Cloning, tissue and ontogenetic expression of the taurine transporter 1319 Table 1 Primer sequences for ampliﬁcation and expression analysis of taurine transporter in S. senegalensis TauT cloning TauT cloning TauT qPCR eEF1a1 qPCR Primer sequence 50 ? 30 ATGGCACAGAAAGAGAAACTCCA (Fw) TTGCTGTTATGTAGACCACTTTCCC (Rev) TCGCTGTGTGGGTCATCTGC (Fw) CCTCTCACAGAGAGCGTTCAC (Rev) CCGAAAGCTGTGTCCATGATG (Fw) CAATAGAGGTGATCTGTCCTTCCA (Rev) GATTGACCGTCGTTCTGGCAAGAAGC (Fw) GGCAAAGCGACCAAGGGGAGCAT (Rev) Ampliﬁcation region (bp) Amplicon length (bp) 1–23 727–751 674–693 1,875–1,895 1,123–1,143 1,220–1,243 – 750 1,222 120 142 TauT taurine transporter, eEF1a1 elongation factor 1 alpha isoform 1, Fw forward primer, Rev reverse primer, qPCR real time quantitative PCR 2, 6, 12, 16, 20 and 30 DAH. The metamorphosis pattern was assessed according to Ferna´ndez-D´ıaz et al. (2001). These authors categorised ﬁve metamorphic stages, according to the eye migration status: 0, pre-metamorphic (symmetric larvae); 1, early metamorphic (beginning of left eye migration); 2, middle metamorphic (left eye touching the midline of the dorsal surface); 3, middle metamorphic (changing in the swimming plane and left eye migration within the ocular side); and 4, later metamorphic (comple- tion of left eye migration and visibility of orbital arch). For gene expression analysis whole larvae were col- lected at the same sampling points as above (2, 6, 12, 16, 20 and 30 DAH). From 12 DAH onwards, additional larvae were dissected to reveal the compartmental distribution of TauT; larvae were separated into three fractions: (1) head (including heart), (2) digestive tract with liver and (3) remaining body. Samples from whole and dissected larvae were rapidly transferred to RNAlater (Ambion) and stored at -80 C for later analysis. For further analysis of the organ and tissue distribution of the taurine transporter, Senegalese sole juveniles (94.1 ± 18.1 g wet weight; 17.6 ± 0.7 cm total length) were killed with a lethal dose of 2-phenoxiethanol (Sigma, St Louis, MO, USA). The ﬁsh were then dissected and the following samples were collected for gene expression analysis: brain, eye, gills, heart, head kidney, spleen, gallbladder, liver, stomach, midgut (divided into four equal fractions) and hindgut (divided into two equal fractions). The samples were stored in RNAlater at -80 C until analysis. Cloning and sequence analysis of Senegalese sole taurine transporter Total RNA (RNA) was extracted from midgut of juvenile Senegalese sole using TRI Reagent (Sigma). First-strand cDNA was subsequently synthesised from RNA (3 lg) using oligo (dT) primer from SuperScript III First-Strand Synthe- sis system for RT-PCR kit (Invitrogen). Ampliﬁcations were performed on the resulting cDNA using primer sets designed on Primer premier 5 (Biosoft international) and based upon Common carp (Cyprinus carpio) mRNA (Genbank acces- sion No. AB006986.1; Table 1). Ampliﬁcation reactions were performed using GoTaq PCR reagents (Promega) according to the manufacturer’s instructions and by the fol- lowing protocol: 95 C for 2 min followed by 32 cycles of 95 C for 30 s, 55 C for 45 s and 72 C for 90 s and a ﬁnal termination step of 72 C for 5 min. The ampliﬁed PCR products were visualised in an agarose gel (1%) stained with GelRed (Biotium), puriﬁed using a QIAquick Gel Extraction Kit (Qiagen) and cloned into the pCR4-TOPO vector (TOPO TA Cloning Kit, Invitrogen). Sequencing was performed at the University of Bergen Sequencing Facility (Bergen, Norway), using BigDye Terminator v3.1 chemistry in a ABI PRISM377 DNA sequencer (Applied Biosystems). Nucle- otide sequences were identiﬁed using the Basic Local Alignment Search Tool (BLAST) from GenBank database. The Senegalese sole TauT gene structure was deduced with Spidey software (http://www.ncbi.nlm.nih.gov/IEB/ Research/Ostell/Spidey) following identiﬁcation of intron/ exon splices boundary consensus sequences (AG/GT). To conﬁrm the predicted gene organisation, comparisons were performed with TauT sequence in other species using Ensembl database (http://www.ensembl.org/). Conserved domains present in protein sequence were identiﬁed using InterProScan (http://www.ebi.ac.uk/Tools/InterProScan/) and putative transmembrane domains were predicted using TMHMM 2.0, which is part of the Simple Modular Architecture Research Tool. Transmembrane protein 2D topology was created using TOPO2 software (Johns 2010). Multiple alignment of TauT AA sequences was per- formed with ClustalX (version 2.0.11) according to the following parameters: Gonnet series matrix, Gap opening penalty 10, Gap extension 0.2 (Thompson et al. 1997; Larkin et al. 2007). Alignments were displayed in Gene- Doc (www.psc.edu/biomed/genedoc), edited and percent- ages of sequence identity and similarity were calculated. 123