Báo cáo khoa học: Hierarchical subfunctionalization of fabp1a, fabp1b and fabp10 tissue-specific expression may account for retention of these duplicated genes in the zebrafish (Danio rerio) genome docx

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. Sharma
1
, Rong-Zong Liu
1
, Christine Thisse
2
, Bernard Thisse
2
, Eileen M. Denovan-
Wright
3
and Jonathan M. Wright
1
1 Department of Biology, Dalhousie University, Halifax, NS, Canada
2 Institut de Ge
´
ne
´
tique et de Biologie Mole
´
culaire et Cellulaire, Department of Developmental Biology, Illkirch, France
3 Department of Pharmacology, Dalhousie University, Halifax, NS, Canada
The fatty acid-binding protein 1 (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]. Origin-
ally, FABPs were named according to their initial
site of isolation, e.g. intestinal-type fatty acid-binding
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:
Database
The following sequences have been submit-
ted to the GenBank database under acces-
sion 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 1 (FABP1), commonly termed liver-type
fatty acid-binding protein (L-FABP), is encoded by a single gene in mam-
mals. We cloned and sequenced cDNAs for two distinct FABP1s in zebra-
fish 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 5 and 8, respectively. Both linkage groups show con-
served syntenies to a segment of mouse chromosome 6, rat chromosome 4
and human chromosome 2 harboring the FABP1 locus. Phylogenetic analy-
sis 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 func-
tion 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.
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
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, how-
ever, 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 desig-
nation, 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 nomen-
clature 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 distribu-
tion 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 nonmamma-
lian vertebrates. Like FABP1, FABP10 has the capa-
city to bind two ligand molecules, unlike all other
iLBPs that bind a single molecule [18], suggesting that
FABP1 and FABP10 are evolutionarily related, possi-
bly 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
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 Infor-
mation (NCBI) DNA sequence database revealed an
expressed sequence tag (EST; GenBank accession num-
ber 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¢-RLM-
RACE 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 isoelec-
tric 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 posi-
tion 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. Align-
ment of the human FABP1 sequence to the deduced
M. K. Sharma et al. Zebrafish duplicated fabp1
FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3217
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. Phy-
logenetic analysis using clustalx [27] strongly sup-
ports 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.
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
A

B
Fig. 1. 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 nucleo-
tides 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 poly-
adenylation 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 duplicated fabp1 M. K. Sharma et al.
3218 FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 transcrip-
tion start sites for the fabp1b gene (see Fig. 4B and dis-
cussion 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 utili-
zed (Fig. 1B, see Fig. 4B). All nucleotide positions for
fabp1b cDNA mentioned here refer to nucleotide posi-
tions in the fabp1a long transcript variant. A putative
polyadenylation signal, ATTAAA, is located from nuc-

leotide 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 cal-
culated 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 arti-
fact of PCR. Alignment of the human FABP1
sequence with the deduced zebrafish FABP1 sequence
using the bioedit sequence alignment editor (blo-
sum62 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 zebra-
fish FABP1a and FABP1b encoded by the sister dupli-
cate genes, fabp1a and fabp1b, do not cluster as closely

as we observed for proteins encoded by other duplica-
ted 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 non-
mammalian vertebrates, suggesting that FABP10 and
FABP1 are paralogs. The presence of fabp1 and fabp10
genes in zebrafish and other nonmammalian verte-
brates 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).
Fig. 2. 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 maxi-
mize alignment. The amino acid sequences that determine higher-order structure are shown in bold and labeled: aI, aII, a helix I and II; bAto
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.
M. K. Sharma et al. Zebrafish duplicated fabp1
FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3219

Fig. 3. Phylogenetic relationship of zebrafish FABP1a, FABP1b and FABP10 in the iLBP multigene family. The bootstrap neighbor-joining phy-
logenetic 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 ze-
brafish (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.
Zebrafish duplicated fabp1 M. K. Sharma et al.
3220 FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS
A
B
C
Fig. 4. 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, ⁄ 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 0 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 zebra-
fish genome database at the Wellcome Trust Sanger Institute (scaffold 1725, assembly Zv4, 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.
M. K. Sharma et al. Zebrafish duplicated fabp1
FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3221
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 eicosa-
noids. 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 net-
work. 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 resi-
due is identical in human FABP1, zebrafish FABP1a
and FABP1b. Residue R122 in human FABP1 corres-
ponds 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 carboxy-
late 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 2 involves K31, A54,
S56, D88¢ and bound water [34]. Residue K31 is identi-
cal 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 mole-
cule and is of lesser importance [34]. Variations in the
residues involved in the binding at site 2 of the human
FABP1, and possibly in zebrafish FABP1a and zebra-
fish 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.
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 data-
base (assembly Zv2) at the Wellcome Trust Sanger
Institute ( />using the zebrafish fabp1a cDNA sequence and identi-
fied a contig, ctg30243.1, containing sequence of the
zebrafish fabp1a gene. Alignment of the 5¢-RLM-
RACE sequence with the zebrafish fabp1a gene
sequence localized the transcription start site 407 bp
upstream of the initiation codon (Fig. 4A). An addi-
tional intron was identified in the 5¢-UTR of the zebra-
fish fabp1a gene by sequence alignment of the cDNA
and genomic sequences, thereby generating a fifth
exon, exon 0. A fifth exon is unprecedented in mem-
bers of the vertebrate iLBP multigene family. (Evi-
dence for an additional intron in the zebrafish fabp1a
gene is provided below.)
Alignment analysis also revealed that the 5¢-RLM-
RACE product contained a string of 10 ‘CA’ repeats,
whereas the genomic sequence had only four ‘CA’
dinucleotide repeats in exon 0 (Fig. 4A). The difference
in the number of ‘CA’ repeats may represent a zebra-
fish strain-specific or individual polymorphism. Dinu-
cleotide 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). Align-
ment 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 transcrip-
tion start sites at 21 and 52 bp upstream of the initi-
ation 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, exclu-
ding 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
Zebrafish duplicated fabp1 M. K. Sharma et al.
3222 FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS
gene at 27 bp upstream of the initiation codon
(Fig. 4C).
Putative TATA boxes were identified using matin-
spector [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 gener-
ating an unprecedented five exons in a vertebrate iLBP
gene, consisting of exons 1–4, plus exon 0 (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 PCR-
amplified DNA fragment of  650 bp was generated
using primers based on a sequence 5¢ upsteam of

exon 0 and a sequence within exon 1, and zebrafish
genomic DNA as template (see Experimental proce-
dures). A product of this size could only be generated
from genomic DNA if it contained an intron of
 350 bp (data not shown).
Intron 0 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
the presence of intron 0 in the zebrafish fabp1a gene is
an insertional mutation that occurred after the whole-
genome duplication event which followed the diver-
gence 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 inde-
pendent of other fish lineages. Analysis of fabp1a and
fabp1b genes in various fish species would likely
resolve the timing of the insertion of intron 0 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 5 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 8 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 6 (position 30 cM), the rat FABP1 gene
on chromosome 4 (position 4q32) and the human
FABP1 gene on chromosome 2 (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 mamma-
lian FABP1 gene, and the two fabp1 genes arose as a
result of a duplication event, most probably the whole-
genome 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
M. K. Sharma et al. Zebrafish duplicated fabp1
FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3223
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 ampli-
fied 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 intes-
tine 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 tissue-
section 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
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,
Table 1. Conserved synteny of zebrafish fabp1a and fabp1b genes with their mammalian ortholog, Fabp1. –, Data not available.
Gene Name
Chromosomal ⁄ linkage
group assignment
Zebrafish Mouse Rat Human
Fabp1 fatty acid binding protein 1, liver 5 (fabp1a)
8 (fabp1b)
6 (30 c
M) 4q32 2p11
Smyd1 SET and MYND domain containing 1 5 6 (30.5 c
M) 4q32-q33 2p11.2
Vamp5 vesicle-associated membrane protein 5 5 6 C1 4q33 2p11.2
Vamp8 vesicle-associated membrane protein 8 5 6 (31.5 c
M) 4q33 2p12-p11.2
Usp39 ubiquitin specific peptidase 39 5 6 (31.5 c
M) 4q33 2p11.2

Mobk1b Mps one binder kinase activator-like 1B 5 6 C3 4q34 2p13.1
Hk2 hexokinase 2 5 6 C3 (34.5 c
M) 4q34 2p13
Pax8 paired box gene 8 5 2 (13.5 c
M) 3p13 2q12-q14
Mcm6 minichromosome maintenance deficient 6 5 1 (66.6 c
M) – 2q21
Adra2b adrenergic receptor, alpha 2b 8 2 (71.0 c
M) 3q36 2p13-q13
Atoh1 atonal homolog 1 8 6 (29.69 c
M) – 4q22
Grid2 glutamate receptor, ionotropic, delta 2 8 6 (29.65 c
M) 4q31 4q22
Xpc xeroderma pigmentosum, complementation group C 8 6 D 4q34 3p25
Brpf1 bromodomain and PHD finger containing, 1 8 6 E3 4q42 3p26-p25
Plxnd1 plexin D1 8 6 E3 4q42 1p32-p31
Tuba8 tubulin, alpha 8 8 6 F1 – 22q11.1
Fig. 5. 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 extrac-
ted from adult zebrafish intestine (I). No fabp1a or fabp1b mRNA-
specific product was generated from adult zebrafish liver (L), heart
(H), muscle (M), ovary (O), skin (S), brain (B), testes (T) or the neg-
ative 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 mRNA-
specific product was generated from all the adult zebrafish tissues

analyzed (4).
Zebrafish duplicated fabp1 M. K. Sharma et al.
3224 FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS
reptiles and birds. Detection of fabp1a and fabp1b gene
transcripts in zebrafish intestine, and fabp10 gene tran-
scripts in zebrafish liver shows a division of gene-
expression patterns, or subfunctionalization [44,45], of
the gene-expression patterns exhibited by the mamma-
lian FABP1 gene. As such, subfunctionalization of the
fabp1 and fabp10 genes may explain the retention of
these paralogous genes, at least in zebrafish, and poss-
ibly 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
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 ( 6 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 morphogen-
esis, 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. 6. 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 5 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 5 days of development showing fabp10 mRNA in the
liver (L).
M. K. Sharma et al. Zebrafish duplicated fabp1
FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3225
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 intes-
tine during the late fetal stage. The mRNA concentra-
tion 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 suck-
ling and weaning period, up to 35 days post partum
[48]. Whole-mount in situ hybridization analyses dem-
onstrated that the zebrafish fabp1a mRNA reported
here is not detected during embryogenesis and there-
fore may not perform a function equivalent to mam-
malian FABP1 during zebrafish embryogenesis. The
distinct patterns of expression for the fabp1b (in the
intestine) and fabp10 (in the liver) genes in the devel-
oping zebrafish combined represent the expression pat-
tern 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 contribu-
ted 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¢; nucleo-
tides 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:
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 0 in the fabp1a gene
Primers based on the sequence 5¢ upstream of exon 0 (sense:
5¢-GTCGTGAGAAAGCGGAAAC-3¢; nucleotides )176 to
)194, Fig. 4) and the sequence of exon 1 (antisense: 5¢-AT
GCCTCAAAGTTCTCGTG-3¢; nucleotides 91–109, Fig. 1A)

were used to amplify intron 0 of the fabp1a gene from
zebrafish genomic DNA using the following conditions:
initial denaturation of genomic DNA at 94 °C for 2 min, fol-
lowed 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 5 min. 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-on-
Hudson, 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.
Zebrafish duplicated fabp1 M. K. Sharma et al.
3226 FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS
Linkage analysis by radiation hybrid mapping
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
CATGTCGTCA GGA-3¢; nucleotides 201 835–201 815 con-
tig 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 5
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 ( />map_search.cgi?taxid ¼ 9606).
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 mRNA-
specific 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 antisen se: 5¢-CTTCCTGATC ATGG TGGT TC-3¢; nucleo-
tides 378–358, GenBank accession number AF254642) were
used in the RT-PCR.
Whole-mount in situ hybridization to zebrafish embryos
was performed using riboprobes based on the sequences of
the fabp10 [17], fabp1a and fabp1b cDNA clones as des-
cribed by Thisse et al. [53].
Acknowledgements
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 Me
´
dicale, Centre National de la
Recherche Scientifique, Hoˆ pital Universitaire de
Strasbourg, Association pour la Recherche sur le
Cancer, Ligue Nationale Contre le Cancer, National
Institute of Health (to CT and BT), the Natural Sci-
ences and Engineering Research Council of Canada
(to JMW), and the Izaak Walton Killam Memorial
Scholarship (to R-ZL and MKS).
References
1 Bernlohr DA, Simpson MA, Hertzel AV & Banaszak
LJ (1997) Intracellular lipid-binding proteins and their
genes. Annu Rev Nutr 17, 277–303.
2 Schaap FG, van der Vusse GJ & Glatz JF (2002) Evolu-
tion of the family of intracellular lipid binding proteins
in vertebrates. Mol Cell Biochem 239, 69–77.
3 Ockner RK, Manning JA, Poppenhausen RB & Ho
WK (1972) A binding protein for fatty acids in cytosol
of intestinal mucosa, liver, myocardium, and other tis-
sues. Science 177, 56–58.
4 Zimmerman AW & Veerkamp JH (2002) New insights
into the structure and function of fatty acid-binding
proteins. Cell Mol Life Sci 59, 1096–1116.
5 Hertzel AV & Bernlohr DA (2000) The mammalian

fatty acid-binding protein multigene family: molecular
and genetic insights into function. Trends Endocrinol
Metab 11, 175–180.
6 Sharma MK, Denovan-Wright EM, Boudreau ME &
Wright JM (2003) A cellular retinoic acid-binding pro-
tein from zebrafish (Danio rerio): cDNA sequence, phy-
logenetic analysis, mRNA expression, and gene linkage
mapping. Gene 311, 119–128.
7 Liu RZ, Sharma MK, Sun Q, Thisse C, Thisse B,
Denovan-Wright EM & Wright JM (2005) Retention of
the duplicated cellular retinoic acid-binding protein 1
genes (crabp1a and crabp1b) in the zebrafish genome by
subfunctionalization of tissue-specific expression.
FEBS J 272, 3561–3571.
8 Liu RZ, Sun Q, Thisse C, Thisse B, Wright JM &
Denovan-Wright EM (2005) The cellular retinol-binding
protein genes are duplicated and differentially tran-
scribed in the developing and adult zebrafish (Danio
rerio). Mol Biol Evol 22, 469–477.
M. K. Sharma et al. Zebrafish duplicated fabp1
FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3227
9 Coe NR & Bernlohr DA (1998) Physiological properties
and functions of intracellular fatty acid-binding pro-
teins. Biochim Biophys Acta 1391, 287–306.
10 Besnard P, Niot I, Poirier H, Clement L & Bernard A
(2002) New insights into the fatty acid-binding protein
(FABP) family in the small intestine. Mol Cell Biochem
239, 139–147.
11 Veerkamp JH & Maatman RG (1995) Cytoplasmic fatty
acid-binding proteins: their structure and genes. Prog

Lipid Res 34, 17–52.
12 Wolfrum C, Ellinghaus P, Fobker M, Seedorf U, Ass-
mann G, Bo
¨
rchers T & Spener F (1999) Phytanic acid is
ligand and transcriptional activator of murine liver fatty
acid binding protein. J Lipid Res 40, 708–714.
13 Thompson J, Winter N, Terwey D, Bratt J & Banaszak
L (1997) The crystal structure of the liver fatty acid-
binding protein. A complex with two bound oleates.
J Biol Chem 272, 7140–7150.
14 Di Pietro SM, Veerkamp JH & Santome
´
JA (1999) Iso-
lation, amino acid sequence determination and binding
properties of two fatty-acid-binding proteins from axo-
lotl (Ambistoma mexicanum) liver. Evolutionary rela-
tionship. Eur J Biochem 259, 127–134.
15 Nemecz G, Hubbell T, Jefferson JR, Lowe JB &
Schroeder F (1991) Interaction of fatty acids with
recombinant rat intestinal and liver fatty acid-binding
proteins. Arch Biochem Biophys 286, 300–309.
16 Richieri GV, Ogata RT & Kleinfeld AM (1994) Equili-
brium constants for the binding of fatty acids with fatty
acid-binding proteins from adipocyte, intestine, heart,
and liver measured with the fluorescent probe ADIFAB.
J Biol Chem 269, 23918–23930.
17 Denovan-Wright EM, Pierce M, Sharma MK & Wright
JM (2000) cDNA sequence and tissue-specific expression
of a basic liver-type fatty acid binding protein in adult

zebrafish (Danio rerio). Biochim Biophys Acta 1492,
227–232.
18 Nichesola D, Perduca M, Capaldi S, Carrizo ME,
Righetti PG & Monaco HL (2004) Crystal structure of
chicken liver basic fatty acid-binding protein complexed
with cholic acid. Biochemistry 43, 14072–14079.
19 Schleicher CH, Co
´
rdoba OL, Santome
´
JA &
Dell’Angelica EC (1995) Molecular evolution of the
multigene family of intracellular lipid-binding proteins.
Biochem Mol Biol Int 36, 1117–1125.
20 Amores A, Force A, Yan YL, Joly L, Amemiya C,
Fritz A, Ho RK, Langeland J, Prince V, Wang YL
et al. (1998) Zebrafish hox clusters and vertebrate gen-
ome evolution. Science 282, 1711–1714.
21 Wittbrodt J, Meyer A & Schartl M (1998) More genes
in fish? Bioessays 20, 511–515.
22 Taylor JS, Braasch I, Frickey T, Meyer A & Van de
Peer Y (2003) Genome duplication, a trait shared by
22000 species of ray-finned fish. Genome Res 13 ,
382–390.
23 Van de Peer Y, Taylor JS & Meyer A (2003) Are all
fishes ancient polyploids? J Struct Funct Genomics 3,
65–73.
24 Jaillon O, Aury JM, Brunet F, Petit JL, Stange-
Thomann N, Mauceli E, Bouneau L, Fischer C,
Ozouf-Costaz C, Bernot A et al. (2004) Genome

duplication in the teleost fish Tetraodon nigroviridis
reveals the early vertebrate proto-karyotype. Nature
431, 946–957.
25 Vandepoele K, De Vos W, Taylor JS, Meyer A & Van
de Peer Y (2004) Major events in the genome evolution
of vertebrates: paranome age and size differ consider-
ably between ray-finned fishes and land vertebrates.
Proc Natl Acad Sci USA 101, 1638–1643.
26 Hall T (1999) BioEdit: a user-friendly biological
sequence alignment editor and analysis program for
Windows 95 ⁄ 98 ⁄ NT. Nucleic Acids Symp Series 41,
95–98.
27 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F
& Higgins DG (1997) The CLUSTAL_X windows inter-
face: flexible strategies for multiple sequence alignment
aided by quality analysis tools. Nucleic Acids Res 25,
4876–4882.
28 Liu RZ, Denovan-Wright EM, Degrave A, Thisse C,
Thisse B & Wright JM (2004) Differential expression of
duplicated genes for brain-type fatty acid-binding pro-
teins (fabp7a and fabp7b) during early development of
the CNS in zebrafish (Danio rerio). Gene Expr Patterns
4, 379–387.
29 Haunerland N, Jagschies G, Schulenberg H & Spener F
(1984) Fatty-acid-binding proteins. Occurrence of two
fatty-acid-binding proteins in bovine liver cytosol and
their binding of fatty acids, cholesterol, and other
lipophilic ligands. Hoppe Seylers Z Physiol Chem 365,
365–376.
30 Hanhoff T, Lu

¨
cke C & Spener F (2002) Insights into
binding of fatty acids by fatty acid binding proteins.
Mol Cell Biochem 239, 45–54.
31 Bo
¨
rchers T & Spener F (1993) Involvement of arginine
in the binding of heme and fatty acids to fatty acid-
binding protein from bovine liver. Mol Cell Biochem
123, 23–27.
32 Thumser AE, Evans C, Worrall AF & Wilton DC
(1994) Effect on ligand binding of arginine mutations
in recombinant rat liver fatty acid-binding protein.
Biochem J 297, 103–107.
33 Thumser AE, Voysey J & Wilton DC (1996) Mutations
of recombinant rat liver fatty acid-binding protein
at residues 102 and 122 alter its structural integrity
and affinity for physiological ligands. Biochem J 314,
943–949.
34 Thompson J, Ory J, Reese-Wagoner A & Banaszak L
(1999) The liver fatty acid binding protein – comparison
of cavity properties of intracellular lipid-binding pro-
teins. Mol Cell Biochem 192, 9–16.
Zebrafish duplicated fabp1 M. K. Sharma et al.
3228 FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS
35 O’Rielly P, Hamilton L, McConnell S & Wright J
(1996) Rapid analysis of genetic variation in Atlantic
salmon (Salmo salar) by PCR multiplexing of dinucleo-
tide and tetranucleotide microsatellites. Can J Fish
Aquat Sci 53, 2292–2298.

36 Carleton KL, Streelman JT, Lee B-Y, Garnhart N,
Kidd M & Kocher TD (2002) Rapid isolation of CA
microsatellites from the tilapia genome. Anim Genet 33,
140–144.
37 Her GM, Yeh YH & Wu JL (2003) 435-bp liver regula-
tory sequence in the liver fatty acid binding protein
(L-FABP) gene is sufficient to modulate liver regional
expression in transgenic zebrafish. Dev Dyn 227, 347–
356.
38 Cartharius K, Frech K, Grote K, Klocke B, Haltmeier
M, Klingenhoff A, Frisch M, Bayerlein M & Werner T
(2005) MatInspector and beyond: promoter analysis
based on transcription factor binding sites. Bioinfor-
matics 21, 2933–2942.
39 Lewin B (2004) Genes VIII. Prentice Hall, Englewood
Cliffs.
40 Breathnach R & Chambon P (1981) Organization and
expression of eucaryotic split genes coding for proteins.
Annu Rev Biochem 50, 349–383.
41 Hukriede NA, Joly L, Tsang M, Miles J, Tellis P,
Epstein JA, Barbazuk WB, Li FN, Paw B, Postlethwait
JH et al. (1999) Radiation hybrid mapping of the zebra-
fish genome. Proc Natl Acad Sci USA 96, 9745–9750.
42 Di Pietro SM, Dell’Angelica EC, Veerkamp JH, Sterin-
Speziale N & Santome
´
JA (1997) Amino acid sequence,
binding properties and evolutionary relationships of the
basic liver fatty-acid-binding protein from the catfish
Rhamdia sapo. Eur J Biochem 249, 510–517.

43 Sweetser DA, Lowe JB & Gordon JI (1986) The nucleo-
tide sequence of the rat liver fatty acid-binding protein
gene. Evidence that exon 1 encodes an oligopeptide
domain shared by a family of proteins which bind
hydrophobic ligands. J Biol Chem 261, 5553–5561.
44 Force A, Lynch M, Pickett FB, Amores A, Yan YL &
Postlethwait J (1999) Preservation of duplicate genes by
complementary, degenerative mutations. Genetics 151,
1531–1545.
45 Prince VE & Pickett FB (2002) Splitting pairs: the
diverging fates of duplicated genes. Nat Rev Genet 3,
827–837.
46 Ober EA, Field HA & Stainier DY (2003) From endo-
derm formation to liver and pancreas development in
zebrafish. Mech Dev 120, 5–18.
47 Her GM, Chiang CC, Chen WY & Wu JL (2003)
In vivo studies of liver-type fatty acid binding protein
(L-FABP) gene expression in liver of transgenic
zebrafish (Danio rerio). FEBS Lett 538, 125–133.
48 Gordon JI, Elshourbagy N, Lowe JB, Liao WS, Alpers
DH & Taylor JM (1985) Tissue specific expression and
developmental regulation of two genes coding for rat
fatty acid binding proteins. J Biol Chem 260, 1995–
1998.
49 Bo
¨
rchers T & Spener F (1994) Fatty acid binding pro-
teins. Current Topics in Membranes (Kleinzeller A &
Benos D, eds), pp. 261–294. Academic Press, New
York.

50 Liu RZ, Denovan-Wright EM & Wright JM (2003)
Structure, linkage mapping and expression of the
heart-type fatty acid-binding protein gene (fabp3)
from zebrafish (Danio rerio). Eur J Biochem 270,
3223–3234.
51 Thompson JD, Higgins DG & Gibson TJ (1994) CLUS-
TAL W: improving the sensitivity of progressive multi-
ple sequence alignment through sequence weighting,
position-specific gap penalties and weight matrix choice.
Nucleic Acids Res 22, 4673–4680.
52 Shi J, Blundell TL & Mizuguchi K (2001) FUGUE:
sequence–structure homology recognition using environ-
ment-specific substitution tables and structure-dependent
gap penalties. J Mol Biol 310, 243–257.
53 Thisse B, Heyer V, Lux A, Alunni V, Degrave A, Seiliez
I, Kirchner J, Parkhill JP & Thisse C (2004) Spatial and
temporal expression of the zebrafish genome by large-
scale in situ hybridization screening. Methods Cell Biol
77, 505–519.
M. K. Sharma et al. Zebrafish duplicated fabp1
FEBS Journal 273 (2006) 3216–3229 ª 2006 The Authors Journal compilation ª 2006 FEBS 3229