1320 Phylogenetic analysis was performed by Maximum Parsimony (Eck and Dayhoff 1966), using MEGA 4.0 software (Tamura et al. 2007) to build a tree with 1,000 bootstrap replicates based on vertebrate TauT AA sequen- ces. The sequence from the Human solute carrier SLC6A13 (neurotransmitter transporter, GABA; Genbank accession No. ENSP00000339260) was used as an outgroup. Taurine transporter expression analysis For analysis of TauT expression, RNA was isolated from Senegalese sole tissues (see ‘‘Fish sampling’’ for further information on sampled tissues). RNA quality was assessed on all samples using Agilent 2100 Bioanalyzer (Agilent Technologies) and only samples with a RNA integrity number (RIN) equal or higher than 7, on a scale of 1–10, were utilised for ﬁrst-strand cDNA synthesis, since a high RNA integrity seems to be crucial for obtaining meaningful gene expression data (Fleige and Pfafﬂ 2006). First-strand cDNA synthesis occurred according to the procedures previously described. Senegalese sole TauT mRNA expression was analysed by qPCR using a Chromo4 Con- tinuous Fluorescence Detector (Bio-Rad). The qPCR was performed in a volume of 25 ll containing cDNA gener- ated from 100 ng of original mRNA template, 300 nM of forward and reverse primers and 12.5 ll of SYBR Green PCR master mix (Applied Biosystems). Primers were designed to hybridise in different exons (Table 1), thereby avoiding contamination by genomical DNA and false positive signal in qPCR (Kubista et al. 2006). Genomic DNA contamination was also monitored in every assay using a sample free of reverse transcribed cDNA (minus RT control). The ampliﬁcation protocol used was: 94 C for 3 min and 40 cycles at 94 C for 30 s, 60 C for 30 s and 72 C for 30 s. Absence of primer dimers and non-speciﬁc products was veriﬁed in every qPCR assay by melting curve analysis (temperature reading every 0.2 C from 60 C until 95 C). qPCR analysis was conducted in triplicate reactions for all samples. Senegalese sole elongation factor 1 alpha isoform 1 (eEF1a1; Genbank accession No. AB326302) was used as an internal reference gene for normalisation of TauT mRNA expression. Ampliﬁcation of eEF1a1 was per- formed according to Infante et al. (2008), while cloning and sequencing occurred as previously described. Standard curves (tenfold dilution series) were generated for Sene- galese sole TauT and eEF1a1 by plotting the cycle threshold (Ct) obtained in qPCR analysis versus the loga- rithm of standard copy numbers (CN) and performing a linear regression (Bustin 2000). These curves were used to estimate efﬁciency in every qPCR assay, showing corre- lation coefﬁcients higher than 0.99 and a mean efﬁciency of 98.8 ± 1.9%. Senegalese sole TauT and eEF1a1 copy 123 W. Pinto et al. numbers were calculated for each sample based on the respective standard curve, using the following expression: CN = (Ct - intercept) 9 (slope)-1. TauT copy numbers were subsequently normalised: CNTauT 9 CNeEF1a1-1. Data analysis Results were expressed as mean ± standard deviation (SD). Data were initially tested by Levene’s test for homogeneity of variances. When homogeneity of variances was observed, detection of group mean differences was tested by one-way ANOVA followed by Tukey’s multiple comparison tests. Data were analysed through Kruskall– Wallis and Mann–Whitney U non parametric tests, when mean variances were signiﬁcantly different across groups. Data regarding Senegalese sole metamorphosis pattern were analysed through Chi-square tests. The signiﬁcance level was P B 0.05. All results expressed as a percentage were based on arcsine transformed data (Ennos 2007). Results Cloning of Senegalese sole taurine transporter The full coding cDNA sequence for Senegalese sole TauT was 1,895 bp long, with an open reading frame of 1,878 bp (Genbank accession No. HQ148721) encoding for a puta- tive protein with 625 AA and a predicted molecular weight of 70 kDa (Fig. 1). TauT structure comparisons and hydropathy analysis predicted 14 exons and 12 putative transmembrane helices, respectively (Supplemental Fig- ure 1). Three signature ﬁngerprints were identiﬁed among the AA residues of Senegalese sole TauT, corresponding to the sodium/chloride-dependent neurotransmitter trans- porter superfamily (eight elements), neurotransmitter symporter family (two elements) and taurine transporters (four elements) (Fig. 1 and Supplemental Figure 1). The multiple AA sequence alignment (Supplemental Figure 2) showed that TauT was very similar among Senegalese sole and the remaining vertebrates, sharing at least 76% identity with mammals and not less than 87% with other teleosts. The intracellular segment 4 (S4) was highly conserved among vertebrates, but changes in AA residues 319 (K) and 322 (C) were found for Senegalese sole (Supplemental Figures 1 and 2). C322 was also found in other ﬁsh, reptiles and birds, but a serine replacement was found in this position for mammals. Two major clades were found in the phylogenetic tree constructed with vertebrate TauT AA sequences, corre- sponding to a division between mammals and other ver- tebrates. Senegalese sole TauT was grouped with other teleost species (Supplemental Figure 3).