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BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-7-1071760892610.1186/1471-2148-7-107Research ArticleComparative sequence analysis of the complete set of 40S ribosomal proteins in the Senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.) (Teleostei: Pleuronectiformes): phylogeny and tissue- and development-specific expression Manchado Manuel patrick02@example.com Carlos patrick02@example.com Esther patrick02@example.com.esCañavate Jose Pedro patrick02@example.com Susan E patrick02@example.com IFAPA Centro El Toruño, Junta de Andalucía Camino Tiro de pichón s/n, 11500 El Puerto de Santa María, Cádiz, Spain2 Institute for Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada2007 3 7 2007 7 107 107 5 3 2007 3 7 2007 Copyright © 2007 Manchado et al; licensee BioMed Central Ltd.2007Manchado et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background
Ribosomal proteins (RPs) 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.

Results
The development of large-scale genomics of Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species, has made possible the identification and systematic analysis of the complete set of RP sequences for the small (40S) 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 RPS27 genes in Senegalese sole and Atlantic halibut, respectively. Phylogenetic analysis clustered RPS27 in two separate clades with their fish and mammalian counterparts. Steady-state transcript levels for eight RPs (RPS2, RPS3a, RPS15, RPS27-1, RPS27-2, RPS27a, RPS28, and RPS29) 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.

Conclusion
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.
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Background
The eukaryotic ribosome is a complex macromolecular structure composed of a large (60S) and a small (40S) 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 [1]. Biochemically, the eukaryotic ribosome is composed of four ribosomal RNA molecules and over 70 ribosomal proteins (RPs) [2]. In mammals, the 60S and 40S subunits are composed of 47 and 32 RPs, respectively [3]. Each mammalian RP is typically encoded by a single gene except RPS4 in human [4], and RPS27 in rat [5] and human [6], which are encoded by two separate genes. In contrast, in the yeast Saccharomyces cerevisiae, the 78 RPs are encoded by 137 genes, 59 of which are duplicated [7]. In fish, the complete set of RPs in Fugu rubripes [8] and Ictalurus punctatus [9] has been described. Of the 32 RPs from the 40S subunit, a duplication of RPS27 in both species and of RPS26 in I. punctatus was observed. In the 47 RPs from the 60S subunit, all of them but one (RPL5 in I. punctatus) appeared to have only one type of mRNA [10].

RPs play a critical role in protein biosynthesis. Cellular levels change as a function of growth rate in bacteria and fungi [11-14]. In fish, mRNA levels increase co-ordinately during embryogenesis and larval development [15-18]. In mammals, certain tumors have substantially increased levels of some RP transcripts [19,20]. However, different RPs have also been associated with various other cellular processes; the so-called extraribosomal functions. For example, in Drosophila, mutations in the RPS2 gene appear to cause arrest of oogenesis [21] and RPS6 functions as a tumor suppressor in the hematopoietic system [22]. Mammalian RPS3 appears to possess apurinic/apyrimidinic endonuclease activity involved in DNA repair functions [23]. Haploinsufficiency of the RPS4 genes has been suggested to contribute to anatomic abnormalities associated with the Turner syndrome in humans [4]. The gene encoding RPS19 seems to participate in embryogenesis due to its capacity to interact with FGF-2, a factor involved in the differentiation process of different cell types [24]. Finally, apoptosis can be induced by inhibiting or activating expression of RPS3a and RPS27L, respectively [6,25].

Senegalese sole, Solea senegalensis (Pleuronectiformes: Soleidae), and Atlantic halibut, Hippoglossus hippoglossus (Pleuronectiformes: Pleuronectidae), 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 12 and 19 days after hatching (DAH) [26]. In Atlantic halibut, metamorphosis begins with the migration of the left eye about 80 DAH [27]. Apoptotic processes induced by thyroxine hormone have been associated with this tissue remodelling in flatfish [28]. In addition, Senegalese sole larvae exhibit two different growth rates during development [26,29]. 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 [9,10]. 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 32 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 RPS27 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 31 out of 32 40S subunit RPs (only RPS28 was absent). RPS28 was obtained from a premetamorphic stage larval library using specific primers (Table 1). Overall, 40S RP genes were not highly represented in the normalized libraries accounting for 252 (2.5%) out of the 10,099 good sequences. The number of clones for each RP ranged between 24 for RPS2 and only 1 for RPS27-2 and RPS29 (Table 2A).

Table 1 Primers used for real-time PCR gene expression analysis. F and R refer to forward and reverse primers, respectively.

Target	Primers			Fragment size (bp)	
			
	Primer pair name	Sequence	5'-position		
RPS2	SserpS2•1	5'-CCAAGCTGTCGATTGTCCCGGTCA-3' (F)	434	127	
	SserpS2•2	5'-CGGGGGGCAGGGATGAGACG-3' (R)	560		
RPS3a	SserpS3a•1	5'-TCAGAAAGACCTCCTACGCCCAGCA-3' (F)	450	94	
	SserpS3a•2	5'-AGATCATTGGTCTGAACCTCACGGGTCA-3' (R)	543		
RPS15	SserpS15•1	5'-CATGGTTGGCGTGTACAATGGCAAA-3' (F)	300	116	
	SserpS15•2	5'-GGCGACCGTGCTTGACTGGCTTG-3'(R)	415		
RPS27-1	SserpS27-1•1	5'-CCCGAGGAGGAGAAGAGGAGGCACA-3' (F)	64	124	
	SserpS27-1•2	5'-CTGTCTGAGCGTGACTGAACACCGTCGT-3' (R)	187		
RPS27-2	SserpS27-2•1	5'-GCTAAAGACCTCCTCCACCCTGCCATT-3' (F)	72	130	
	SserpS27-2•2	5'-ACACAGTTGTGATTTTGTAGCAGCCTGGAC-3' (R)	201		
RPS27a	SserpS27a•1	5'-GCGTGAGTGTCCGGCTGACGA-3' (F)	431	87	
	SserpS27a•2	5'-GTGAGGCAGCACTTCCCGCAGT-3' (R)	517		
RPS28	SserpS28•1	5'-CGATAGTTCCCGCTGAAGCTGTGAGGTG-3' (F)	206	95	
	SserpS28•2	5'-GAGAATGTGAGGGATGTCCGCCGTTG-3' (R)	300		
RPS29	SserpS29•1	5'-AGGCAGTACGCTAAAGACATCGGCTTCGTG-3' (F)	135	121	
	SserpS29•2	5'-GTGCTGAATTATCCCATCATCTTGGCTGGT-3' (R)	255		
GAPDH	SseGAPDH231•1	5'-AGCCACCGTGTCGCCGACCT-3' (F)	1001	107	
	SseGAPDH231•2	5'-AAAAGAGGAGATGGTGGGGGGTGGT-3' (R)	1107		
Ubiquitin	SseUB•1	5'-AGCTGGCCCAGAAATATAACTGCGACA-3' (F)	289	93	
	SseUB•2	5'-ACTTCTTCTTGCGGCAGTTGACAGCAC-3' (R)	381		
Table 2 Structural characteristics of the cDNAs encoding RPs of (A) Senegalese sole, Solea senegalensis and (B) Atlantic halibut, Hippoglossus hippoglossus. Lengths of coding regions, available 5'-UTR, 3'-UTR and poly (A) tail distances from poly(A) signals are indicated. Asterisk (*) denotes RPS coding sequences that were derived partially or completely from sequences present in GenBank. ND, not detected.

(A)							
Gene	# clones	Accession #	Coding region	5'-UTR	3'-UTR	Poly(A) from poly(A)Signal	
	
RPSa	15	AB291586	942	60	41	17	
RPS2	24	AB291554	843	ND	39	12	
RPS3	16	AB291555	738	9	84	17	
RPS3a	13	AB291556	801	1	41	17	
RPS4	7	AB291557	792	ND	57	17	
RPS5	5	AB291558	612	29	62	37	
RPS6	14	AB291559	750	23	39	22	
RPS7	19	AB291560	585	51	36	13	
RPS8	6	AB291561	627	ND	52	24	
RPS9	6	AB291562	585	46	67	14	
RPS10	9	AB291563	501	5	44	15	
RPS11	7	AB291564	486	11	77	26	
RPS12	16	AB291565	399	54	23	11	
RPS13	4	AB291566	456	3	37	13	
RPS14	2	AB291567	456	37	43	13	
RPS15	4	AB291568	438	24	43	16	
RPS15a	4	AB291569	393	34	38	16	
RPS16	3	AB291570	441	18	39	14	
RPS17	2	AB291571	405	21	39	11	
RPS18	10	AB291572	459	5	39	15	
RPS19	7	AB291573	444	5	22	22	
RPS20	6	AB291574	360	98	54	24, 29	
RPS21	4	AB291575	252	92	44	7	
RPS23	5	AB291576	451	19	41	14	
RPS24	9	AB291577	396	17	84	22	
RPS25	5	AB291578	372	6	66	24	
RPS26	6	AB291579	348	23	47	15	
RPS27-1	7	AB291580	255	30	200	20	
RPS27-2	1	AB291581	255	62	190	13	
RPS27a	7	AB291582	471	77	36	19	
RPS28	-	AB291583	210	1	126	21	
RPS29	1	AB291584	171	5	103	14	
RPS30	8	AB291585	402	35	96	15	
	
(B)							
	
Gene	# clones	Accession #	Coding region	5'-UTR	3'-UTR	Poly(A) from poly(A) Signal	
	
RPSa	3	EB034722, EB039353	930	84	41	17	
RPS2*	4	EB029719, EB030051	858	3	41	12	
RPS3	2	EB034826, EB036030	741	25	126	12	
RPS3a	6	EB035413, EB031090	801	23	41	14	
RPS4	8	EB032598	792	41	57	20	
RPS5	2	EB032359 EB036495	612	44	44	14	
RPS6	2	EB031692, EB032095	750	40	33	14	
RPS7	7	EB032431	585	37	33	14	
RPS8	5	EB035663	627	24	39	11	
RPS9	2	EB040140, EB032308	585	46	60	19	
RPS10	2	EB031080	501	39	41	15	
RPS11	5	EB035513	486	23	67	11	
RPS12	1	EB029693	399	65	23	11	
RPS13	1	EB034224	456	21	41	14	
RPS14	3	EB040078	456	43	38	12	
RPS15	3	EB036829, EB029855	438	25	42	18	
RPS15a	2	EB036744	393	212	36	14	
RPS16	1	EB038832	441	33	44	19	
RPS17	1	EB035783	405	24	38	12	
RPS18	6	EB035452	459	25	46	14	
RPS19	2	EB030274	444	27	21	20	
RPS20	2	EB038732	360	97	34	12	
RPS21	3	EB030651	252	71	45	8	
RPS23	5	EB035807	432	59	42	16, 21	
RPS24	1	EB034880	399	34	81	19	
RPS25	3	EB040606, EB040657	396	21	49	12	
RPS26	7	EB036835	348	29	49	11	
RPS27-1	4	EB039502	255	73	207	11	
RPS27-2*		DN794622	255	52	192	8	
RPS27-3	5	EB039943	255	73	>635	>451	
RPS27-4	1	EB040256	249	84	>402	>74	
RPS27a	4	EB038389	471	52	43	17	
RPS28	1	EB037921	210	22	123	16	
RPS29*		DN792676	171	30	>103	>15	
RPS30	4	EB032882	402	59	96	15	
* GenBank sequences comprise final contig

Gene sizes for the complete set of 40S RPs ranged between 279 and 1,043 bp for RPS29 and RPSa, respectively. Only RPS2, RPS4 and RPS8 had partial sequences missing the 5'-ends. All cDNA sequences have been deposited in the GenBank/EMBL/DDBJ with accession numbers from AB291554 to AB291586 (Table 2A). Most RPs (63.6%) used TAA as termination codon. Only RPS8, RPS11, RPS12, RPS15, RPS17, RPS27-1, RPS28, and RPS29 used TAG, and RPS6 and RPS24, TGA. The 3'-UTRs were highly AT-rich. All RPs had a canonical AATAAA polyadenylation signal between 7–37 nucleotides from the poly(A) tail.

In halibut, sequences for all except RPS29 and RPS27-2 were identified from the Pleurogene database (Table 2B). 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 RPS29 and RPS27-2 sequences presented in this analysis derive from Atlantic halibut ESTs in GenBank [GenBank:DN792676, GenBank:DN794622]. In addition, only a partial sequence for RPS2 was obtained and the 5'-end was completed by the addition of an Atlantic halibut EST in GenBank [GenBank:CF931586]. 108 ESTs from a total of 12,675 sequences in the database encoded RPs (0.9%), 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 RPS20, which had two possible non-canonical sites (AtTAAA and AATgAA), and RPS23, which had two possible canonical sites. The polyadenylation signal for RPS19 overlapped the stop signal and no polyadenylation signal could be identified in RPS27-3. As with Senegalese sole, most 40S subunit RPs used TAA (59%) as a stop codon, followed by TAG (26%) and TGA (15%) and the 3'-UTRs were very AT-rich. Two ESTs comprising RPS14 and RPS27a contigs differed in the lengths of their 5'-UTRs by over 100 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 (92.1%) as determined by deduced amino acid sequences (Table 3). With respect to the other species, Senegalese sole overall similarities ranged between 92.8 and 95.4% with human and F. rubripes, respectively. For halibut, these values were 89.8 and 92.5% with human and F. rubripes, respectively. The most conserved 40S RPs in the five species (overall mean >99%) were RPS23 and RPS27a; and the most divergent ones were RPS30 (86.9%), RPS25 (87.9%), and RPSa (89.0%).

Table 3 Amino acid comparisons of the RPs from S. senegalensis (Sse) and H. hippoglossus (Hhi) with those of I. punctatus (Ipu), F. rubripes (Fru) and human (Hsa). Similarity values for Senegalese sole and Atlantic halibut are separated by "/".

	Number of aminoacids	Similarity (%)	
			
Gene	Sse	Hhi	Ipu	Fru	Hsa	Sse/Hhi	Ipu	Fru	Hsa	
Sa	313	309	317	306	295	91.3	88.5/88.0	90.5/89.5	86.8/88.1	
S2	280	285	278	279	293	94.0	96.3/91.0	97.3/92.0	90.0/85.0	
S3	245	246	245	245	243	97.6	98.8/96.3	99.2/96.7	95.9/96.3	
S3a	266	266	266	266	264	96.6	94.7/94.7	96.2/95.5	93.9/93.6	
S4	263	263	263	263	263
263	96.6	95.8/93.9	95.8/94.3	93.5/92.8a
89.4/88.2b	
S5	203	203	203	203	204	99.5	99.0/98.5	97.0/96.6	97.5/97.0	
S6	249	249	249	249	249	98.4	95.6/94.0	98.4/97.2	95.6/94.4	
S7	194	194	194	194	194	98.5	96.4/95.9	98.5/99.0	96.9/96.4	
S8	208	208	208	208	208	98.1	89.4/88.9	96.2/94.2	94.2/93.8	
S9	194	194	194	194	194	97.9	95.9/96.9	97.4/96.6	95.9/96.9	
S10	166	166	166	166	165	97.6	96.4/94.6	97.0/95.8	90.3/89.7	
S11	161	161	159	161	158	98.1	90.6/91.8	97.5/96.9	89.2/89.2	
S12	132	132	132	132	132	97.7	97.7/95.5	98.5/97.7	97.7/96.2	
S13	151	151	151	151	151	98.0	98.7/98.0	100/98.0	98.7/96.7	
S14	151	151	151	151	151	96.0	95.4/99.3	96.0/100	96.0/100	
S15	145	145	145	145	145	98.6	96.6/96.6	99.3/99.3	94.5/94.5	
S15a	130	130	130	130	130	100	96.9/96.9	99.2/99.2	97.7/97.7	
S16	146	146	146	146	146	97.9	96.6/97.3	95.2/95.2	96.6/95.9	
S17	134	134	134	134	135	92.5	92.5/96.3	91.8/97.0	92.5/96.3	
S18	152	152	152	152	152	98.7	99.3/98.0	100/98.7	98.0/98.0	
S19	147	147	147	146	145	92.5	89.1/87.1	95.2/95.9	86.9/84.1	
S20	119	119	119	119	119	98.3	98.3/97.5	100/98.3	98.3/97.5	
S21	83	83	83	83	83	92.8	92.8/90.4	95.2/89.2	94.0/95.2	
S23	143	143	143	143	143	100	99.3/99.3	100/100	98.6/98.6	
S24	131	132	131	131	133	98.5	96.9/96.2	95.4/97.7	90.1/90.9	
S25	123	131	124	123	125	95.1	83.1/82.3	96.7/95.1	82.4/80.8	
S26	115	115	115	115	115	98.3	93.0/94.8c
93.9/94.8d	96.5/98.3	93.9/95.7	
S27-1	84	84	84	84	84	See Table 4				
S27-2	84	84	84	84	84					
S27-3		84								
S27-4		82								
S27a	156	156	156	156	156	100	100/100	100/100	98.7/98.7	
S28	69	69	69	69	69	98.6	100/98.6	97.1/95.7	95.7/94.2	
S29	56	56	56	56	56	98.2	100/98.2	100/98.2	98.2/96.4	
S30	133	133	133	133	133	94.7	85.8/85.0	90.3/91.0	80.6/81.2	
Overall						92.1	94.0/90.9	95.4/92.5	92.8/89.8	
a and b refer to similarities to H. sapiens RPS4X and RPS4Y isoforms, respectively.

c and d refer to similarities to I. punctatus RPS26-1 and RPS26-2 isoforms, respectively.

The number of amino acids was highly conserved throughout evolution. Of the 32 40S subunit RP cDNAs, 21 had ORFs with identical number of amino acids in the five species compared; RPS3 and RPS24 showed different sizes in Atlantic halibut and human. Four RPs had the same number of amino acids among fish species but different from human (RPS3a, RPS5, RPS10, and RPS17). In addition, three others varied in more than two species (RPSa, RPS2, and RPS25) (Table 3). Senegalese sole RPSa and RPS2 showed the highest differences in size being 18 amino acids longer and 13 amino acids shorter than in human, respectively.

Phylogenetic analysis of RPS27 genes
Two and four cDNAs encoding RPS27 were found in Senegalese sole (referred to as RPS27-1 and RPS27-2) and Atlantic halibut (referred to as RPS27-1, RPS27-2, RPS27-3, and RPS27-4), respectively (Figure 1A). In Senegalese sole, the total lengths of RPS27-1 and RPS27-2 were 485 and 507 nucleotides (nt), respectively. They were represented by 7 and 1 clones, respectively (Table 2A). In Atlantic halibut, total lengths ranged between 499 and 963 nt for RPS27-2 and RPS27-3, respectively. The Atlantic halibut RPS27-4 had a slightly shorter coding region (249 nt), whereas no putative polyadenylation signal could be identified in RPS27-3. RPS27-1 and RPS27-3 were represented by 4 and 5 clones in the halibut libraries, respectively, and RPS27-4 by only 1 EST.

Figure 1 RPS27 genes from S. senegalensis (Sse) and H. hippoglossus (Hhi). (A) Coding sequence alignment. (B) Amino acid alignment. Dots indicate identity and hyphens represent indels.

Both Senegalese sole paralogs showed a high divergence at the nucleotide level when complete cDNAs (48.4% identity) or coding regions (73.7%) were aligned. At the amino acid level, they differed in 9 residues with a sequence identity of 89.3% (Figure 1B; Table 4). Similarly, low sequence similarity (36.5–49.4%) was detected among Atlantic halibut full-length sequences. These values ranged between 66.7 (RPS27-1 and RPS27-4) and 82.0% (RPS27-1 and RPS27-3) in the coding regions. At the protein level, RPS27-4 was 2 amino acids shorter that the other paralogs. Amino acid similarities ranged between 74.4 (RPS27-3 and RPS27-4 with 21 amino acid changes) and 95.2% (RPS27-1 and RPS27-3 with 4 residue differences). Among species, S. senegalensis RPS27-1 and H. hippoglossus RPS27-1 were the closest evolutionary homologues with 82.5% similarity using full-length sequence, 92.5% in coding sequence and 100% in amino acid sequence (Table 4).

Table 4 Amino acid similarities of RPS27 from S. senegalensis (Sse) and H. hippoglossus (Hhi) with those of I. punctatus (Ipu), F. rubripes (Fru) and H. sapiens (Hsa).

	Sse RPS27-2	Hhi RPS27-1	Hhi RPS27-2	Hhi RPS27-3	Hhi RPS27-4	Ipu RPS27-1	Ipu RPS27-2	Fru RPS27-1	Hsa RPS27-1	Hsa RPS27-L	
SseRPS27-1	89.3	100.0	91.7	95.2	79.3	96.4	97.6	100.0	98.8	95.2	
SseRPS27-2		89.3	96.4	85.7	74.4	90.5	90.5	89.3	89.3	86.9	
HhiRPS27-1			91.7	95.2	79.3	96.4	97.6	100.0	98.8	95.2	
HhiRPS27-2				88.1	76.8	92.9	92.9	91.7	91.7	89.3	
HhiRPS27-3					74.4	91.7	92.9	95.2	94.0	91.7	
HhiRPS27-4						79.3	79.3	79.3	78.0	75.6	
IpuRPS27-1							98.8	96.4	95.2	92.9	
IpuRPS27-2								97.6	96.4	92.9	
FruRPS27-1									98.8	95.2	
HsaRPS27-1										96.4	
A phylogenetic analysis based on RPS27 coding sequences using the NJ, MP and ML methods showed that fish RPS27 genes grouped mainly in two distinct clades (Figure 2). Both Senegalese sole and Atlantic halibut RPS27-2 genes clustered with their fish counterparts. The RPS27-4 appeared more closely related to RPS27-2 than the other two Atlantic halibut RPS27 genes. Moreover, RPS27-2 and RPS27L genes in rat and human, respectively, formed a sister clade sharing a common ancestor with fish RPS27-2 genes (bootstrap values higher than 50%). The other fish RPS27 gene copies appeared linked in a clade that was not well resolved. Both Senegalese sole and Atlantic halibut RPS27-1 grouped with P. flesus and T. nigroviridis RPS27-1 (bootstrap values higher than 70%). Curiously, this clade contained both I. punctatus RPS27 genes.

Figure 2 Phylogenetic relationships of RPS27 genes from S. senegalensis (Sse), H. hippoglossus (Hhi) H. sapiens (Hsa), R. norvegicus (Rno), I. punctatus (Ipu), P. flesus (Pfl), D. rerio (Dre) and T. nigroviridis (Tni). Xenopus laevis RPS27 was used as outgroup to root tree. Bootstrap values using NJ/MP/ML are indicated on each branch.

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 (RPS2, RPS3a, RPS15, RPS27-1, RPS27-2, RPS27a, RPS28, and RPS29) 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 (Figure 3). RPS2 transcripts were the most abundant except in brain, where RPS27-1 showed the highest values (1.82-fold higher than RPS2). On the other hand, RPS27-2 was expressed at the lowest level in all tissues analyzed.

Figure 3 Gene expression of RPS2, RPS3a, RPS15, RPS27-1, RPS27-2 RPS27a, RPS28 and RPS29 in tissues from Senegalese sole. Expression ratios of each RP mRNA relative to ubiquitin mRNA ± SEM are shown. Values with the same superscript are not significantly different (P < 0.05).

RP genes were expressed differentially among tissues. RPS2, RPS15, and RPS28 exhibited lower expression levels in brain and RPS3a transcripts were reduced in brain, heart, and skin. In contrast, RPS27-2 was expressed more highly in intestine and stomach, and RPS29 in heart. If we calculate the mean ribosomal expression ratio as a global RP expression index, all tissues showed similar values (0.74–0.99) except brain with only 0.48.

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 2 to 22 DAH (Figure 4). Expression levels of each RP gene were normalized to that of GAPDH. RPS2, RPS27-1, RPS27a, RPS28, and RPS29 showed higher transcript levels than RPS3a, RPS15 and RPS27-2 during early (2 to 3 DAH) larval development in Senegalese sole.

Figure 4 Gene expression of RPS2, RPS3a, RPS15, RPS27-1, RPS27-2 RPS27a, RPS28 and RPS29 during larval development in Senegalese sole. Expression ratios of each RP mRNA relative to GAPDH mRNA ± SEM are shown. Values with the same superscript are not significantly different (P < 0.05).

All RP mRNAs increased from 2 to 3 DAH, 24 hours after first external feeding (2.1-fold as global mean). Fold induction values ranged between 1.7 for both RPS27-1 and RPS28 and 3.0 for RPS27-2. These levels were reduced at 9 DAH, two days before the onset of eye migration, with the lowest values at the first metamorphic stages (13 DAH). The number of mRNA molecules for the 8 RPs analyzed declined approximately 3.5-fold as global mean from 3 (pre-metamorphosis) to 13 DAH (metamorphosis). The fold reduction values ranged between 1.7 and 10.3 for RPS28 and RPS27-2, respectively. RPS27-1 was expressed at a higher level than RPS27-2 during all pre-metamorphic, metamorphic and post-metamorphic stages (28-fold higher on average).

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 (EST) 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 [30]. 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 10,000 characters has allowed the reconstruction of phylogenetic relationships between animal, fungal, and plant kingdoms [31]. With regard to this, Pleuronectiformes comprises a broad taxonomic group with 11 families and about 500 species worldwide, some of them of high commercially interest both in fisheries and aquaculture [32-34]. 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 [35-38]. Most phylogenetic studies focused on relationships among Pleuronectiformes have been based on partial mitochondrial DNA sequences [39-41]. 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 (neofunctionalization) or divide the ancestral function between the paralogs (subfunctionalization) [42,43].

Three rounds of large-scale gene duplications (referred to as 1R, 2R, and 3R or fish-specific genome duplication) have been identified in fish [44,45]. These duplications are responsible, at least in part, for their speciation, adaptive radiation and high morphological complexity [45]. 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 [44], Hox genes [46,47] and hormones and their receptors [48,49]. Similarly, different gene copies have been described for some RPs. In human, two different RPS4 genes exist, one encoded on the X and one on the Y chromosome. Rat possesses two distinct RPS27 transcripts that are expressed differentially in the hypothalamus [5]. In fish, I. punctatus has two paralogous genes of RPS26 and RPS27 [9]. In S. senegalensis and H. hippoglossus, two and four different RPS27 genes have been detected, respectively. Phylogenetic analysis revealed that RPS27-1 and RPS27-2 sequences grouped in two separate clades supported by significant bootstrap values. Although Thomas et al. [5] proposed the RPS27-2 as a mammalian-specific isoform, the identification of orthologous sequences for both RPS27 genes in different fish species supports the hypothesis of at least two RPS27 paralogs as a common feature in fish as well. Moreover, two additional RPS27 genes (referred to as RPS27-3 and RPS27-4) 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 RPS27 genes in I. punctatus grouped together in the same clade with S. senegalensis RPS27-1 and H. hippoglossus RPS27-1 and RPS27-3. This clustering suggests the existence of, at least, a third RPS27 gene in I. punctatus orthologous to fish RPS27-2. Such a hypothesis is also supported by the fact that both I. punctatus RPS27 paralogs were expressed at a similar level (represented by 7 and 10 clones for RPS27-1 and RPS27-2, respectively) [9], whereas in S. senegalensis RPS27-2 was expressed at a much lower level than RPS27-1 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 [16-18]. In addition, in Atlantic halibut up to 40 and 41 RPs increase mRNA levels from embryos to 1 day-old yolk sac larvae and fast skeletal muscle in juveniles, respectively [15]. 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 [50]. 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 [51] and digestive enzymes [52]. 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 (about 50% of RNA polymerase II transcription in rapidly growing yeast cells [53]), most of which are considered as house-keeping genes, indicates that even small induction values, as observed in this survey (1.7–3.0 fold), can play an important physiological role.

Although RP transcript levels increased after first feeding, they dropped at the first metamorphic stages (13 DAH). 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 [26,29]. The lower growth rate at metamorphosis has been correlated with reduced IGF-II expression levels [54], an activator of the 70-kDa ribosomal S6 kinase (S6K1), a serine/threonine protein kinase that plays a central role in cell growth and proliferation. This kinase mediates the phosphorylation of RPS6, thereby enabling efficient translation of 5'-terminal oligopyrimidine tract (5'-TOP) mRNAs. Since RPs and translation elongation factors are encoded by 5'-TOP mRNAs, signalling along the S6K1 pathway may regulate ribosome biogenesis and therefore the response to growth conditions [55,56]. Moreover, apoptosis has been shown to play an important role in the organ-rebuilding process during flatfish metamorphosis [57] and some RPs have been associated with apoptotic processes [25,58]. 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 [9]. Ribosome formation can vary in response to cellular demands and their protein biosynthetic requirements [59] 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. RPS27-2 mRNA levels were up to 41.9 and 54.7-fold lower than RPS27-1 and RPS2, respectively. These differences in relative mRNA abundance among RPs were also observed in I. punctatus and H. hippoglossus larvae and juveniles [9,15]. 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 [60]. In our study, some RPs exhibited different tissue expression patterns. For instance, RPS2 was highly expressed in all tissues except in brain where RPS27-1 transcripts were the highest. These data agree with those described for I. punctatus where the number of ESTs corresponding to RPS2 and RPS27 were 10-fold lower and 3-fold higher, respectively, in brain than in skin and head kidney [9]. In addition, RPS3a transcripts were reduced in brain, heart, and skin, and RPS27-2 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.

Conclusion
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.

Methods
Identification of RP cDNAs in Senegalese sole and Atlantic halibut
Ten cDNA libraries were constructed from different larval stages and adult tissues of Senegalese sole using the ZAP Express® cDNA Syntesis kit and Zap Express cDNA Gigapack® III Gold Cloning kit (Stratagene) following the manufacturer's protocol (Cerdà et al., in preparation). The libraries were pooled and normalized, and approximately 11,000 randomly selected clones were sequenced from the 3'-end. Expressed sequence tags (ESTs) encoding RPs were identified after EST annotation. For RPS28 isolation, we designed specific primers (Table 1) using a partial sequence from a suppression subtractive hybridization library. RPS28 was amplified from the premetamorphic larval development library using combination of specific primers and the universal primers T3 and T7.

In halibut, normalised cDNA libraries were constructed for five different larval time points (hatching, mouth-opening, midway to metamorphosis, premetamorphosis, and postmetamorphosis) and eight adult tissues (testis, ovary, liver, head kidney, spleen, skin, gill, and intestine) [61], incorporated into the Pleurogene database  and provisionally annotated using AUTOFACT [62] implemented on the database.

Fish sampling
Juvenile Senegalese sole individuals (n = 3) were obtained from IFAPA Centro El Toruño facilities (El Puerto Santa María, Cádiz, Spain). They were sacrificed by immersion in tricaine methanesulfonate (MS-222). Liver, spleen, intestine, stomach, head kidney, gills, muscle, brain, heart, and skin were rapidly dissected, frozen in liquid nitrogen and stored at -80°C until use.

For larval studies, fertilized eggs from a naturally spawning Senegalese sole broodstock (IFAPA Centro El Toruño) were collected. They were incubated in a 150 L tank at 19–21°C for two days. Newly hatched larvae were transferred to a 400 L tank at an initial density from 45 to 50 larvae L-1 with a 16L:8D photoperiod and a light intensity of 600–800 lux. Larvae were fed rotifers (Brachionus plicatilis) 3 DAH till 9 DAH. From 7 DAH enriched artemia metanauplii were fed until the end of the experiment. Pools of larvae from 2 to 22 DAH (n = 3) were collected, washed with DEPC water, frozen in liquid nitrogen and stored at -80°C until analysis.

RNA isolation and gene expression analysis
Homogenization of juvenile tissues and larvae was carried out using Lysing Matrix D (Q-BioGene) for 40 s at speed setting 6 in the Fastprep FG120 instrument (Bio101). Total RNA was isolated from 50 mg of S. senegalensis tissues or pools of larvae using the RNeasy Mini Kit (Qiagen). All RNA isolation procedures were performed in accordance with the manufacturer's protocol. In all cases, total RNA was treated twice with DNase I using the RNase-Free DNase kit (Qiagen) for 30 min in order to avoid amplification of contaminated genomic DNA. RNA sample quality was checked using Experion (Bio-Rad) and quantification was performed spectrophotometrically. Total RNA (1 μg) from each sample was reverse-transcribed using the iScript™ cDNA Synthesis kit (Bio-Rad). Reverse transcription reactions were performed in duplicate. Lack of genomic DNA contamination was confirmed by PCR amplification of RNA samples in the absence of cDNA synthesis.

Real-time analysis was carried out on an iCycler (Bio-Rad). Reactions were performed in a 25 μl volume containing cDNA generated from 10 ng of original RNA template, 300 nM each of specific forward (F) and reverse (R) primers (Table 1), and 12.5 μl of iQ™ SYBR Green Supermix (Bio-Rad). Matching oligonucleotide primers were designed using Oligo v6.89 software (Medprobe). The amplification protocol used was as follows: initial 7 min denaturation and enzyme activation at 95°C, 40 cycles of 95°C for 15 s and 70°C for 30 s. Each assay was performed in duplicate. For normalization of cDNA loading, all samples were run in parallel with a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH; [DDBJ:AB291587]) or ubiquitin ([DDBJ:AB291588]) for larval development or juvenile tissues, respectively). To estimate efficiencies, a standard curve was generated for each primer pair based on known quantities of cDNA (10-fold serial dilutions corresponding to cDNA transcribed from 100 to 0.01 ng of total RNA). All calibration curves exhibited correlation coefficients higher than 0.99 and the corresponding real-time PCR efficiencies were 0.90–0.95. Relative mRNA expression of RPs was determined using the Ct method (value obtained by subtracting the Ct value of GAPDH or ubiquitin mRNA from the Ct value of the target mRNA). Data was expressed as the ratio (calculated using 1.93-(ΔCt)) of target mRNA to reference (GAPDH or ubiquitin) mRNA. Owing to the small differences among amplicons both in size (87–139 bp) and %GC (45.4–65.4), the relative sensitivity factor KRS was assumed to be one.

Results were expressed as mean ± SEM. Comparisons among groups were performed with one-way analysis of variance, followed by a Tukey test for identification of the statistically distinct groups. Significance was accepted for P < 0.05.

Sequence and phylogenetic analysis
Alignments of sequences were carried out and the sequence similarities calculated by the MegAlign program from the LASERGENE software suite. For phylogenetic analysis, sequences of RPS27 from different species including Homo sapiens ([GenBank:HSU57847, GenBank:NM_015920]; [6,63]), Rattus norvegicus ([GenBank:AF184893, EMBL:X59375]; [5,64]), Xenopus laevis ([GenBank:BC053815]; [65]), Ictalurus punctatus ([GenBank:AF402836, GenBank:AF402837]; [9]), Platichthys flesus ([GenBank:DV566302 and GenBank:DV567451]; unpublished), Danio rerio ([GenBank:BQ077524, GenBank:BC114281]; unpublished) and Tetraodon nigroviridis ([EMBL:CR722207 and EMBL:CR642405]; unpublished) were employed. Coding sequences were aligned using MegAlign software. Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses were carried out using PAUPv4beta10 software [66]. The TrNef + G model of sequence evolution was the most appropriate as selected by MODELTEST v3.5 [67]. The parameters of ML methods were R(a) = 1.0000, R(b) = 3.2865, R(c) = 1.0000, R(d) = 1.0000, and R(e) = 6.057. The gamma distribution shape parameter was estimated to be 0.3234. The degree of confidence assigned to nodes in trees was achieved by bootstrapping with 1,000 replicates.

Authors' contributions
MM designed the study, carried out the phylogenetic analyses, and drafted the manuscript. CI carried out the gene expression analysis and helped to draft the manuscript. EA performed the Senegalese sole cultures and samplings. JPC participated in the study design and coordination and helped to draft the manuscript. SED participated in sequence analysis and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements
This work has been financially supported by PLEUROGENE project funded by the Genome Canada-Genoma España joint program. Sequencing by the Atlantic Genome Centre , Halifax, Nova Scotia, Canada a partnership between Genome Atlantic and the National Research Council of Canada Institute for Marine Biosciences, is gratefully acknowledged. This is NRC publication number 42688.
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Zarivach R Bashan A Berisio R Harms J Auerbach T Schluenzen F Bartels H Baram D Pyetan E Sittner A  Functional aspects of ribosomal architecture: symmetry, chirality and regulation J Phys Org Chem 2004 17 901 912 10.1002/poc.831 
Wool IG Endo Y Chan YL Glück A  Hill W, Dahlberg A, Garrett R, Moore P, Schlessinger D, Warner J  Studies of the structure, function and evolution of mammalian ribosomes Ribosome Structure, Function and Evolution 1990 Washington DC: Society for Microbiology 203 214 
Wool IG Chan YL Glück A  Structure and evolution of mammalian ribosomal proteins Biochem Cell Biol 1995 73 933 947 8722009 
Fisher EM Beer-Romero P Brown LG Ridley A McNeil JA Lawrence JB Willard HF Bieber FR Page DC  Homologous ribosomal protein genes on the human X and Y chromosomes: escape from X inactivation and possible implications for Turner syndrome Cell 1990 63 1205 1218 2124803-17-0737/0092-8674(90)90416-C 
Thomas EA Alvarez CE Sutcliffe JG  Evolutionarily distinct classes of S27 ribosomal proteins with differential mRNA expression in rat hypothalamus J Neurochem 2000 74 2259 2267 10820803-17-0737/j.1471-4159.2000.0742259.x 
He H Sun Y  Ribosomal protein S27L is a direct p53 target that regulates apoptosis Oncogene 2803-17-0737 2716 17057803-17-0737/sj.onc.1210073 
Planta RJ Mager WH  The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae Yeast 1998 14 471 477 9559803-17-0737/(SICI)1097-0061(19980330)14:5<8b7c:f320:99b9:690f:4595:cd17:293a:c069AID-YEA241>3.0.CO;2-U 
Ribosomal Protein Gene Database 
Karsi A Patterson A Feng J Liu Z  Translational machinery of channel catfish: I. A transcriptomic approach to the analysis of 32 40S ribosomal protein genes and their expression Gene 2002 291 177 186 12095803-17-0737/S0378-1119(02)00595-4 
Patterson A Karsi A Feng J Liu Z  Translational machinery of channel catfish: II. Complementary DNA and expression of the complete set of 47 60S ribosomal proteins Gene 2003 305 151 160 12609803-17-0737/S0378-1119(02)01183-6 
Cujec TP Tyler BM  Nutritional and growth control of ribosomal protein mRNA and rRNA in Neurospora crassa Nucleic Acids Res 1996 24 943 950 8600803-17-0737/nar/24.5.943 
Herruer MH Mager WH Woudt LP Nieuwint RT Wassenaar GM Groeneveld P Planta RJ  Transcriptional control of yeast ribosomal protein synthesis during carbon-source upshift Nucleic Acids Res 1987 15 10133 10144 3320961 10.1093/nar/15.24.10133 
Milne AN Mak WW Wong JT  Variation of ribosomal proteins with bacterial growth rate J Bacteriol 1975 122 89 92 1091633 
Waldron C Jund R Lacroute F  Evidence for a high proportion of inactive ribosomes in slow-growing yeast cells Biochem J 1977 168 409 415 343781 
Bai J Solberg C Fernandes JM Johnston IA  Profiling of maternal and developmental-stage specific mRNA transcripts in Atlantic halibut Hippoglossus hippoglossus Gene 2006 386 202 210 17118803-17-0737/j.gene.2006.09.012 
Linney E Dobbs-McAuliffe B Sajadi H Malek RL  Microarray gene expression profiling during the segmentation phase of zebrafish development Comp Biochem Physiol C Toxicol Pharmacol 2004 138 351 362 15533803-17-0737/j.cca.2004.08.008 
Lo J Lee S Xu M Liu F Ruan H Eun A He Y Ma W Wang W Wen Z  15000 unique zebrafish EST clusters and their future use in microarray for profiling gene expression patterns during embryogenesis Genome Res 2003 13 455 466 12618803-17-0737/gr.885403 
Mathavan S Lee SG Mak A Miller LD Murthy KR Govindarajan KR Tong Y Wu YL Lam SH Yang H  Transcriptome analysis of zebrafish embryogenesis using microarrays PLoS Genet 2005 1 260 276 16132803-17-0737/journal.pgen.0010029 
Kowalczyk P Woszczynski M Ostrowski J  Increased expression of ribosomal protein S2 in liver tumors, posthepactomized livers, and proliferating hepatocytes in vitro Acta Biochim Pol 2002 49 615 624 12422231 
Pogue-Geile K Geiser JR Shu M Miller C Wool IG Meisler AI Pipas JM  Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein Mol Cell Biol 1991 11 3842 3849 1712897 
Cramton SE Laski FA  String of pearls encodes Drosophila ribosomal protein S2, has Minute-like characteristics, and is required during oogenesis Genetics 1994 137 1039 1048 7982558 
Watson KL Konrad KD Woods DF Bryant PJ  Drosophila homolog of the human S6 ribosomal protein is required for tumor suppression in the hematopoietic system Proc Natl Acad Sci USA 1992 89 11302 11306 1454803-17-0737/pnas.89.23.11302 
Kim J Chubatsu LS Admon A Stahl J Fellous R Linn S  Implication of mammalian ribosomal protein S3 in the processing of DNA damage J Biol Chem 1995 270 13620 13629 7775803-17-0737/jbc.{{PHONE+SOCIAL_SECURITY_NUMBER}} 
Draptchinskaia N Gustavsson P Andersson B Pettersson M Willig TN Dianzani I Ball S Tchernia G Klar J Matsson H  The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia Nat Genet 1999 21 169 175 9988803-17-0737/5951 
Naora H Takai I Adachi M Naora H  Altered cellular responses by varying expression of a ribosomal protein gene: sequential coordination of enhancement and suppression of ribosomal protein S3a gene expression induces apoptosis J Cell Biol 1998 141 741 753 9566973 10.1083/jcb.141.3.741 
Fernández-Díaz C Yúfera M Cañavate JP Moyano FJ Alarcón FJ Díaz M  Growth and physiological changes during metamorphosis of Senegal sole reared in the laboratory J Fish Biol 2001 58 1 13 10.1111/j.1095-8649.2001.tb00557.x 
Haug T  Biology of the Atlantic halibut, Hippoglossus hippoglossus (L. 1758) Adv Mar Biol 1990 26 2 70 
Power DM Llewellyn L Faustino M Nowell MA Bjornsson BT Einarsdottir IE Canario AV Sweeney GE  Thyroid hormones in growth and development of fish Comp Biochem Physiol C Toxicol Pharmacol 2001 130 447 459 11738803-17-0737/S1532-0456(01)00271-X 
Parra G Yúfera M  Comparative energetics during early development of two marine fish species, Solea senegalensis (KAUP) and Sparus aurata (L.) J Exp Biol 2001 204 2175 2183 11441059 
Nakao A Yoshihama M Kenmochi N  RPG: the Ribosomal Protein Gene database Nucleic Acids Res 2004 32 D168 170 14681803-17-0737/nar/gkh004 
Veuthey AL Bittar G  Phylogenetic relationships of fungi, plantae, and animalia inferred from homologous comparison of ribosomal proteins J Mol Evol 1998 47 81 92 9664803-17-0737/PL00006365 
Nelson JS  Fishes of the world 1994 3 New York: John Wiley & Sons 
Helfman G Collette B Facey D  The diversity of fishes 1997 Malden, MA: Blackwell Science 
Froese R Pauly D  FishBase World Wide Web electronic publication version (01/2007) 2007 
Chapleau F  Pleuronectiform relationships: a cladistic reassessment Bull Mar Sci 1993 52 516 540 
Cooper JA Chapleau F  Monophyly and intrarelationships of the family Pleuronectidae (Pleuronectiformes), with a revised classification Fish Bull 1998 96 686 726 
Hensley DA  An overview of the systematics and biogeography of the flatfishes J Sea Res 1997 37 187 803-17-0737/S1385-1101(97)00017-8 
Hoshino K  Monophyly of the Citharidae (Pleuronectoidei: Pleuronectiformes: Teleostei) with considerations of pleuronectoid phylogeny Ichthyol Res 2001 48 391 803-17-0737/s10228-001-8163-0 
Pardo BG Machordom A Foresti F Porto-Foresti F Azevedo MFC Bañón R Sánchez L Martínez P  Phylogenetic analysis of flatfish (Order Pleuronectiformes) based on mitochondrial 16S rDNA sequences Sci Mar 2005 69 531 543 
Infante C Catanese G Manchado M  Phylogenetic relationships among ten sole species (Soleidae, Pleuronectiformes) from the Gulf of Cadiz (Spain) based on mitochondrial DNA sequences Mar Biotechnol (NY) 2004 6 612 624 15747803-17-0737/s10126-004-3081-6 
Berendzen PB Dimmick WW  Phylogenetic relationships of Pleuronectiformes based on molecular evidence Copeia 2002 3 642 803-17-0737/0045-8511(2002)002[0642:PROPBO]2.0.CO;2 
Force A Lynch M Pickett FB Amores A Yan YL Postlethwait J  Preservation of duplicate genes by complementary, degenerative mutations Genetics 1999 151 1531 1545 10101175 
Sidow A  Gen(om)e duplications in the evolution of early vertebrates Curr Opin Genet Dev 1996 6 715 722 8994803-17-0737/S0959-437X(96)80026-8 
Steinke D Hoegg S Brinkmann H Meyer A  Three rounds (1R/2R/3R) of genome duplications and the evolution of the glycolytic pathway in vertebrates BMC Biol 2006 4 16 16756803-17-0737/1741-7007-4-16 
Volff JN  Genome evolution and biodiversity in teleost fish Heredity 2005 94 280 294 15674803-17-0737/sj.hdy.6800635 
Hoegg S Brinkmann H Taylor JS Meyer A  Phylogenetic timing of the fish-specific genome duplication correlates with the diversification of teleost fish J Mol Evol 2004 59 190 203 15486803-17-0737/s00239-004-2613-z 
Hoegg S Meyer A  Hox clusters as models for vertebrate genome evolution Trends Genet 2005 21 421 424 15967803-17-0737/j.tig.2005.06.004 
Moncaut N Somoza G Power DM Canario AV  Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships J Mol Endocrinol 2005 34 767 779 15956803-17-0737/jme.1.01757 
Sherwood NM Adams BA  Melamed P, Sherwood NM  Gonadotropin-Releasing hormone in fish: Evolution, expression and regulation of the GnRH gene Hormones and Their Receptors in Fish Reproduction 2005 4 Singapore: World Scientific Publishing Company 1 39 
Ribeiro L Sarasquete C Dinis MT  Histological and histochemical development of the digestive system of Solea senegalensis (Kaup, 1858) larvae Aquaculture 1999 171 293 803-17-0737/S0044-8486(98)00496-7 
Fernández-Díaz C Kopecka J Cañavate JP Sarasquete C Solé M  Variations on development and stress defences in Solea senegalensis larvae fed on live and microencapsulated diets Aquaculture 2006 251 573 803-17-0737/j.aquaculture.2005.06.014 
Gudmundsdottir A Palsdottir HM  Atlantic cod trypsins: from basic research to practical applications Mar Biotechnol (NY) 2005 7 77 88 15759803-17-0737/s10126-004-0061-9 
Warner JR  The economics of ribosome biosynthesis in yeast Trends Biochem Sci 1999 24 437 440 10542803-17-0737/S0968-0004(99)01460-7 
Funes V Asensio E Ponce M Infante C Cañavate JP Manchado M  Insulin-like growth factors I and II in the sole Solea senegalensis: cDNA cloning and quantitation of gene expression in tissues and during larval development Gen Comp Endocrinol 2006 149 166 172 16836995 
Jefferies HB Fumagalli S Dennis PB Reinhard C Pearson RB Thomas G  Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k EMBO J 1997 16 3693 3704 9218803-17-0737/emboj/16.12.3693 
Terada N Patel HR Takase K Kohno K Nairn AC Gelfand EW  Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins Proc Natl Acad Sci USA 1994 91 11477 11481 7972803-17-0737/pnas.91.24.11477 
Bao-Long B Gui-Mei Y Da-Ming R  Apoptosis in the metamorphosis of Japanese flounder Paralichthys olivaceus Acta Zool Sin 2006 52 355 361 
Bushell M Stoneley M Sarnow P Willis AE  Translation inhibition during the induction of apoptosis: RNA or protein degradation? Biochem Soc Trans 2004 32 606 610 15270803-17-0737/BST0320606 
Woolford JL JrWarner JR  Broach JR, Pringle JR, Jones EW  The ribosome and its synthesis The Molecular Biology and Cellular Biology of the Yeast Saccharomyces cerevisiae 1991 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 587 626 
Wool IG  Extraribosomal functions of ribosomal proteins Trends Biochem Sci 1996 21 164 165 8871803-17-0737/0968-0004(96)20011-8 
Douglas SE Knickle LC Kimball J Reith ME  Comprehensive EST analysis of Atlantic halibut (Hippoglossus hippoglossus), a commercially relevant aquaculture species BMC genomics  
Koski LB Gray MW Lang BF Burger G  AutoFACT: an automatic functional annotation and classification tool BMC Bioinformatics 2005 6 151 15960803-17-0737/1471-2105-6-151 
Tsui SK Lee SM Fung KP Waye MM Lee CY  Primary structures and sequence analysis of human ribosomal proteins L39 and S27 Biochem Mol Biol Int 1996 40 611 616 8908372 
Chan YL Suzuki K Olvera J Wool IG  Zinc finger-like motifs in rat ribosomal proteins S27 and S29 Nucleic Acids Res 1993 21 649 655 8441803-17-0737/nar/21.3.649 
Klein SL Strausberg RL Wagner L Pontius J Clifton SW Richardson P  Genetic and genomic tools for Xenopus research: The NIH Xenopus initiative Dev Dyn 2002 225 384 391 12454917 10.1002/dvdy.10174 
Swofford DL  PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods) 2003 Version 4 Sunderland, Massachusetts: Sinauer Associates 
Posada D Crandall KA  Modeltest: testing the model of DNA substitution Bioinformatics 1998 14 817 818 9918953 10.1093/bioinformatics/14.9.817


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