Origin and evolution of vertebrate ABCA genes: A story from Amphioxus
Guang Lia, Qiu-Jin Zhanga,b, Zhi-Liang Jia, Yi-Quan Wanga,⁎
aKey Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, 361005, China
bCollege of Life Sciences, Fujian Normal University, Fuzhou, 350108, China
Received 2 June 2007; received in revised form 20 September 2007; accepted 25 September 2007
Available online 4 October 2007
Received by J.G. Zhang
Previous studies showed that the vertebrate ABCA subfamily, one subgroup of the ATP-binding-cassette superfamily, has evolved rapidly in
terms of gene duplication and loss. To further uncover the evolutionary history of the ABCA subfamily, we characterized ABCA members of two
amphioxus species (Branchiostoma floridae and B. belcheri), the closest living invertebrate relative to vertebrates. Phylogenetic analysis indicated
that these two species have the same set of ABCA genes (both containing six members). Five of these genes have clear orthologs in vertebrate,
including one cephalochordate-specific duplication and one vertebrate-specific duplication. In addition, it is found that human orthologs of
amphioxus ABCA1/4/7 and its neighboring genes mainly localize on chromosome 1, 9, 19 and 5. Considering that most of analyzed amphioxus
genes have clear orthologs in zebrafish, we conclude these four human paralogous regions might derive from a common ancestral region by
genome duplication occurred prior to teleost/tetrapod split. Therefore, the present results provide new evidence for 2R hypothesis.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Amphioxus; Ortholog; Homolog; Comparative genomics; Genome duplication
ATP-binding-cassette (ABC) transporters constitute one of
the largest protein superfamilies in all cellular organisms. Most
ABC proteins anchor in the plasma membrane or membranes of
cellular organelles and transport a wide range of substrates
protein structure, the transporters can be divided into 2 classes:
1) full-transporters containing 2 transmembrane domains
(TMD) and 2 nucleotide binding domains (NBD), and 2) half-
transporters composed of single TMD and NBD (Klein et al.,
1999). The NBD is highly conserved, which contains Walker A,
Walker B and signature C motifs. It binds and hydrolyzes ATP
to provide energy for transporting. The TMD is relatively di-
versified, and it was generally thought to determine the spec-
ificity of substrate transportation (Rosenberg et al., 2003).
In vertebrates, the ABC superfamily can be classified into
8 subfamilies according to their sequence similarities, designated
ABCA∼ABCH (Dean et al., 2001; Annilo et al., 2006). Among
them, the ABCA subfamily is involved in the transmembrane
transport of endogenous lipids. Mutations of ABCA genes may
lead to developmental disorders such as Tangier disease
(Fitzgerald et al., 2004), fatal surfactant deficiency (Shulenin
et al., 2004) and Stargardt's macular dystrophy (Cideciyan et al.,
2004). It is interesting that ABCA subfamily has experienced a
high rate of gene gain and loss during vertebrate evolution. To
date, a total of approximately 30ABCA genes have been reported
in vertebrates (Dean and Annilo, 2005; Annilo et al., 2006; Li
et al., 2007), seven of which (or 23%) are conserved among all
identified in the genomes of invertebrates, e.g. in Drosophila and
C. elegans. However, most of these invertebrate ABCA genes
appear to have derived from frequent species-specific gene
duplication events, which rendered them having no orthologs to
any vertebrate ABCA genes. Thus, the proper transitional species
Available online at www.sciencedirect.com
Gene 405 (2007) 88–95
Abbreviations: ABC, ATP-binding-cassette; N-NBD, N-terminal nucleotide
binding domain; C-NBD, C-terminal nucleotide binding domain; ECD,
⁎Corresponding author. Tel.: +86 952 2184427; fax: +86 952 2181015.
E-mail address: firstname.lastname@example.org (Y.-Q. Wang).
0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
are desired to track the origin and evolution of vertebrate ABCA
Amphioxus, as the closest living invertebrate relative to the
vertebrate, has been widely used for exploring the origin of
vertebrate gene families owing to its simple and prototypical
body-plan and pre-duplicated genome. Thus, characterization of
amphioxus ABCA subfamily might shed light on the subfamily
evolution in vertebrates. In the present study, we characterized
ABCA genes in two amphioxus species, Branchiostoma floridae
and B. belcher, searched ABCA genes from Ciona intestinalis
(Urochordata) and Strongylocentrotus purpuratus (Echinoder-
mata) genome databases, and further performed phylogenetic
analysisontheABCAsubfamilyofvertebrates andtheir relatives.
Moreover, we performed comparative genomic analysis on the
amphioxus ABCA1/4/7-surrounding region and its vertebrate rel-
2. Materials and methods
2.1. Identification of ABCA genes from B. floridae genome
To identify ABCA genes from B. floridae, orthologs of 7
vertebrate-conserved human ABCA protein sequences were
screened against its genome database at http://genome.jgi-psf.
org/Brafl1/Brafl1.home.html using tblastn program (Altschul
et al., 1990). It is noted that the current version of the B. floridae
genome database contains sequences of two haplotypes. To
distinguish alleles from different gene copies, we examined the
relationships of their neighboring genes (5 upstream and 5
downstream genes). If the neighboring genes were parallel and
showed N90% identity at the nucleotide level, we defined these
twogenes asalleles,and,iftheywerenot parallelordidnot show
N90% at the nucleotide level, we treated them as different gene
copies. Because several ABCA genes are not correctly predicted
ABCA genes using GENEWISE (http://www.ebi.ac.uk/Wise2/).
The detailed annotation process has been well described in
references (Yang et al., 2005; Shi and Zhang, 2006). In addition,
we also screened ciona (Ciona intestinalis) and sea urchin
(Strongylocentrotus purpuratus) genome databases to character-
ize their ABCA genes at JGI (http://genome.jgi-psf.org/Cioin2/
Cioin2.home.html) and NCBI (http://www.ncbi.nlm.nih.gov/),
2.2. Cloning and sequencing of ABCA cDNA from B. belcheri
Total RNAs were isolated from the gut, gill, muscle and
gonad of Chinese amphioxus B. belcheri (identified according
to Zhang et al., 2006) using the Trizol kit (Tiangen Co., China)
according to the manufacturer's instructions. The 3′RACE kit
(Takara Co., Japan) was used to reverse-transcribe the extracted
mRNA. A set of degenerated primers, designed based on the
sequences of vertebrate homologs, were used to amplify ABCA
genes from the cDNA. The products were sequenced after
being cloned into pMD 18-T vector (Takara Co., Japan). The
sequencing analysis was performed on a CEQ8000 Automatic
Sequencer (Beckman Coulter Co., USA) and the sequences of
fragments were further assembled using program CAP3 (Huang
and Madan, 1999).
2.3. Exon/intron structure comparison between amphioxus
ABCA1/4/7 and human homologues
The exon/intron structure of B. floridae ABCA1/4/7 was
belcheri ABCA1/4/7 protein sequence using GENEWISE (http://
www.ebi.ac.uk/Wise2/). Gene structures of human ABCA1,
ABCA4 and ABCA7 were directly retrieved from the Ensembl
database. The longest transcriptingenes withmultipletranscripts
was selected for analysis.
2.4. Identification of human orthologs of B. floridae ABCA1/4/7
The Scaffold_28 (S28) and Scaffold_318 (S318) containing
the two alleles of B. floridae ABCA1/4/7 are approximately
2 Gb and 950 kb long respectively. The nucleotide sequences of
all genes on S318 (annotated by JGI) were blated (Kent, 2002)
against B. floridae genome database. In most cases, the query
sequence generally hits two genes: one on the S318 and the
other on the S28. Moreover, the order of the hit genes on the
S318 is the same on the S28 (Supplementary Fig. 1). These
results suggested that the common part of two scaffolds was
correctly assembled. To ensure the reliability of the data sets,
only those genes whose two alleles were separately mapped on
The distribution of B. floridae ABCA on Scaffolds
GeneABCA1/4/7 ABCA2 ABCA3 ABCA5a ABCA5b ABCA3-like
Two ABCA3 haptotypes were found in the Scaffold_5 due to incorrect assembly
and two copies of ABCA5 were named ABCA5a and ABCA5b respectively.
The ABCA proteins of two amphioxus species
ABCA1/4/7 ABCA2 ABCA3 ABCA3-like ABCA5a ABCA5b
Bf vs Bbd
Hs vs Mmd88.2%
aLengths of the protein sequences deduced from the partially cloned B.
belcheri ABCA genes.
bLengths of B. floridae ABCA proteins annotated by JGI (the longer allele
cThe percentage of length of the determined B. belcheri ABCA protein to
that of B. floridae.
dThe identities of B. floridae–B. belcheri and human–mouse ABCA
proteins. It is notable that the identities were calculated based on the complete-
deletion pattern after each gene protein sequences of the four species were
aligned. ABCA4, rather than ABCA4 and ABCA7, was chose in the
comparison with amphioxus ABCA1/4/7, and for ABCA5a and ABCA5b,
human and mouse ABCA5 were compared to them.
89G. Li et al. / Gene 405 (2007) 88–95
the S28 and S318 were chosen, and the longer alleles (annotated
by JGI) were adopted for the subsequent analysis. Each selected
gene was used to search for its homologs in several divergent
representative organisms in NCBI Refseq and JGI databases
using the blastp program (Altschul et al., 1990). These or-
ganisms include Homo sapiens, Mus musculus, Gallus gallus,
Xenopus tropicalis, Danio rerio, Ciona intestinalis, Strongylo-
centrotus purpuratus and Drosophila melanogaster. The blastp
result for each gene was separately displayed for each species
with 10 descriptions and E value=10−5. The retrieved human
protein sequences were used to blastp back to B. floridae
database and the first ten hit sequences were recorded. The
acquired sequences were aligned using CLUSTAL_X (Thomp-
son et al., 1997). To determine the major groups, we constructed
Neighbor–Joining (NJ) trees (Saitou and Nei, 1987) with
Poisson Correction distance and pairwise deletion comparison
(Nei and Kumar, 2000). The groups were defined by high
distance values and high bootstrap proportions (Felsenstein,
1985). The sequences in the group containing amphioxus gene
were retained and were used in final phylogenetic analysis using
the NJ method (Saitou and Nei, 1987) with Poisson Correction
distance and complete deletion comparison (Nei and Kumar,
2000). If the final tree was supported by high bootstrap values,
we assigned the human genes in the group as orthologs of the
corresponding amphioxus gene. The BLAT program at UCSC
was used to map the human genes (http://www.genome.ucsc.
2.5. Real-time quantitative PCR (RTQPCR)
Total RNA extracted from eight different organs, including
Fig. 1. Phylogenetic relationship of ABCA genes in Chordata. The tree was reconstructed by the neighbor-joining method with protein Poisson distances and
complete-deletion pattern. Bootstrap percentages are shown on interior branches. Accession numbers the genes are listed in Supplementary Table 1.
90 G. Li et al. / Gene 405 (2007) 88–95
Fig. 2. Comparison of exon/intron structure of ABCA1/4/7 and human ABCA1, ABCA4, and ABCA7. Boxes represent exons and numbers in the boxes denote exon sizes (nt). Bold lines mean that exon/intron structures
inthoseregionsareconservedacrossall genes, whereasboldbrokenlines meanthatexon/intron structuresareconservedatleastbetweentwogenes.The diagramatthe bottomshowsthe maindomainsof ABCAproteins
(after Illing et al., 1997) and the numbers under it represent mean identities of the main domains. Numbers 1-7 represent 7 transmembrane segments.
G. Li et al. / Gene 405 (2007) 88–95
d(t) primers (TaKaRa Co., Japan). SYBR Green real-time PCR
reactions were carried out on the Rotor-Gene 3000 (Corbett
Robotics, Australia), and conditions were 94 °C for 1 min,
followed by 40 cycles of 94 °C for 30 s, 58 °C for 15 s and 72 °C
for 20 s. Normalization was performed using β-actin as an
internal control. We examined only the expression profile of
amphioxus ABCA1/4/7 gene. The primer sequences and related
information are available on request.
3.1. ABCA genes in two amphioxus species
TBLASTN program was implemented to screen the B.
floridae genome sequence. Twelve gene models representing 6
distinct ABCA genes were identified, and their locations on the
from the ciona genome and this result is consistent with the
vertebrates. For example, there are 12 and 16 functional ABCA
genes in human and mouse genomes respectively (Ban et al.,
in zebrafish (Dean and Annilo, 2005). There are at least eleven
sequences representingABCAgenes inthe newly-built sea urchin
genome database, but only four of them are fully sequenced. Of
the rest, two sequences are relatively longer and have at least two
of the four main ABCA domains, and the others are all less than
have six to eleven ABCA genes.
To further investigate the evolution of ABCA subfamily in
cephalochordates and perform a comparison between different
amphioxus species, the ABCA genes were amplified from the
cDNA of another amphioxus species B. belcheri using PCR
method. Six ABCA genes were characterized in this species.
Constrained by the very large size of ABCA proteins (N1500 aa),
these genes were partially sequenced for ascertaining the category
of B. belcheri ABCA genes. As shown in Table 2, four of them
covered more than 50% of their B. floridae orthologous genes
(being compared to the longer JGI-annotating alleles). The
nucleotide sequences of the 6 ABCA genes from B. belcheri
were blated against the B. floridae genome database. The results
ABCA2, ABCA3, ABCA3-like, ABCA5a and ABCA5b (data not
shown), suggesting that these genes are one-to-one orthologs.
Moreover, we applied 5 degenerated primers (two forward and
three backward) corresponding to the N-terminal nucleotide-
binding region of vertebrate ABCA12 genes in an attempt to
amplify B. belcheri ABCA12 gene, but failed to generate the gene
segment. It suggests thatB. belcheri might also have no orthologs
of vertebrate ABCA12 gene as in B. floridae (see Section 3.2).
3.2. Phylogeny of amphioxus ABCA genes
To determine the evolutionary relationship of amphioxus
ABCA genes, a phylogenetic tree was constructed using the
protein sequences of ABCA genes from amphioxus and four
representative animals including human, mouse, ciona and sea
urchin (Fig. 1). Since most vertebrate ABCA genes derived from
multiple duplications occurring after the lineage divergence,
only seven conserved ABCA genes, ABCA1–ABCA5, ABCA7
and ABCA12, from human and mouse were adopted. And since
B. floridae and B. belcheri contain an identical gene set, the
incomplete B. belcheri ABCA protein sequences were not
sequences were applied in the analysis to ensure enough
informative sites. As shown in Fig. 1, five of six amphioxus
ABCA genes were found with human orthologs: ABCA2 and
ABCA3 in a one-to-one pattern, ABCA1/4/7 in a one-to-three
pattern, and ABCA5 in a two-to-one pattern. In contrast,
ABCA3-like in amphioxus has no cognate in human genes, and
human ABCA12 is not orthologous to any amphioxus ABCA
3.3. Exon/intron structure of amphioxus ABCA1/4/7 gene
Phylogenetic analysis indicates that vertebrate ABCA1,
ABCA4 and ABCA7 derived from ABCA1/4/7 at the birth of
vertebrates by multiple duplications (Fig. 1). To further address
Fig. 3. Map positions of amphioxus ABCA1/4/7 and its surrounding genes, and their human orthologs. Numbers represent the assigned orders of amphioxus genes
analyzed. Amphioxus genes are shown in their order, but their human orthologs are not drawn strictly in order. The human gene nomenclature was set according to the
UCSCdatabase. Amphioxusgenes withoutvertebrate homologs(forgene 8,9, 11and19) or well resolvedphylogeneticrelationships(forgene12) are notshownhere.
Human orthologs of gene 7 and 13 are not mapped on the four main regions. It is notable that human orthologs, SPATA6 and C9orf68, of amphioxus gene 4 were
determined by their reciprocal blast hits rather than by phylogenetic analysis.
92G. Li et al. / Gene 405 (2007) 88–95
the origin of vertebrate ABCA1, ABCA4 and ABCA7, we char-
acterized the exon/intron structure of amphioxus ABCA1/4/7 by
aligning the B. belcheri ABCA1/4/7 protein sequence to its B.
it with that of human ABCA1, ABCA4 and ABCA7 (Fig. 2). The
results illustrated that many exons of these four genes are
which suggests these three vertebrate ABCA genes arose from
its progenitor ABCA1/4/7 by genome duplication, rather than by
retroposition (Zhang, 2003).
3.4. Map comparison of ABCA1/4/7 neighboring genes and
their human orthologs
along with ABCA1/4/7, we applied their protein sequences to
search for orthologs in several divergent animals using the blastp
program and phylogenetic analysis (see Section 2.4 and
Supplementary Fig. 2). Due to inadequate genome information,
only 21 ABCA1/4/7 neighboring genes (see Section 2.4 and
Supplementary Fig. 1) were selected in the present analysis (their
assigned orders and model names are listed in Supplementary
Table2). Theapplications revealedthat17selectedgenes showed
significant similarity with known genes in the examined ver-
tebrates (Supplementary Fig. 2). Among those 17 genes, 15 have
apparent orthologs in humans with five being in a one-to-two
pattern and 10 being in a one-to-one pattern. Of these 20 human
orthologs, 14 genes are mapped on chromosomes 1, 9 and 19
wherehumanABCA4,ABCA1andABCA7are located(Fig. 3).In
addition, there are 3 human orthologs on chromosome 5, which
might indicate that this region would be another orthologous
region of amphioxus genome containing ABCA1/4/7 and their
linked genes. These results, together with the finding that almost
all of the 15 B. floridae genes also have orthologs in zebrafish
(Supplementary Fig. 2), suggested region ofABCA1/4/7 and their
linked genes had been duplicated at least twice in the vertebrate
evolution just after its separation from the cephalochordate. This
conclusion is consistent with the findings of Hox genes (Holland
et al., 1994), MHC (Abi-Rached et al., 2002), NK homeobox
genes (Luke et al., 2003) and Fox genes (Wotton and Shimeld,
2006), thus providing a new evidence for the hypothesis of two-
round (2R) whole genome duplication.
3.5. Expression profile of amphioxus ABCA1/4/7 gene
In order to know if the expression of the amphioxus ABCA1/
4/7 is similar to that of its mammalian paralogs, we performed a
real-time quantitive PCR to access the gene expression profile
in eight tissues. The results (Fig. 4.) demonstrated that ABCA1/
4/7 mRNA exists in all examined tissues with especially high
levels in the pharyngeal gill, segmental muscles, hepatic diver-
ticulum and notochord, and relatively low levels in ovaries,
testes, intestines and metapleural folds. This expression pattern
is more similar to that of vertebrate ABCA1 gene than that of
ABCA4 or ABCA7 (see discussion).
The ABCA subfamily belongs to ABC multigene superfamily
and its evolution has been studied in several distantly related
species, including Drosophila (Dean et al., 2001), C. elegans
(Sheps et al., 2004), zebrafish, chicken and humans (Dean and
Annilo, 2005; Li et al., 2007). Possibly due to their distant
relationship or the rapid evolution of ABCA genes, no apparent
human ABCA orthologs were identified in Drosophila and C.
elegans genomes (Dean et al., 2001; Sheps et al., 2004). In the
present study, we found that the vertebrate ABCA2, ABCA3 and
ABCA5 also existed in the genomes of B. floridae, ciona and
sea urchins, which suggested that these three genes arose before
the divergence of deuterostome. In contrast, the ABCA1, ABCA4
and ABCA7 appeared to have derived just before the appearance
of the vertebrate lineage, since only their progenitor ABCA1/4/7
identified in amphioxus, ciona or sea urchins. Its position at the
base of ABCA1/4/7 and ABCA2 (bootstrap value=76%, Fig. 1)
indicated that it arose before the last common ancestor of
chordates, urochordates and echinoderms. However, it appears to
be more parsimonious to consider that ABCA12 diverged at the
early stage of vertebrate evolution if the low possibility was
considered that ABCA12 was lost in Cephalochordata, Urochor-
separation at the birth of Deuterostomia.
Moreover, six ABCA genes from Chinese amphioxus B.
belcheriwere cloned in this study.They showed apparently one-
to-one orthologous relationships with B. floridae ABCA genes
by sequence analysis using the BLAT program. Therefore, it is
inferred that these two amphioxus species should have a same
ABCA gene set, although they diverged about 120 million
years ago (Zhong et al., unpublished data), almost 1.5 times
longer ago than the primate-rodent split. Six genes are different
between the mouse and human ABCA subfamilies, five of
which are caused by gene loss (one in mouse and four in human)
and one by gene duplication (in mouse) (Annilo et al., 2006).
These results indicate that the ABCA subfamily evolved more
slowly in amphioxus than in mammals. Furthermore, as it
showed in Table 2, all ABCA genes showed significantly higher
levels of sequence identities between B. floridae and B. belcheri
Fig. 4. Relative expression levels of amphioxus ABCA1/4/7 gene in eight
different tissues. Abbreviations: PhG — pharyngeal gill; MeF — metapleural
fold; SeM — segmental muscles; HeD — hepatic diverticulum; Nt —
93G. Li et al. / Gene 405 (2007) 88–95
than that between mouse and human (z-test, p=0.014), which
suggests that each amphioxus ABCA gene might slow down its
evolutionary rates after the cephalochordate-vertebrate split.
Our phylogenetic analysis and exon/intron structure com-
parison suggests that vertebrate ABCA1, ABCA4 and ABCA7
genes derive from the founder gene ABCA1/4/7 by genome
duplication. In vertebrates, ABCA1 mediated efflux of cellular
cholesterol and phospholipids to apoA-I, and its mutation are
protein is proposed to be involved in the transport of all-trans-
retinal aldehyde. Its mutation was suggested to be responsible
recessive CRD and autosomal recessive RP (Cideciyan et al.,
2004). ABCA7, the closest homolog of ABCA1, can also
(Wang et al., 2003). Moreover, these three genes exhibited
distinct expression profiles. For example, ACBA1 mRNA was
expressed at a high level in the liver, lung, small intestine and
ABCA7 was found mainly in myelo-lymphatic tissues including
peripheral leukocytes, thymus, spleen and bone marrow
(Kaminski and Orsó, 2000). ABCA4 is more tissue-specific
and its expression was restricted to the retina (Rust and Rosier,
1999). Based on these observations, we could infer that these
three vertebrate ABCA genes have functionally diverged by
alteration of expression pattern or substrate specificity, or by a
combination of the two methods. Interestingly, the function of
ABCA1 appeared more ancient than that of the other two genes,
since its expression pattern is most similar to that of amphioxus
ABCA1/4/7. Homology analysis revealed (Fig. 2) the high
conservation (N56% identity) of both NBDs and TMs, which
suggests that they are functionally conserved. In contrast to
NBD and TM, the extracellar regions of the ABCA genes are
relatively divergent (N-ECD=34%, C-ECD=43%). Thus they
are probably responsible for substrate specificity if they have
the function of amphioxus ABCA1/4/7 in a further study.
Expansion of gene number is assumed to be associated with
the increasing complexity of vertebrate organs and life process.
Two competing hypotheses have been proposed to interpret the
gene number increase in vertebrates: One theory asserts that this
increase was caused by two rounds (2R) of whole genome
duplication in the early vertebrate evolution (Ohno, 1970;
Holland et al., 1994; Abi-Rached et al., 2002; Luke et al., 2003;
Dehal and Boore, 2005; Wotton and Shimeld, 2006),whereas the
second considers that continuous gene duplication occurring
throughout vertebrate evolution has played a crucial role in
the increase of vertebrate gene numbers (Hughes, 1998, 1999;
Friedman and Hughes, 2001; Martin, 2001). Comparisons of
extended Hox gene clusters (Holland et al., 1994), MHC genes
(Abi-Rached et al., 2002) and the recently reported Fox gene
cluster (Wotton and Shimeld, 2006), have provided important
provided for the 2R hypothesis by comparing the amphioxus
ABCA1/4/7 region with its human paralogous region. In addition,
our study also showed that genes with a one-to-one orthologous
pattern between amphioxus and humans can also provide im-
portant implications for two rounds of entire genome duplication
(Fig. 3). This could be because the amphioxus and human ge-
nomes did not undergo extensive rearrangement (Minguillon
et al., 2005) and that paralogous genes in some segments of
chromosomes were retained randomly after two rounds genome
The authorsthank Dr.Peng Shifor his comments on theearly
manuscript and Ms. Deborah Jane Lowe at School of Medicine,
Boston University for her excellent proofreading. We are also
grateful to two anonymous reviewers for their constructive
30470938 & 30570208) and Natural Science Foundation of
Fujian Province, China (No. D0510002).
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