BACKGROUND
salmonids are used as models for studies in environmental toxicology, physiology, comparative immunology, growth, gametogenesis, olfaction and osmoregulation  <cit> .
although considerable knowledge of the basic biology of salmonids exists  <cit> , the characterization of salmonid genomes will better enable scientific decisions on the conservation and enhancement of wild stocks, improve knowledge of fish health and increase the commercial viability of aquaculture.
sequencing of salmonid genomes also permits investigators to study fundamental questions concerning genome evolution, genome duplication and rearrangements, repeat-rich structures, transposon activity, gene silencing, and the re-establishment of a more stable diploid genome from a pseudotetraploid state.
growth hormone  plays a very important role in many regulatory, metabolic and developmental processes in various vertebrate tissues  <cit> .
in salmonids, gh is the principle stimulator of skeletal growth and plays a key role in lipid mobilization, protein synthesis and feeding behaviour  <cit> .
in various fishes, gh also manifests some functions not found in all vertebrates, such as activities that influence sexual maturation and saltwater adaptation  <cit> .
the gh gene  in salmonids is represented by duplicated, non-allelic isoforms designated as gh <dig> and gh <dig>  <cit> , which diverged at least  <dig> million years  ago  <cit> .
in vertebrates, the major source of gh is the somatotrophic cells in the pituitary, from which it is secreted into and borne by the plasma to act on receptors throughout the organism  <cit> .
splice variants have also been isolated from extrapituitary tissue in humans  and chicken  .
in trout, gh transcripts have been detected in the pituitary, liver, head kidney, spleen, thymus, intestine and leukocytes and gut  <cit> .
improved understanding of the chromosomal environment in which gh genes reside in salmonids will assist our understanding of the transcriptional controls regulating these genes.
by analyzing two paralogous genes  and doing so in two species , identification of common conserved regions with presumed broad functional importance can be achieved.
further, regions which are found to be conserved only between species in one paralogue type may be important for differential regulatory control between the paralogues, while differences between species within a single paralogue type may help identify regions not important to regulation.
regions which are conserved between paralogues in only one species are candidates for regions which have undergone gene conversion subsequent to divergence between the two species.
while gene conversion does not appear to have occurred at salmonid gh loci when examined at the gene level  <cit> , an analysis of gene conversion has not been examined at larger scales now possible with genomic analyses.
understanding these sequence relationships has important practical ramifications since complete genomic resources are being developed for atlantic salmon.
it is important to determine the degree of conservation of this information with other important salmonid species such as chinook salmon for extrapolation of emerging genomic information from one species to another.
bacterial artificial chromosomes  containing gh <dig> and gh <dig> from both atlantic and chinook salmon were assembled, annotated, and compared to each other in their coding, intronic, regulatory, and flanking regions.
a core proximal promoter for transcription of both gh <dig> and gh <dig> is conserved between the two species of salmon.
a  <dig> bp insertion of a tc1-like dna transposon sequence was found within the promoter region of both gh <dig> genes, but not in the promoter of the gh <dig> genes.
furthermore, a polinton- <dig> transposon is inserted in only the promoter for chinook salmon gh <dig>  other differences within the promoters and intronic and 3'-flanking regions of the four genes provide evidence that supports the notion that they are regulated differently and thus may possess different functions.
intriguingly, chinook salmon gh <dig> has undergone more than twice as many changes than any of the other ghs; changes not reflected in the surrounding non-coding dna.
RESULTS
confirmation that the atlantic salmon bacs we isolated were gh-containing bacs was performed by comparing hindiii-digested bac fragment profiles to profiles on the internet contig explorer version  <dig>   database  <cit> .
the gene for the skeletal muscle sodium channel  was also identified by pcr for each isolated bac, suggesting that each bac contained the 5'-region upstream of each gh gene.
gh type  was determined for bacs in both atlantic and chinook salmon by paralogue-specific pcr.
bac and gh comparisons
atlantic salmon  and chinook salmon gh loci were analyzed using digit  <cit> , which identified the presence, location, and direction of putative genes on each bac.
each of these putative genes were assessed by blastx  <cit>  to protein databases.
bacs containing the gh genes also bore the genes for scn oriented in the same direction .
the sequences of the genes for interferon alpha- <dig> , myosin alkali light chain  and microtubule associated protein tau  were also identified, and found in opposite orientations relative to gh <dig> and gh <dig> .
the chinook salmon gh <dig> and gh2-containing bacs are similarly organized.
an overall comparison of atlantic and chinook salmon gh <dig> and gh <dig> loci are shown in vista plots  <cit>  .
this figure shows many regions of high similarity separated by gaps that represent repeat sequences, deletions and insertions.
visually orthologous comparisons  appear more similar than paralogous comparisons .
using repeatmasker  <cit>  and a new salmonid repeat database  <cit> , 30% of the cs gh <dig>  28% of the cs gh <dig>  33% of as gh <dig> and 35% of as gh <dig> are repeated elements or simple repeats .
with repeats removed from the genomic sequences, paralogous comparisons showed that atlantic salmon gh <dig> and gh <dig> have  <dig> % identity over  <dig>  bp, and chinook salmon gh <dig> and gh <dig> showed  <dig> % identity over  <dig>  bp.
these alignments also revealed numerous insertions and deletions among these loci.
in comparisons of atlantic and chinook salmon bacs, gh <dig> showed  <dig> % identity over  <dig>  bp and gh <dig> showed  <dig> % identity over  <dig>  bp.
estimates of 93% identity between atlantic salmon and chinook salmon are similar to the 94% identity found between atlantic salmon and rainbow trout  over  <dig> kb  <cit> .
comparisons of the  <dig> amino acid residues of gh show very strong similarity .
atlantic salmon gh <dig> and gh <dig> shows  <dig> % identity, while chinook salmon gh <dig> and gh <dig> shows  <dig> % identity, and atlantic salmon and chinook salmon gh <dig> and gh <dig> shows  <dig> % identity and  <dig> % identity, respectively.
an analysis of these relationships indicates that the cs gh <dig> has undergone more than twice as many changes than for any of the other ghs.
this change in cs gh <dig> is not apparent in the surrounding non-coding sequences and indicates there may have been a shift of natural selection pressures on the coding region of the cs gh <dig>
gene annotation
the gh paralogs were fully annotated and the genes consist of  <dig> exons and  <dig> introns.
all upstream positions we document are presented in relationship to the translation start site  and, due to variations in insertions and deletions between the gh genes within their intronic regions, all downstream information is based on nucleotide  numbering as presented in additional file  <dig>
gh promoter and intron analysis
a tata box for all four gh genes is found at position - <dig> to - <dig> .
each gene has an asymmetric camp-response element   at -217/- <dig> in the proximal promoter and a palindromic cre at position 3563/ <dig> within intron  <dig>  but the gh <dig> genes have single nt substitutions in the fourth base at this location .
there are at least four potential pituitary transcriptional activator- <dig>  binding elements  that are shared by each gene and two upstream, reverse-oriented pit- <dig> binding motifs in only the gh <dig> promoters .
a retinoic acid receptor/retinoid × receptor  binding element is also located here at position - <dig> to - <dig>  a potential estrogen response element  can be found in each promoter , roughly  <dig>  bp and  <dig>  bp upstream of the transcription start site in both the gh <dig> and gh <dig> genes, respectively.
another potential ere , found in only the gh <dig> promoters, is located about  <dig>  bp and  <dig>  bp upstream of the cs and as gh genes, respectively.
intron  <dig> of the gh <dig> genes are much shorter than the corresponding intron for the gh <dig> paralogs, partly accounted for by insertion of sssp <dig> microsatellites within them.
intron  <dig> of cs gh <dig> is at least  <dig> nts longer than for any of the other genes.
also, numerous polymorphic loci exist in intron  <dig> of the gh genes that have been previously characterized for salmonids, including as and cs   <cit> .
the sequences from position  <dig> to  <dig> match identically , but we show that there is one nucleotide difference in the as gh <dig> paralogue at position  <dig>  all of the other polymorphic loci match identically.
there are three polyadenylation ) tail signal sequences  beginning at positions  <dig>   <dig> and  <dig> .
all four gh genes have at least one poly signal sequence, but only the gh <dig> genes potentially possess three canonical signal sequences.
transposon integration analysis
a tc1-like dna transposon sequence  is inserted into the promoter region of both as and cs gh <dig>  approximately  <dig> bp upstream of the transcription initiation site  .
this transposon is not found in the promoters of gh <dig> for either species.
this places the insertion of this transposon between the time of the gene duplication and when the two species diverged.
a second tc1-like transposon, dtssa <dig> , is inserted approximately  <dig> kb upstream of the initiation site in only the as gh <dig> promoter.
in addition, a  <dig>  kb fragment of ssart.
<dig>  a non-ltr long interspersed nuclear element  sequence identified in as , is inserted approximately  <dig> bp 5' of the transcription start site in cs gh <dig> .
this fragment is truncated at the 3'-end, consistent with partial retrotranscription.
furthermore, an unusual insertion  <dig> kb upstream of only the cs gh <dig> gene was found .
this insertion is approximately  <dig> kb in length containing a  <dig> kb palindromic sequence which is part of a polinton- <dig> transposon   <cit> .
tissue expression pcrs
to examine the functionality of the genes identified on each gh-containing bac, primers specific for each gene were designed and used in pcrs on a panel of tissues for each species .
this was not done for genes that were clearly pseudogenic or for genes for which information was incomplete, such as the genes that encode dynein heavy chain  and microtubule associated protein tau.
microtubule associated protein tau is pseudogenic due to frameshifts and stop codons within the gene in each species .
we conducted tissue expression studies of the  <dig> genes we had characterized in the bacs to determine if similarities existed in expression pattern between the two species; if expression was confined to specific tissues and whether indeed each of these genes were functional.
of the  <dig> genes examined, the expression of the gh paralogues was the least diverse.
the expression of gh <dig> and gh <dig> was restricted to only muscle, brain, gill or eye .
expression of the scn genes was not restricted to just skeletal muscle, but indeed was found in every tissue except the skin.
it is also evident that ifna <dig> has fairly broad expression in the tissues examined here.
myosin alkali light chain  <dig> also was expressed in a large range of different tissues.
however, myosin alkali light chain  <dig>  appears to not be expressed at detectable levels in any of the tissues we examined in either species, despite in silico analysis indicating the viability of the mlc <dig> transcript.
each gene that encodes mlc <dig> appears intact, with no obvious problems for transcription or translation.
discussion
gh transcription
transactivation and footprinting studies have been conducted on the gh promoters of both chinook salmon and rainbow trout  <cit> .
assembly of at least part of the transcriptional machinery was delineated to binding elements between positions - <dig> to - <dig>  it was demonstrated that cyclic-amp/pka regulation of these promoters required nucleation and recruitment of both pituitary transcriptional activator- <dig>  and camp-response element  binding proteins   <cit> .
we note strong sequence identity of potential pit- <dig>  creb, and retinoic acid receptor /retinoid × receptor  binding elements that form the core of the proximal gh promoters of both atlantic salmon  and chinook salmon .
an asymmetric cre  at -217/- <dig> is flanked by at least four pit- <dig> binding elements .
the more distal pit- <dig> binding elements follow more strictly the core consensus sequence for pit- <dig>  that is conserved in fish and mammals  <cit> .
the potential rar/rxr response element present at position -190/- <dig> could hypothetically permit interaction of chicken ovulbumin upstream promoter transcription factor , or retinoic acid, estrogen or thyroid hormone receptors with an assembled pit-1/creb complex.
different variations of gtct-rich blocks immediately upstream of each proximal promoter, as well as within the introns of each gene, may make regulatory or structural contributions to gh transcription.
for example, some of the ctct blocks found in these regions could serve as binding elements for gaga-binding factors  <cit> .
however, differences do exist between the as and cs gh <dig> and gh <dig> promoters.
for example, there is the potential for pit- <dig> to bind a reverse-oriented response element roughly  <dig> bp further upstream in each gh <dig> promoter that is not present in the gh <dig> promoters.
no palindromic cres are found within  <dig> kb upstream of the gh genes, but one is present in intron  <dig> of the gh <dig> genes .
the different cre, ere and pit- <dig> motif locations suggest that the gh <dig> and gh <dig> paralogues may have distinct regulatory modes and possibly act to function differently and/or at different times of salmonid development.
gh expression and function
the more traditional models that have regarded divergence among duplicate genes as a means for acquisition of new functions have met with recent challenges.
for example, it has been demonstrated that mutation in many duplicated genes of rice is much slower than in 'singletons' and less drastic in terms of amino acid change  <cit> .
conservation of the apparent crucial functionality of the duplicates outweighs processes that lead to neofunctionalizaton.
indeed, it may be more common for gene novelty to arise from preserved duplicates that are subfunctionalized  <cit> .
at the amino acid level, cs gh <dig> appears to be undergoing change much faster than any of the other ghs .
in terms of retention of amino acid residue identity, the cs gh <dig> orf has more changes than any of the other translated regions .
these changes do alter the polarity and charge of the proteins and therefore may subtly effect the kinetics and affinity of hormone binding to their receptor.
we did find colocalized expression of both cdnas in the cs gill and brain .
interestingly, both gh receptors have been found in various regions of the brain and in the gill filaments of trout  <cit> .
furthermore, although the very limited amount of change between all four genes indicates that their functions are conserved, we do observe that gh <dig> and gh <dig> may potentially have different subfunctions.
our rt-pcr work demonstrates that some tissue expression segregation has occurred, whereby only gh <dig> appears to be expressed in muscle or eye .
to determine the function of locally expressed gh <dig> in the gill, it will be necessary to elucidate the specific cells gh synthesis is associated with and to determine whether local production is a component of known osmoregulatory processes or some other activity .
it is also possible that the gh <dig> expression observed in the gill is from leukocyte accumulation as leukocytes have been shown to produce both gh <dig> and gh <dig>  with gh <dig> the predominant transcript  <cit> .
expression of genes linked with gh
much less is known about the expression of the skeletal muscle sodium channel , microtubule associated protein tau , myosin alkali light chain  and interferon alpha- <dig>  genes in salmonids.
to the best of our knowledge, for the other genes examined in this study, only the promoters of two ifn genes have been characterized in salmon.
two different as type  <dig> ifn genes have been demonstrated to each possess two promoters that respond to different phases of infection or are tissue-specific in their transcriptional contexts  <cit> .
the scn gene has been characterized in zebrafish  <cit>  and fugu  <cit> .
some subfunctional partitioning of the four sets of duplicated zebrafish scn genes does exist, but there is still expression overlap across neuronal and muscle tissues  <cit> .
the expression of the scn genes in the salmonids examined here also are not restricted to skeletal muscle, but their activities are found in a large number of different tissues .
there appears to be a number of ifn genes in both the atlantic and chinook salmon gh <dig> bacs analyzed .
at least one functional ifna <dig> gene and two to five different ifna genes  exist at these loci for each species.
it should be noted, however, that this region was difficult to sequence and assemble due to repetitive regions, therefore we cannot conclude unequivocally on the number of ifn genes present at these loci.
a genomic bac clone containing the ifna <dig> gene and seven other ifn genes has been characterized in atlantic salmon .
however, to date, we find no other ifna genes present in our database other than ifna <dig> to enable us to conclude that expression of these other ifn genes does occur.
zebrafish also has two to three ifn genes in the 3'-flanking region and the scn gene in the 5'-flanking region of the gh gene on chromosome  <dig>  <cit> .
genome rearrangements
the gh <dig> genes have undergone some rearrangements in comparison to the gh <dig> paralogues .
part of this may be due to chromosome structure or positioning  <cit> .
also, some of the genomic variations observed between the gh <dig> and gh <dig> paralogues might be due to vestigial effects caused by a rearrangement that led to the rise of the y-linked gh <dig> pseudogene  <cit> .
whether the additional transposon insertions found in the as gh <dig> promoter can account for why we do see gh-y in cs, but not in as  <cit> , will require characterization of other pacific salmon gh regions.
it should be noted, as an example, that dramatic differences have also been found between the promoters of the salmon form of gonadotropin-releasing hormone in as and sockeye salmon  <cit> .
an indication of the changes that can occur when sequences of various transposon families integrate into the promoters of genes are shown in figure  <dig>  insertions such as these may help to shape the dynamism of genomes by influencing modes of transcription through the introduction of new regulatory elements, by changing the distances between functional binding elements or by disabling them.
it can be envisaged how alterations to transcription through these integration events could lead to changes in the function of their translated products over time.
for example, note there are at least two different transposon sequences found within the as gh <dig> proximal regulatory sequences that are not present in any of the other gh promoters .
these integrated sequences might impact the observed specific expression of gh <dig> in as compared to cs .
also, the presence of a tss transposon in the 5' sequence of each gh <dig> and an hpai short interspersed nuclear element  in intron  <dig> of each gh <dig> is consistent with speciation events occurring after the gene or genome duplication events .
vertebrate genomes contain large numbers of transposon sequences.
the mouse and human genome contain approximately  <dig> million such sequences and zebrafish approximately  <dig>  million.
however, transposons occur in certain genome regions more frequently than in so-called transposon-free regions , frequently associated with developmental genes  <cit> .
this association with such genes suggests that transposons are deleterious to gene integrity possibly through increased mutagenesis or recombination, or by changing regulation.
the presence of the polinton sequence found upstream of the cs gh <dig> gene might therefore affect its genomic stability.
CONCLUSIONS
this is one of the first in a series of studies that are needed to document coding and non-coding changes that have occurred subsequent to a whole genome duplication.
as gh has been the focus of biotechnological advances in aquaculture, it is important to investigate expression changes and genomic organizational changes in important economic traits such as growth.
in this paper, we report on the impact of repeat elements and transposon integrations and show that in oncorhynchus species the gh <dig> duplicate has undergone a higher rate of change.
genomes, particularly salmonid genomes, are dynamic.
part of the dynamism is the result of the purported whole genome duplication and the past integration of sines and lines into them.
lines and sines introduce repetitive elements that may misalign causing unequal recombination to occur, and thereby introduce deletions and insertions among duplicated genomes.
coupled with this is the integration of a wide assortment of transposon sequences as described here and elsewhere  <cit> .
over time, these genomic rearrangements have led to the creation of pseudogenes, structural differences between duplicated genes and to differential regulation of paralogues.
in as and cs, we show that transposon insertions have occurred differently in the promoters of gh, within and between each species.
the organization of transposon families in the bacs containing gh <dig> and in the bacs containing gh <dig>  is very similar within orthologous regions .
however, much less conservation is apparent in comparisons between the gh1- and gh2-containing paralogous bacs for the two fish species .
the appearance of repeated elements and differential rates of change in the gh <dig> and gh <dig> regions is consistent with the hypothesis that a burst of transposition activity occurred during the speciation events which led to atlantic and pacific salmon  <cit> .
genome and gene duplication  has taken place much earlier than the speciation  and these data suggest that after gene duplication, gh <dig> in oncorhynchus has evolved much faster than gh <dig>  possibly because the genomic region for gh <dig> has undergone more reorganization compared to the region containing gh <dig>

 salmonids are used as models for studies in environmental toxicology, physiology, comparative immunology, growth, gametogenesis, olfaction and osmoregulation. although considerable knowledge of the basic biology of salmonids exists, the characterization of salmonid genomes will better enable scientific decisions on the conservation and enhancement of wild stocks, improve knowledge of fish health and increase the commercial viability of aquaculture. sequencing of salmonid genomes also permits investigators to study fundamental questions concerning genome evolution, genome duplication and rearrangements, repeat-rich structures, transposon activity, gene silencing, and the re-establishment of a more stable diploid genome from a pseudotetraploid state. growth hormone plays a very important role in many regulatory, metabolic and developmental processes in various vertebrate tissues. in salmonids, gh is the principle stimulator of skeletal growth and plays a key role in lipid mobilization, protein synthesis and feeding behaviour. in various fishes, gh also manifests some functions not found in all vertebrates, such as activities that influence sexual maturation and saltwater adaptation. the gh gene in salmonids is represented by duplicated, non-allelic isoforms designated as gh and gh, which diverged at least million years ago. in vertebrates, the major source of gh is the somatotrophic cells in the pituitary, from which it is secreted into and borne by the plasma to act on receptors throughout the organism. splice variants have also been isolated from extrapituitary tissue in humans and chicken. in trout, gh transcripts have been detected in the pituitary, liver, head kidney, spleen, thymus, intestine and leukocytes and gut. improved understanding of the chromosomal environment in which gh genes reside in salmonids will assist our understanding of the transcriptional controls regulating these genes. by analyzing two paralogous genes and doing so in two species, identification of common conserved regions with presumed broad functional importance can be achieved. further, regions which are found to be conserved only between species in one paralogue type may be important for differential regulatory control between the paralogues, while differences between species within a single paralogue type may help identify regions not important to regulation. regions which are conserved between paralogues in only one species are candidates for regions which have undergone gene conversion subsequent to divergence between the two species. while gene conversion does not appear to have occurred at salmonid gh loci when examined at the gene level, an analysis of gene conversion has not been examined at larger scales now possible with genomic analyses. understanding these sequence relationships has important practical ramifications since complete genomic resources are being developed for atlantic salmon. it is important to determine the degree of conservation of this information with other important salmonid species such as chinook salmon for extrapolation of emerging genomic information from one species to another. bacterial artificial chromosomes containing gh and gh from both atlantic and chinook salmon were assembled, annotated, and compared to each other in their coding, intronic, regulatory, and flanking regions. a core proximal promoter for transcription of both gh and gh is conserved between the two species of salmon. a bp insertion of a tc1-like dna transposon sequence was found within the promoter region of both gh genes, but not in the promoter of the gh genes. furthermore, a polinton- transposon is inserted in only the promoter for chinook salmon gh other differences within the promoters and intronic and 3'-flanking regions of the four genes provide evidence that supports the notion that they are regulated differently and thus may possess different functions. intriguingly, chinook salmon gh has undergone more than twice as many changes than any of the other ghs; changes not reflected in the surrounding non-coding dna.  confirmation that the atlantic salmon bacs we isolated were gh-containing bacs was performed by comparing hindiii-digested bac fragment profiles to profiles on the internet contig explorer version database. the gene for the skeletal muscle sodium channel was also identified by pcr for each isolated bac, suggesting that each bac contained the 5'-region upstream of each gh gene. gh type was determined for bacs in both atlantic and chinook salmon by paralogue-specific pcr. bac and gh comparisons atlantic salmon and chinook salmon gh loci were analyzed using digit, which identified the presence, location, and direction of putative genes on each bac. each of these putative genes were assessed by blastx to protein databases. bacs containing the gh genes also bore the genes for scn oriented in the same direction. the sequences of the genes for interferon alpha-, myosin alkali light chain and microtubule associated protein tau were also identified, and found in opposite orientations relative to gh and gh. the chinook salmon gh and gh2-containing bacs are similarly organized. an overall comparison of atlantic and chinook salmon gh and gh loci are shown in vista plots. this figure shows many regions of high similarity separated by gaps that represent repeat sequences, deletions and insertions. visually orthologous comparisons appear more similar than paralogous comparisons. using repeatmasker and a new salmonid repeat database, 30% of the cs gh 28% of the cs gh 33% of as gh and 35% of as gh are repeated elements or simple repeats. with repeats removed from the genomic sequences, paralogous comparisons showed that atlantic salmon gh and gh have % identity over bp, and chinook salmon gh and gh showed % identity over bp. these alignments also revealed numerous insertions and deletions among these loci. in comparisons of atlantic and chinook salmon bacs, gh showed % identity over bp and gh showed % identity over bp. estimates of 93% identity between atlantic salmon and chinook salmon are similar to the 94% identity found between atlantic salmon and rainbow trout over kb. comparisons of the amino acid residues of gh show very strong similarity. atlantic salmon gh and gh shows % identity, while chinook salmon gh and gh shows % identity, and atlantic salmon and chinook salmon gh and gh shows % identity and % identity, respectively. an analysis of these relationships indicates that the cs gh has undergone more than twice as many changes than for any of the other ghs. this change in cs gh is not apparent in the surrounding non-coding sequences and indicates there may have been a shift of natural selection pressures on the coding region of the cs gh gene annotation the gh paralogs were fully annotated and the genes consist of exons and introns. all upstream positions we document are presented in relationship to the translation start site and, due to variations in insertions and deletions between the gh genes within their intronic regions, all downstream information is based on nucleotide numbering as presented in additional file gh promoter and intron analysis a tata box for all four gh genes is found at position - to -. each gene has an asymmetric camp-response element at -217/- in the proximal promoter and a palindromic cre at position 3563/ within intron but the gh genes have single nt substitutions in the fourth base at this location. there are at least four potential pituitary transcriptional activator- binding elements that are shared by each gene and two upstream, reverse-oriented pit- binding motifs in only the gh promoters. a retinoic acid receptor/retinoid × receptor binding element is also located here at position - to - a potential estrogen response element can be found in each promoter, roughly bp and bp upstream of the transcription start site in both the gh and gh genes, respectively. another potential ere, found in only the gh promoters, is located about bp and bp upstream of the cs and as gh genes, respectively. intron of the gh genes are much shorter than the corresponding intron for the gh paralogs, partly accounted for by insertion of sssp microsatellites within them. intron of cs gh is at least nts longer than for any of the other genes. also, numerous polymorphic loci exist in intron of the gh genes that have been previously characterized for salmonids, including as and cs. the sequences from position to match identically, but we show that there is one nucleotide difference in the as gh paralogue at position all of the other polymorphic loci match identically. there are three polyadenylation ) tail signal sequences beginning at positions and. all four gh genes have at least one poly signal sequence, but only the gh genes potentially possess three canonical signal sequences. transposon integration analysis a tc1-like dna transposon sequence is inserted into the promoter region of both as and cs gh approximately bp upstream of the transcription initiation site. this transposon is not found in the promoters of gh for either species. this places the insertion of this transposon between the time of the gene duplication and when the two species diverged. a second tc1-like transposon, dtssa, is inserted approximately kb upstream of the initiation site in only the as gh promoter. in addition, a kb fragment of ssart. a non-ltr long interspersed nuclear element sequence identified in as, is inserted approximately bp 5' of the transcription start site in cs gh. this fragment is truncated at the 3'-end, consistent with partial retrotranscription. furthermore, an unusual insertion kb upstream of only the cs gh gene was found. this insertion is approximately kb in length containing a kb palindromic sequence which is part of a polinton- transposon. tissue expression pcrs to examine the functionality of the genes identified on each gh-containing bac, primers specific for each gene were designed and used in pcrs on a panel of tissues for each species. this was not done for genes that were clearly pseudogenic or for genes for which information was incomplete, such as the genes that encode dynein heavy chain and microtubule associated protein tau. microtubule associated protein tau is pseudogenic due to frameshifts and stop codons within the gene in each species. we conducted tissue expression studies of the genes we had characterized in the bacs to determine if similarities existed in expression pattern between the two species; if expression was confined to specific tissues and whether indeed each of these genes were functional. of the genes examined, the expression of the gh paralogues was the least diverse. the expression of gh and gh was restricted to only muscle, brain, gill or eye. expression of the scn genes was not restricted to just skeletal muscle, but indeed was found in every tissue except the skin. it is also evident that ifna has fairly broad expression in the tissues examined here. myosin alkali light chain also was expressed in a large range of different tissues. however, myosin alkali light chain appears to not be expressed at detectable levels in any of the tissues we examined in either species, despite in silico analysis indicating the viability of the mlc transcript. each gene that encodes mlc appears intact, with no obvious problems for transcription or translation. discussion gh transcription transactivation and footprinting studies have been conducted on the gh promoters of both chinook salmon and rainbow trout. assembly of at least part of the transcriptional machinery was delineated to binding elements between positions - to - it was demonstrated that cyclic-amp/pka regulation of these promoters required nucleation and recruitment of both pituitary transcriptional activator- and camp-response element binding proteins. we note strong sequence identity of potential pit- creb, and retinoic acid receptor /retinoid × receptor binding elements that form the core of the proximal gh promoters of both atlantic salmon and chinook salmon. an asymmetric cre at -217/- is flanked by at least four pit- binding elements. the more distal pit- binding elements follow more strictly the core consensus sequence for pit- that is conserved in fish and mammals. the potential rar/rxr response element present at position -190/- could hypothetically permit interaction of chicken ovulbumin upstream promoter transcription factor, or retinoic acid, estrogen or thyroid hormone receptors with an assembled pit-1/creb complex. different variations of gtct-rich blocks immediately upstream of each proximal promoter, as well as within the introns of each gene, may make regulatory or structural contributions to gh transcription. for example, some of the ctct blocks found in these regions could serve as binding elements for gaga-binding factors. however, differences do exist between the as and cs gh and gh promoters. for example, there is the potential for pit- to bind a reverse-oriented response element roughly bp further upstream in each gh promoter that is not present in the gh promoters. no palindromic cres are found within kb upstream of the gh genes, but one is present in intron of the gh genes. the different cre, ere and pit- motif locations suggest that the gh and gh paralogues may have distinct regulatory modes and possibly act to function differently and/or at different times of salmonid development. gh expression and function the more traditional models that have regarded divergence among duplicate genes as a means for acquisition of new functions have met with recent challenges. for example, it has been demonstrated that mutation in many duplicated genes of rice is much slower than in'singletons' and less drastic in terms of amino acid change. conservation of the apparent crucial functionality of the duplicates outweighs processes that lead to neofunctionalizaton. indeed, it may be more common for gene novelty to arise from preserved duplicates that are subfunctionalized. at the amino acid level, cs gh appears to be undergoing change much faster than any of the other ghs. in terms of retention of amino acid residue identity, the cs gh orf has more changes than any of the other translated regions. these changes do alter the polarity and charge of the proteins and therefore may subtly effect the kinetics and affinity of hormone binding to their receptor. we did find colocalized expression of both cdnas in the cs gill and brain. interestingly, both gh receptors have been found in various regions of the brain and in the gill filaments of trout. furthermore, although the very limited amount of change between all four genes indicates that their functions are conserved, we do observe that gh and gh may potentially have different subfunctions. our rt-pcr work demonstrates that some tissue expression segregation has occurred, whereby only gh appears to be expressed in muscle or eye. to determine the function of locally expressed gh in the gill, it will be necessary to elucidate the specific cells gh synthesis is associated with and to determine whether local production is a component of known osmoregulatory processes or some other activity. it is also possible that the gh expression observed in the gill is from leukocyte accumulation as leukocytes have been shown to produce both gh and gh with gh the predominant transcript. expression of genes linked with gh much less is known about the expression of the skeletal muscle sodium channel, microtubule associated protein tau, myosin alkali light chain and interferon alpha- genes in salmonids. to the best of our knowledge, for the other genes examined in this study, only the promoters of two ifn genes have been characterized in salmon. two different as type ifn genes have been demonstrated to each possess two promoters that respond to different phases of infection or are tissue-specific in their transcriptional contexts. the scn gene has been characterized in zebrafish and fugu. some subfunctional partitioning of the four sets of duplicated zebrafish scn genes does exist, but there is still expression overlap across neuronal and muscle tissues. the expression of the scn genes in the salmonids examined here also are not restricted to skeletal muscle, but their activities are found in a large number of different tissues. there appears to be a number of ifn genes in both the atlantic and chinook salmon gh bacs analyzed. at least one functional ifna gene and two to five different ifna genes exist at these loci for each species. it should be noted, however, that this region was difficult to sequence and assemble due to repetitive regions, therefore we cannot conclude unequivocally on the number of ifn genes present at these loci. a genomic bac clone containing the ifna gene and seven other ifn genes has been characterized in atlantic salmon. however, to date, we find no other ifna genes present in our database other than ifna to enable us to conclude that expression of these other ifn genes does occur. zebrafish also has two to three ifn genes in the 3'-flanking region and the scn gene in the 5'-flanking region of the gh gene on chromosome. genome rearrangements the gh genes have undergone some rearrangements in comparison to the gh paralogues. part of this may be due to chromosome structure or positioning. also, some of the genomic variations observed between the gh and gh paralogues might be due to vestigial effects caused by a rearrangement that led to the rise of the y-linked gh pseudogene. whether the additional transposon insertions found in the as gh promoter can account for why we do see gh-y in cs, but not in as, will require characterization of other pacific salmon gh regions. it should be noted, as an example, that dramatic differences have also been found between the promoters of the salmon form of gonadotropin-releasing hormone in as and sockeye salmon. an indication of the changes that can occur when sequences of various transposon families integrate into the promoters of genes are shown in figure insertions such as these may help to shape the dynamism of genomes by influencing modes of transcription through the introduction of new regulatory elements, by changing the distances between functional binding elements or by disabling them. it can be envisaged how alterations to transcription through these integration events could lead to changes in the function of their translated products over time. for example, note there are at least two different transposon sequences found within the as gh proximal regulatory sequences that are not present in any of the other gh promoters. these integrated sequences might impact the observed specific expression of gh in as compared to cs. also, the presence of a tss transposon in the 5' sequence of each gh and an hpai short interspersed nuclear element in intron of each gh is consistent with speciation events occurring after the gene or genome duplication events. vertebrate genomes contain large numbers of transposon sequences. the mouse and human genome contain approximately million such sequences and zebrafish approximately million. however, transposons occur in certain genome regions more frequently than in so-called transposon-free regions, frequently associated with developmental genes. this association with such genes suggests that transposons are deleterious to gene integrity possibly through increased mutagenesis or recombination, or by changing regulation. the presence of the polinton sequence found upstream of the cs gh gene might therefore affect its genomic stability.  this is one of the first in a series of studies that are needed to document coding and non-coding changes that have occurred subsequent to a whole genome duplication. as gh has been the focus of biotechnological advances in aquaculture, it is important to investigate expression changes and genomic organizational changes in important economic traits such as growth. in this paper, we report on the impact of repeat elements and transposon integrations and show that in oncorhynchus species the gh duplicate has undergone a higher rate of change. genomes, particularly salmonid genomes, are dynamic. part of the dynamism is the result of the purported whole genome duplication and the past integration of sines and lines into them. lines and sines introduce repetitive elements that may misalign causing unequal recombination to occur, and thereby introduce deletions and insertions among duplicated genomes. coupled with this is the integration of a wide assortment of transposon sequences as described here and elsewhere. over time, these genomic rearrangements have led to the creation of pseudogenes, structural differences between duplicated genes and to differential regulation of paralogues. in as and cs, we show that transposon insertions have occurred differently in the promoters of gh, within and between each species. the organization of transposon families in the bacs containing gh and in the bacs containing gh is very similar within orthologous regions. however, much less conservation is apparent in comparisons between the gh1- and gh2-containing paralogous bacs for the two fish species. the appearance of repeated elements and differential rates of change in the gh and gh regions is consistent with the hypothesis that a burst of transposition activity occurred during the speciation events which led to atlantic and pacific salmon. genome and gene duplication has taken place much earlier than the speciation and these data suggest that after gene duplication, gh in oncorhynchus has evolved much faster than gh possibly because the genomic region for gh has undergone more reorganization compared to the region containing gh 
we show that transposon insertions have occurred differently in the promoters of gh, within and between each species. other differences within the promoters and intronic and 3'-flanking regions of the four gh genes provide evidence that they have distinct regulatory modes and possibly act to function differently and/or during different times of salmonid development. growth hormone is an important regulator of skeletal growth, as well as other adapted processes in salmonids. the gh gene in salmonids is represented by duplicated, non-allelic isoforms designated as gh and gh we have isolated and characterized gh-containing bacterial artificial chromosomes of both atlantic and chinook salmon in order to further elucidate our understanding of the conservation and regulation of these loci. a core proximal promoter for transcription of both gh and gh is conserved between the two species of salmon. these bacs also contain the genes for skeletal muscle sodium channel oriented in the same direction. this is consistent with the hypothesis that a burst of transposition activity occurred during the speciation events which led to atlantic and pacific salmon. nevertheless, transposon integration and regulatory element differences do exist between the promoters of gh and gh additionally, organization of transposon families into the bacs containing gh and for the bacs containing gh are very similar within orthologous regions, but much less clear conservation is apparent in comparisons between the gh1- and gh2-containing paralogous bacs for the two fish species. the sequences of the genes for interferon alpha- myosin alkali light chain and microtubule associated protein tau were also identified, and found in opposite orientations relative to gh and gh viability of each of these genes was examined by pcr. bacs containing gh and gh from both atlantic and chinook salmon were assembled, annotated, and compared to each other in their coding, intronic, regulatory, and flanking regions.
 growth hormone is an important regulator of skeletal growth, as well as other adapted processes in salmonids. the gh gene in salmonids is represented by duplicated, non-allelic isoforms designated as gh and gh we have isolated and characterized gh-containing bacterial artificial chromosomes of both atlantic and chinook salmon in order to further elucidate our understanding of the conservation and regulation of these loci.  bacs containing gh and gh from both atlantic and chinook salmon were assembled, annotated, and compared to each other in their coding, intronic, regulatory, and flanking regions. these bacs also contain the genes for skeletal muscle sodium channel oriented in the same direction. the sequences of the genes for interferon alpha- myosin alkali light chain and microtubule associated protein tau were also identified, and found in opposite orientations relative to gh and gh viability of each of these genes was examined by pcr. we show that transposon insertions have occurred differently in the promoters of gh, within and between each species. other differences within the promoters and intronic and 3'-flanking regions of the four gh genes provide evidence that they have distinct regulatory modes and possibly act to function differently and/or during different times of salmonid development.  a core proximal promoter for transcription of both gh and gh is conserved between the two species of salmon. nevertheless, transposon integration and regulatory element differences do exist between the promoters of gh and gh additionally, organization of transposon families into the bacs containing gh and for the bacs containing gh are very similar within orthologous regions, but much less clear conservation is apparent in comparisons between the gh1- and gh2-containing paralogous bacs for the two fish species. this is consistent with the hypothesis that a burst of transposition activity occurred during the speciation events which led to atlantic and pacific salmon. the chinook and other oncorhynchus gh1s are strikingly different in comparison to the other ghs and this change is not apparent in the surrounding non-coding sequences. 