diet
containing elevated proportions of saturated fats (see Table
1). The saturated fat diet contained (in
%dry weight) fish meal (5%), Soya bean protein concentrate (39%),
potato starch (47.5%), coconut oil (3.8%), inosine (0.2%),
carboxymethylcellulose as binder (1%), vitamins (1.5%), minerals
(0.5%), and calcium phosphate (1.5%). Fish fed control and the
saturated fat diets were fed for 14 days at 30°C. One tank for each
diet treatment was then cooled at 1°C/h to 23°C on day 1 and to 15°C on day 2 at which temperature they were
maintained for a further 14 days. At each of the indicated times,
replicate fish from each treatment group were killed and their livers
excised for RNA extraction. Transcript levels for Cds1 and
Cds2 were compared between groups of fish at a given time
point using the nonparametric Wilcoxon's signed rank test, which makes
no assumptions about the shape of the data.
Yeast strains and growth conditions.
Yeast strain Aw3a (MATa, leu2-3, leu2-112, trp1-1,
can1-100, ura3-1, ade2-1,
ole1::LEU2) was kindly provided by
Prof. C. Martin (21) and strain FY251 (MATa, ura3-52,
his3200, leu21, trp163) by Dr. A. Platt. Yeast was grown on
synthetic complete drop-out (SCDO) media, and all physical
manipulations, including protein extraction and transformation, were
performed as described by Adams (1). Aw3a were grown on
SCDO containing 0.5 mM palmitoleic acid and 0.5 mM oleic acid plus 1%
(vol/vol) tergitol type NP-40 (Sigma Chemicals). Heterologous
expression of Cds1 and Cds2 as fusion proteins
was achieved using the S. cerevisiae expression vector
pXY213 (R&D Systems, Abingdon, UK). Oligonucleotide primers (Csd1
BamHI, 5'-GGGATCCTGACAGGGACATCAAATCTCCA-3'; Csd2 BamHI, 5'-GGGATCCAGACAGGGAAATCAAATCTCC-3') were used to introduce a
BamHI restriction site into the second codon of the
Cds1 and Cds2 reading frames by PCR. PCR was
carried out using the Accurase Taq Polymerase (Biogene)
according to the manufacturer's recommendations, and the resulting
products were cloned into pGEM-TEasy (Promega) and sequenced to confirm
sequence fidelity. The BamHI sites were then used to excise
the Cds coding regions. This allowed the Cds1 and Cds2 fragments to be ligated into the pXY213 expression
vector in frame with the translation initiation site to create
pXY::Cds1 and pXY::Cds2,
respectively. The yeast ole1 mutant strain Aw3a was
transformed with either pXY::Cds1,
pXY::Cds2, or the empty expression vector
pXY213::MBV and transformants selected on SCDO plus glucose
plus oleic and palmitoleic fatty acids. Mutants expressing a functional
9-desaturase were selected by their growth on SCDO plus galactose as
sole carbon source in the absence of fatty acid supplementation.
Fatty acid composition.
Ura+ cells were isolated and grown for 120 h on SCDO medium plus
galactose as sole carbon source in the presence of 0, 0.1, and 1 mM
linoleic acid. Yeast cells were washed into 100 mM phosphate-buffered saline by repeated centrifugation. Total lipid fraction was extracted from the resulting pellet as described previously (4).
Fatty acids were saponified, methylated, identified, and quantified by
capillary gas liquid chromatography as described (17).
General molecular genetic techniques.
Standard molecular techniques were performed as described
(26). Southern and Northern transfers and hybridization
were performed using Zeta Probe GT membrane (Bio-Rad) according to
manufacturer's instructions. For low-stringency probing,
posthybridization washes were conducted at 50°C. SCD1
homologs were isolated by screening a commercial carp liver cDNA
library (Stratagene) as described previously (32). RNA was
isolated from carp liver as described by Chomzynski and Sacchi
(6). Northern blots were quantified using the STORM 840 and ImageQuant software (Molecular Dynamics).
Genomic DNA was isolated from carp erythrocytes by a modified
extraction protocol (M. Hughes, personal communication). Washed erythrocytes from 0.5 ml blood were hypotonically lysed with 5 ml of a
solution containing 5 mM MgCl2 and 10 mM CaCl2.
The nuclei and cell debris were washed and resuspended in 9 ml
buffer B (0.1 M NaCl, 40 mM EDTA, 50 mM
Tris · HCl, pH 8.0) in a sterile Oakridge test
tube. One-half milliliter of a solution containing 5% SDS (wt/vol) and
4 mg/ml proteinase K was added, and the mixture was incubated overnight
at 50°C. This was mixed with an equal volume of saturated phenol (pH
8.0), overlaid with 2 ml of L phase lock gel (5 Prime > 3 Prime,
Boulder, CO), and the tube was centrifuged at 13,000 g for 5 min. The resulting supernatant was removed and extracted against equal
volumes of phenol-chloroform and chloroform-isoamyl alcohol, again
using phase lock gel. DNA was precipitated from this solution with 0.1 vol of 3 M sodium acetate (pH 5.2) and 0.7 vol isopropanol before
washing with 70% ethanol followed by resuspension in TE buffer
(10 mM Tris · HCl, 1 mM EDTA, pH 8).
Plasmid DNA was prepared using Wizard Miniprep kits (Promega) according
to the manufacturer's instructions. cDNAs and DNA inserts were
sequenced using the ABI 373A sequencer, and the resulting sequences
were compiled and analyzed using DNAStar (LaserGene). Computer analysis
of predicted protein sequences was performed using the PROSITE
program, while homology searches were performed using the
TFAST and BLITZ programs. All homology programs were accessed via the
EBI website (http://www.ebi.ac.uk/).
DNA probes for Southern and Northern hybridization were labeled by
random priming using High Prime (Boehringer) as per manufacturer's instructions. Probes for the 3'-untranslated region (UTR) of
Cds1 and Cds2 comprised the HindIII
fragment spanning nucleotides 1388-2500 (Cds1), and a
XhoI fragment spanning nucleotides 1167-1994
(Cds2). The open reading frame (ORF) of Cds1 was
excised using an ApaI, HindIII double digest and
comprised nucleotides 347-1320 of Cds1. The 18S probe
was a human 18S rRNA gene (Ambion) and was excised from the plasmid
with an EcoRI digest.
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RESULTS |
Identification of a second carp desaturase, Cds2.
A putative 9-desaturase gene had previously been identified by
screening a commercial carp hepatic cDNA library with the rat
SCD1 (32). This gene has now been designated
Cds1 (carp desaturase 1, GenBank CC31864). The original cDNA
clone isolated, pcDsL-7, was used to reprobe the same cDNA library
under conditions of low stringency to determine if any additional
homologs were present. One hundred sixty eight cDNAs were isolated, and
their DNA was dot blotted as a sublibrary onto charged nylon membrane. This sublibrary was probed sequentially, at high stringency, with the
coding region, the 3'-UTR, and the 5'-UTR of pcDsL-7.
Seven clones were identified that cross-hybridized to the coding region
of pcDsL-7 but not the 3'-UTR of this clone. All seven clones were
sequenced. The longest clone contained a single long ORF encoding a
putative protein of 325 amino acids (Fig.
1). This protein shows 62% identity to
rat SCD1 and 93% identity to the putative product of Cds1
(32). The gene encoding this second putative carp
9-desaturase has been named Cds2 (EMBL AJ249259). A
second Cds2 sequence was found with an identical ORF but
with the addition of a 269-nt sequence in the 3'-UTR, this extra
sequence being present in the genomic copy of Cds2 (data not
shown). The genomic sequence excised from the shorter cDNA was flanked
by splice sites. The two cDNAs are therefore likely to represent alternatively spliced transcripts. This is consistent with a Northern analysis that revealed two alternative Cds2 transcripts
(Fig. 2).
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Fig. 2.
Northern analysis of total hepatic RNA extracts from carp
subjected to chronic cooling. Carp were previously acclimated for >60
days at 30°C and subjected to a standard cooling regime as described
in EXPERIMENTAL PROCEDURES. RNA was probed after Northern
blotting with probes specific to either Cds1,
Cds2, or 18S rRNA transcripts. Each lane represents a single
fish.
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Forty-five of the remaining 161 clones cross-hybridized with the coding
region and the 3'-UTR of pcDsL-7. Sequencing of the largest of these
clones revealed the presence of a 152-nt sequence that was absent in
pcDsL-7. This sequence was also present in the genomic sequence of
Cds1 (data not shown). PCR on all 45 of the
Cds1-derived cDNA clones using primers flanking this region showed that it was absent only in clone pcDsL-7. This clone may therefore represent a very rare or incorrectly spliced transcript or
alternatively a deletion within this specific clone. The revised sequence extends the putative product of Cds1 by 39 amino
acids at the COOH terminus of the predicted protein (Fig. 1)
(32). pcDsL-7 was also found to be carrying a sequence
fused onto the 5'-end of Cds1 that was not present on the
genomic sequence. The sequence has subsequently been
isolated from the cDNA library as an independent transcript, confirmed
by Northern analysis, which shows high identity to the yeast
transcription initiation factor SUI 1 (S. D. Polley, unpublished
observation). Primer extension was used to identify the transcription
initiation start sites for both Cds1 and Cds2
(data not shown), and this confirmed that full-length cDNAs had been isolated.
Southern analysis of carp genomic DNA showed the presence of at least
three sequences, which cross hybridized to the coding region of
Cds1 at low stringency (data not shown). Cds1
remained bound to only two of these sequences when the hybridization
was repeated at high stringency. These were shown to
correspond to Cds1 and Cds2 by hybridization to
their respective 3'-UTRs (Fig. 3), which
share only 67% identity and were used as probes to distinguish the two
genes.
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Fig. 3.
Southern analysis of desaturase isoforms in carp genomic
DNA. Each lane contains DNA digested with the indicated restriction
enzyme with the exception of the DNA ladders (HP). The DNA in the
left and right panels was probed with the
3'-untranslated region (UTR) probes for Cds1 and
Cds2, respectively. Sizes of the resulting bands are
indicated.
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Expression of both Cds1 and Cds2 is temperature dependent.
Total RNA was extracted from the livers of fish subjected to the
cooling regime described previously (32) and probed under high stringency with probes prepared from the 3'-UTRs of
Cds1 and Cds2. As a control each probe was
cross-hybridized to various cDNAs to confirm its specificity for a
single transcript (data not shown). Under this regime Cds1
was expressed in fish at 30°C, before cooling, but was repressed on
cooling to 15°C (Fig. 2). A separate cooling experiment has shown
that even a modest temperature reduction to 23°C is sufficient to
repress its expression (data not shown). By contrast, the
Cds2 probe revealed two transcripts that were shown by
Northern analysis to be present in only one of three control fish
acclimated to 30°C and then only at a very low level. Cooling to
17°C caused a significant increase in amounts of both Cds2
transcripts, which reached a maximum on day 3, by which time
the fish had been cooled to 10°C. After day 3 Cds2 transcript levels partially decreased, reaching
approximately one-half their maximum level on day 6. The
relative abundance of the Cds1 and Cds2
transcripts over the time course of cold induction was quantified using
the coding region of Cds1 as a probe. This probe binds
Cds1 and Cds2 with equal intensity. In warm-acclimated animals, Cds1 accounted for >90% of
Cds1-like transcripts, a situation that was reversed in
10°C carp. Similarly, the relative levels of the two Cds2
transcripts changed over the time course of cold treatment, with the
smaller transcript responding most strongly to cold induction, such
that its abundance increased from 0.5 times (day 0) to over
two times that of the larger species (day 3). From these
data, it is clear that expression of Cds2 and not
Cds1 is induced during cold acclimation and that
Cds2 is subject to temperature-dependent differential splicing.
Dietary regulation of Cds1 and Cds2.
We have explored the differential regulation of Cds1 and
Cds2 in response to combined dietary and thermal
manipulation. Groups of 16 carp sampled randomly from a common
preacclimated stock were placed in each of four identical 1,000-liter
tanks at 30°C. Carp in two of the four tanks were fed a normal trout
pellet while the remaining animals were fed a pelleted diet enriched in
saturated fats at the expense of polyunsaturated fats. Fish were killed and sampled for transcript analysis at 0 and 14 days. At day
14, a subsample of animals from both dietary regimes was cooled to 15°C and sampled, together with control fish maintained at 30°C, at
4 days (day 18) and 10 days (day 24) after
cooling. Figure 4, A and
B, shows the transcript levels of Cds1 and
Cds2 in replicate carp at each of the sampling times while
Fig. 4, C-J, plots their amounts relative to 18S rRNA.
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Fig. 4.
Expression of Cds1 and Cds2 in carp
subjected to dietary and chronic cooling treatments. A and
B: total hepatic RNA extracts from individual carp fed an
unsaturated and saturated diet, respectively, and analyzed for
Cds1, Cds2, and 18S rRNA transcript levels. For
each diet, a random sample of fish was cooled to 10°C on day
14 as described in EXPERIMENTAL PROCEDURES and sampled
on days 18 and 24. C-J quantify
the transcript amounts of both Cds1 and Cds2
relative to the 18S rRNA, and the results are plotted against time.
C and D illustrate results for carp fed the
normal unsaturated diet (Unsat) from day 0, while
E and F relate to carp fed a saturated diet (Sat)
throughout. For these 4 plots, open squares indicate fish held at
30°C and closed squares fish transferred on day 14 to
15°C and held at that temperature to the end of the experiment.
G-J compare results for carp fed saturated and
unsaturated diets for a given temperature regime, 30°C (G
and H) and 15°C (I and J). For
G-J, closed circles indicate a saturated
diet while open circles indicate an unsaturated diet. For I
and J, only the last 2 time points occur in fish held at
15°C; earlier time points (30°C) are included for continuity and
indicated by a gray triangle. * Significant differences between
animals experiencing 30°C and 15°C or saturated and unsaturated
diets (in relevant panels) as calculated by a Wilcoxon's signed rank
test.
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By comparing animals sampled from the same time point and dietary
regime but subjected to different temperature profiles, we can evaluate
the effect that temperature has on Cds1 and Cds2 transcript levels. When we considered only those fish fed the normal
unsaturated diet, at 30°C a low but somewhat variable expression of
both isoforms was seen (as had been observed previously) (Fig. 4,
A and B). These carp express both Cds1
and Cds2 transcripts simultaneously, with the ratio between
the two isoforms being similar from one carp to another. After
progressive cooling of animals over days 14 and
15, down from 30°C to 15°C, the Cds1 transcript (Fig. 4C) showed no significant cold-induced
increase in amounts at day 18 in cold-acclimated animals
(15°C) compared with warm-acclimated (30°C) controls
(P = 0.686). A significant difference was observed on
day 24 (P = 0.029), but overall transcript amounts were very low. For Cds2 transcript amounts (Fig.
4D), a consistent and substantial increase was seen in
cooled fish (15°C) compared with warm-acclimated (30°C) controls,
which is significant at day 18 (P = 0.029)
and tending toward significance on day 24 (P = 0.057). Similar results were obtained for those animals transferred
to a saturated diet on day 0; Cds1 amounts (Fig.
4E) were reduced by cooling at day 18 or
day 24, and Cds2 amounts (Fig. 4F)
showed substantial increases compared with warm-acclimated (30°C)
controls at day 18 (P = 0.029). These
results confirm previous results that it was Cds2 and not
Cds1 that was cold induced, and this was unaffected by
dietary treatment.
By comparing carp sampled from the same time points and thermal regime
but fed either a saturated or unsaturated diet (Fig. 4,
G-J), we can determine the effects of diet on
Cds1 and Cds2 transcript amounts. Feeding the
saturated diet to carp maintained at 30°C led to a progressive and
significant increase in the transcript levels of Cds1 (Fig.
4G) on days 14 (P = 0.029) and
18 (P = 0.029) compared with unsaturated
diet controls, but not on day 24 (P = 0.057). Cooling of carp to 15°C (Fig. 4I) abolished this
effect (P = 0.342 on day 18 and 0.685 on
day 24). Regarding Cds2, we see a
modest but statistically significant increase in transcript amounts in
fish held at 30°C (Fig. 4H) and fed a saturated diet on
days 14 (P = 0.029) and 18 (P = 0.028), but not on day 24 (P = 0.342), compared with unsaturated controls. This
dietary induced increase in Cds2 transcript levels was
maintained in fish cooled to 15°C (Fig. 4J,
P = 0.029, day 18 and P = 0.342, day 24). We conclude, first, that Cds1 is
substantially elevated and Cds2 is slightly elevated in
response to feeding a saturated diet, and, second, for Cds1
this effect can be prevented by cooling.
Tissue-specific expression of desaturase isoforms.
The tissue specificity of Cds1 and Cds2
expression has been examined in response to cooling. Replicate
warm-acclimated carp were killed on day 0 and day
2 of the standard cooling regime, and total RNA extracts from a
range of tissues were prepared and pooled for each time point. Figure
5 shows Northern blots probed with the
Cds1 ORF, which under the conditions used will hybridize to
both the Cds1 and Cds2 transcripts.
Cds1 homologous transcripts were evident in carp cooled to
17°C but not in control fish held throughout at 30°C. High levels
of transcript were only observed in the liver, although faint bands
were seen in several other tissues, including brain and spleen. These
data suggest that the liver is the principal tissue for the expression
of both desaturase isoforms.
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Fig. 5.
The tissue-specific expression of Cds1 and
Cds2 in carp. Northern analysis of RNA isolated from various
tissues of warm-acclimated and cold-treated carp. The blots were probed
with the coding sequence of Cds1, which is unable to
discriminate between Cds1 and Cds2. ORF, open
reading frame.
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Complementation of the S. cerevisiae ole1 mutation by both Cds1 and
Cds2.
The suggested functions of Cds1 and Cds2 were
based on their similarity to the rat 9-desaturase structural gene
SCD1. We have sought to confirm this by testing the ability
of both Cds1 and Cds2 to complement the
ole1 mutation in the yeast S. cerevisiae. This
mutation disrupts the endogenous yeast 9-desaturase making the
growth of mutant strains dependent on provision of unsaturated fatty
acids in the culture medium (30). Functional
complementation of this mutation by a heterologous gene has previously
been used to demonstrate that SCD1 encodes a 9-desaturase
(31). We have developed an inducible construct containing
either Cds1 or Cds2 to allow the activity of the
enzyme to be directly tested.
Both Cds1 and Cds2 were introduced into a
galactose-inducible, glucose-suppressible yeast expression vector.
pXY::Cds1 and pXY::Cds2 were engineered
to express the carp Cds1 and Cds2 transcripts, respectively. Both expression constructs and the empty expression vector pXY213::MBV were transformed separately into the
ole1 strain Aw3a. Transformants were selected on the basis
of uracil prototrophy in the presence of oleic and palmitoleic acid.
These transformants were then transferred to uracil-deficient plates
either in the presence or absence of monounsaturated fatty acid
supplementation and using either glucose or galactose as sole carbon
source (Fig. 6).
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Fig. 6.
Complementation test of putative carp desaturases in the
ole1 mutant of the yeast Saccharomyces
cerevisiae. pXY::Cds1 and
pXY::Cds2 were transformed into the Aw3a strain of
yeast as described in EXPERIMENTAL PROCEDURES.
Transformants were grown in the presence of glucose, which acts as a
repressor, and galactose, which acts as an inducer of the
GAL1 promoter, which is responsible for the transcription of
the 2 carp genes. The presence or absence of monounsaturated fatty acid
(MUFA) supplementation in the medium was as indicated.
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pXY::Cds1 and pXY::Cds2
transformed cells showed definite growth in the absence of oleic and
palmitoleic acids in the presence of galactose, as sole carbon source,
but failed to grow in the presence of glucose on a medium lacking in
these fatty acids (Fig. 6). Only on the addition of oleic and
palmitoleic acid to the medium were these transformants able to grow in
presence of glucose. pXY213::MBV transformed cells failed to
show any growth in the absence of monounsaturated fatty acid
supplementation using either galactose or glucose as sole carbon source.
Although S. cerevisiae does not form linoleic acid (18:2
9-12) under normal growth conditions, it is a strong repressor
of OLE1 expression and is preferentially incorporated into
the membrane lipids of wild-type cells when added into the medium, to
replace the 16:1 and 18:1 products of the 9-desaturase activity
(20). ole1 strains show good growth on media
not containing monounsaturated fatty acids but supplemented with
linoleic acid (Fig. 6). The pXY::Cds1,
pXY::Cds2, and pXY::MBV transformed
cells were grown on media supplemented with varying levels of linoleic
acid using galactose as sole carbon source and then harvested for
analysis of their total fatty acids. The traces showed an abundance of 18:2 fatty acids in pXY213::MBV transformed cells grown on
0.1 µM linoleic acid, but no 16:1 or 18:1 peaks (Fig.
7). By contrast, the
pXY::Cds1 transformants showed peaks corresponding
to the 9-desaturation products, 16:1 and 18:1, that increased in
relative magnitude as the level of linoleic acid supplementation was
reduced.
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Fig. 7.
Gas-liquid chromatograms of fatty acid methyl esters
prepared from total lipid fraction of yeast transformed with
pXY::Cds1. Yeast strain Aw3a was transformed with
the constructs pXY213::MBV (Aw3a::MBV) and
pXY213::Cds1 (Aw3a::Cds1) as
indicated. Yeast was grown on a medium supplemented with 1, 0.1, or
0.01 mM 18:2 linolenic acid. Identical results were obtained for the
pXY::Cds2 transformed yeast.
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These data demonstrate that both Cds1 and Cds2
encode functional 9-desaturases.
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DISCUSSION |
A revised structure for Cds1.
We have previously cloned a homolog of rat SCD1 from a
commercial carp liver cDNA library (32). The original
clone, designated pcDsL-7, possessed a single long ORF encoding a
putative protein of 292 amino acid residues with 55 and 53% identity
with rat and mouse SCD1 9-desaturases, respectively. However, the
predicted protein product of pcDsL-7 was ~30 residues shorter at the
COOH-terminal end than the putative product of SCD1, and the
pcDsL-7 transcript possessed an unexpectedly long 5'-UTR of 520 nucleotides. We now show through characterization of other
SCD1 homologous clones that pcDsL-7 appears to contain an
internal deletion within the ORF and a distinct cDNA sequence
erroneously fused to the 5' end of Cds1. The ORF contained
by the other cDNAs encoded a putative protein of 327 amino acid
residues and a molecular mass of 37.7 kDa both of which more closely
match the COOH terminal sequences of the rat and yeast homologs. This
revised carp gene has been redesignated Cds1.
Relationship of Cds1 and Cds2.
Rescreening the carp liver cDNA library revealed a group of transcripts
with high identity to the coding sequence of Cds1 but not
with the corresponding 3'-UTR. Sequencing of these clones revealed a
second putative desaturase gene with high sequence similarity to the
putative protein products of Cds1 (93%) and mouse
SCD1 (62%). This new gene has been designated
Cds2. Both isoforms are expressed in liver and not in any
other tissue, at least in amounts detectable by our methodology. We
have also identified and isolated the genomic sequences encoding these
genes and confirmed their identity by sequencing (S. D. Polley, H. Evans, B. Cossins, and P. E. Tiku, unpublished data).
Mouse also expresses two 9-desaturase genes that are 89% identical
at the amino acid level; one (SCD1) is expressed constitutively in
adipose tissue and induced in liver by dietary treatment
(23) and the other (SCD2) is expressed in brain but not in
liver (16). An important question is whether these
isoforms are related to the two hepatic isoforms in carp or have arisen
independently. Figure 8 shows a
dendrogram based on similarity analysis from which it is evident that
the two carp isoforms are more similar to each other than either is to
the mouse or rat isoforms. Moreover, of the 33 amino acid substitutions
between the two mouse homologs, only five coincided with substitutions
between the two carp homologs and only two of these involved similar
substitutions, indicating no relationship between the respective mouse
and carp homologs. Because of this and the different tissue-specific
patterns of expression, we conclude that the two carp isoforms have a
phylogenetic origin different from the two mouse 9-desaturases and
are therefore likely to have a different physiological significance
compared with those observed in the mammals.
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Fig. 8.
An unrooted additive tree illustrating the amino acid
identity between the 2 carp desaturase proteins CDS1 (accession number
U31864) and CDS2 (AJ249259), the desaturases for the grass carp
(Ctenopharyngodon idella, AJ243835), the Antarctic
Chiondraco hamatus (AJ249579), the milk fish (Chanos
chanos AY082003), and the mammalian homologs, mouse SCD1
(AF509567), mouse SCD2 (M26270), rat SCD1 (AB032243), and rat SCD2
(AF509569), together with an insect homolog as an outgroup
(Epiphyas postvittana, AY061988). The tree was generated by
the least-squares method program Fitch, using the Dayhoff PAM matrix to
calculate pairwise distances. Bootstrap analysis with 100 replicates
gave values of 99% or above for all branches. Fitch is part of
Felsenstein's Phylip program (25).
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Complementation of yeast ole1 mutation by Cds1 and Cds2.
Although both the carp putative desaturases have a high sequence
identity with the rat SCD1, it was important to determine whether either or both sequences code for a functional 9-desaturase. We have tested both genes by complementation of a yeast strain (ole1) that is deficient in its endogenous 9-desaturase
activity and is auxotrophic for monounsaturated fatty acids
(30). We have shown that both genes restored growth to
yeast cultures when their expression is induced by growth with
galactose as sole carbon source but not when it is repressed by glucose
(Fig. 6). Moreover, cultures complemented with either Cds1
or Cds2 produced monounsaturated fatty acids demonstrating
unequivocally that these genes code for 9-desaturases.
Temporal and spatial regulation of the two isoforms during chronic
cooling.
Previous work has shown that the enzymatic activity of the hepatic
desaturase was low in 30°C-acclimated carp but increased 8- to
10-fold in the 4-5 days after cooling to 10°C (32).
This was associated with a transient increase in amounts of
Cds transcript levels, caused at least in part by an
increased rate of transcription. We now show that the transcript
evident in 30°C-acclimated carp is mainly that encoded by
Cds1 (Fig. 2). Cooling of fish down to 10°C over 3 days
led to a substantial increase in the level of Cds2
transcripts while the level of Cds1 was reduced. Thus only
Cds2 is cold inducible while Cds1 expression is
transiently repressed by cold.
Despite the low levels of Cds1 transcript in the liver of
30°C-acclimated carp, we have previously shown by Western immunoassay that these animals possess significant amounts of largely inactive desaturase protein whose enzymatic activity increases two- to fourfold
during the first 2 days of cooling (32). The prevalence of
Cds1 transcript in these animals suggests that this protein is largely, if not entirely, composed of CDS1, and that this isoform is
subjected to activation. Because cooling leads specifically to
increased levels of the Cds2 transcript, it follows that the subsequent increase in desaturase protein abundance observed in Western
immunoassays is solely due to CDS2. Thus during the early period of chronic cooling the population of desaturase proteins comprises a mixture of the two isoforms with CDS2 possibly becoming predominant after prolonged cooling. At present we are unable to
confirm this scenario first because the polyclonal antibody we have is
unable to discriminate between the two isoforms and second because
little is known about the degradation of desaturase proteins in fish.
It is known, however, that the degradation of desaturases in the
bacterium Escherichia coli is markedly temperature dependent
(10).
Dietary regulation of hepatic desaturases.
The presence of two desaturase isoforms in carp liver may permit the
differentiated regulation of desaturase activity to different stimuli,
perhaps as part of different response systems. It is well known that
mammalian hepatic 9-desaturases, most notably in rat and mouse, are
greatly induced by a dietary regime of starvation followed by refeeding
with a fat-free diet (23, 29). More significantly in the
present context, Wodtke and Cossins (37) have demonstrated
a long-lasting increase in hepatic desaturase activity in carp fed a
commercial diet containing elevated proportions of saturated fatty acids.
We have therefore tested the effects of manipulation of dietary lipid
saturation on desaturase expression by feeding carp a pelleted diet
containing elevated levels of coconut oil and reduced levels of fish
oil. Feeding this diet to carp held at 30°C over a 2-wk period led to
a statistically significant increase in the level of Cds1
transcript (10-fold over that observed with an unsaturated diet) with a
much smaller absolute effect on Cds2. Cooling from 30 to
15°C abolished the diet-induced increase in Cds1
transcript observed at 30°C but instead caused a substantial induction of Cds2, a response to cooling that was more
marked in animals fed the saturated diet.
This complex experiment supports the idea that the two desaturase
isoforms respond quite differently to physiological stimuli, and there
is comparatively little cross-talk between them. Cds1 responds to the modified diet, which is substantially more saturated than the trout diet. The main difference in these two diets is the
proportion of C14:0 myristate, which could account for the effect we
have seen in isolation or in conjunction with the other saturated fatty
acids. Although the level of Cds1 transcript was elevated
after 10 days of cooling, this response was relatively small and
occurred well after the cold-induced changes in fatty acid composition
of microsomal phospholipids (32, 33). These compositional
changes, representing the acute phase of membrane lipid restructuring,
can therefore be attributed exclusively to elevated expression of the
CDS2 isoform. Whether the elevation of CDS1 over the longer term has
any impact on membrane lipid composition or on the composition of other
fatty acid pools is not clear. However, the original report of
cold-induced desaturase activity by Schünke and Wodtke
(27) claimed a biphasic response to cold with peak
enzymatic activities occurring at 5 and 10 days. This may represent the
quite separate inductions of Cds2 and Cds1, respectively. Finally, the ~10-fold greater representation of Cds1 clones in the commercial cDNA library compared with
Cds2 clones is consistent with the animals used for library
construction having experienced warm conditions and a relatively
saturated diet. This together with the use of a coding sequence probe
probably accounts for the fact that our initial screen identified only Cds1.
Temperature-specific desaturase isoforms?
Although we demonstrate that both Cds1 and Cds2
code for 9-desaturases, it is much less certain that the resulting
proteins are functionally identical. Functionally important changes to proteins can result from very few amino acid substitutions, so the 21 substitutions between CDS1 and CDS2 may well have a functional significance. One possibility is that the two isoforms might be catalytically most effective over a different range of temperatures such that their temperature-specific expression is temperature adaptive
as well as being associated with two different response systems. This
phenomenon is well documented for the regulation of skeletal muscle
contractile activity in summer- and winter-acclimated carp by the
differential expression of functionally distinct forms of the myosin
heavy chain molecule (11, 15, 34). However, expressing
both isoforms in ole1-deficient yeast allowed growth at
30°C, and there was no noticeable difference in the growth characteristics of the two complemented yeast strains.
Recent evidence from the analysis of homeobox genes has indicated that
teleost fish experienced a genomic duplication event followed by
subsequent selective reduction in the repertoire of expressed genes
(2). Furthermore, some groups of fish, including some
cyprinid fish, have undergone additional, more recent duplications, and
having double the number of chromosomes are thus regarded as tetraploid
(8, 25). Figure 8 shows a dendrogram containing the known
teleost 9-desaturases, including the closely related grass carp, for
which there is only one gene (5), and the Antarctic Chionodraco. The dendrogram uses an insect desaturase as an
outgroup. This shows that the two carp isoforms are more similar to
each other than either is to the 9-desaturases of other species,
including grass carp, indicating that gene duplication and divergence
occurred more recently than the evolutionary divergence of the grass
and common carp. By contrast, rat SCD1 shows higher identity to the orthologous protein in mouse (SCD1) than it does to the other rat
desaturase (SCD2), indicating that gene duplication and divergence occurred before the divergence of two species. High bootstrap values
indicate a robust phylogeny. Even though the existence of a second
desaturase cannot be discounted in grass carp by sequencing of cDNAs,
Southern analysis with grass carp genomic DNA supports the existence of
only one (H. Evans, personal communication). A BLAST search of the
genome of the Japanese pufferfish Fugu rubripes (http://Fugu.jgi-psf.org) reveals the existence of two SCD1
homologs in scaffolds 64 and 5415. It is likely however that these two genes result from a very ancient duplication because they show a much
higher level of divergence than the two carp desaturases at both the
synonymous and nonsynonymous levels, the latter evident as a 70.5%
level of identity between the putative protein products of two fugu
paralogs. The role of these two SCD1 homologs is unknown at present.
In many tetraploid species the nonallelic gene copies are functional
but appear to be fully redundant (25). Divergence in the
regulatory sequences might, however, alter the spatial or temporal
pattern of expression, giving rise to novel expression characteristics.
This has been observed within a developmental context in mice where the
homologs Hoxa3 and Hoxd3 encode proteins with an identical biological
activity but with different expression patterns within the embryo
(13). Within an environmental context, the additional
complement of genes might provide a more plastic physiology, capable of
tolerating wider environmental conditions than other related groups of
fish, giving rise to the flexible genome concept (35). On
the other hand, duplicated genes can be fixed by the partitioning of
ancestral functions rather than the evolution of new functions per se
(9). Although the divergence of the regulatory regions of
Cds1 and Cds2 causes them to exhibit differentiated responses to cooling and dietary manipulation, it is not
clear whether the ancestral desaturase responded to both stimuli.
Distinguishing between these two contrasting models requires analysis
of outgroup species corresponding more closely to the ancestral
unduplicated gene, including perhaps the grass carp,
Ctenophanyngodon idella. This might indicate whether
possession of two desaturase isoforms and the partitioning of
responsiveness to different stimuli offer any selective advantage with
respect to environmental stress.
| |
ACKNOWLEDGEMENTS |
We thank Dr. O. Day (Centre for Environment, Fisheries and
Aquaculture, Weymouth) for providing the saturated fat diet
and Prof. C. Martin and Dr. A. Platt for providing the yeast mutants.
| |
FOOTNOTES |
This work was supported by grants from the Natural Environmental
Research Council (NERC; UK) and from Scotia Holdings. R. T. Trueman was supported by a postgraduate studentship from NERC.
Present address for S. D. Polley: London School of Hygiene and
Tropical Medicine, Unit of Infectious and Tropical Diseases, Dept. of
Parasite Molecular Biology and Biochemistry, Keppel Street, London WC1E
7H, UK.
Address for reprint requests and other correspondence:
A. R. Cossins, School of Biological Sciences, University of
Liverpool, Liverpool L69 7ZB, United Kingdom (E-mail:
cossins{at}liv.ac.uk).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
September 12, 2002;10.1152/ajpregu.00263.2002
Received 10 May 2002; accepted in final form 10 September 2002.
| |
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ABSTRACT
INTRODUCTION
