BACKGROUND
in living cells, most energy is produced in the mitochondria through oxidative phosphorylation.
in this process, the electron flow from reduced substrates to oxygen generates an electrochemical proton gradient across the inner membrane.
this force drives the proton back into the matrix and results in atp synthesis from adp and pi.
uncoupling proteins , which are members of the superfamily of mitochondrial anion-carrier proteins, are capable of dissipating the proton gradient across the inner mitochondrial membrane to generate heat while reducing the efficiency of atp synthesis  <cit> .
the archetypical ucp <dig> is expressed in brown adipose tissue of mammals and is involved in non-shivering thermogenesis  <cit> .
ucp <dig> mrna has been recently found in ectothermic organisms such as carp, zebrafish and pufferfish  <cit> .
homologues of ucp <dig>  have been identified from various tissues in vertebrates  <cit>  and plants  <cit> .
ucp <dig> has been described in previous studies to play a role in various physiological processes such as body weight control  <cit> , fatty acid metabolism  <cit> , control of reactive oxygen species  <cit> , and negative regulation of insulin secretion  <cit> .
no clear thermogenic function has been identified for ucp <dig>  <cit>  but increases of muscle ucp <dig> mrna in response to fasting has been reported in rat  <cit> , human  <cit>  and marsupials  <cit> .
ucp <dig> appeared along with ucp <dig> to affect energy partitioning, feed efficiency, body mass index and obesity  <cit> .
the present study was designed to characterize ucp <dig> genes in the rainbow trout and investigate their potential as candidate genes affecting traits associated with energy balance and nutrition.
to this end, we analyzed the genomic structure, phylogenetic relationships with other ucps, tissue distribution and expression in muscle of ucp <dig> in response to fasting.
RESULTS
analysis of cdna and amino acid sequences
we identified two similar tentative consensus sequences  by homology search for ucp <dig> in the tigr rainbow trout gene index .
both tc <dig> and tc <dig> were found to contain full-length coding sequences from clones tcad0009a.o <dig> and tcad0008a.b <dig>  respectively.
an additional cdna clone  containing est ca <dig> which is assigned to tc <dig> was also picked, purified and sequenced.
full sequences of tcad0009a.o <dig>  rt84b <dig> and tcad0008a.b <dig> were deposited to genbank and assigned accession numbers .
similarity analyses between cdna sequences revealed that the  <dig> -bp 1rt84b <dig> was 90% identical with  <dig> -bp tcad0009a.o <dig>  an 157-bp insert in 1rt84b <dig> accounted for the 10% difference between both sequences.
the cdna clone tcad0008a.b <dig> was  <dig> bp and was 78% and 88% similar to 1rt84b <dig> and tcad0009a.o <dig>  respectively.
for ease of identification, tcad0009a.o <dig> and tcad0008a.b <dig> were dubbed ucp2a and ucp2b, respectively.
the deduced amino acid sequences of ucp2a and ucp2b consist of  <dig> and  <dig> amino acid residues, respectively .
the peptide sequence deduced from 1rt84b <dig> is a truncated form of that obtained from tcad0009a.o <dig>  the deduced protein, which consisted only of transmembrane domain i and one proton carrier signature, most likely lacks the proton dissipation function as it has been demonstrated that the second transmembrane domain of ucp genes is essential for the anion channel formation  <cit> .
this sequence was therefore discarded from further analysis.
we found 93% similarity between the ucp2a and ucp2b peptide sequences with both containing six transmembrane domains and three proton carrier signatures which define the general triplicate structure of mitochondrial uncoupling proteins  <cit> .
the purine-binding domain involved in the control of coupling efficiency was also identified.
over the protein conserved domains rainbow trout ucp2a and ucp2b diverged only by one amino-acid at position  <dig> in transmembrane domain iv.
the switch from alanine  to valine  should not make any difference in the three-dimensional structure of the peptides as these are both hydrophobic amino acids.
on average, both rainbow trout ucp2s showed 83% amino acid similarity with zebrafish ucp <dig> and 78% amino acid similarity with human, rat and mouse ucp2s.
ucp <dig> and ucp <dig> have been described to be fairly similar in their peptide sequences and their physiological functions  <cit> .
our results showed 69% amino acid similarity between rainbow trout ucp2s and mammalian ucp3s.
the identification of two copies of ucp <dig> genes in rainbow trout genome is consistent with the ancestral whole genome duplication  <cit>  and re-diploidization of salmonidae species.
several genes and microsatellite loci in rainbow trout have been previously described to be present in multiple copies  <cit>  and palti et al.
estimated that approximately two-thirds of the rainbow trout genes remained duplicated  <cit> .
phylogenetic relationships
we performed a phylogenetic analysis to classify rainbow trout ucp2a and ucp2b and determine their relationships to ucps from other species .
the tree generated included  <dig> peptide sequences from the ucp gene family retrieved from public databases.
trees with similar topology to that of figure  <dig> were drawn when either minimum evolution or unweighted pair group algorithms were performed.
both rainbow trout ucp <dig> duplicates were grouped together with their ucp <dig> orthologs in the same clade.
fish  and frog ucp <dig> peptides were assigned to a specific cluster, with a high bootstrap , as opposed to mammalian ucp <dig> genes.
our data showed a close relationship between ucp <dig> and ucp <dig> in agreement with previous expression patterns and amino acid sequence similarity suggesting that ucp <dig> and ucp <dig> are paralogs  <cit> .
moreover both genes have been mapped on the same chromosome in endothermic vertebrates such as human, bovine, porcine and mouse  <cit>  and more recently the same evidence was found in ectothermic vertebrates like zebrafish.
<cit> .
overall, ucp genes were clustered in three major branches at 100% bootstrap value leading to three groups consisting of ucp1- <dig>  ucp- <dig> and ucp- <dig>  we suggest an early divergence of the ucp family gene into three major clades in agreement with the phylogeny described by sokolova and sokolov  <cit> .
these authors also demonstrated that this subdivision could be correlated with a functional specialization among the ucp superfamily.
genomic organization of rainbow trout ucp2
we identified  <dig> total bac clones putatively containing an ucp <dig> gene of which  <dig> where specific to ucp2a and  <dig> were specific to ucp2b.
after fingerprinting, most of these bac clones were assembled into three contigs c <dig>  c <dig> and c <dig> containing  <dig>   <dig> and  <dig> clones, respectively.
c <dig> consisted of ucp2a-containing bacs while c <dig> and c <dig> consisted of ucp2b-containing bacs.
two representative bacs were selected from each contig for direct sequencing.
two bac clones, 243p <dig> and 398g <dig>  were selected from c <dig> and c <dig> for further analysis based on their sequence similarities with cdna clones tcad0009a.o <dig>  and tcad0008a.b <dig> , respectively.
genomic sequences for ucp2a and ucp2b genomic sequences have been deposited at ncbi .
alignment between cdna and bac genomic sequences revealed the exon-intron structures of ucp2a and ucp2b .
in each alignment a tata box was identified on genomic dna  <dig> and  <dig> bp upstream of the 5'end of ucp2a and ucp2b, respectively.
ucp2a and ucp2b span  <dig>  kb and  <dig>  kb, respectively.
both rainbow trout ucp2s contain  <dig> exons and  <dig> introns.
coding sequences for both genes covered  <dig> exons as described for mammalian ucp <dig>  <cit> , ucp <dig>  <cit>  and ucp <dig>  <cit>  and each exon encodes one transmembrane domain of the protein.
the analysis of genomic structure showed that 1rt84b <dig> is a splice variant of tcad0009a.o <dig>  exon  <dig> was 157-bp longer and intron  <dig> was subsequently 157-bp shorter in 1rt84b <dig> than in tcad0009a.o <dig> .
exon  <dig> of 1rt84b <dig> is not fully translated as a stop codon is encountered in the first  <dig> nucleotides.
in human where ucp <dig> has been well characterized, snp and insertion/deletion variants of the gene were identified  <cit>  and no alternative splice variants have been reported to date.
similarity between ucp2a and ucp2b was low in the introns with 60% similarity on average while the average similarity in the exon sequences was 86%.
the number and the size of the exons and introns of rainbow trout, human, mouse and zebrafish ucp2s are represented in table  <dig>  the number of exons was identical in these vertebrate organisms although exon lengths were observed to be somewhat variable.
translated exons  <dig>   <dig>   <dig> and  <dig> were the most conserved in size between species and within rainbow trout.
exon  <dig> had the same size in all species.
these translated exons showed 93% average sequence similarity between rainbow trout ucp2a and ucp2b while untranslated exons were 79% identical.
mutations apparently occur at a slower rate in the coding region than in the untranslated region of duplicated genes.
these results show conservation of the structure of ucp <dig> genes coding exons within vertebrates confirming the hypothesis of a common ancestry for ucp genes.
we examined introns  <dig> and  <dig>  untranslated exons  <dig>   <dig>  untranslated part of exon  <dig> and approximately  <dig> bp of genomic sequence upstream of each gene's transcription initiation site as the putative promoter regions.
this represented  <dig>  and  <dig>  kb-long sequence upstream of the translation-initiation site  of ucp2a and ucp2b, respectively.
similarity was 59% between overlaps suggesting differentiation between regulatory regions of ucp2a and ucp2b.
the promoter regions of rainbow trout ucp2a and ucp2b, and the upstream non-coding sequences identified in human , mouse  and zebrafish  ucp <dig> were searched for regulatory element binding sites using the tfsearch engine version  <dig> .
the results for ten transcription factors frequently identified are summarized in tables  <dig> and  <dig>  the identification of regulatory conserved motifs upstream of the five genes is consistent with the phylogenetic relationships previously described between the ucp <dig> genes in human, mouse, zebrafish and rainbow trout.
the binding sites of adr <dig>  cap, cdxa and hsf were identified in upstream region of all five genes although at different frequency.
aml1a , mzf  usf  binding sites were identified in mouse and human sequences and absent in fish .
among the fish ucp <dig> genes, the rainbow trout ucp2a regulatory region appeared to be the most unique with almost half of the binding motifs identified in ucp2b and zebrafish ucp <dig>  among the regulatory motifs we identified for ucp <dig> genes, usf and creb have been shown to regulate the expression of ucp <dig> and ucp <dig>  respectively  <cit> .
our results suggest, in agreement with previous data in the literature  <cit> , that some transcription factor binding sites are conserved across species while others are not.
adr1: alcohol dehydrogenase gene regulator  <dig> , activator.
aml-1a: acute myeloid leukemia 1; ; may act as a negative regulator.
cap: cap signal for transcription initiation signal , activator.
cdxa: chick; chicken chox-cad gene for caudal homologue, activator.
creb: cyclic-amp response element-binding protein, transactivator.
hsf: heat shock transcription factor , activator.
mzf1: myeloid zinc finger protein mzf <dig> ; possibly involved in hemopoietic development.
nkx2: murine homeobox gene  modest activator probably important for myocardial development.
sry: sex-determining region y gene product .
usf: upstream stimulatory transcription, activator .
tissue distribution and response to fasting
the distribution of relative expression of rainbow trout ucp2a and ucp2b was investigated on a panel of  <dig> tissues from adult fish .
both ucp2a and ucp2b genes appeared ubiquitously expressed in the body as described in marsupials  <cit> , human  <cit> , carp  <cit>  and mouse  <cit>  making the genes likely to influence several physiological processes.
the highest relative expression was observed in ovary, peripheral blood leucocytes , gill, testis, spleen and trunk kidney.
in all these tissues except ovary, ucp2b mrna relative amounts were at least twice that of ucp2a.
in brain, fat, pituitary and red blood cells, ucp2a mrna was more abundant than ucp2b mrna.
high amounts of ucp <dig> mrnas in reproductive organs and in tissues rich in macrophages like pbls, spleen, and kidney have been reported in several studies.
arsenijevic et al.
<cit>  and kizaki et al.
<cit>  suggested a role for ucp <dig> in immunity or inflammatory responsiveness through the regulation of reactive oxygen species in macrophages.
in mouse ovary, ucp <dig> mrna levels have been described to increase during ovulation as a result of the increasing number of macrophages and neutrophils  <cit> .
in trout, ovaries in maturation are characterized by the presence of many macrophages inside the cytoplasm of the oocytes  <cit> .
the ovaries used in our tissue distribution panel were developing.
therefore, we suggest the high amount of ucp <dig> mrna in trout ovary could be due to the macrophage-rich developmental stage of the tissue used.
the initial average weight of the fish used in the current study was  <dig> mg .
following day  <dig>  the fed fish weighed  <dig> mg and the fasted fish were  <dig> mg. at day  <dig> of fasting, termination of the fasted group, the average weight of the fish was  <dig> mg,  <dig> mg and  <dig> mg for the fed, fasted and refed groups, respectively.
rainbow trout ucp2a and ucp2b were differentially expressed in response to fasting and refeeding in fry .
ucp2a mrna expression was similar between all groups at the beginning of the experiment.
three days after food deprivation, ucp2a expression was three times higher in fed fish than in fasted fish.
the amount of gene expression remained significantly higher in fed fish than in fasted fish until day  <dig> of the experiment.
refeeding of fasted animals started at day  <dig> and resulted in a 2-fold increase of ucp2a at day  <dig>  at day  <dig>  refed and fed fish had the same amount of ucp2a expression.
at day  <dig>  ucp2b expression is similar in all fry.
at day  <dig>  expression was 3-fold augmented in fasted fish while no significant increase was found in the fed fish.
ucp2b expression drastically decreased in fasted fish to the level observed in fed fish at day  <dig> before a significant increase was observed again at day  <dig>  at days  <dig> and  <dig>  ucp2b was expressed higher in fasted fry than in fed fry.
in fed animals, no significant differences were noted between ucp2b expression levels throughout the experiment, except at day  <dig>  refed fish showed the same level of expression of the gene as the fasted fish at day  <dig>  ucp2b expression then decreased in refed fish to reach the same level expressed in fed animals at days  <dig> and  <dig>  the expression pattern of ucp2b in response to fasting is consistent with what has been described for other species in the literature.
the food deprivation induced-increase in mrna expression is one of the most spectacular features of ucp <dig> and ucp <dig> in rodent and human  <cit> .
the hypothesis is that during fasting when glucose is limiting, upregulation of muscle ucp <dig> is associated with the shift in muscle substrate utilization primarily towards lipid.
on refeeding with a low-fat diet, ucp <dig> is down regulated allowing the re-use of glucose as major substrate.
it has been also demonstrated that a high-fat diet induces up-regulation of ucp <dig> and ucp <dig> thus promoting fat utilization over storage  <cit> .
the actual mechanism of ucp involvement in fatty acid metabolism is not yet fully understood, but samec et al.
<cit>  established a close relationship between expression of ucp <dig> and ucp <dig> and well-known key regulators of lipid oxidation.
our study, unlike most of experiments in the literature, monitored ucp <dig> mrna levels for a relatively long fasting time .
our results suggest that increase of ucp2b mrna is maintained during the whole fasting time, although the higher expression is detected early.
the overall amount of expression of ucp2b genes in fry muscle were much lower than what we observed for ucp2a.
five times more cdna was included in real-time pcr for ucp2b primers than was used for ucp2a to detect readable amplification signals.
as a result, the highest detected amount of ucp2b gene ef1-normalized expression is  <dig>  compared with that of ucp2a which was  <dig> .
in adult fish, as described above ucp2b mrna expression was much higher relatively to ucp2a mrna in some of the tissues.
in light of the expression data in response to fasting, ucp2b is likely the homolog of the ucp <dig> described in other vertebrates.
ucp2a is a duplicate form of the gene that has undergone a different evolutionary process leading to a different expression pattern.
this is illustrated by the sequence divergence and different cis-regulatory elements identified in the promoter regions of both genes.
further functional characterization of the promoter region and identification of regulatory motifs could help address the differences between mrna expression of ucp2a and ucp2b.
CONCLUSIONS
we have identified two ucp <dig> genes in rainbow trout.
both genes were highly similar at the amino acid level and widely expressed in the body.
analyses of the genomic organization and phylogeny data based on protein sequence similarity suggest a close relationship between members of the ucp superfamily.
in rainbow trout fry ucp2a and ucp2b appeared downregulated and upregulated on fasting, respectively.
further characterization of both genes in different physiological and dietary conditions, and developmental stages will assist in better testing and understanding hypothesis related to their specific functions and differentiation.

 in living cells, most energy is produced in the mitochondria through oxidative phosphorylation. in this process, the electron flow from reduced substrates to oxygen generates an electrochemical proton gradient across the inner membrane. this force drives the proton back into the matrix and results in atp synthesis from adp and pi. uncoupling proteins, which are members of the superfamily of mitochondrial anion-carrier proteins, are capable of dissipating the proton gradient across the inner mitochondrial membrane to generate heat while reducing the efficiency of atp synthesis. the archetypical ucp is expressed in brown adipose tissue of mammals and is involved in non-shivering thermogenesis. ucp mrna has been recently found in ectothermic organisms such as carp, zebrafish and pufferfish. homologues of ucp have been identified from various tissues in vertebrates and plants. ucp has been described in previous studies to play a role in various physiological processes such as body weight control, fatty acid metabolism, control of reactive oxygen species, and negative regulation of insulin secretion. no clear thermogenic function has been identified for ucp but increases of muscle ucp mrna in response to fasting has been reported in rat, human and marsupials. ucp appeared along with ucp to affect energy partitioning, feed efficiency, body mass index and obesity. the present study was designed to characterize ucp genes in the rainbow trout and investigate their potential as candidate genes affecting traits associated with energy balance and nutrition. to this end, we analyzed the genomic structure, phylogenetic relationships with other ucps, tissue distribution and expression in muscle of ucp in response to fasting.  analysis of cdna and amino acid sequences we identified two similar tentative consensus sequences by homology search for ucp in the tigr rainbow trout gene index. both tc and tc were found to contain full-length coding sequences from clones tcad0009a.o and tcad0008a.b respectively. an additional cdna clone containing est ca which is assigned to tc was also picked, purified and sequenced. full sequences of tcad0009a.o rt84b and tcad0008a.b were deposited to genbank and assigned accession numbers. similarity analyses between cdna sequences revealed that the -bp 1rt84b was 90% identical with -bp tcad0009a.o an 157-bp insert in 1rt84b accounted for the 10% difference between both sequences. the cdna clone tcad0008a.b was bp and was 78% and 88% similar to 1rt84b and tcad0009a.o respectively. for ease of identification, tcad0009a.o and tcad0008a.b were dubbed ucp2a and ucp2b, respectively. the deduced amino acid sequences of ucp2a and ucp2b consist of and amino acid residues, respectively. the peptide sequence deduced from 1rt84b is a truncated form of that obtained from tcad0009a.o the deduced protein, which consisted only of transmembrane domain i and one proton carrier signature, most likely lacks the proton dissipation function as it has been demonstrated that the second transmembrane domain of ucp genes is essential for the anion channel formation. this sequence was therefore discarded from further analysis. we found 93% similarity between the ucp2a and ucp2b peptide sequences with both containing six transmembrane domains and three proton carrier signatures which define the general triplicate structure of mitochondrial uncoupling proteins. the purine-binding domain involved in the control of coupling efficiency was also identified. over the protein conserved domains rainbow trout ucp2a and ucp2b diverged only by one amino-acid at position in transmembrane domain iv. the switch from alanine to valine should not make any difference in the three-dimensional structure of the peptides as these are both hydrophobic amino acids. on average, both rainbow trout ucp2s showed 83% amino acid similarity with zebrafish ucp and 78% amino acid similarity with human, rat and mouse ucp2s. ucp and ucp have been described to be fairly similar in their peptide sequences and their physiological functions. our results showed 69% amino acid similarity between rainbow trout ucp2s and mammalian ucp3s. the identification of two copies of ucp genes in rainbow trout genome is consistent with the ancestral whole genome duplication and re-diploidization of salmonidae species. several genes and microsatellite loci in rainbow trout have been previously described to be present in multiple copies and palti et al. estimated that approximately two-thirds of the rainbow trout genes remained duplicated. phylogenetic relationships we performed a phylogenetic analysis to classify rainbow trout ucp2a and ucp2b and determine their relationships to ucps from other species. the tree generated included peptide sequences from the ucp gene family retrieved from public databases. trees with similar topology to that of figure were drawn when either minimum evolution or unweighted pair group algorithms were performed. both rainbow trout ucp duplicates were grouped together with their ucp orthologs in the same clade. fish and frog ucp peptides were assigned to a specific cluster, with a high bootstrap, as opposed to mammalian ucp genes. our data showed a close relationship between ucp and ucp in agreement with previous expression patterns and amino acid sequence similarity suggesting that ucp and ucp are paralogs. moreover both genes have been mapped on the same chromosome in endothermic vertebrates such as human, bovine, porcine and mouse and more recently the same evidence was found in ectothermic vertebrates like zebrafish.. overall, ucp genes were clustered in three major branches at 100% bootstrap value leading to three groups consisting of ucp1- ucp- and ucp- we suggest an early divergence of the ucp family gene into three major clades in agreement with the phylogeny described by sokolova and sokolov. these authors also demonstrated that this subdivision could be correlated with a functional specialization among the ucp superfamily. genomic organization of rainbow trout ucp2 we identified total bac clones putatively containing an ucp gene of which where specific to ucp2a and were specific to ucp2b. after fingerprinting, most of these bac clones were assembled into three contigs c c and c containing and clones, respectively. c consisted of ucp2a-containing bacs while c and c consisted of ucp2b-containing bacs. two representative bacs were selected from each contig for direct sequencing. two bac clones, 243p and 398g were selected from c and c for further analysis based on their sequence similarities with cdna clones tcad0009a.o and tcad0008a.b, respectively. genomic sequences for ucp2a and ucp2b genomic sequences have been deposited at ncbi. alignment between cdna and bac genomic sequences revealed the exon-intron structures of ucp2a and ucp2b. in each alignment a tata box was identified on genomic dna and bp upstream of the 5'end of ucp2a and ucp2b, respectively. ucp2a and ucp2b span kb and kb, respectively. both rainbow trout ucp2s contain exons and introns. coding sequences for both genes covered exons as described for mammalian ucp, ucp and ucp and each exon encodes one transmembrane domain of the protein. the analysis of genomic structure showed that 1rt84b is a splice variant of tcad0009a.o exon was 157-bp longer and intron was subsequently 157-bp shorter in 1rt84b than in tcad0009a.o. exon of 1rt84b is not fully translated as a stop codon is encountered in the first nucleotides. in human where ucp has been well characterized, snp and insertion/deletion variants of the gene were identified and no alternative splice variants have been reported to date. similarity between ucp2a and ucp2b was low in the introns with 60% similarity on average while the average similarity in the exon sequences was 86%. the number and the size of the exons and introns of rainbow trout, human, mouse and zebrafish ucp2s are represented in table the number of exons was identical in these vertebrate organisms although exon lengths were observed to be somewhat variable. translated exons and were the most conserved in size between species and within rainbow trout. exon had the same size in all species. these translated exons showed 93% average sequence similarity between rainbow trout ucp2a and ucp2b while untranslated exons were 79% identical. mutations apparently occur at a slower rate in the coding region than in the untranslated region of duplicated genes. these results show conservation of the structure of ucp genes coding exons within vertebrates confirming the hypothesis of a common ancestry for ucp genes. we examined introns and untranslated exons untranslated part of exon and approximately bp of genomic sequence upstream of each gene's transcription initiation site as the putative promoter regions. this represented and kb-long sequence upstream of the translation-initiation site of ucp2a and ucp2b, respectively. similarity was 59% between overlaps suggesting differentiation between regulatory regions of ucp2a and ucp2b. the promoter regions of rainbow trout ucp2a and ucp2b, and the upstream non-coding sequences identified in human, mouse and zebrafish ucp were searched for regulatory element binding sites using the tfsearch engine version. the results for ten transcription factors frequently identified are summarized in tables and the identification of regulatory conserved motifs upstream of the five genes is consistent with the phylogenetic relationships previously described between the ucp genes in human, mouse, zebrafish and rainbow trout. the binding sites of adr cap, cdxa and hsf were identified in upstream region of all five genes although at different frequency. aml1a, mzf usf binding sites were identified in mouse and human sequences and absent in fish. among the fish ucp genes, the rainbow trout ucp2a regulatory region appeared to be the most unique with almost half of the binding motifs identified in ucp2b and zebrafish ucp among the regulatory motifs we identified for ucp genes, usf and creb have been shown to regulate the expression of ucp and ucp respectively. our results suggest, in agreement with previous data in the literature, that some transcription factor binding sites are conserved across species while others are not. adr1: alcohol dehydrogenase gene regulator, activator. aml-1a: acute myeloid leukemia 1;; may act as a negative regulator. cap: cap signal for transcription initiation signal, activator. cdxa: chick; chicken chox-cad gene for caudal homologue, activator. creb: cyclic-amp response element-binding protein, transactivator. hsf: heat shock transcription factor, activator. mzf1: myeloid zinc finger protein mzf; possibly involved in hemopoietic development. nkx2: murine homeobox gene modest activator probably important for myocardial development. sry: sex-determining region y gene product. usf: upstream stimulatory transcription, activator. tissue distribution and response to fasting the distribution of relative expression of rainbow trout ucp2a and ucp2b was investigated on a panel of tissues from adult fish. both ucp2a and ucp2b genes appeared ubiquitously expressed in the body as described in marsupials, human, carp and mouse making the genes likely to influence several physiological processes. the highest relative expression was observed in ovary, peripheral blood leucocytes, gill, testis, spleen and trunk kidney. in all these tissues except ovary, ucp2b mrna relative amounts were at least twice that of ucp2a. in brain, fat, pituitary and red blood cells, ucp2a mrna was more abundant than ucp2b mrna. high amounts of ucp mrnas in reproductive organs and in tissues rich in macrophages like pbls, spleen, and kidney have been reported in several studies. arsenijevic et al. and kizaki et al. suggested a role for ucp in immunity or inflammatory responsiveness through the regulation of reactive oxygen species in macrophages. in mouse ovary, ucp mrna levels have been described to increase during ovulation as a result of the increasing number of macrophages and neutrophils. in trout, ovaries in maturation are characterized by the presence of many macrophages inside the cytoplasm of the oocytes. the ovaries used in our tissue distribution panel were developing. therefore, we suggest the high amount of ucp mrna in trout ovary could be due to the macrophage-rich developmental stage of the tissue used. the initial average weight of the fish used in the current study was mg. following day the fed fish weighed mg and the fasted fish were mg. at day of fasting, termination of the fasted group, the average weight of the fish was mg, mg and mg for the fed, fasted and refed groups, respectively. rainbow trout ucp2a and ucp2b were differentially expressed in response to fasting and refeeding in fry. ucp2a mrna expression was similar between all groups at the beginning of the experiment. three days after food deprivation, ucp2a expression was three times higher in fed fish than in fasted fish. the amount of gene expression remained significantly higher in fed fish than in fasted fish until day of the experiment. refeeding of fasted animals started at day and resulted in a 2-fold increase of ucp2a at day at day refed and fed fish had the same amount of ucp2a expression. at day ucp2b expression is similar in all fry. at day expression was 3-fold augmented in fasted fish while no significant increase was found in the fed fish. ucp2b expression drastically decreased in fasted fish to the level observed in fed fish at day before a significant increase was observed again at day at days and ucp2b was expressed higher in fasted fry than in fed fry. in fed animals, no significant differences were noted between ucp2b expression levels throughout the experiment, except at day refed fish showed the same level of expression of the gene as the fasted fish at day ucp2b expression then decreased in refed fish to reach the same level expressed in fed animals at days and the expression pattern of ucp2b in response to fasting is consistent with what has been described for other species in the literature. the food deprivation induced-increase in mrna expression is one of the most spectacular features of ucp and ucp in rodent and human. the hypothesis is that during fasting when glucose is limiting, upregulation of muscle ucp is associated with the shift in muscle substrate utilization primarily towards lipid. on refeeding with a low-fat diet, ucp is down regulated allowing the re-use of glucose as major substrate. it has been also demonstrated that a high-fat diet induces up-regulation of ucp and ucp thus promoting fat utilization over storage. the actual mechanism of ucp involvement in fatty acid metabolism is not yet fully understood, but samec et al. established a close relationship between expression of ucp and ucp and well-known key regulators of lipid oxidation. our study, unlike most of experiments in the literature, monitored ucp mrna levels for a relatively long fasting time. our results suggest that increase of ucp2b mrna is maintained during the whole fasting time, although the higher expression is detected early. the overall amount of expression of ucp2b genes in fry muscle were much lower than what we observed for ucp2a. five times more cdna was included in real-time pcr for ucp2b primers than was used for ucp2a to detect readable amplification signals. as a result, the highest detected amount of ucp2b gene ef1-normalized expression is compared with that of ucp2a which was. in adult fish, as described above ucp2b mrna expression was much higher relatively to ucp2a mrna in some of the tissues. in light of the expression data in response to fasting, ucp2b is likely the homolog of the ucp described in other vertebrates. ucp2a is a duplicate form of the gene that has undergone a different evolutionary process leading to a different expression pattern. this is illustrated by the sequence divergence and different cis-regulatory elements identified in the promoter regions of both genes. further functional characterization of the promoter region and identification of regulatory motifs could help address the differences between mrna expression of ucp2a and ucp2b.  we have identified two ucp genes in rainbow trout. both genes were highly similar at the amino acid level and widely expressed in the body. analyses of the genomic organization and phylogeny data based on protein sequence similarity suggest a close relationship between members of the ucp superfamily. in rainbow trout fry ucp2a and ucp2b appeared downregulated and upregulated on fasting, respectively. further characterization of both genes in different physiological and dietary conditions, and developmental stages will assist in better testing and understanding hypothesis related to their specific functions and differentiation. 
the genomic organization and phylogeny analysis support the hypothesis of a common ancestry between the vertebrate ucps. the converse expression pattern was observed for ucp2a mrna which decreased during fasting, suggesting different metabolic roles for ucp2a and ucp2b in rainbow trout muscle. these genes are 93% similar in their predicted amino acid sequences and display the same genomic structure as other vertebrates spanning kb and kb, respectively. we characterized two ucp genes in rainbow trout with similar genomic structures, amino acid sequences and distribution profiles. in fry muscle we observed an increase in ucp2b expression in response to fasting and a decrease after refeeding in agreement with previous studies in human, mouse, rat, and marsupials. these genes appeared to be differentially regulated in response to fasting and refeeding in fry muscle. it has been determined that ucp plays a role in several physiological processes such as energy expenditure, body weight control and fatty acid metabolism in several vertebrate species. uncoupling protein belongs to the superfamily of mitochondrial anion carriers that dissociate the respiratory chain from atp synthesis. two ucp genes were identified in the rainbow trout genome, ucp2a and ucp2b. phylogenetic analysis including other genes from the ucp core family located rainbow trout ucp2a and ucp2b with their orthologs and suggested an early divergence of vertebrate ucps from a common ancestor gene. ucp2a and ucp2b were widely expressed in all tissues of the study with a predominant level in macrophage-rich tissues and reproductive organs. we report the first characterization of ucp2s in rainbow trout.
 uncoupling protein belongs to the superfamily of mitochondrial anion carriers that dissociate the respiratory chain from atp synthesis. it has been determined that ucp plays a role in several physiological processes such as energy expenditure, body weight control and fatty acid metabolism in several vertebrate species. we report the first characterization of ucp2s in rainbow trout.  two ucp genes were identified in the rainbow trout genome, ucp2a and ucp2b. these genes are 93% similar in their predicted amino acid sequences and display the same genomic structure as other vertebrates spanning kb and kb, respectively. ucp2a and ucp2b were widely expressed in all tissues of the study with a predominant level in macrophage-rich tissues and reproductive organs. in fry muscle we observed an increase in ucp2b expression in response to fasting and a decrease after refeeding in agreement with previous studies in human, mouse, rat, and marsupials. the converse expression pattern was observed for ucp2a mrna which decreased during fasting, suggesting different metabolic roles for ucp2a and ucp2b in rainbow trout muscle. phylogenetic analysis including other genes from the ucp core family located rainbow trout ucp2a and ucp2b with their orthologs and suggested an early divergence of vertebrate ucps from a common ancestor gene.  we characterized two ucp genes in rainbow trout with similar genomic structures, amino acid sequences and distribution profiles. these genes appeared to be differentially regulated in response to fasting and refeeding in fry muscle. the genomic organization and phylogeny analysis support the hypothesis of a common ancestry between the vertebrate ucps. 