BACKGROUND
the eukaryotic ribosome is a complex macromolecular structure composed of a large  and a small  subunit.
the large ribosomal subunit catalyses peptide bond formation and is responsible for channelling the nascent proteins through their exit tunnel.
the small ribosomal subunit binds mrna and is responsible for the fidelity of translation by ensuring the correct base pairing between aminoacyl-trnas and codons of the mrna in the decoding centre  <cit> .
biochemically, the eukaryotic ribosome is composed of four ribosomal rna molecules and over  <dig> ribosomal proteins   <cit> .
in mammals, the 60s and 40s subunits are composed of  <dig> and  <dig> rps, respectively  <cit> .
each mammalian rp is typically encoded by a single gene except rps <dig> in human  <cit> , and rps <dig> in rat  <cit>  and human  <cit> , which are encoded by two separate genes.
in contrast, in the yeast saccharomyces cerevisiae, the  <dig> rps are encoded by  <dig> genes,  <dig> of which are duplicated  <cit> .
in fish, the complete set of rps in fugu rubripes  <cit>  and ictalurus punctatus  <cit>  has been described.
of the  <dig> rps from the 40s subunit, a duplication of rps <dig> in both species and of rps <dig> in i. punctatus was observed.
in the  <dig> rps from the 60s subunit, all of them but one  appeared to have only one type of mrna  <cit> .
rps play a critical role in protein biosynthesis.
cellular levels change as a function of growth rate in bacteria and fungi  <cit> .
in fish, mrna levels increase co-ordinately during embryogenesis and larval development  <cit> .
in mammals, certain tumors have substantially increased levels of some rp transcripts  <cit> .
however, different rps have also been associated with various other cellular processes; the so-called extraribosomal functions.
for example, in drosophila, mutations in the rps <dig> gene appear to cause arrest of oogenesis  <cit>  and rps <dig> functions as a tumor suppressor in the hematopoietic system  <cit> .
mammalian rps <dig> appears to possess apurinic/apyrimidinic endonuclease activity involved in dna repair functions  <cit> .
haploinsufficiency of the rps <dig> genes has been suggested to contribute to anatomic abnormalities associated with the turner syndrome in humans  <cit> .
the gene encoding rps <dig> seems to participate in embryogenesis due to its capacity to interact with fgf- <dig>  a factor involved in the differentiation process of different cell types  <cit> .
finally, apoptosis can be induced by inhibiting or activating expression of rps3a and rps27l, respectively  <cit> .
senegalese sole, solea senegalensis , and atlantic halibut, hippoglossus hippoglossus , are two commercially important flatfish species.
during larval development, both species change from a symmetrical morphology to an asymmetric, benthic juvenile.
this metamorphic process involves dramatic morphological and physiological changes.
in senegalese sole, metamorphosis occurs very early during larval development, between  <dig> and  <dig> days after hatching   <cit> .
in atlantic halibut, metamorphosis begins with the migration of the left eye about  <dig> dah  <cit> .
apoptotic processes induced by thyroxine hormone have been associated with this tissue remodelling in flatfish  <cit> .
in addition, senegalese sole larvae exhibit two different growth rates during development  <cit> .
because of the key role rps play in cellular growth and proliferation and in some cases apoptosis, it is important to elucidate the expression pattern of rps during flatfish development.
rps are highly represented in cdna libraries  <cit> .
the development of large-scale genomics on senegalese sole and atlantic halibut has made possible an efficient and systematic analysis of rp sequences in both species.
in this work, we report the complete set of  <dig> 40s subunit rp cdnas for both senegalese sole and atlantic halibut and describe their main characteristics.
comparative sequence analysis revealed the existence of two and four rps <dig> genes in senegalese sole and atlantic halibut, respectively.
real-time pcr analysis revealed different rp expression patterns during larval development and in tissues in sole.
RESULTS
characteristics of the 40s rps
sequence analysis of normalized libraries for senegalese sole allowed the identification of  <dig> out of  <dig> 40s subunit rps .
rps <dig> was obtained from a premetamorphic stage larval library using specific primers .
overall, 40s rp genes were not highly represented in the normalized libraries accounting for  <dig>  out of the  <dig>  good sequences.
the number of clones for each rp ranged between  <dig> for rps <dig> and only  <dig> for rps27- <dig> and rps <dig> .
* genbank sequences comprise final contig
gene sizes for the complete set of 40s rps ranged between  <dig> and  <dig>  bp for rps <dig> and rpsa, respectively.
only rps <dig>  rps <dig> and rps <dig> had partial sequences missing the 5'-ends.
all cdna sequences have been deposited in the genbank/embl/ddbj with accession numbers from ab <dig> to ab <dig> .
most rps  used taa as termination codon.
only rps <dig>  rps <dig>  rps <dig>  rps <dig>  rps <dig>  rps27- <dig>  rps <dig>  and rps <dig> used tag, and rps <dig> and rps <dig>  tga.
the 3'-utrs were highly at-rich.
all rps had a canonical aataaa polyadenylation signal between 7– <dig> nucleotides from the poly tail.
in halibut, sequences for all except rps <dig> and rps27- <dig> were identified from the pleurogene database .
in most cases, the complete coding sequences were obtained, but 3'-end sequencing was performed for all rp sequences to confirm the 3'ends, particularly of the long ests.
the rps <dig> and rps27- <dig> sequences presented in this analysis derive from atlantic halibut ests in genbank .
in addition, only a partial sequence for rps <dig> was obtained and the 5'-end was completed by the addition of an atlantic halibut est in genbank .
<dig> ests from a total of  <dig>  sequences in the database encoded rps , an indication of the excellent normalization in these cdna libraries.
sizes of coding sequences, 5'- and 3'-utrs and positions of polyadenylation signals are given in table 2b.
all rps had a single polyadenylation signal with the exception of rps <dig>  which had two possible non-canonical sites , and rps <dig>  which had two possible canonical sites.
the polyadenylation signal for rps <dig> overlapped the stop signal and no polyadenylation signal could be identified in rps27- <dig>  as with senegalese sole, most 40s subunit rps used taa  as a stop codon, followed by tag  and tga  and the 3'-utrs were very at-rich.
two ests comprising rps <dig> and rps27a contigs differed in the lengths of their 5'-utrs by over  <dig> nucleotides due to the presence of unspliced introns in this region.
comparison of the senegalese sole and atlantic halibut 40s subunit rps
the complete set of 40s rps in senegalese sole and atlantic halibut had a high overall similarity  as determined by deduced amino acid sequences .
with respect to the other species, senegalese sole overall similarities ranged between  <dig>  and  <dig> % with human and f. rubripes, respectively.
for halibut, these values were  <dig>  and  <dig> % with human and f. rubripes, respectively.
the most conserved 40s rps in the five species  were rps <dig> and rps27a; and the most divergent ones were rps <dig> , rps <dig> , and rpsa .
a and b refer to similarities to h. sapiens rps4x and rps4y isoforms, respectively.
c and d refer to similarities to i. punctatus rps26- <dig> and rps26- <dig> isoforms, respectively.
the number of amino acids was highly conserved throughout evolution.
of the  <dig> 40s subunit rp cdnas,  <dig> had orfs with identical number of amino acids in the five species compared; rps <dig> and rps <dig> showed different sizes in atlantic halibut and human.
four rps had the same number of amino acids among fish species but different from human .
in addition, three others varied in more than two species  .
senegalese sole rpsa and rps <dig> showed the highest differences in size being  <dig> amino acids longer and  <dig> amino acids shorter than in human, respectively.
phylogenetic analysis of rps <dig> genes
two and four cdnas encoding rps <dig> were found in senegalese sole  and atlantic halibut , respectively .
in senegalese sole, the total lengths of rps27- <dig> and rps27- <dig> were  <dig> and  <dig> nucleotides , respectively.
they were represented by  <dig> and  <dig> clones, respectively .
in atlantic halibut, total lengths ranged between  <dig> and  <dig> nt for rps27- <dig> and rps27- <dig>  respectively.
the atlantic halibut rps27- <dig> had a slightly shorter coding region , whereas no putative polyadenylation signal could be identified in rps27- <dig>  rps27- <dig> and rps27- <dig> were represented by  <dig> and  <dig> clones in the halibut libraries, respectively, and rps27- <dig> by only  <dig> est.
both senegalese sole paralogs showed a high divergence at the nucleotide level when complete cdnas  or coding regions  were aligned.
at the amino acid level, they differed in  <dig> residues with a sequence identity of  <dig> % .
similarly, low sequence similarity  was detected among atlantic halibut full-length sequences.
these values ranged between  <dig>   and  <dig> %  in the coding regions.
at the protein level, rps27- <dig> was  <dig> amino acids shorter that the other paralogs.
amino acid similarities ranged between  <dig>   and  <dig> % .
among species, s. senegalensis rps27- <dig> and h. hippoglossus rps27- <dig> were the closest evolutionary homologues with  <dig> % similarity using full-length sequence,  <dig> % in coding sequence and 100% in amino acid sequence .
a phylogenetic analysis based on rps <dig> coding sequences using the nj, mp and ml methods showed that fish rps <dig> genes grouped mainly in two distinct clades .
both senegalese sole and atlantic halibut rps27- <dig> genes clustered with their fish counterparts.
the rps27- <dig> appeared more closely related to rps27- <dig> than the other two atlantic halibut rps <dig> genes.
moreover, rps27- <dig> and rps27l genes in rat and human, respectively, formed a sister clade sharing a common ancestor with fish rps27- <dig> genes .
the other fish rps <dig> gene copies appeared linked in a clade that was not well resolved.
both senegalese sole and atlantic halibut rps27- <dig> grouped with p. flesus and t. nigroviridis rps27- <dig> .
curiously, this clade contained both i. punctatus rps <dig> genes.
gene expression analysis
we used a quantitative approach based on reverse transcription followed by real-time pcr amplification to investigate the steady-state levels of eight sole rp transcripts  in liver, spleen, intestine, stomach, head kidney, gills, muscle, brain, heart, and skin.
relative gene expression levels were normalized by measuring ubiquitin levels.
all eight rp genes were expressed in detectable amounts in all tissues .
rps <dig> transcripts were the most abundant except in brain, where rps27- <dig> showed the highest values .
on the other hand, rps27- <dig> was expressed at the lowest level in all tissues analyzed.
rp genes were expressed differentially among tissues.
rps <dig>  rps <dig>  and rps <dig> exhibited lower expression levels in brain and rps3a transcripts were reduced in brain, heart, and skin.
in contrast, rps27- <dig> was expressed more highly in intestine and stomach, and rps <dig> in heart.
if we calculate the mean ribosomal expression ratio as a global rp expression index, all tissues showed similar values  except brain with only  <dig> .
we also investigated the expression pattern of rp genes during sole larval development.
mrna levels were determined in samples extracted from whole larvae pools collected from  <dig> to  <dig> dah .
expression levels of each rp gene were normalized to that of gapdh.
rps <dig>  rps27- <dig>  rps27a, rps <dig>  and rps <dig> showed higher transcript levels than rps3a, rps <dig> and rps27- <dig> during early  larval development in senegalese sole.
all rp mrnas increased from  <dig> to  <dig> dah,  <dig> hours after first external feeding .
fold induction values ranged between  <dig>  for both rps27- <dig> and rps <dig> and  <dig>  for rps27- <dig>  these levels were reduced at  <dig> dah, two days before the onset of eye migration, with the lowest values at the first metamorphic stages .
the number of mrna molecules for the  <dig> rps analyzed declined approximately  <dig> -fold as global mean from  <dig>  to  <dig> dah .
the fold reduction values ranged between  <dig>  and  <dig>  for rps <dig> and rps27- <dig>  respectively.
rps27- <dig> was expressed at a higher level than rps27- <dig> during all pre-metamorphic, metamorphic and post-metamorphic stages .
discussion
in this work, we describe the complete set of 40s rps in the senegalese sole and atlantic halibut.
sequences were generated from normalized cdna libraries constructed for expressed sequence tag  analysis.
the rapid development of genomics in all biological research areas, including aquaculture, and the high representation of rps in the cdna libraries have favoured the availability of an increasing number of rp sequences from different organisms  <cit> .
this fact has motivated their proposed use as appropriate molecular markers for phylogenetic analysis.
in fact, concatenation of orthologous rp amino acid sequences to form a single one of more than  <dig>  characters has allowed the reconstruction of phylogenetic relationships between animal, fungal, and plant kingdoms  <cit> .
with regard to this, pleuronectiformes comprises a broad taxonomic group with  <dig> families and about  <dig> species worldwide, some of them of high commercially interest both in fisheries and aquaculture  <cit> .
all flatfish species share in common an asymmetrical body development and a bottom-dwelling mode of life.
however, their high phenotypic similarity has invoked great differences in the number and nomenclature of taxa depending on the relevance assigned to morphologic features  <cit> .
most phylogenetic studies focused on relationships among pleuronectiformes have been based on partial mitochondrial dna sequences  <cit> .
the description of the complete set of rps in one pleuronectidae and soleidae species provides new molecular markers to investigate the taxonomy and phylogeny among pleuronectiformes.
also, the existence of paralogous genes exhibiting differential expression patterns in tissues, and even more important during larval development, particularly in metamorphosis, suggests rps as interesting molecular markers to investigate flatfish genome evolution in terms of gain and loss of paralogous genes and the availabilty to acquire new functions  or divide the ancestral function between the paralogs   <cit> .
three rounds of large-scale gene duplications  have been identified in fish  <cit> .
these duplications are responsible, at least in part, for their speciation, adaptive radiation and high morphological complexity  <cit> .
although the majority of these gene duplicates have been lost or silenced during evolution, several gene copies have been described for some group of genes including glycolytic enzymes  <cit> , hox genes  <cit>  and hormones and their receptors  <cit> .
similarly, different gene copies have been described for some rps.
in human, two different rps <dig> genes exist, one encoded on the x and one on the y chromosome.
rat possesses two distinct rps <dig> transcripts that are expressed differentially in the hypothalamus  <cit> .
in fish, i. punctatus has two paralogous genes of rps <dig> and rps <dig>  <cit> .
in s. senegalensis and h. hippoglossus, two and four different rps <dig> genes have been detected, respectively.
phylogenetic analysis revealed that rps27- <dig> and rps27- <dig> sequences grouped in two separate clades supported by significant bootstrap values.
although thomas et al.
<cit>  proposed the rps27- <dig> as a mammalian-specific isoform, the identification of orthologous sequences for both rps <dig> genes in different fish species supports the hypothesis of at least two rps <dig> paralogs as a common feature in fish as well.
moreover, two additional rps <dig> genes  were identified in h. hippoglossus.
these two paralogous genes might have appeared in the 3r or fish-specific genome duplication, in this respect, we should highlight that both rps <dig> genes in i. punctatus grouped together in the same clade with s. senegalensis rps27- <dig> and h. hippoglossus rps27- <dig> and rps27- <dig>  this clustering suggests the existence of, at least, a third rps <dig> gene in i. punctatus orthologous to fish rps27- <dig>  such a hypothesis is also supported by the fact that both i. punctatus rps <dig> paralogs were expressed at a similar level   <cit> , whereas in s. senegalensis rps27- <dig> was expressed at a much lower level than rps27- <dig> in all tissues and during larval development as determined by real-time pcr.
during embryogenesis, after mid-blastula transition in zebrafish, rp genes co-ordinately increase their expression  <cit> .
in addition, in atlantic halibut up to  <dig> and  <dig> rps increase mrna levels from embryos to  <dig> day-old yolk sac larvae and fast skeletal muscle in juveniles, respectively  <cit> .
in this study, we provide evidence that one day after first feeding, the eight rps analyzed by real-time pcr increase their expression levels in senegalese sole also.
during this period, larvae undergo important physiological and morphological changes such as the opening of the mouth and anus.
when live prey are provided for feeding, different organs such as the liver, pancreas, and the digestive tract are activated, promoting larval metabolism  <cit> .
larval rearing is a critical period during which different aspects concerning important anatomical and physiological traits in the juvenile stage are modulated.
there are reports on biomarkers for fish larvae fed different diets that focused specifically on oxidative stress  <cit>  and digestive enzymes  <cit> .
the co-ordinate changes in rp expression under important physiological events such as the first feeding suggest rps might be considered as biomarkers that could provide broader information about the general physiological condition in fish.
in addition, the high abundance of these rps , most of which are considered as house-keeping genes, indicates that even small induction values, as observed in this survey , can play an important physiological role.
although rp transcript levels increased after first feeding, they dropped at the first metamorphic stages .
in this respect, we should take into account that senegalese sole exhibits two different growth rates during larval development.
at pre-metamorphosis, larvae grow at almost twice the rate as at metamorphosis and accumulate energy reserves in tissues to be used during this important period  <cit> .
the lower growth rate at metamorphosis has been correlated with reduced igf-ii expression levels  <cit> , an activator of the 70-kda ribosomal s <dig> kinase , a serine/threonine protein kinase that plays a central role in cell growth and proliferation.
this kinase mediates the phosphorylation of rps <dig>  thereby enabling efficient translation of 5'-terminal oligopyrimidine tract  mrnas.
since rps and translation elongation factors are encoded by 5'-top mrnas, signalling along the s6k <dig> pathway may regulate ribosome biogenesis and therefore the response to growth conditions  <cit> .
moreover, apoptosis has been shown to play an important role in the organ-rebuilding process during flatfish metamorphosis  <cit>  and some rps have been associated with apoptotic processes  <cit> .
the reduction in rp gene expression, especially at the beginning of sole metamorphosis, suggests they could also be involved in the control of apoptosis during metamorphosis.
we evaluated gene expression of eight rps in ten different sole tissues.
overall, all tissues except brain expressed rps at a similar level.
these data agree with those obtained in i. punctatus using a transcriptomic approach.
representation of rps was reduced in brain compared with skin and head kidney  <cit> .
ribosome formation can vary in response to cellular demands and their protein biosynthetic requirements  <cit>  and these differences in the steady-state number of rp transcripts might reflect the distinct metabolic activity of tissues.
rps exhibited different expression levels in different sole tissues.
rps27- <dig> mrna levels were up to  <dig>  and  <dig> -fold lower than rps27- <dig> and rps <dig>  respectively.
these differences in relative mrna abundance among rps were also observed in i. punctatus and h. hippoglossus larvae and juveniles  <cit> .
such difference suggests a translational regulation to facilitate the correct assembly of ribosomes.
moreover, there is increasing evidence that rps modulate a variety of cellular activities independent of their own involvement in the protein biosynthesis such as replication, transcription, rna processing, dna repair, and inflammation  <cit> .
in our study, some rps exhibited different tissue expression patterns.
for instance, rps <dig> was highly expressed in all tissues except in brain where rps27- <dig> transcripts were the highest.
these data agree with those described for i. punctatus where the number of ests corresponding to rps <dig> and rps <dig> were 10-fold lower and 3-fold higher, respectively, in brain than in skin and head kidney  <cit> .
in addition, rps3a transcripts were reduced in brain, heart, and skin, and rps27- <dig> showed the highest expression levels in intestine and stomach.
all these data underscore the necessity for new studies to elucidate the regulation of these rps in tissues and their possible extraribosomal function.
CONCLUSIONS
in this work we have identified and characterized the complete set of 40s rps in two pleuronectiformes: senegalese sole and atlantic halibut.
these data provide new molecular markers to investigate genome evolution and phylogenetic relationships among flatfish.
also, gene expression studies in senegalese sole have revealed a co-ordinate response after first feeding in larvae suggesting a possible role of rps as general condition biomarkers to estimate larval physiological status in response to changing environmental conditions.
moreover, the differential expression patterns in tissues suggest that rps might perform other functions distinct from protein biosynthesis.

 the eukaryotic ribosome is a complex macromolecular structure composed of a large and a small subunit. the large ribosomal subunit catalyses peptide bond formation and is responsible for channelling the nascent proteins through their exit tunnel. the small ribosomal subunit binds mrna and is responsible for the fidelity of translation by ensuring the correct base pairing between aminoacyl-trnas and codons of the mrna in the decoding centre. biochemically, the eukaryotic ribosome is composed of four ribosomal rna molecules and over ribosomal proteins. in mammals, the 60s and 40s subunits are composed of and rps, respectively. each mammalian rp is typically encoded by a single gene except rps in human, and rps in rat and human, which are encoded by two separate genes. in contrast, in the yeast saccharomyces cerevisiae, the rps are encoded by genes, of which are duplicated. in fish, the complete set of rps in fugu rubripes and ictalurus punctatus has been described. of the rps from the 40s subunit, a duplication of rps in both species and of rps in i. punctatus was observed. in the rps from the 60s subunit, all of them but one appeared to have only one type of mrna. rps play a critical role in protein biosynthesis. cellular levels change as a function of growth rate in bacteria and fungi. in fish, mrna levels increase co-ordinately during embryogenesis and larval development. in mammals, certain tumors have substantially increased levels of some rp transcripts. however, different rps have also been associated with various other cellular processes; the so-called extraribosomal functions. for example, in drosophila, mutations in the rps gene appear to cause arrest of oogenesis and rps functions as a tumor suppressor in the hematopoietic system. mammalian rps appears to possess apurinic/apyrimidinic endonuclease activity involved in dna repair functions. haploinsufficiency of the rps genes has been suggested to contribute to anatomic abnormalities associated with the turner syndrome in humans. the gene encoding rps seems to participate in embryogenesis due to its capacity to interact with fgf- a factor involved in the differentiation process of different cell types. finally, apoptosis can be induced by inhibiting or activating expression of rps3a and rps27l, respectively. senegalese sole, solea senegalensis, and atlantic halibut, hippoglossus hippoglossus, are two commercially important flatfish species. during larval development, both species change from a symmetrical morphology to an asymmetric, benthic juvenile. this metamorphic process involves dramatic morphological and physiological changes. in senegalese sole, metamorphosis occurs very early during larval development, between and days after hatching. in atlantic halibut, metamorphosis begins with the migration of the left eye about dah. apoptotic processes induced by thyroxine hormone have been associated with this tissue remodelling in flatfish. in addition, senegalese sole larvae exhibit two different growth rates during development. because of the key role rps play in cellular growth and proliferation and in some cases apoptosis, it is important to elucidate the expression pattern of rps during flatfish development. rps are highly represented in cdna libraries. the development of large-scale genomics on senegalese sole and atlantic halibut has made possible an efficient and systematic analysis of rp sequences in both species. in this work, we report the complete set of 40s subunit rp cdnas for both senegalese sole and atlantic halibut and describe their main characteristics. comparative sequence analysis revealed the existence of two and four rps genes in senegalese sole and atlantic halibut, respectively. real-time pcr analysis revealed different rp expression patterns during larval development and in tissues in sole.  characteristics of the 40s rps sequence analysis of normalized libraries for senegalese sole allowed the identification of out of 40s subunit rps. rps was obtained from a premetamorphic stage larval library using specific primers. overall, 40s rp genes were not highly represented in the normalized libraries accounting for out of the good sequences. the number of clones for each rp ranged between for rps and only for rps27- and rps. * genbank sequences comprise final contig gene sizes for the complete set of 40s rps ranged between and bp for rps and rpsa, respectively. only rps rps and rps had partial sequences missing the 5'-ends. all cdna sequences have been deposited in the genbank/embl/ddbj with accession numbers from ab to ab. most rps used taa as termination codon. only rps rps rps rps rps rps27- rps and rps used tag, and rps and rps tga. the 3'-utrs were highly at-rich. all rps had a canonical aataaa polyadenylation signal between 7– nucleotides from the poly tail. in halibut, sequences for all except rps and rps27- were identified from the pleurogene database. in most cases, the complete coding sequences were obtained, but 3'-end sequencing was performed for all rp sequences to confirm the 3'ends, particularly of the long ests. the rps and rps27- sequences presented in this analysis derive from atlantic halibut ests in genbank. in addition, only a partial sequence for rps was obtained and the 5'-end was completed by the addition of an atlantic halibut est in genbank. ests from a total of sequences in the database encoded rps, an indication of the excellent normalization in these cdna libraries. sizes of coding sequences, 5'- and 3'-utrs and positions of polyadenylation signals are given in table 2b. all rps had a single polyadenylation signal with the exception of rps which had two possible non-canonical sites, and rps which had two possible canonical sites. the polyadenylation signal for rps overlapped the stop signal and no polyadenylation signal could be identified in rps27- as with senegalese sole, most 40s subunit rps used taa as a stop codon, followed by tag and tga and the 3'-utrs were very at-rich. two ests comprising rps and rps27a contigs differed in the lengths of their 5'-utrs by over nucleotides due to the presence of unspliced introns in this region. comparison of the senegalese sole and atlantic halibut 40s subunit rps the complete set of 40s rps in senegalese sole and atlantic halibut had a high overall similarity as determined by deduced amino acid sequences. with respect to the other species, senegalese sole overall similarities ranged between and % with human and f. rubripes, respectively. for halibut, these values were and % with human and f. rubripes, respectively. the most conserved 40s rps in the five species were rps and rps27a; and the most divergent ones were rps, rps, and rpsa. a and b refer to similarities to h. sapiens rps4x and rps4y isoforms, respectively. c and d refer to similarities to i. punctatus rps26- and rps26- isoforms, respectively. the number of amino acids was highly conserved throughout evolution. of the 40s subunit rp cdnas, had orfs with identical number of amino acids in the five species compared; rps and rps showed different sizes in atlantic halibut and human. four rps had the same number of amino acids among fish species but different from human. in addition, three others varied in more than two species. senegalese sole rpsa and rps showed the highest differences in size being amino acids longer and amino acids shorter than in human, respectively. phylogenetic analysis of rps genes two and four cdnas encoding rps were found in senegalese sole and atlantic halibut, respectively. in senegalese sole, the total lengths of rps27- and rps27- were and nucleotides, respectively. they were represented by and clones, respectively. in atlantic halibut, total lengths ranged between and nt for rps27- and rps27- respectively. the atlantic halibut rps27- had a slightly shorter coding region, whereas no putative polyadenylation signal could be identified in rps27- rps27- and rps27- were represented by and clones in the halibut libraries, respectively, and rps27- by only est. both senegalese sole paralogs showed a high divergence at the nucleotide level when complete cdnas or coding regions were aligned. at the amino acid level, they differed in residues with a sequence identity of %. similarly, low sequence similarity was detected among atlantic halibut full-length sequences. these values ranged between and % in the coding regions. at the protein level, rps27- was amino acids shorter that the other paralogs. amino acid similarities ranged between and %. among species, s. senegalensis rps27- and h. hippoglossus rps27- were the closest evolutionary homologues with % similarity using full-length sequence, % in coding sequence and 100% in amino acid sequence. a phylogenetic analysis based on rps coding sequences using the nj, mp and ml methods showed that fish rps genes grouped mainly in two distinct clades. both senegalese sole and atlantic halibut rps27- genes clustered with their fish counterparts. the rps27- appeared more closely related to rps27- than the other two atlantic halibut rps genes. moreover, rps27- and rps27l genes in rat and human, respectively, formed a sister clade sharing a common ancestor with fish rps27- genes. the other fish rps gene copies appeared linked in a clade that was not well resolved. both senegalese sole and atlantic halibut rps27- grouped with p. flesus and t. nigroviridis rps27-. curiously, this clade contained both i. punctatus rps genes. gene expression analysis we used a quantitative approach based on reverse transcription followed by real-time pcr amplification to investigate the steady-state levels of eight sole rp transcripts in liver, spleen, intestine, stomach, head kidney, gills, muscle, brain, heart, and skin. relative gene expression levels were normalized by measuring ubiquitin levels. all eight rp genes were expressed in detectable amounts in all tissues. rps transcripts were the most abundant except in brain, where rps27- showed the highest values. on the other hand, rps27- was expressed at the lowest level in all tissues analyzed. rp genes were expressed differentially among tissues. rps rps and rps exhibited lower expression levels in brain and rps3a transcripts were reduced in brain, heart, and skin. in contrast, rps27- was expressed more highly in intestine and stomach, and rps in heart. if we calculate the mean ribosomal expression ratio as a global rp expression index, all tissues showed similar values except brain with only. we also investigated the expression pattern of rp genes during sole larval development. mrna levels were determined in samples extracted from whole larvae pools collected from to dah. expression levels of each rp gene were normalized to that of gapdh. rps rps27- rps27a, rps and rps showed higher transcript levels than rps3a, rps and rps27- during early larval development in senegalese sole. all rp mrnas increased from to dah, hours after first external feeding. fold induction values ranged between for both rps27- and rps and for rps27- these levels were reduced at dah, two days before the onset of eye migration, with the lowest values at the first metamorphic stages. the number of mrna molecules for the rps analyzed declined approximately -fold as global mean from to dah. the fold reduction values ranged between and for rps and rps27- respectively. rps27- was expressed at a higher level than rps27- during all pre-metamorphic, metamorphic and post-metamorphic stages. discussion in this work, we describe the complete set of 40s rps in the senegalese sole and atlantic halibut. sequences were generated from normalized cdna libraries constructed for expressed sequence tag analysis. the rapid development of genomics in all biological research areas, including aquaculture, and the high representation of rps in the cdna libraries have favoured the availability of an increasing number of rp sequences from different organisms. this fact has motivated their proposed use as appropriate molecular markers for phylogenetic analysis. in fact, concatenation of orthologous rp amino acid sequences to form a single one of more than characters has allowed the reconstruction of phylogenetic relationships between animal, fungal, and plant kingdoms. with regard to this, pleuronectiformes comprises a broad taxonomic group with families and about species worldwide, some of them of high commercially interest both in fisheries and aquaculture. all flatfish species share in common an asymmetrical body development and a bottom-dwelling mode of life. however, their high phenotypic similarity has invoked great differences in the number and nomenclature of taxa depending on the relevance assigned to morphologic features. most phylogenetic studies focused on relationships among pleuronectiformes have been based on partial mitochondrial dna sequences. the description of the complete set of rps in one pleuronectidae and soleidae species provides new molecular markers to investigate the taxonomy and phylogeny among pleuronectiformes. also, the existence of paralogous genes exhibiting differential expression patterns in tissues, and even more important during larval development, particularly in metamorphosis, suggests rps as interesting molecular markers to investigate flatfish genome evolution in terms of gain and loss of paralogous genes and the availabilty to acquire new functions or divide the ancestral function between the paralogs. three rounds of large-scale gene duplications have been identified in fish. these duplications are responsible, at least in part, for their speciation, adaptive radiation and high morphological complexity. although the majority of these gene duplicates have been lost or silenced during evolution, several gene copies have been described for some group of genes including glycolytic enzymes, hox genes and hormones and their receptors. similarly, different gene copies have been described for some rps. in human, two different rps genes exist, one encoded on the x and one on the y chromosome. rat possesses two distinct rps transcripts that are expressed differentially in the hypothalamus. in fish, i. punctatus has two paralogous genes of rps and rps. in s. senegalensis and h. hippoglossus, two and four different rps genes have been detected, respectively. phylogenetic analysis revealed that rps27- and rps27- sequences grouped in two separate clades supported by significant bootstrap values. although thomas et al. proposed the rps27- as a mammalian-specific isoform, the identification of orthologous sequences for both rps genes in different fish species supports the hypothesis of at least two rps paralogs as a common feature in fish as well. moreover, two additional rps genes were identified in h. hippoglossus. these two paralogous genes might have appeared in the 3r or fish-specific genome duplication, in this respect, we should highlight that both rps genes in i. punctatus grouped together in the same clade with s. senegalensis rps27- and h. hippoglossus rps27- and rps27- this clustering suggests the existence of, at least, a third rps gene in i. punctatus orthologous to fish rps27- such a hypothesis is also supported by the fact that both i. punctatus rps paralogs were expressed at a similar level, whereas in s. senegalensis rps27- was expressed at a much lower level than rps27- in all tissues and during larval development as determined by real-time pcr. during embryogenesis, after mid-blastula transition in zebrafish, rp genes co-ordinately increase their expression. in addition, in atlantic halibut up to and rps increase mrna levels from embryos to day-old yolk sac larvae and fast skeletal muscle in juveniles, respectively. in this study, we provide evidence that one day after first feeding, the eight rps analyzed by real-time pcr increase their expression levels in senegalese sole also. during this period, larvae undergo important physiological and morphological changes such as the opening of the mouth and anus. when live prey are provided for feeding, different organs such as the liver, pancreas, and the digestive tract are activated, promoting larval metabolism. larval rearing is a critical period during which different aspects concerning important anatomical and physiological traits in the juvenile stage are modulated. there are reports on biomarkers for fish larvae fed different diets that focused specifically on oxidative stress and digestive enzymes. the co-ordinate changes in rp expression under important physiological events such as the first feeding suggest rps might be considered as biomarkers that could provide broader information about the general physiological condition in fish. in addition, the high abundance of these rps, most of which are considered as house-keeping genes, indicates that even small induction values, as observed in this survey, can play an important physiological role. although rp transcript levels increased after first feeding, they dropped at the first metamorphic stages. in this respect, we should take into account that senegalese sole exhibits two different growth rates during larval development. at pre-metamorphosis, larvae grow at almost twice the rate as at metamorphosis and accumulate energy reserves in tissues to be used during this important period. the lower growth rate at metamorphosis has been correlated with reduced igf-ii expression levels, an activator of the 70-kda ribosomal s kinase, a serine/threonine protein kinase that plays a central role in cell growth and proliferation. this kinase mediates the phosphorylation of rps thereby enabling efficient translation of 5'-terminal oligopyrimidine tract mrnas. since rps and translation elongation factors are encoded by 5'-top mrnas, signalling along the s6k pathway may regulate ribosome biogenesis and therefore the response to growth conditions. moreover, apoptosis has been shown to play an important role in the organ-rebuilding process during flatfish metamorphosis and some rps have been associated with apoptotic processes. the reduction in rp gene expression, especially at the beginning of sole metamorphosis, suggests they could also be involved in the control of apoptosis during metamorphosis. we evaluated gene expression of eight rps in ten different sole tissues. overall, all tissues except brain expressed rps at a similar level. these data agree with those obtained in i. punctatus using a transcriptomic approach. representation of rps was reduced in brain compared with skin and head kidney. ribosome formation can vary in response to cellular demands and their protein biosynthetic requirements and these differences in the steady-state number of rp transcripts might reflect the distinct metabolic activity of tissues. rps exhibited different expression levels in different sole tissues. rps27- mrna levels were up to and -fold lower than rps27- and rps respectively. these differences in relative mrna abundance among rps were also observed in i. punctatus and h. hippoglossus larvae and juveniles. such difference suggests a translational regulation to facilitate the correct assembly of ribosomes. moreover, there is increasing evidence that rps modulate a variety of cellular activities independent of their own involvement in the protein biosynthesis such as replication, transcription, rna processing, dna repair, and inflammation. in our study, some rps exhibited different tissue expression patterns. for instance, rps was highly expressed in all tissues except in brain where rps27- transcripts were the highest. these data agree with those described for i. punctatus where the number of ests corresponding to rps and rps were 10-fold lower and 3-fold higher, respectively, in brain than in skin and head kidney. in addition, rps3a transcripts were reduced in brain, heart, and skin, and rps27- showed the highest expression levels in intestine and stomach. all these data underscore the necessity for new studies to elucidate the regulation of these rps in tissues and their possible extraribosomal function.  in this work we have identified and characterized the complete set of 40s rps in two pleuronectiformes: senegalese sole and atlantic halibut. these data provide new molecular markers to investigate genome evolution and phylogenetic relationships among flatfish. also, gene expression studies in senegalese sole have revealed a co-ordinate response after first feeding in larvae suggesting a possible role of rps as general condition biomarkers to estimate larval physiological status in response to changing environmental conditions. moreover, the differential expression patterns in tissues suggest that rps might perform other functions distinct from protein biosynthesis. 
their high representation in cdna libraries has resulted in the increase of rp sequences available from different organisms and their proposal as appropriate molecular markers for phylogenetic analysis. the development of large-scale genomics of senegalese sole and atlantic halibut, two commercially important flatfish species, has made possible the identification and systematic analysis of the complete set of rp sequences for the small ribosome subunit. amino acid sequence comparisons showed a high similarity both between these two flatfish species and with respect to other fish and human. their levels can vary as a function of organism growth and development; however, some rps have been associated with other cellular processes or extraribosomal functions. steady-state transcript levels for eight rps in sole were quantitated during larval development and in tissues, using a real-time pcr approach. ribosomal proteins are key components of ribosomes, the cellular organelle responsible for protein biosynthesis in cells. phylogenetic analysis clustered rps in two separate clades with their fish and mammalian counterparts. on the contrary, rp transcripts increased co-ordinately after first larval feeding reducing progressively during the metamorphic process. est analysis revealed the existence of two and four rps genes in senegalese sole and atlantic halibut, respectively.
 ribosomal proteins are key components of ribosomes, the cellular organelle responsible for protein biosynthesis in cells. their levels can vary as a function of organism growth and development; however, some rps have been associated with other cellular processes or extraribosomal functions. their high representation in cdna libraries has resulted in the increase of rp sequences available from different organisms and their proposal as appropriate molecular markers for phylogenetic analysis.  the development of large-scale genomics of senegalese sole and atlantic halibut, two commercially important flatfish species, has made possible the identification and systematic analysis of the complete set of rp sequences for the small ribosome subunit. amino acid sequence comparisons showed a high similarity both between these two flatfish species and with respect to other fish and human. est analysis revealed the existence of two and four rps genes in senegalese sole and atlantic halibut, respectively. phylogenetic analysis clustered rps in two separate clades with their fish and mammalian counterparts. steady-state transcript levels for eight rps in sole were quantitated during larval development and in tissues, using a real-time pcr approach. all eight rps exhibited different expression patterns in tissues with the lowest levels in brain. on the contrary, rp transcripts increased co-ordinately after first larval feeding reducing progressively during the metamorphic process.  the genomic resources and knowledge developed in this survey will provide new insights into the evolution of pleuronectiformes. expression data will contribute to a better understanding of rp functions in fish, especially the mechanisms that govern growth and development in larvae, with implications in aquaculture. 