Furthermore, we propose that the FABP4–FABP5–FABP8–FABP9 PERF15 gene cluster on a single chromosome in the tetrapod genome and the fabp11 genes in the zebrafish genome originated from a c
Trang 1copies of the zebrafish fabp11 gene and the tetrapod
FABP4, FABP5, FABP8 and FABP9 genes
Santhosh Karanth1, Eileen M Denovan-Wright2, Christine Thisse3, Bernard Thisse3and Jonathan
M Wright1
1 Department of Biology, Dalhousie University, Halifax, Canada
2 Department of Pharmacology, Dalhousie University, Halifax, Canada
3 Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, VA, USA
The multigene family coding for vertebrate
intra-cellular lipid-binding proteins (iLBPs) consists of the
fatty acid-binding protein (FABP), cellular retinoic
acid-binding protein (CRABP) and cellular
retinol-binding protein (CRBP) genes FABPs bind selectively
to fatty acids, CRABPs bind to retinoic acid, and
CRBPs bind to retinol [1] For many iLBPs, the
precise physiological function(s) is not completely understood or remains unknown However, it is clear that iLBPs are involved in cellular uptake and intracel-lular transport of long-chain fatty acids, bile salts and retinoids, protection of cellular structures from the detergent effects of fatty acids by sequestering them until required in various metabolic processes, interaction
Keywords
Fabp11; retinal development; spinal cord;
tandem gene duplication; whole genome
duplication
Correspondence
J M Wright, Department of Biology,
Dalhousie University, Halifax, NS, Canada
B3H 4J1
Fax: +1 902 494 3736
Tel: +1 902 494 6468
E-mail: jmwright@dal.ca
Website: http://www.dal.ca/biology2/
(Received 23 January 2008, revised 8 March
2008, accepted 9 April 2008)
doi:10.1111/j.1742-4658.2008.06455.x
We describe the structure of a fatty acid-binding protein 11 (fabp11b) gene and its tissue-specific expression in zebrafish The 3.4 kb zebrafish fabp11b
is the paralog of the previously described zebrafish fabp11a, with a deduced amino acid sequence for Fabp11B exhibiting 65% identity with that of Fabp11A Whole mount in situ hybridization of a riboprobe to embryos and larvae showed that zebrafish fabp11b transcripts were restricted solely
to the retina and were first detected at 24 h postfertilization In situ hybrid-ization revealed fabp11b transcripts along the spinal cord in adult zebrafish However, the highly sensitive RT-PCR assay detected fabp11b transcripts
in the brain, heart, ovary and eye in adult tissues By contrast, fabp11a transcripts had been previously detected in the liver, brain, heart, testis, muscle, ovary and skin of adult zebrafish Using the LN54 radiation hybrid panel, we assigned zebrafish fabp11b to linkage group 16 Phylogenetic analysis and conserved gene synteny with tetrapod genes indicated that the emergence of two copies of fabp11 in the zebrafish genome may have resulted from a fish-specific whole genome duplication event Furthermore,
we propose that the FABP4–FABP5–FABP8–FABP9 (PERF15) gene cluster on a single chromosome in the tetrapod genome and the fabp11 genes in the zebrafish genome originated from a common ancestral gene, which, following their divergence, gave rise to the fabp11 genes of zebrafish, and the progenitor of the FABP4, FABP5, FABP8 and FABP9 genes in tetrapods after the separation of the fish and tetrapod lineages
Abbreviations
CRABP, cellular retinoic acid-binding protein; CRBP, cellular retinol-binding protein; EST, expressed sequence tag; FABP, fatty acid-binding protein; hpf, hours postfertilization; iLBP, intracellular lipid-binding protein; LG, linkage group; mya, million years ago; WGD, whole genome duplication.
Trang 2transcriptional regulation of specific genes [2–6] By
functioning in the transport and metabolism of retinol
and retinoic acid, CRBPs and CRABPs may play an
important role in development, growth and
reproduc-tion, primarily by making retinoids available to
recep-tors in the nucleus to regulate specific gene
transcription [7] Originally, FABPs and their genes
were named on the basis of the tissue in which they
were first isolated Later, Hertzel & Bernlohr [8]
pro-posed a different nomenclature, in which FABPs are
numbered according to the temporal order of their
identification (e.g fabp1 and fabp2) We chose, for the
sake of clarity, to use here the nomenclature adopted
by Hertzel & Bernlohr [8]
Thus far, iLBPs have only been found in species
from the animal kingdom, suggesting that a single
ancestral iLBP gene emerged in animals after their
divergence from plants and fungi [9] The diversity of
the iLBP multigene family is thought to have arisen
through a series of gene duplication events followed by
their sequence divergence [10] Estimates of the earliest
iLBP gene duplication vary between 930 and 1000
million years ago (mya) [9–11] Schaap et al [9] and
Schleicher et al [10] have suggested that 600–700 mya,
before the invertebrate and vertebrate lineages split,
the gene(s) that would give rise to the FABP1–
FABP2–FABP6 clade had already diverged from the
gene(s) that would give rise to the FABP4–FABP8
clade CRBPs and CRABPs, which are absent in
inver-tebrates, might have diverged from the FABP1 clade
after the vertebrates and invertebrates split To date,
paralogs of 11 genes coding for FABPs have been
described in vertebrates [12]
Zebrafish have attracted the attention of
evolu-tionary molecular biologists partly because of the
abundance of genetic and biological resources for this
model organism for developmental studies, but
partic-ularly for the whole genome duplication (WGD) event
that occurred in ray-finned fishes some 250–400 mya
[13], which has led to investigations on the genesis and
fate of duplicated genes Gene duplication has been
proposed by Ohno [14] as a major evolutionary force
in driving the increasing complexity of life In addition
to WGD, tandem duplication of individual genes by
the process of unequal crossing-over during meiosis
may also account for the increase in the number of
genes in eukaryotes [15]
Vayda et al [16] described a fabp gene from four
Antarctic fishes, termed Had-FABP, which they suggest
is the ortholog of mammalian FABP4 In a previous
communication [17], we described an fabp4 gene from
zebrafish that showed greatest sequence identity to,
Antarctic fish Had-FABP Recently, however, Agulleiro
et al [12] recognized that the Fabps reported by Vayda
et al [16] and by us [17] constitute a new FABP clade that is probably restricted to fishes, and renamed the novel fish gene and protein fabp11 and Fabp11, respec-tively [12] In this article, we report the charac-terization of a duplicated Fabp11 gene (fabp11b) from the zebrafish genome, the distribution of fabp11b mRNA transcripts in adult tissues and during embry-onic and larval development, and linkage group (LG) (chromosome) assignment of fabp11b by radiation hybrid mapping On the basis of phylogenetic analysis and conserved gene syntenies of zebrafish fabp11a and fabp11b with other vertebrate FABP genes, we propose that the duplicated fabp11 genes in fishes and the FABP4–FABP5–FABP8–FABP9 gene cluster in tetra-pods arose from a single progenitor gene
Results and Discussion
Identification of a duplicated fabp11 gene from zebrafish
A paralogous gene to the previously described zebra-fish fabp11 (fabp4, but now referred to as fabp11a) [17] was identified from a blast search of the zebrafish genome sequence database at the Wellcome Trust Sanger Institute (version Zv6, http://www.ensembl.org/ Danio_rerio/index.html), using as query the GenBank sequence AY628221 The 3.4 kb duplicated fabp11 (hereafter referred to as fabp11b) consists of four exons and three introns (Fig 1), a FABP gene organization seen for most vertebrate iLBP genes [1] The four exons of fabp11b code for 24, 59, 34 and 17 amino acids, respectively (Fig 1), identical to what is seen for zebrafish fabp11a [17] For fabp11b, the splice junc-tions for intron 1 and intron 2 conform to the GT⁄ AG intron⁄ exon rule [18], whereas the splice junction for intron 3 is TA⁄ AG A putative binding site for a GATA transcription factor was identified at position )471 to )483 in the 5¢-upstream region of fabp11b (Fig 1) Divine et al [19] reported that GATA-4, GATA-5 and GATA-6 act cooperatively in activating FABP1 transcription in the murine small intestine A putative TATA box is located at position)30 to )24
Distribution of fabp11b transcripts in the retina
of developing zebrafish embryo and larvae
To determine the spatial and temporal distribution of fabp11b transcripts during zebrafish embryonic and larval development, we conducted whole mount in situ
Trang 3hybridization to zebrafish embryos and larvae at
different developmental stages (Fig 2) fabp11b
tran-scripts were first detected in the retina of developing
embryos at 24 h postfertilization (hpf) in the
pig-mented epithelium, starting in the proximal part of the
retina fabp11b transcripts spread across this layer of
the retina by 30 hpf (Fig 2B) A homogeneous
distri-bution of fabp11b transcripts throughout the
pig-mented epithelium was observed at 48 hpf (Fig 2C)
In contrast to fabp11b, at 24 hpf and 36 hpf the
fabp11a transcripts were detected in the lens and diencephalon [17] In 5-day-old larvae, the hybridiza-tion signal for fabp11b transcripts was still observed in the same layer of the retina (Fig 2D) Sellner [20] reported a similar trend, whereby retinal FABP appears to be maximally expressed in the chicken ret-ina around the ninth day of embryonic development and declines at later stages Embryonic development in zebrafish spans 3 days, whereas embryonic develop-ment in chicken extends over 20–21 days Two other FABP transcripts, transcripts for fabp3 and fabp7b, have been detected in the developing retina of zebra-fish embryos [17,21] It has been proposed that FABPs are involved in sequestering of fatty acids during retinal differentiation [20]
Tissue-specific distribution of fabp11b transcripts
in adult zebrafish
In situ hybridization was performed on sections of adult zebrafish to determine the tissue-specific distribu-tion of fabp11b transcripts fabp11b transcripts were detected along the vertebra in the spinal cord of adult zebrafish (Fig 3A shows the hybridization signal in a sagittal section, and Fig 3B a transverse section) While describing the distribution of FABP8 expression
in the rabbit spinal cord, Narayanan et al [22] noted that the spinal cord has a high rate of fatty acid biosynthesis RT-PCR detected fabp11b transcripts in total RNA extracted from the brain, heart, ovary and
Fig 1 The sequence of zebrafish fabp11b
and its 5¢-upstream promoter region Exons
are shown in upper-case letters, with the
coding sequences of each exon underlined
and the deduced amino acid sequence
indi-cated below The stop codon is indiindi-cated by
the diamond symbol Only partial nucleotide
sequences are shown for introns 2 and 3,
where the dotted lines indicate interruption
in the sequence +1 indicates the
transcrip-tion start site A putative polyadenylatranscrip-tion
signal, AATAAA, is highlighted in bold and
underlined A putative TATA box and a site
for GATA-binding factor are highlighted in
bold and underlined The in situ hybridization
probe (isp) used for detection of fabp11b
transcripts in adult zebrafish tissues, and
PCR primers used for radiation hybrid
map-ping (rhf, rhr) and for RT-PCR detection of
fabp11b transcripts in RNA extracted from
adult zebrafish tissues (rtf, rtr), are either
underlined or overlined.
Fig 2 Spatiotemporal distribution of fabp11b transcripts during
zebrafish embryonic and larval development was determined by
whole mount in situ hybridization fabp11b transcripts were first
detected in the pigmented epithelium of the retina (Re) at 24 hpf
(A) The distribution of fabp11b transcripts had spread across the
retina by 30 hpf (B) and 48 hpf (C) In 5-day-old larvae, fabp11b
transcripts were restricted to the circumference of the pigmented
epithelium of the retina (D).
Trang 4eye of adult zebrafish (Fig 4) fabp11b transcripts were
not detected in total RNA extracted from the liver,
intestine, kidneys, gills or muscle of adult zebrafish As
a positive control for the quality of RNA in each
tissue, transcripts for the constitutively expressed ef1a
gene were assayed by RT-PCR and detected in all
tissues examined (Fig 4) The difference observed in
the tissue-specific distribution of fabp11b transcripts
using in situ hybridization and RT-PCR is probably
due to the sensitivities of the two assays; RT-PCR is
far more sensitive than in situ hybridization In
con-trast to zebrafish fabp11b transcripts, fabp11a
tran-scripts were detected in liver, intestine, brain, heart
and muscle using RT-PCR [17] Abundant fabp11
transcripts were detected in liver, adipose tissue and
the vitellogenic ovary, transcripts were detected to a
lesser extent in the previtellogenic ovary, heart, kidney,
and muscle, and trace amounts were detected in testis
by RT-PCR using total RNA extracted from tissues of
adult Senegalese sole [12] In adult Antarctic fishes
[16], fabp11 (Had-FABP) transcripts were detected in
muscle, kidney, heart and brain by the less sensitive
assay of northern blot and hybridization
Duplicate copies of fabp11 in zebrafish may have
arisen by a fish-specific WGD event
Multiple sequence alignments of selected zebrafish and
mouse FABP amino acid sequences and the prototypic
Fabp11 from the Senegalese sole [12] were performed
using clustalw [23] Zebrafish Fabp11b showed the highest sequence identity and similarity (65% and 84%, respectively) with zebrafish Fabp11a, and the next highest sequence identity and similarity with the Senegalese sole Fabp11 (63% and 82%, respectively) (Fig 5) The sequence identity and similarity of zebra-fish Fabp11 decreased with paralogous FABP⁄ Fabps from zebrafish and mouse (Fig 5)
Radiation hybrid mapping using the LN54 panel [24] assigned zebrafish fabp11b to LG (chromosome) 16 at a distance of 26.59 cR from the marker fc09b04 with
a logarithm (base 10) of odds (LOD) score of 10 Zebrafish fabp11a had previously been assigned to LG (chromosome) 19 by the same LN54 radiation hybrid panel [17] Conserved gene synteny on zebrafish LGs (chromosomes) 16 and 19 [17] with genes on human chromosome 8 (Table 1) suggest that fabp11a and fabp11b may have arisen by the teleost fish-specific WGD event that occurred approximately 250–400 mya [13] Both fabp11a (LG 19) and fabp11b (LG 16), and two other duplicated genes on these LGs, showed conserved gene synteny with the FABP4–FABP5– FABP8–FABP9 gene cluster on human chromosome 8 For duplicated genes to be retained in the genome, Force et al [25] have proposed that either both dupli-cated genes undergo subfunctionalization, in which the functions of the ancestral gene are subdivided between the sister duplicate genes, or one of the duplicates acquires a new function, called neofunctionalization Force et al [25] further proposed that subfunctional-ization of duplicated genes arises owing to the accumu-lation of mutations in the regulatory elements of duplicated genes, which leads to divergence in their tissue-specific patterns of expression fabp11a mRNA transcripts were detected in ovary, liver, skin, intestine, brain, heart, testis and muscle in adult zebrafish [17] During larval development, fabp11a transcripts were detected in the lens and diencephalon [17] In contrast, fabp11b transcripts were detected in brain, heart, ovary
Fig 3 Tissue-specific distribution of fabp11b mRNA in adult
zebra-fish sections determined by in situ hybridization Sagittal (A) and
transverse (B) sections of adult zebrafish were hybridized to an
[ 33 P]dATP[aP] 3¢-end-labeled fabp11b antisense probe The
hybrid-ization signal of the antisense probe was limited to the spinal cord
(Sc) of adult zebrafish.
Fig 4 RT-PCR detection of fabp11b transcripts in RNA extracted from adult tissues of zebrafish using fabp11b cDNA-specific prim-ers fabp11b transcripts were detected by RT-PCR in RNA extracted from the brain (B), heart (H), ovary (O), and eye (E) No fabp11b transcripts were detected in RNA extracted from the liver (L), gills (G), intestine (I), muscle (M) or kidney (K) of adult zebrafish (upper panel) As a positive control, constitutively expressed elongation factor 1a (ef1a) transcripts were detected by RT-PCR in RNA extracted from all adult tissues examined (lower panel).
Trang 5and eye by RT-PCR (Fig 4) and in the spinal chord
by in situ hybridization (Fig 3) in adult zebrafish, and
in the retina during larval development (Fig 2)
Although fabp11a and fabp11b transcripts exhibit
strik-ingly different patterns of tissue distribution in
devel-oping embryos, larvae, and adult zebrafish, it is not
possible to ascertain whether the duplicated copies of
fabp11 have been retained in the zebrafish genome by
either subfunctionalization or neofunctionalization, as
there is no readily apparent ortholog of fish fabp11 in
tetrapods or other species studied thus far [12]
fabp11, FABP4, FABP5, FABP8 and FABP9 evolved
from a common ancestral gene on a single
progenitor chromosome
We constructed a neighbor-joining phylogenetic tree
using the amino acid sequences of selected vertebrate
FABPs (Fig 6) Zebrafish Fabp11b and zebrafish Fabp11a formed a clade with other teleost Fabp11s (Fig 6), a clade distinct from the frog, chicken and mammalian FABP4–FABP5–FABP8–FABP9 clade, as previously shown by Agulleiro et al [12] The zebrafish Fabp11a and Fabp11b amino acid sequences have diverged sufficiently from each other that they are not linked by a common node on the tree Similarly, the putative fugu and stickleback Fabp11a and Fabp11b
do not share a common node with their sister duplicates either, but are all clustered in the same clade with all other teleost fish Fabp11s
The sequence identity of zebrafish Fabp11b with mouse FABP4, FABP5, FABP8 and FABP9 (also known as PERF15) varied from 43% to 47% (Fig 5) The sequence identity of the Senegalese sole Fabp11 with human FABP4, FABP5, FABP8 and FABP9 varied from 52.2% to 54.5% [12] To date,
Fig 5 Zebrafish (D rerio, Dr) Fabp11b is aligned with: zebrafish Fabp11a (deduced from AY628221), Fabp3 (AAL40832), Fabp7a (AAH55621) and Fabp7b (AAQ92970); Senegalese sole (Solea senegalensis, Sos) Fabp11 (CAM58515); mouse (M musculus, Mm) FABP4 (AAH02148), FABP5 (NP_034764), FABP8 (XP_485204), and FABP9 (NP_035728) Dashes specify gaps and dots indicate amino acid identity The percentage sequence identity and similarity of zebrafish, Senegalese sole and mouse FABP sequences with zebrafish Fabp11b are shown at the end of each sequence.
Trang 6we have no evidence for orthologs of tetrapod
FABP4, FABP5, FABP8 and FABP9 in teleost
fishes This is based on tblastn searches of
com-plete or nearly comcom-plete sequences for several fish
genomes (e.g Danio rerio, Takifugu rubrifus and
Tet-raodon nigroviridis) in ensembl (http://www.ensembl
org) and in the extensive expressed sequence tag
(EST) databases (e.g salmonids) in GenBank [26]
Agulleiro et al [12] were the first to suggest that
fabp11 formed a novel clade among vertebrate
FABPs and that this gene is unique to teleost fishes
In addition to fabp11 of the Senegalese sole,
homol-ogous fabp genes from the Antarctic fishes [16], and
the zebrafish fabp genes reported here and previously
[17], appear to belong to a sister group of FABP4,
FABP5, FABP8 and FABP9 of mammals, and
tetra-pod FABP4, FABP5, FABP8 and FABP9, and the
teleost fish fabp11 genes might have arisen from a
common ancestral gene [12]
On the basis of the evidence obtained from
phylo-genetic analysis, linkage mapping and conserved gene
synteny, we propose the following model for the evolution of fabp11 in fishes and the FABP4– FABP5–FABP8–FABP9 gene cluster in tetrapods (Fig 7) First, fabp4, fabp5, fabp8 and fabp9 are absent in teleost fishes Second, an ancestral gene diverged to give rise to the progenitors of fabp11 in fishes and FABP4, FABP5, FABP8 and FABP9 in tetrapods, probably before the fish–tetrapod split some 450 mya [27] Third, the FABP4–FABP5– FABP8–FABP9 gene cluster in tetrapods arose by successive tandem duplications and divergence of a single ancestral gene, as has been suggested for the evolution of some of the globin gene clusters [28] Last, the ancestral gene of fabp11 and the FABP4– FABP5–FABP8–FABP9 gene cluster resided on a chromosome, or a part of it, that was the progenitor
of zebrafish LGs (chromosomes) 16 and 19, chicken chromosome 2, mouse chromosome 3, human chro-mosome 8, and frog scaffold 225 (see Experimental procedures for details of retrieval of genomic sequence data)
Gene name
Gene symbol LG position (cM) Mapping panel Gene symbol Chromosomal location
NADH dehydrogenase (ubiquinone)-1beta
subcomplex 9
TAF2 RNA polymerase II, TATA box-binding
protein (TBP)-associated factor
Eukaryotic translation initiation factor 3,
subunit 3 (gamma)
Protein tyrosine phosphatase
type IVA, member 3
Trang 7Tetrapod FABP5, FABP8, FABP9 and FABP4 are
tandemly arrayed and probably arose by unequal
crossing-over – which gene was duplicated first?
Although it is speculative, it is possible to provide a
parsimonious scheme for the tandem duplication
events that gave rise to the cluster of four FABP
genes on a single mammalian chromosome In the
frog genome, fabp4, fabp4-like and fabp5 are
tan-demly arrayed on scaffold 225 (Fig 7) Owing to
sequence identity and their relationship in the
phylo-genetic tree (Fig 6), fabp4 and fabp4-like may have
arisen in the frog genome as a result of tandem
duplication of an ancestral gene on this
chromo-some It is not readily apparent, however, whether
the tandem duplication that gave rise to frog fabp5 occurred before or after the tandem duplication that produced fabp4 and fabp4-like The phylogenetic analysis would favor the fabp4⁄ fabp4-like duplication occurring after the duplication event that generated fabp5 Orthologs of mammalian FABP8 and FABP9 have not yet been identified, or more likely are not present, in the frog genome
Chicken FABP5, FABP8 and FABP4 are also tan-demly arrayed on chromosome 2; on the basis of data-base searches, it seems that FABP9 is absent from the chicken genome Therefore, chicken FABP8 most probably arose from the tandem duplication of either FABP4 or FABP5 Again, the phylogenetic tree (Fig 6) suggests that chicken FABP8 may have origi-nated from tandem duplication of FABP4 rather than FABP5, as FABP8 and FABP4 form a common clade, whereas FABP5 was placed in a different clade Our model is consistent with the time-scale for these dupli-cation events based on synonymous⁄ nonsynonymous amino acid substitution rates in FABP4, FABP5, FABP8 and FABP9, and the topology of the phylo-genetic tree reported by Schaap et al [9]
Fig 6 Phylogenetic tree of selected vertebrate FABPs, showing the relationship between zebrafish Fabp11a and Fabp11b The neighbor-joining tree was constructed using H sapiens LCN1 (NP_002288) as outgroup The bootstrap values (per 100 duplicates) are indicated above or under each node The teleost Fabp11s formed a common clade, which is indicated by a bracket Amino acid sequences used in this analysis include: D rerio (zebrafish) (Dr) Fabp11a (derived from AY628221), and Fabp11b (ENS-DARP0000002311); Gobionotothen gibberifrons (Gog) Fabp11 (H6-Fabp, AAC60354); Notothenia coriiceps (Nc) Fabp11 (H6-(H6-Fabp, AAC60352); Parachaenichthys charcoti (Pc) Fabp11 (H6- Fabp, AAC60355); Ta rubripes (takifugu) (Fr) Fabp11a (deduced from AL837220), and Fabp11b (deduced from AL836636); Te nigroviridis (Tn) Fabp11 (deduced from CR723700); Oryzias latipes (medaka) (Ol) Fabp11 (deduced from BJ899828); Cyprinus carpio (common carp) (Cc) Fabp11 (deduced from CF661735); So senegalensis (Senegalese sole) (Sos) Fabp11 (CAM58515); Gasterosteus aculea-tus (stickleback) (Ga) putative Fabp11a (ENSGACP00000004532), and putative Fabp11b (ENSGACP00000011538); H sapiens (Hs) FABP4 (CAG33184), FABP5 (AAH70303), and FABP8 (AAH34997); FABP9 (PERF15) (Uniprot ID Q0Z7S8); M musculus (mouse) (Mm) FABP4 (AAH02148), FABP5 (NP_034764), FABP8 (XP_485204), and FABP9 (NP_035728); Rattus norvegicus (rat) (Rn) FABP4 (NP_445817), FABP5 (NP_665885), and FABP9 (NP_074045); Sus -scrofa (pig) (Ss) FABP4 (CAC95166); Gal gallus (chicken) (Gg) FABP4 (NP_989621), FABP5 (ENSGALP00000025375), and FABP8 (ENSGALP00000025382); X tropicalis (African frog) (Xt), putative FABP4 (ENSXETP00000022878), putative FABP4-like (NP_ 001096256.1), and putative FABP5 (ENSXETP00000022879); Pan troglodytes (chimpanzee) (Pt) FABP9 (PERF15) (ENS-PTRP00000053126).
Trang 8Fabp5, Fabp8, Fabp9 and Fabp4 are arranged in
sequential order on mouse chromosome 3, and the
FABP5–FABP8–FABP9–FABP4 gene cluster is present
on human chromosome 8 Tandem duplication of
FABP8may have resulted in the formation of FABP9,
as FABP8 and FABP9 formed a common clade (Fig 6)
Experimental procedures
Husbandry of zebrafish
Adult zebrafish were purchased from a local aquarium store
and maintained according to established procedures [29]
Experimental protocols were reviewed by the Animal Care
Committee of Dalhousie University in accordance with the
Canadian Committee on Animal Care
Nucleotide sequence of zebrafish fabp11b cDNA
and gene
We retrieved a novel ensembl gene (ENSDARG000000023
11) from a blastn search of the zebrafish genome sequence
database at the Wellcome Trust Sanger Institute
(ver-sion Zv6; http://www.ensembl.org/Danio_rerio/index.html),
using the fabp11a cDNA sequence as a query [17] The
novel ensembl gene (ENSDARG00000002311) exhibited
most sequence identity and similarity to zebrafish fabp11a,
hereafter referred to as zebrafish fabp11b We confirmed the
sequence of the coding region for fabp11b by comparison
with fabp11b ESTs obtained from the blastn search of
GenBank at the National Center for Biotechnology Infor-mation All ESTs were identical to the coding region of zebrafish fabp11b
Phylogenetic analysis
blosum62 matrix and clustalw [23] were used to align FABP sequences from zebrafish and other vertebrates The bootstrap neighbor-joining tree was constructed using mega4 software [30] Human LCN1 (NP_002288) was used
as the outgroup
Radiation hybrid mapping of zebrafish fabp11b
A detailed protocol for radiation hybrid mapping of zebra-fish genes is described by Hukriede et al [24] PCR reactions were carried out using the forward primer rhf (5¢-GT GTTGTGATTTTCGGTGG-3¢; nucleotide positions 33–51), and the reverse primer rhr (5¢-TTCTGTCATCTGCTG TCGTC-3¢; nucleotide positions 396–423), as shown in Fig 1 PCR conditions were initial denaturation at 94C for
2 min, followed by 30 cycles at 94C for 30 s (denaturation), 54.5C for 30 s (primer annealing) and 72 C for 1 min (elongation), with a final elongation step at 72C for 5 min
In situ hybridization to whole mount embryos and larvae, and to sections of adult zebrafish
Whole mount in situ hybridization to zebrafish embryos and larvae was performed using a riboprobe synthesized
Fig 7 Evolutionary relationship between the duplicated copies of fabp11 in fishes and FABP4, FABP5, FABP8 and FABP9 (PERF15) in frog, chicken, mouse, and human In zebrafish, fabp11a is found on LG 19, and fabp11b is on LG 16 In frog (X tropicalis), fabp4, fabp4-like (fabp4l) and fabp5 are found on scaffold 225, where fabp4l and fabp5 are immediately adjacent to each other Chicken (Gal gallus) FABP5, FABP8 and FABP4 are located next to each other on chromosome 2 FABP5, FABP8, FABP9 (PERF15) and FABP4 are tandemly arrayed on human (H sapiens) chromosome 8 and mouse (M musculus) chromosome 3 Gaps in white indicate the presence of an additional gene between two FABP genes on a particular chromosome.
Trang 9from fabp11b cDNA clone zgc:110029 (GenBank accession
number BC095142), as described by Thisse & Thisse [31]
In situhybridization of an oligonucleotide probe to sections
of adult zebrafish followed the protocol of Denovan-Wright
et al [32] Briefly, sagittal and transverse sections of adult
zebrafish were hybridized to [33P]dATP[aP] 3¢-end-labeled
fabp11bantisense probe, isf (5¢-CACAACACAAGACGTT
TGACAGATAATAGC-3¢; nucleotide positions 11–40),
shown in Fig 1 Following hybridization and
autoradio-graphy, tissue sections were stained with cresyl violet to
identify specific tissues
RT-PCR detection of fabp11b transcripts in adult
zebrafish tissues
RT-PCR was employed for the tissue-specific detection of
fabp11btranscripts in RNA extracted from tissues of adult
zebrafish Following synthesis of cDNA from RNA samples
using the Omniscript RT kit (Qiagen, Mississauga, Ontario,
Canada), fabp11b cDNA was PCR-amplified by the
GACCCTGGA-3¢; nucleotide positions 337–363) and the
reverse primer rtr (5¢-ACCATCCGCAAGGCTCATAGTA
GT-3¢; nucleotide positions 1369–1393), shown in Fig 1
PCR conditions for the amplification of fabp11b transcripts
comprised an initial denaturation step at 94C for 2 min,
followed by 30 cycles at 94C for 30 s (denaturation),
56C for 30 s (primer annealing) and 72 C for 1 min
(elongation), with a final elongation step at 72C for
5 min PCR primers used for detection of elongation
fac-tor 1a (ef1a) transcripts in total zebrafish RNA are
described in Pattyn et al [33] The PCR conditions were
an initial denaturation step at 94C for 2 min, followed by
30 cycles at 94C for 30 s (denaturation), 62 C for 30 s
(primer annealing) and 72C for 1 min (elongation), with a
final elongation step of 72C for 5 min
Database searches of genome sequences and
identification of transcription factor-binding sites
The genomic organization of tetrapod FABP4, FABP5,
FABP8and FABP9 was derived from the Xenopus
tropical-is, Gallus gallus, Mus musculus and Homo sapiens genome
sequence databases at http://www.ensembl.org Putative
transcription factor-binding sites in the 5¢-upstream region
of zebrafish fabp11b were identified using alibaba 2.1
soft-ware [34]
Acknowledgements
The authors thank Mark Soric and Fernanda
Alves-Costa for technical assistance, and David R Smith
and Dr Tudor Borza for helpful comments This work
was supported by funds from the Natural Sciences and
Engineering Research Council of Canada (to
J M Wright), Canadian Institutes of Health Research (to E Denovan-Wright), and the National Institutes of Health, the European Commission as part of the ZF-Models integrated project in the 6th Framework Programme (to B Thisse and C Thisse) S Karanth is
a recipient of a Faculty of Graduate Studies Scholar-ship from Dalhousie University
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 Corsico B, Liou HL & Storch J (2004) The alpha-helical domain of liver fatty acid binding protein is responsible for the diffusion-mediated transfer of fatty acids to phospholipid membranes Biochemistry 43, 3600–3607
3 Ho SY, Delgado L & Storch J (2002) Monoacylglycerol metabolism in human intestinal Caco-2 cells: evidence for metabolic compartmentation and hydrolysis J Biol Chem 277, 1816–1823
4 Murota K & Storch J (2005) Uptake of micellar long-chain fatty acid and sn-2-monoacylglycerol into human intestinal Caco-2 cells exhibits characteristics of protein-mediated transport J Nutr 135, 1626–1630
5 Storch J, Veerkamp JH & Hsu KT (2002) Similar mech-anisms of fatty acid transfer from human and rodent fatty acid-binding proteins to membranes: liver, intes-tine, heart muscle and adipose tissue FABPs Mol Cell Biochem 239, 25–33
6 Veerkamp JH & van Moerkerk HTB (1993) Fatty acid-binding protein and its relation to fatty acid oxidation Mol Cell Biochem 123, 101–106
7 Ong DE, Newcomer ME & Chytil F (1994) Cellular retinoid-binding proteins In The Retinoids: Biology, Chemistry and Medicine, 2nd edn (Sporn MB, Roberts
AB & Goodman DS, eds), pp 283–317 Raven Press, New York, NY
8 Hertzel AV & Bernlohr DA (2000) The mammalian fatty acid-binding protein multigene family: molecular and genetic insights into function Trends Endocrine Metab 11, 175–180
9 Schaap FG, van der Vusse GJ & Glatz JFC (2002) Evo-lution of the family of intracellular lipid binding pro-teins in vertebrates Mol Cell Biochem 239, 69–77
10 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
11 Chan L, Wei C-F, Li W-H, Yang C-Y, Ratner P, Pownall H, Gotto AM Jr & Smith LC (1985) Human liver fatty acid binding protein cDNA and amino acid
Trang 10J Biol Chem 260, 2629–2632.
12 Agulleiro MJ, Andre´ M, Morais S, Cerda` J & Babin PJ
(2007) High transcript level of fatty acid-binding
pro-tein 11 but not of very low-density lipopropro-tein receptor
is correlated to ovarian follicle atresia in a teleost fish
(Solea senegalensis) Biol Reprod 77, 504–516
13 Furlong RF & Holland PW (2002) Were vertebrates
octoploid? Phil Trans R Soc Lond B Biol Sci 357, 531–
544
14 Ohno S (1970) Evolution by Gene Duplication Springer,
New York, NY
15 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 considerably between ray-finned fishes and
land vertebrates Proc Natl Acad Sci USA 101, 1638–
1643
16 Vayda ME, Londraville RL, Cashon RE, Costello L &
Sidell BD (1998) Two distinct types of fatty
acid-bind-ing protein are expressed in heart ventricle of Antarctic
teleost fishes Biochem J 330, 375–382
17 Liu R-Z, Saxena V, Sharma MK, Thisse C, Thisse B,
Denovan-Wright EM & Wright JM (2007) The fabp4
gene of zebrafish (Danio rerio) – genomic homology
with the mammalian FABP4 and divergence from the
zebrafish fabp3 in developmental expression FEBS J
274, 1621–1633
18 Breathnach R & Chambon P (1981) Organization and
expression of eukaryotic split genes coding for proteins
Annu Rev Biochem 50, 349–383
19 Divine JK, Staloch LJ, Haveri H, Rowley CW,
Hei-kinheimo M & Simon TC (2006) Cooperative
interac-tions among intestinal GATA factors in activating the
rat liver fatty acid binding protein gene Am J
Physiol-Gastr L 291, G297–306
20 Sellner P (1994) Developmental regulation of fatty acid
binding protein in neural tissue Dev Dynam 200, 333–
339
21 Liu R-Z, 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
Structure of the mouse myelin P2 protein gene J Neu-rochem 57, 75–80
23 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F
& Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence align-ment aided by quality analysis tools Nucleic Acids Res
25, 4876–4882
24 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
25 Force A, Lynch M, Pickett FB, Amores A, Yan YL & Postlethwait JH (1999) Preservation of duplicate genes
by complementary, degenerative mutations Genetics
151, 1531–1545
26 Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J & Wheeler DL (2005) GenBank Nucleic Acids Res 33, D34–D38
27 Kumar S & Hedges SB (1998) A molecular timescale for vertebrate evolution Nature 392, 917–920
28 Hardison R (1998) Hemoglobins from bacteria to man: evolution of different patterns of gene expression J Exp Biol 201, 1099–1117
29 Westerfield M (2000) The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio) Univer-sity of Oregon Press, Eugene, OR
30 Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) soft-ware version 4.0 Mol Biol Evol 24, 1596–1599
31 Thisse C & Thisse B (2008) High-resolution in situ hybridization to whole-mount zebrafish embryos Nat Protoc 3, 59–69
32 Denovan-Wright EM, Newton RA, Armstrong JM, Babity JM & Robertsin HA (1998) Acute administra-tion of cocaine, but not amphetamine, increase the level
of synaptotgmin IV mRNA in the dorsal striatum of rat Mol Brain Res 55, 350–354
33 Pattyn F, Robbrecht P, Speleman F, De Paepe A & Vandesompele J (2006) RTPrimerDB: the real-time PCR primer and probe database, major update 2006 Nucleic Acids Res 34, D684–D688
34 Grabe N (2002) Alibaba2: context specific identification
of transcription factor binding sites In Silico Biol 2, S1–S15