Hierarchical subfunctionalization of fabp1a, fabp1b and fabp10 tissue-specific expression may account for retention of these duplicated genes in the zebrafish (Danio rerio) genome Mukesh K Sharma1, Rong-Zong Liu1, Christine Thisse2, Bernard Thisse2, Eileen M DenovanWright3 and Jonathan M Wright1 Department of Biology, Dalhousie University, Halifax, NS, Canada ´ ´ ´ Institut de Genetique et de Biologie Moleculaire et Cellulaire, Department of Developmental Biology, Illkirch, France Department of Pharmacology, Dalhousie University, Halifax, NS, Canada Keywords embryonic development; FABP1; gene duplication; linkage mapping; subfunctionalization Correspondence J M Wright, Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4J1 Fax: +1 902 494 3736 Tel: +1 902 494 6468 E-mail: jmwright@dal.ca Database The following sequences have been submitted to the GenBank database under accession numbers DQ062095 (fabp1a cDNA), DQ062096 (fabp1b cDNA, long transcript variant) and DQ474062 (fabp1b cDNA, short transcript variant) (Received 30 January 2006, revised 10 April 2006, accepted 18 May 2006) doi:10.1111/j.1742-4658.2006.05330.x Fatty acid-binding protein type (FABP1), commonly termed liver-type fatty acid-binding protein (L-FABP), is encoded by a single gene in mammals We cloned and sequenced cDNAs for two distinct FABP1s in zebrafish coded by genes designated fabp1a and fabp1b The zebrafish proteins, FABP1a and FABP1b, show highest sequence identity and similarity to the human protein FABP1 Zebrafish fabp1a and fabp1b genes were assigned to linkage groups and 8, respectively Both linkage groups show conserved syntenies to a segment of mouse chromosome 6, rat chromosome and human chromosome harboring the FABP1 locus Phylogenetic analysis further suggests that zebrafish fabp1a and fabp1b genes are orthologs of mammalian FABP1 and most likely arose by a whole-genome duplication event in the ray-finned fish lineage, estimated to have occurred 200–450 million years ago The paralogous fabp10 gene encoding basic L-FABP, found to date in only nonmammalian vertebrates, was assigned to zebrafish linkage group 16 RT-PCR amplification of mRNA in adults, and in situ hybridization to whole-mount embryos to fabp1a, fabp1b and fapb10 mRNAs, revealed a distinct and differential pattern of expression for the fabp1a, fabp1b and fabp10 genes in zebrafish, suggesting a division of function for these orthogolous and paralogous gene products following their duplication in the vertebrate genome The differential and complementary expression patterns of the zebrafish fabp1a, fapb1b and fabp10 genes imply a hierarchical subfunctionalization that may account for the retention of both the duplicated fabp1a and fabp1b genes, and the fabp10 gene in the zebrafish genome The fatty acid-binding protein (FABP1), commonly termed liver-type fatty acid-binding protein (L-FABP), is a low molecular mass (14 kDa) polypeptide that belongs to the multigene family of intracellular lipid- binding proteins (iLBP) [1] At least 16 members of the iLBP multigene family have been described [2] Originally, FABPs were named according to their initial site of isolation, e.g intestinal-type fatty acid-binding Abbreviations EST, expressed sequence tag; FABP1, liver-type fatty acid-binding protein type 1; FABP10, basic liver-type fatty acid-binding protein; hpf, hours post fertilization; iLBP, intracellular lipid-binding proteins; mya, million years ago; 5¢-RLM-RACE, 5¢ RNA ligase-mediated-RACE; YSL, yolk syncytial layer 3216 FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS M K Sharma et al protein (I-FABP), heart-type fatty acid-binding protein (H-FABP), liver-type fatty acid-binding protein (L-FABP), etc [3] Many studies have shown, however, that different types of FABP are present in the same tissue [4] Moreover, some orthologous FABPs from different species exhibit different tissue-specific patterns of distribution Owing to the many names for the same or orthologous proteins, Hetzel & Bernlohr [5] have suggested an alternative nomenclature in which each FABP is referred to only by its gene designation, presumably reflecting the chronological order of their discovery, i.e FABP1 (liver-type), FABP2 (intestinal-type), FABP3 (heart ⁄ muscle-type), etc More recently, individual iLBPs have been classified according to phylogenetic analyses based on the amino acid sequences [6–8] We prefer the FABP nomenclature of Herzel & Bernlohr [5], and therefore use it here However, where appropriate, we also include the common name for FABPs Upper case letters are used to designate the protein, e.g FABP1, and lower case italic letters (e.g fabp1) and upper case italic letters (FABP1) are used to designate a gene coding for a particular FABP in zebrafish and mammals, respectively FABP1 is thought to be involved in the uptake of fatty acids [5], the modulation of enzyme activity by altering lipid levels [9], the sequestering of fatty acids to protect cells against the harmful detergent effects of excess free fatty acids [10], regulation of the expression of specific genes, and the control of cell growth and differentiation [11] In addition to long chain fatty acids, FABP1 binds lysophospholipids, prostaglandins, phytanic acid, eicosanoids, heme and acyl-CoAs [4,9,12] Several studies have shown that FABP1 binds two fatty acids per molecule [13–15], whereas other iLBPs bind only a single ligand [15,16] Previously, we reported the cDNA sequence for FABP10, the basic liver-type fatty acid-binding protein, and the distribution of the fabp10 gene transcripts in adult zebrafish [17] To date, the FABP10 protein, fabp10 gene and its encoded mRNA have been found only in nonmammalian vertebrates Like FABP1, FABP10 has the capacity to bind two ligand molecules, unlike all other iLBPs that bind a single molecule [18], suggesting that FABP1 and FABP10 are evolutionarily related, possibly by a gene duplication event Phylogenetic analysis of available iLBPs places FABP10 as a separate branch of an iLBP subfamily that includes FABP1 and FABP6, the ileal-type fatty acid-binding protein [19] It is estimated that the fabp1, fabp6 and fabp10 genes diverged from their last common ancestral gene  679 million years ago (mya) [2] We therefore anticipated that the zebrafish genome may harbor a functional Zebrafish duplicated fabp1 fabp1 gene Moreover, as a whole-genome duplication event is thought to have occurred in the ray-finned fish lineage after their divergence from the lobe-finned fish lineage 200–450 mya [20–25], we further predicted that duplicate copies of the fabp1 gene may be present in the zebrafish genome Here, we describe the cloning and sequence of cDNAs coded by the duplicated zebrafish genes for FABP1, hereafter referred to as cDNAs encoded by the fabp1a and fabp1b genes These duplicated fabp1 genes were assigned to different linkage groups using radiation hybrid mapping, and tissue-specific patterns of distribution for the fabp1a and fabp1b mRNAs were determined during embryonic and larval development, and in adulthood Results and Discussion Sequence of a zebrafish FABP1a cDNA Searches of the National Centre for Biological Information (NCBI) DNA sequence database revealed an expressed sequence tag (EST; GenBank accession number BI846703) coding for a protein described as being similar to human FABP1 3¢-RACE and 5¢ RNA ligase-mediated-RACE (5¢-RLM-RACE) using primers based on this EST were performed to obtain the cDNA sequence A single product of 759 bp, excluding the 20 bp adapter sequence and the poly(A) tail, was obtained in 3¢-RACE and a single major product of 91 bp (excluding the 38 bp 5¢-RLM-RACE adapter sequence) was obtained in 5¢-RLM-RACE (data not shown) Three clones for each 3¢-RACE and 5¢-RLMRACE product were sequenced on both strands Sequence analysis showed that the zebrafish fabp1a cDNA was 827 bp, excluding the poly(A) tail (Fig 1A) An ORF of 384 bp from nucleotide 58 to 441, including the stop codon, was identified that codes for a polypeptide of 127 amino acids with a molecular mass of  14.1 kDa and a calculated isoelectric point of 4.97 The 5¢- and 3¢-UTR were 57 and 386 bp, respectively A polyadenylation signal (AAT AAA) was located from nucleotide 809 to 814 In one of the 5¢-RLM-RACE clones, the nucleotide at position 70 was an adenine in place of guanine resulting in a change in the encoded amino acid from Gly to Arg (Fig 1A) This nucleotide difference at position 70 in the 5¢-RLM-RACE clones for FABP1a may represent a polymorphism or a PCR artifact Analysis of zebrafish fabp1a cDNA using blastx revealed sequence similarity to FABP1 sequences from other species available in the NCBI database Alignment of the human FABP1 sequence to the deduced FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3217 Zebrafish duplicated fabp1 M K Sharma et al A B Fig Sequences of cDNAs coding for zebrafish FABP1a and FABP1b (A) The 827 bp fabp1a cDNA sequence, excluding the poly(A) tail, was determined by cloning and sequencing of 3¢-RACE and 5¢-RLM-RACE products The cDNA sequence contained an ORF of 384 nucleotides coding for a polypeptide of 127 amino acids with the identity of the amino acid sequence shown below the nucleotide sequence The stop codon is underlined A variation between sequenced 5¢-RLM-RACE products is shown in bold with the variation indicated above A polyadenylation signal sequence, AATAAA, is in bold italic font The NCBI GenBank accession number for this sequence is DQ062095 (B) The 498 bp (long transcript variant) or 467 bp (short transcript variant) fabp1b cDNA sequences were determined by cloning and sequencing of 3¢-RACE and 5¢-RLM-RACE products The ORF of 387 bp encodes a polypeptide of 128 amino acids with the amino acid sequence shown below the nucleotide sequence Variation between the 5¢-RLM-RACE products is shown in bold with the variation indicated above A polyadenylation signal sequence, ATTAAA, is in bold italics The two transcription start sites determined from the mature and capped mRNA using 5¢-RLM-RACE are indicated by * The stop codon is underlined The NCBI GenBank accession number for these nucleotide sequences are DQ062096 (long transcript variant) and DQ474062 (short transcript variant) zebrafish FABP1a sequence (Fig 2) using bioedit sequence alignment editor (blosum62 similarity matrix, v 5.0.9) [26] revealed 64% sequence identity and 83% sequence similarity to human FABP1, suggesting that the cDNA clone codes for a FABP1 in zebrafish Phylogenetic analysis using clustalx [27] strongly supports the inclusion of zebrafish FABP1a in the same clade as FABP1s from other species (boot strap value ¼ 1000; Fig 3) suggesting that the zebrafish fabp1a gene is an ortholog of the mammalian FABP1 gene 3218 Identification of a cDNA sequence coding for a second zebrafish FABP1, FABP1b The cDNA sequence coded by a duplicated fabp1 gene in zebrafish was identified and determined using 3¢-RACE and 5¢-RLM-RACE with mRNA-specific primers based on a zebrafish EST (GenBank accession number BQ075349) described as being similar to the human FABP1, hereafter referred to as the fabp1b cDNA A single product of 378 bp, excluding the 20 bp adapter sequence and the poly(A) tail, was FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS M K Sharma et al Zebrafish duplicated fabp1 Fig Sequence alignment of zebrafish FABP1a and FABP1b with human FABP1 The amino acid sequences of the human FABP1 (Hu-FABP1; GenBank accession number P07148), zebrafish FABP1a (Zf-FABP1a) and zebrafish FABP1b (Zf-FABP1b) were aligned using the BLOSUM62 similarity matrix in BIOEDIT sequence alignment editor (v 5.0.9) [25] Dots indicate identity and dashes were introduced to maximize alignment The amino acid sequences that determine higher-order structure are shown in bold and labeled: aI, aII, a helix I and II; bA to J, b sheet A to J The percentage sequence identity and percentage sequence similarity between the human FABP1 and zebrafish FABP1a, and FABP1b are shown at the end of each sequence obtained in 3¢-RACE Two products of 343 and 312 bp (excluding the 38 bp 5¢-RLM-RACE adapter sequence) were obtained in 5¢-RLM-RACE for the fabp1b mRNA owing to the presence of two transcription start sites for the fabp1b gene (see Fig 4B and discussion below) Three clones were sequenced for each 3¢-RACE and 5¢-RLM-RACE product Sequence analysis of the fabp1b 3¢-RACE and 5¢-RLM-RACE products revealed that the fabp1b cDNA was either 498 bp (long transcript variant) or 467 bp (short transcript variant), excluding the poly(A) tail, depending on the transcription initiation site utilized (Fig 1B, see Fig 4B) All nucleotide positions for fabp1b cDNA mentioned here refer to nucleotide positions in the fabp1a long transcript variant A putative polyadenylation signal, ATTAAA, is located from nucleotide 479 to 484 (Fig 1B) An ORF of 387 bp from nucleotide 53 to 439, including the stop codon, was identified that codes for a polypeptide of 128 amino acids with a molecular mass of  14.1 kDa and a calculated isoelectric point of 5.53 In one of the 5¢-RLM-RACE clones, the nucleotide at position 99 was thymine in place of adenine, resulting in a change in the encoded amino acid from Glu to Val (Fig 1B) The difference in sequence between the 5¢-RLM-RACE clones for FABP1b may be a polymorphism or an artifact of PCR Alignment of the human FABP1 sequence with the deduced zebrafish FABP1 sequence using the bioedit sequence alignment editor (blosum62 similarity matrix, v 5.0.9) [26] revealed 60% identity and 78% similarity with human FABP1 (Fig 2), suggesting that the cDNA clone codes for a second FABP1 in zebrafish, referred to here as FABP1b As with zebrafish FABP1a, phylogenetic analysis using clustalx [27] strongly supports the inclusion of zebrafish FABP1b in the same clade as FABP1s from other species (boot strap value ¼ 1000; Fig 3) This suggests that zebrafish fabp1a and fabp1b genes are orthologs of the mammalian FABP1 gene, and the duplicated copies of the fapb1 genes in zebrafish most likely arose as a result of a whole-genome duplication event in the ray-finned fish lineage [20–25] We noted that the amino acid sequences for zebrafish FABP1a and FABP1b encoded by the sister duplicate genes, fabp1a and fabp1b, not cluster as closely as we observed for proteins encoded by other duplicated gene copies in this zebrafish multigene family, e.g FABP7a and FABP7b, or CRABP1a and CRABP1b [7,28] An explanation for this may be a reflection of the different rates of amino acid substitution in FABP1a compared with FABP1b (or vice versa) owing to reduced selective pressure on one of the duplicated genes Alternatively, the topography of the tree may change with the addition of other fish FABP1s in a phylogenetic analysis when these sequences become available Zebrafish FABP10 (basic liver-type FABP) formed a separate clade, along with FABP10s of other nonmammalian vertebrates, suggesting that FABP10 and FABP1 are paralogs The presence of fabp1 and fabp10 genes in zebrafish and other nonmammalian vertebrates is likely due to a duplication event, presumably predating the divergences of mammals, birds, fishes, reptiles and amphibians [2] Absence of the FABP10 gene in mammals is likely due to loss of the FABP10 gene function in mammals, which was subsequently acquired by the FABP1 gene (see below) FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3219 Zebrafish duplicated fabp1 M K Sharma et al Fig Phylogenetic relationship of zebrafish FABP1a, FABP1b and FABP10 in the iLBP multigene family The bootstrap neighbor-joining phylogenetic tree was constructed with CLUSTALX [26] using human Von Ebner’s gland protein (Hu-LCN1, GenBank accession number NP_002288) that belongs to the lipocalin family of the calycins as an out-group Bootstrap values supporting the branch points are shown as number per 1000 duplicates Branch points supported by a bootstrap value of at least 700 are indicated The inclusion of zebrafish FABP1a (Zf-FABP1a) and FABP1b (Zf-FABP1b) in the FABP1 clade is highly supported *Indicates the duplication event and subsequent divergence of the fabp1, fabp6 and fabp10 genes 679 mya; ** indicates the whole-genome duplication event giving rise to the fabp1a and fabp1b in zebrafish (ray-finned fishes)  200–450 mya and the subsequent subfunctionalization of gene function The sequences used in the analysis include: human FABP2 (Hu-I-FABP; GenBank accession number P12104), zebrafish FABP2 (Zf-FABP2; AF180921), cow FABP5 (Co-FABP5; P55052), human FABP5 (Hu-FABP5; Q01469), cow FABP4 (Co-FABP4; P48035), human FABP4 (Hu-FABP4; P15090), cow FABP3 (Co-FABP3; CAA31212), human FABP3 (Hu-FABP3; P05413), human FABP7 (Hu-FABP7; O15540), zebrafish FABP7a (Zf-FABP7a; AF237712), mouse FABP9 (Mo-FABP9; O08716), rat FABP9 (Ra-FABP9; P55054), human CRABP1 (Hu-CRABP1; NM_004378), human CRABP2 (Hu-CRABP2; M68867), human CRBP1 (Hu-CRBP1; NP_002890), human CRBP2 (Hu-CRBP2; P50120), pufferfish FABP1 (Pf-FABP1; AAC60290), orange-spotted grouper FABP1 (Og-FABP1; AAM22208), cow FABP1 (Co-FABP1; P80425), human FABP1 (Hu-FABP1; P07148), pig FABP1 (Pi-FABP1; P49924), rat FABP1 (Ra-FABP1; P02692), mouse FABP1 (Mo-FABP1; Y14660), chicken FABP1 (Ch-FABP1; AAK58095), salamander FABP (Sa-FABP1; P81399), pig FABP6 (Pi-FABP6; P10289), human FABP6 (Hu-FABP6; NP51161), mouse FABP6 (Mo-FABP6; NP51162), rat FABP6 (Ra-FABP6; P80020), lungfish FABP10 (Lf-FABP10; P82289), salamander FABP10 (Sa-FABP10; P81400), toad FABP10 (To-FABP10; P83409), iguana FABP10 (Ig-FABP10; U28756), chicken FABP10 (Ch-FABP10; P80226), zebrafish FABP10 (Zf-FABP10; AF254642), catfish FABP10 (Cf-FABP10; P80856) and shark FABP (Sh-FABP10; P81653) Scale bar ¼ 0.1 substitutions per site 3220 FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS M K Sharma et al Zebrafish duplicated fabp1 A B C Fig Transcription start sites for the zebrafish fabp1a, fabp1b and fabp10 genes (A) The 5¢ upstream sequence of the initiation codon for the zebrafish fabp1a gene was obtained from zebrafish genome database at the Wellcome Trust Sanger Institute (contig: ctg30243.1, assembly Zv2, http://www.sanger.ac.uk ⁄ Projects ⁄ D_rerio ⁄ ) The transcription start site was identified from the capped and mature fabp1a mRNA by 5¢-RLM-RACE and is marked by an arrow over the * sign An A-rich region that may serve a similar function as a TATA box is in bold font and underlined The initiation codon is boxed, the sequence of intron is in and lower case italics, and the sequence 5¢ upstream of the transcription start site is in lower case The six CA dinucleotide repeats not present in the genomic sequence, but present in the 5¢RLM-RACE sequence, are underlined A single nucleotide variation between the 5¢-RLM-RACE sequence and the genomic sequence is in bold with the variation indicated above (B) The zebrafish fabp1b sequence, 5¢ upstream of the initiation codon, was obtained from the zebrafish genome database at the Wellcome Trust Sanger Institute (scaffold 1725, assembly Zv4, http://www.sanger.ac.uk/Projects/D_rerio/) The two transcription start sites obtained from the sequences of 5¢-RLM-RACE using capped and mature fabp1b mRNA are marked by arrows over the * sign Putative TATA boxes are in bold and underlined The initiation codon is boxed and the sequence 5¢ upstream of the second transcription start site is in lower case A single nucleotide variation between the 5¢-RLM-RACE sequence and the genomic sequence is in bold above the sequence (C) The single transcription start site of the zebrafish fabp10 gene was determined by amplifying the capped and mature fabp10 mRNA by 5¢-RLM-RACE and aligning the sequence with the genomic sequence obtained from GenBank (accession number AF512998) The transcription start site is marked by an arrow over the * sign A putative TATA box is in bold and underlined The initiation codon is boxed and the sequence 5¢ upstream of the transcription start site is in lower case FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3221 Zebrafish duplicated fabp1 M K Sharma et al Amino acid substitutions involved in ligand binding by FABP1 FABP1 has been reported to bind a broad range of ligands including heme, bilirubin and certain eicosanoids FABP1 is the only iLBP that forms a complex with two fatty acid molecules at the same time [13,15,29] One ligand molecule, located at the bottom of the protein cavity in a bent conformation, is coordinated via an extensive hydrogen-bonding network Residues S39, R122 and S124 have been shown to be involved in the network [30–33] Based on amino acid sequence alignment (Fig 2), the S39 residue is identical in human FABP1, zebrafish FABP1a and FABP1b Residue R122 in human FABP1 corresponds to residue R121 in zebrafish FABP1a and R122 in zebrafish FABP1b Residue S124 corresponds to S123 in zebrafish FABP1a and S124 in zebrafish FABP1b (Fig 2) Therefore, all three residues shown to be involved in ligand binding to the primary site (site 1) of FABP1 have been conserved in human and zebrafish FABP1s The second ligand molecule in FABP1 adopts a rather linear shape, with the solvent-exposed carboxylate end sticking out of the fatty acid portal The poorly delimited ‘portal’ region consists of a helix II and the turns connecting b strands C and D, as well as E and F (Fig 2) The hydrogen-bonding network involved in ligand binding at site involves K31, A54, S56, D88¢ and bound water [34] Residue K31 is identical in human FABP1 and zebrafish FABP1a and FABP1b Residue A54 corresponds to A54 in zebrafish FABP1a and to T54 in zebrafish FABP1b The human FABP1 residue S56 corresponds to T56 in zebrafish FABP1a and S56 in FABP1b There are no residues in zebrafish FABP1a and FABP1b that correspond to residue D88¢ The interaction of amino acid D88¢ is due to crystal packing from a symmetry-related molecule and is of lesser importance [34] Variations in the residues involved in the binding at site of the human FABP1, and possibly in zebrafish FABP1a and zebrafish FABP1b, may reflect differences in the binding affinities of the zebrafish FABP1a and zebrafish FABP1b to the ligand molecules Transcription start sites for fabp1a, fabp1b and fabp10 genes Amplification of the capped and mature 5¢-ends of the fabp1a, fabp1b and fabp10 mRNA transcripts from adult zebrafish RNA by 5¢-RLM-RACE identified the transcription start site(s) for the zebrafish fabp1a, fabp1b and fabp10 genes 3222 One abundant product of 91 bp (excluding the 38 bp 5¢-RLM-RACE adapter sequence) was detected in the reaction with CIP ⁄ TAP-treated RNA amplified in nested PCR with the adapter primer and the fabp1a mRNA-specific primer (data not shown) In order to determine the transcription start site of the zebrafish fabp1a gene, we searched the zebrafish genome database (assembly Zv2) at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/D_rerio/) using the zebrafish fabp1a cDNA sequence and identified a contig, ctg30243.1, containing sequence of the zebrafish fabp1a gene Alignment of the 5¢-RLMRACE sequence with the zebrafish fabp1a gene sequence localized the transcription start site 407 bp upstream of the initiation codon (Fig 4A) An additional intron was identified in the 5¢-UTR of the zebrafish fabp1a gene by sequence alignment of the cDNA and genomic sequences, thereby generating a fifth exon, exon A fifth exon is unprecedented in members of the vertebrate iLBP multigene family (Evidence for an additional intron in the zebrafish fabp1a gene is provided below.) Alignment analysis also revealed that the 5¢-RLMRACE product contained a string of 10 ‘CA’ repeats, whereas the genomic sequence had only four ‘CA’ dinucleotide repeats in exon (Fig 4A) The difference in the number of ‘CA’ repeats may represent a zebrafish strain-specific or individual polymorphism Dinucleotide repeats are highly polymorphic that have been used to detect genetic variation in a number of fish species [35,36] Two transcription start sites for the zebrafish fabp1b gene were mapped by 5¢-RLM-RACE Sequencing of 5¢-RLM-RACE products for the fabp1b gene revealed that the two products are 343 and 312 bp (excluding the 38 bp 5¢-RLM-RACE adapter sequence) Alignment of the 5¢ upstream sequence of the zebrafish fabp1b gene (scaffold 1725, assembly Zv4, http:// www.sanger.ac.uk/Projects/D_rerio/) with the fabp1b 5¢-RLM-RACE sequences localized the two transcription start sites at 21 and 52 bp upstream of the initiation codon (Fig 4B) A single band of  125 bp was produced by 5¢-RLM-RACE using primers specific for the fabp10 mRNA (data not shown) The band was isolated and purified following size-fractionation by agarose gel electrophoresis Sequencing of randomly selected clones revealed that the cDNA end was 90 bp, excluding the 38 bp 5¢-RLM-RACE adapter sequence Alignment of the fabp10 5¢-RLM-RACE product and fabp10 genomic sequence reported by Her et al [37] (GenBank accession number AF512998) identified the transcription start site of the zebrafish fabp10 FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS M K Sharma et al gene at 27 bp upstream of the initiation codon (Fig 4C) Putative TATA boxes were identified using matinspector [38] and visual inspection at position 24 to 30 bp 5¢ upstream of the first transcription start site that is nearest to the initiation codon in fabp1b and at 22 to 29 bp upstream of the second transcription start site that is located further from the initiation codon in fabp1b (Fig 4B) A TATA box is present at position 25 to 31 bp 5¢ upstream of transcription start site in fabp10 (Fig 4C) No TATA-like box was identified at  25 bp upstream of the transcription start site in fabp1a (Fig 4A) The absence of a TATA box in the promoter region of eukaryotic genes is not uncommon, with  50% or more gene promoters lacking TATA boxes [39] An A-rich region, however, is evident in the fabp1a gene 25–31 bp 5¢ upstream of transcription start site that may well serve a similar function as a TATA box Evidence for a fifth exon in the zebrafish fabp1a gene An additional intron of 351 bp was identified in the 5¢-UTR of the zebrafish fabp1a gene, thereby generating an unprecedented five exons in a vertebrate iLBP gene, consisting of exons 1–4, plus exon (Fig 4A) Evidence to support this contention is: (a) none of the expressed sequence tags coding for zebrafish FABP1a in the NCBI database (GenBank accession numbers DN899828, DN893893, DN893404, BI846703, DN893348, DN893665 and CR929576) contains this 351 bp sequence; (b) the exon ⁄ intron splice junctions conform to the GT ⁄ AG rule [40]; and (c) a PCRamplified DNA fragment of  650 bp was generated using primers based on a sequence 5¢ upsteam of exon and a sequence within exon 1, and zebrafish genomic DNA as template (see Experimental procedures) A product of this size could only be generated from genomic DNA if it contained an intron of  350 bp (data not shown) Intron identified in the 5¢-UTR of the zebrafish fabp1a gene was not present in either the fabp1b or fabp10 genes (Fig 4A–C) The rat FABP1 gene, the only FABP1 for which a transcript start site has been mapped to provide for a full cDNA and gene sequence [30], does not contain an additional exon within the 5¢-UTR All vertebrate iLBP genes sequenced to date contain four exons interrupted by three introns, with each exon exhibiting a similar coding capacity [1,2] No intron has been reported in the 5¢-UTR for any other iLBP genes As an additional intron is not found in the zebrafish fabp1b gene, a possible explanation for Zebrafish duplicated fabp1 the presence of intron in the zebrafish fabp1a gene is an insertional mutation that occurred after the wholegenome duplication event which followed the divergence of the ray- and lobe-finned fish lineages [19–24] An alternative explanation is that the insertion in the 5¢-UTR of the fabp1a gene occurred in zebrafish independent of other fish lineages Analysis of fabp1a and fabp1b genes in various fish species would likely resolve the timing of the insertion of intron in the fabp1a gene Linkage group assignment of zebrafish fabp1a, fabp1b and fabp10 genes using radiation hybrid mapping The fabp1a gene was assigned to linkage group at a distance of 10.2 cR from marker Z22208 with a LOD of 14.5 using the LN54 panel of radiation hybrids [41] and primers specific to the fabp1a gene Using the same radiation hybrid panel and gene-specific primers, the fabp1b gene was assigned to linkage group at a distance of 4.81 cR from marker Z10731 with a LOD of 16.2, and the fabp10 gene was assigned to linkage group 16 at a distance of 8.23 cR from marker Z10256 with a LOD of 17.1 [mapping data available at ZFIN (http://zfin.org/) for fabp10 and upon request for fabp1a and fabp1b] Both zebrafish fabp1 genes showed conserved syntenies with the mouse FABP1 gene on chromosome (position 30 cM), the rat FABP1 gene on chromosome (position 4q32) and the human FABP1 gene on chromosome (position 2p11) [2] (Table 1) This provides further evidence that the duplicated zebrafish fabp1a and fabp1b genes are orthologs of the mammalian FABP1 gene Based on sequence similarities, phylogenetic analysis, linkage group assignment and conserved syntenies, we can draw the following conclusions regarding the fabp1a, fabp1b and fabp10 genes: (a) the zebrafish fabp1a and fabp1b genes are orthologs of the mammalian FABP1 gene, and the two fabp1 genes arose as a result of a duplication event, most probably the wholegenome duplication that occurred in the ray-finned fish lineage after their divergence from lobe-finned fishes some 200–450 mya [20–25]; (b) the zebrafish fabp1 genes are the paralogs of the fabp10 gene and the proto-fabp1 gene diverged from the fabp10 gene before the fish-tetrapod split,  679 mya [2] Tissue-specific distribution of the fabp1a, fabp1b and fabp10 gene transcripts in adult zebrafish We analyzed the tissue distribution of the zebrafish fabp1a and fabp1b gene transcripts by RT-PCR using FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3223 Zebrafish duplicated fabp1 M K Sharma et al Table Conserved synteny of zebrafish fabp1a and fabp1b genes with their mammalian ortholog, Fabp1 –, Data not available Chromosomal ⁄ linkage group assignment Zebrafish Gene Name Fabp1 fatty acid binding protein 1, liver Smyd1 Vamp5 Vamp8 Usp39 Mobk1b Hk2 Pax8 Mcm6 Adra2b Atoh1 Grid2 Xpc Brpf1 Plxnd1 Tuba8 SET and MYND domain containing vesicle-associated membrane protein vesicle-associated membrane protein ubiquitin specific peptidase 39 Mps one binder kinase activator-like 1B hexokinase paired box gene minichromosome maintenance deficient adrenergic receptor, alpha 2b atonal homolog glutamate receptor, ionotropic, delta xeroderma pigmentosum, complementation group C bromodomain and PHD finger containing, plexin D1 tubulin, alpha total RNA extracted from various adult tissues as a template A fabp1a- and a fabp1b-specific product were generated by RT-PCR from total RNA extracted from adult zebrafish intestine No fabp1a- or fabp1b-specific RT-PCR product was generated from total RNA extracted from liver, brain, testis, muscle, heart, skin or ovary of the adult zebrafish (Fig 5) Product from the constitutively expressed rack1 mRNA was amplified by RT-PCR and used as a positive control for each RNA tissue sample assayed (Fig 5) Di Pietro et al [42] studied the expression of the fabp1 gene in catfish tissues by western blot analysis using antibodies raised against rat FABP1 Catfish fabp1 expression is restricted to the intestine as was observed by RT-PCR analysis of fabp1a and fabp1b mRNA in the adult zebrafish FABP1 mRNA is found in adult rat intestine and liver [43], whereas in adult zebrafish, no fabp1a or fabp1b gene transcripts were detected in the liver (Fig 5) A fabp10-specific product was generated from total RNA extracted from liver, intestine and testis of adult zebrafish (Fig 5) Total RNA extracted from brain, muscle, heart, ovary and skin did not generate a fabp10-specific product by RT-PCR (Fig 5) In an earlier report, we described the detection of fabp10 mRNA in the liver of adult zebrafish using tissuesection in situ hybridization [17] The highly sensitive RT-PCR assay, however, also detected fabp10 gene transcripts in intestine and testis fabp1 (FABP1), a paralogous gene phylogenetically closely related to fabp10, is believed to have arisen by duplication of an 3224 (fabp1a) (fabp1b) 5 5 5 5 8 8 8 Mouse Rat Human (30 cM) 4q32 2p11 6 6 6 2 6 6 6 4q32-q33 4q33 4q33 4q33 4q34 4q34 3p13 – 3q36 – 4q31 4q34 4q42 4q42 – 2p11.2 2p11.2 2p12-p11.2 2p11.2 2p13.1 2p13 2q12-q14 2q21 2p13-q13 4q22 4q22 3p25 3p26-p25 1p32-p31 22q11.1 (30.5 cM) C1 (31.5 cM) (31.5 cM) C3 C3 (34.5 cM) (13.5 cM) (66.6 cM) (71.0 cM) (29.69 cM) (29.65 cM) D E3 E3 F1 Fig RT-PCR analysis of tissue-specific distribution of the fabp1a, fabp1b and fabp10 mRNAs in adult zebrafish RT-PCR generated a fabp1a and fabp1b mRNA-specific product from total RNA extracted from adult zebrafish intestine (I) No fabp1a or fabp1b mRNAspecific product was generated from adult zebrafish liver (L), heart (H), muscle (M), ovary (O), skin (S), brain (B), testes (T) or the negative control (–) lacking total RNA derived from a whole zebrafish in the RT-PCR (1 and 2) RT-PCR generated a fabp10 mRNA-specific product from total RNA extracted from adult zebrafish liver (L), intestine (I) and testis (T) No fabp10 mRNA-specific product was generated from adult zebrafish heart (H), muscle (M), ovary (O), skin (S), brain (B), or the negative control (–) (3) A rack1 mRNAspecific product was generated from all the adult zebrafish tissues analyzed (4) ancestral gene,  679 mya [2], and is expressed in the intestine and liver of adult rats [43] The fabp10 gene or its product has not been detected in mammals However, both fabp1 and fabp10 are expressed in nonmammalian vertebrates including fish, amphibians, FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS M K Sharma et al reptiles and birds Detection of fabp1a and fabp1b gene transcripts in zebrafish intestine, and fabp10 gene transcripts in zebrafish liver shows a division of geneexpression patterns, or subfunctionalization [44,45], of the gene-expression patterns exhibited by the mammalian FABP1 gene As such, subfunctionalization of the fabp1 and fabp10 genes may explain the retention of these paralogous genes, at least in zebrafish, and possibly in many nonmammalian genomes Developmental expression of fabp1b and fabp10 genes during zebrafish embryogenesis Whole-mount in situ hybridization to zebrafish embryos did not detect fabp1a mRNA in any of the embryonic stages investigated (data not shown) fabp1b gene transcripts were detected at the beginning of somitogenesis, i.e 11 h post fertilization (hpf), in the yolk syncytial layer (YSL) (Fig 6A) An increase in the intensity of the hybridization signal suggested that fabp1b transcripts were more abundant in the YSL Zebrafish duplicated fabp1 at 17 hpf (Fig 6B), at 24 hpf (Fig 6C) and then at 36 hpf (Fig 6D) At 48 hpf, fabp1b transcripts were detected in the intestinal bulb and the YSL (Fig 6E,F) In 5-day-old larvae, fabp1b transcripts continued to be abundant in the intestinal bulb (Fig 6G,H), but were also detected in the anterior part of intestine (Fig 6G) No fabp1b transcripts were detected in the liver of the developing zebrafish An antisense RNA probe prepared from zebrafish fabp10 cDNA did not detect fabp10 transcripts in the embryos from the gastrula ( hpf) up to 36 hpf (data not shown) This suggests that the fabp10 gene product, FABP10, most likely does not play an important role in the early stages of liver morphogenesis, at least not for a few hours after the developing hepatocytes have aggregated ( 28 hpf) [46] The weak detection of fabp10 gene transcripts in the ventral endoderm (near the heart chamber) of the 36 hpf zebrafish embryos [47] was not observed in this study The difference between the first detection of fabp10 mRNA reported here and that of Her et al [47] may Fig Detection of fabp1b and fabp10 mRNAs by whole-mount in situ hybridization during zebrafish embryogenesis and larval development fabp1b mRNA was detected in: (A) the YSL at the 3-somite stage (11 hpf); (B) 16-somite stage (17 hpf) embryo in the YSL; (C) in the YSL at 24 hpf embryo; increased levels in the YSL (D) at 36 hpf; and (E) at 48 hpf embryo compared with (C); (F) dorsal view of embryo shown in (E) showing expression in YSL and intestinal bulb (Ib); (G) larvae at days of development showing fabp1b mRNA restricted to intestinal bulb and anterior part of intestine (Apoi); and (H) the dorsal view of embryo shown in (G) showing localization of fabp1b mRNA in intestinal bulb fabp10 mRNA was detected in (I) liver (L) of the 48 hpf zebrafish embryos (J) Dorsal view of 48 hpf embryos showing expression in the liver (L); (K) in the liver (L) of 5-day-old larvae; and (L) dorsal view of larvae at days of development showing fabp10 mRNA in the liver (L) FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3225 Zebrafish duplicated fabp1 M K Sharma et al reflect zebrafish strain-specific differences At 48 hpf, the approximate time when the liver budding process is complete [46], fabp10 transcripts were detected in the liver of the zebrafish embryos (Fig 6I,J) Abundant fabp10 transcripts were also detected in the liver of 5-day-old larvae (Fig 6K,L) In the intestine and liver of the rat embryo, FABP1 mRNA is first detected at day 17–19 of gestation (late fetal life) [48,49] A proximal-to-distal gradient in the levels of rat FABP1 mRNA is established in the intestine during the late fetal stage The mRNA concentration in the intestine increases sharply by three- to four-fold within 24 h of birth and increases another two-fold during the suckling period In hepatocytes, FABP1 mRNA is induced during the first postnatal day, but remains relatively constant during the suckling and weaning period, up to 35 days post partum [48] Whole-mount in situ hybridization analyses demonstrated that the zebrafish fabp1a mRNA reported here is not detected during embryogenesis and therefore may not perform a function equivalent to mammalian FABP1 during zebrafish embryogenesis The distinct patterns of expression for the fabp1b (in the intestine) and fabp10 (in the liver) genes in the developing zebrafish combined represent the expression pattern of the mammalian FABP1 gene, presumably the expression pattern of the ancestral gene Again, this suggests subfunctionalization [44,45] of the ancestral gene following duplication, which may have contributed to the retention of the duplicated fabp1 and fabp10 genes at least in the zebrafish genome, and possibly in the genome of many other nonmammalian vertebrates Experimental procedures 3¢-RACE and 5¢-RLM-RACE to obtain the fabp1a and fabp1b cDNA sequences 3¢-RACE was employed [39] using total RNA extracted from adult zebrafish and primers specific to the zebrafish fabp1a cDNA sequence (outer: 5¢-GGGATCTCCTGA AGCTGAAC-3¢; nucleotides 9–28, Fig 1A and inner: 5¢-TGGGAAATATCAGCTGGAGTC-3¢; nucleotides 69–89, Fig 1A), or to the zebrafish fabp1b cDNA (outer: 5¢-AG CTGGAGAGTCAAGAGGG-3¢; nucleotides 75–93, Fig 1B and inner: 5¢-TCTTCCTGACGACATGATTG-3¢; nucleotides 121–140, Fig 1B) PCR products from 3¢-RACE were cloned into the pGEM-T vector system (Promega, Madison, WI) and sequenced as described in Liu et al [50] 5¢-RLM-RACE was employed [50] using total RNA extracted from adult zebrafish and primers specific to the zebrafish fabp1a cDNA (outer: 5¢-CGTCTGCTGATCCT CTTGTAG-3¢; nucleotides 431–411, Fig 1A and inner: 3226 5¢-CGACCTCATCATCCGGCAC-3¢; nucleotides 145–127, Fig 1A), to the zebrafish fabp1b cDNA (outer: 5¢-GTGTGTTTGCTCAGCTCATG-3¢; nucleotides 462–443, Fig 1B and inner: 5¢-GATTCTGTTCAGCACCACCT-3¢; nucleotides 343–324, Fig 1B), or to zebrafish fabp10 cDNA (outer: 5¢-TGGTGGTGATTTCAGCCTC-3¢; nucleotides 232–214, GenBank accession number AF254642 and inner: 5¢-GGCTCTGAGAAACTCCTCGT-3¢; nucleotides 75–56, GenBank accession number AF254642) PCR products from 5¢-RLM-RACE were cloned into the pGEM-T vector system (Promega) and sequenced as described in Liu et al [50] PCR amplification of intron in the fabp1a gene Primers based on the sequence 5¢ upstream of exon (sense: 5¢-GTCGTGAGAAAGCGGAAAC-3¢; nucleotides )176 to )194, Fig 4) and the sequence of exon (antisense: 5¢-AT GCCTCAAAGTTCTCGTG-3¢; nucleotides 91–109, Fig 1A) were used to amplify intron of the fabp1a gene from zebrafish genomic DNA using the following conditions: initial denaturation of genomic DNA at 94 °C for min, followed by 30 cycles of 94 °C for 30 s for DNA denaturation, 55.6 °C for 30 s for primer annealing, and DNA synthesis elongation at 72 °C for 30 s A final elongation step after the 30 cycles was maintained at 72 °C for PCR products were fractionated by 1% agarose gel electrophoresis cDNA sequence analysis The complete fabp1a and fabp1b cDNA sequences were determined by aligning the overlapping sequences obtained from 3¢-RACE and 5¢-RLM-RACE using clustalw [51] A blastx search of the cDNA sequence was performed at NCBI The cDNA sequences were analyzed for ORFs, protein molecular mass and isoelectric point using gene runner v 3.05 (Hastings Software, Inc., Hastings-onHudson, NY) The deduced amino acid sequence of the ORF for each cDNA was aligned with the human FABP1 sequence using the blosum62 similarity matrix in bioedit sequence alignment editor (version 5.0.9) and an output of percentage sequence identity and percent sequence similarity was generated [26] Location of the b strands (bA- bJ) and the a helices (aI and aII) in zebrafish FABP1a and FABP1b was determined by aligning the deduced amino acid sequences for FABP1a and FABP1b amino acid sequence with FABPs and cellular retinoid (retinol and retinoic acid) binding protein available at fugue [52] clustalx [27] was used to generate a bootstrap neighbor-joining phylogenetic tree to test the position of zebrafish FABP1a and FABP1b among various iLBP sequences obtained from NCBI Human Von Ebner’s gland protein (LCN1, GenBank accession number NP_002288) that belongs to the lipocalin family of the calycins was used as the out-group FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS M K Sharma et al Zebrafish duplicated fabp1 Linkage analysis by radiation hybrid mapping Acknowledgements The LN54 panel of radiation hybrids was used to map the fabp1a, fabp1b and fabp10 genes to specific zebrafish linkage groups as described previously [6] Primers specific to zebrafish fabp1a cDNA (sense 5¢-AAGCAGGAAGTT CTCATCGG-3¢; nucleotides 100 209–100 189, contig ctg30243.1 assembly Zv2 and antisense 5¢-AATCCCCTT GACAAACGCTG-3¢; nucleotides 99 693–99 712, contig ctg30243.1 assembly Zv2), fabp1b cDNA (sense 5¢-ACCA CAGTCTAATGCCTTCC-3¢; nucleotides 201 542–201 561, contig ctg9967 assembly Zv2 and antisense 5¢-TTCTCAAT CATGTCGTCAGGA-3¢; nucleotides 201 835–201 815 contig ctg9967 assembly Zv2) and fabp10 cDNA (sense: 5¢-GCCAGAAGAGGTCATTAAAC-3¢; nucleotides 84– 103, GenBank accession number AF254642 and antisense: 5¢-TGGTGGTGATTTCAGCCTC-3¢; nucleotides 232–214, GenBank accession number AF254642) were used to amplify a genomic DNA segment of the zebrafish fabp1a, fabp1b and fabp10 genes from the 93 mouse-zebrafish cell hybrids of LN54 panel The radiation hybrid panel was scored and analyzed according to the directions at http:// mgchd1.nichd.nih.gov:8000/zfrh/beta.cgi Conserved synteny between zebrafish linkage groups and 8, and mammalian chromosomes was determined by obtaining the list of genes mapped on zebrafish linkage groups from ZFIN (http://zfin.org/) and their cytogenetic location on human, mouse and rat chromosomes provided by Entrez Genome (http://www.ncbi.nlm.nih.gov/mapview/ map_search.cgi?taxid ¼ 9606) We thank Dr Marc Ekker for providing us with DNA from the LN54 collection of radiation hybrids We are grateful for technical assistance provided by Santhosh Karanth and Vishal Saxena during these studies This work was supported by research grants from Canadian Institutes of Health Research (to ´ ED-W), from the Institut National de la Sante et de ´ la Recherche Medicale, Centre National de la Recherche Scientifique, Hopital Universitaire de ˆ Strasbourg, Association pour la Recherche sur le Cancer, Ligue Nationale Contre le Cancer, National Institute of Health (to CT and BT), the Natural Sciences and Engineering Research Council of Canada (to JMW), and the Izaak Walton Killam Memorial Scholarship (to R-ZL and MKS) Tissue-specific expression of fabp1a, fapb1b and fabp10 mRNAs during embryonic and larval development, and in adult zebrafish The tissue-specific distribution of fabp1a, fabp1b and fabp10 gene transcripts in adult zebrafish was assayed by RT-PCR as described in Sharma et al [6] The fabp1a mRNAspecific primers (sense: 5¢-TGGGAAATATCAGCTG GAGTCT-3¢; nucleotides 69–90, Fig 1A and antisense: 5¢-CGTCTGCTGATCCTCTTGTAG-3¢; nucleotides 431– 410, Fig 1A), fabp1b mRNA-specific primers (sense: 5¢-AGCTGGAGAGTCAAGAGGG-3¢; nucleotides 75–93, Fig 1B and antisense: 5¢-GTGTGTTTGCTCAGCTCATG-3¢; nucleotides 462–443, Fig 1B) and fabp10 mRNA-specific primers (sense: 5¢-TTACGCTCAGGAGAACTACG-3¢; nucleotides 39–58, GenBank accession number AF254642 and antisense: 5¢-CTTCCTGATCATGGTGGTTC-3¢; nucleotides 378–358, GenBank 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& Thisse C (2004) Spatial and temporal expression of the zebrafish genome by largescale in situ hybridization screening Methods Cell Biol 77, 505–519 FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3229 ... Variations in the residues involved in the binding at site of the human FABP1, and possibly in zebrafish FABP1a and zebrafish FABP1b, may reflect differences in the binding affinities of the zebrafish. .. during embryogenesis and therefore may not perform a function equivalent to mammalian FABP1 during zebrafish embryogenesis The distinct patterns of expression for the fabp1b (in the intestine) and. .. Analysis of fabp1a and fabp1b genes in various fish species would likely resolve the timing of the insertion of intron in the fabp1a gene Linkage group assignment of zebrafish fabp1a, fabp1b and fabp10