Origin and evolution of vertebrate ABCA genes: A story from Amphioxus

Available online at www.sciencedirect.com

Gene 405 (2007) 88 – 95 www.elsevier.com/locate/gene

Origin and evolution of vertebrate ABCA genes: A story from Amphioxus Guang Li a , Qiu-Jin Zhang a,b , Zhi-Liang Ji a , Yi-Quan Wang a,⁎ a

Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, 361005, China b College 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

Abstract 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

1. Introduction 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 (Croop, 1998; Klein et al., 1999; Dean et al., 2001). Based on the 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) halftransporters 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-

Abbreviations: ABC, ATP-binding-cassette; N-NBD, N-terminal nucleotide binding domain; C-NBD, C-terminal nucleotide binding domain; ECD, exocytoplasmic domain. ⁎ Corresponding author. Tel.: +86 952 2184427; fax: +86 952 2181015. E-mail address: [email protected] (Y.-Q. Wang). 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.09.018

versified, and it was generally thought to determine the specificity 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 30 ABCA 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 vertebrate genomes. Also, members of the ABCA subfamily were 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

G. Li et al. / Gene 405 (2007) 88–95 Table 1 The distribution of B. floridae ABCA on Scaffolds Gene

ABCA1/4/7 ABCA2 ABCA3 ABCA5a ABCA5b ABCA3-like

No. of 318 and scaffold 28

11 and 630

5

112 and 17

269 and 520

251 and 559

Two ABCA3 haptotypes were found in the Scaffold_5 due to incorrect assembly and two copies of ABCA5 were named ABCA5a and ABCA5b respectively.

are desired to track the origin and evolution of vertebrate ABCA genes. 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 (Echinodermata) genome databases, and further performed phylogenetic analysis on the ABCA subfamily of vertebrates and their relatives. Moreover, we performed comparative genomic analysis on the amphioxus ABCA1/4/7-surrounding region and its vertebrate relative regions. 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 two genes as alleles, and, if they were not parallel or did not show N 90% at the nucleotide level, we treated them as different gene copies. Because several ABCA genes are not correctly predicted in the current amphioxus genome database, we re-predicted all the 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 characterize their ABCA genes at JGI (http://genome.jgi-psf.org/Cioin2/ Cioin2.home.html) and NCBI (http://www.ncbi.nlm.nih.gov/), respectively. 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)

89

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 determined by aligning the genome sequence with the deduced B. 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 transcript in genes with multiple transcripts was selected for analysis. 2.4. Identification of human orthologs of B. floridae ABCA1/4/7 neighboring genes 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

Table 2 The ABCA proteins of two amphioxus species Amphioxus ABCA1/4/7 ABCA2 ABCA3 ABCA3-like ABCA5a ABCA5b ABCA gene Bb (aa) a Bf (aa) b Bb/Bf c Bf vs Bb d Hs vs Mm d

2174 2241 97.0% 92.1% 88.2%

1300 2460 52.8% 97.2% 95.6%

1041 1594 65.3% 94.0% 88.6%

104 2130 4.9% – –

1188 1620 73.3% 90.6% 89.7%

177 2160 8.2% 93.7% 90.2%

a Lengths of the protein sequences deduced from the partially cloned B. belcheri ABCA genes. b Lengths of B. floridae ABCA proteins annotated by JGI (the longer allele was adopted). c The percentage of length of the determined B. belcheri ABCA protein to that of B. floridae. d The identities of B. floridae–B. belcheri and human–mouse ABCA proteins. It is notable that the identities were calculated based on the completedeletion 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.

90

G. 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 organisms include Homo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalis, Danio rerio, Ciona intestinalis, Strongylocentrotus 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 (Thompson 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. edu/). 2.5. Real-time quantitative PCR (RTQPCR) Total RNA extracted from eight different organs, including pharyngeal gill, segmental muscles, hepatic diverticulum, ovary, testis, intestine, notochord and metapleural fold (the fold of body wall), was reverse-transcribed to cDNA using random and oligo-

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.

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 in those regions are conserved across all genes, whereas bold broken lines mean that exon/intron structures are conserved at least between two genes. The diagram at the bottom shows the main domains of ABCA proteins (after Illing et al., 1997) and the numbers under it represent mean identities of the main domains. Numbers 1-7 represent 7 transmembrane segments.

91

92

G. Li et al. / Gene 405 (2007) 88–95

d(t) primers (TaKaRa Co., Japan). SYBR Green real-time PCR master mix (TaKaRa Co., Japan) was used for PCR reaction. The 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. Results

(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 showed that they most closely matched the B. floridae ABCA1/4/7, 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 nucleotidebinding region of vertebrate ABCA12 genes in an attempt to amplify B. belcheri ABCA12 gene, but failed to generate the gene segment. It suggests that B. belcheri might also have no orthologs of vertebrate ABCA12 gene as in B. floridae (see Section 3.2).

3.1. ABCA genes in two amphioxus species 3.2. Phylogeny of amphioxus ABCA genes 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 Scaffolds are shown in Table 1. Three ABCA genes were extracted from the ciona genome and this result is consistent with the previous report (Annilo et al., 2006). It is obvious that members of the ABCA subfamily in sea squirt and amphioxus are fewer than in vertebrates. For example, there are 12 and 16 functional ABCA genes in human and mouse genomes respectively (Ban et al., 2005; Dean and Annilo, 2005), and 9 ABCA genes were identified in zebrafish (Dean and Annilo, 2005). There are at least eleven sequences representing ABCA genes in the 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 600 aa in length. Therefore, we inferred that the sea urchin should 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 (N 1500 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

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 included in the analysis. Only six sea urchin ABCA genes of long 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 genes. 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 UCSC database. Amphioxus genes without vertebrate homologs (for gene 8, 9, 11 and 19) or well resolved phylogenetic relationships (for gene 12) are not shown here. 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.

G. Li et al. / Gene 405 (2007) 88–95

the origin of vertebrate ABCA1, ABCA4 and ABCA7, we characterized the exon/intron structure of amphioxus ABCA1/4/7 by aligning the B. belcheri ABCA1/4/7 protein sequence to its B. floridae ortholog genome sequence (S318), and further compared it with that of human ABCA1, ABCA4 and ABCA7 (Fig. 2). The results illustrated that many exons of these four genes are conserved in size, especially in TMs and NBDs encoding regions, 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 To examine whether the neighboring genes had also duplicated 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 Table 2). The applications revealed that 17 selected genes showed significant similarity with known genes in the examined vertebrates (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 where human ABCA4, ABCA1 and ABCA7 are 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 of ABCA1/4/7 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,

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 — notochord.

93

2006), thus providing a new evidence for the hypothesis of tworound (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 diverticulum 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). 4. 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 was retrieved from the above species. Vertebrate ABCA12 was not 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, Urochordata and Echinodermata, and only preserved in Vertebrata after its separation at the birth of Deuterostomia. Moreover, six ABCA genes from Chinese amphioxus B. belcheri were cloned in this study. They showed apparently oneto-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

94

G. 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 comparison 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 associated with Tangier disease (Fitzgerald et al., 2004). ABCA4 protein is proposed to be involved in the transport of all-transretinal aldehyde. Its mutation was suggested to be responsible for a spectrum of retinal dystrophies including STGD, autosomal recessive CRD and autosomal recessive RP (Cideciyan et al., 2004). ABCA7, the closest homolog of ABCA1, can also regulate the release of cellular cholesterol and phospholipids like ABCA1; however, it does not promote cellular cholesterol efflux (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 adrenal glands, and at a low level in the testis, ovary, and skeletal muscles (Langmann and Aslanidis, 1999), while the transcript of 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 (N 56% 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 different spectrum of substrates. It may be of interest to investigate 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 several gene clusters between amphioxus and vertebrates, such as 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 indications for the 2R hypothesis. In this paper, new evidence was 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 important implications for two rounds of entire genome duplication (Fig. 3). This could be because the amphioxus and human genomes 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 duplication. Acknowledgements The authors thank Dr. Peng Shi for his comments on the early 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 comments. This work was supported by grants from NSFC (Nos. 30470938 & 30570208) and Natural Science Foundation of Fujian Province, China (No. D0510002). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gene.2007.09.018. References Abi-Rached, L., Gilles, A., Shiina, T., Pontarotti, P., Inoko, H., 2002. Evidence of en bloc duplication in vertebrate genomes. Nat. Genet. 31, 100–105. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Annilo, T., et al., 2006. Evolution of the vertebrate ABC gene family: analysis of gene birth and death. Genomics 88, 1–11. Ban, N., Sasaki, M., Sakai, H., Ueda, K., Inagaki, N., 2005. Cloning of ABCA17, a novel rodent sperm-specific ABC (ATP-binding cassette) transporter that regulates intracellular lipid metabolism. Biochem. J. 389, 577–585. Cideciyan, A.V., et al., 2004. Mutations in ABCA4 result in accumulation of lipofuscin before slowing of the retinoid cycle: a reappraisal of the human disease sequence. Hum. Mol. Genet. 13, 525–534. Croop, J.M., 1998. Evolutionary relationships among ABC transporters. Methods Enzymol. 292, 101–116. Dean, M., Annilo, T., 2005. Evolution of the ATP-binding cassette (ABC) transporter superfamily in vertebrates. Annu. Rev. Genomics Hum. Genet. 6, 123–142. Dean, M., Rzhetsky, A., Allikmets, R., 2001. The human ATP-binding cassette (ABC) transporter superfamily. Genome Res. 11, 1156–1166. Dehal, P., Boore, J.L., 2005. Two rounds of whole genome duplication in the ancestral vertebrate. PLoS Biol. 3, e314. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39. Fitzgerald, M.L., Okuhira, K., Short III, G.F., Manning, J.J., Bell, S.A., Freeman, M.W., 2004. ATP-binding cassette transporter A1 contains a novel C-terminal VFVNFA motif that is required for its cholesterol efflux and ApoA-I binding activities. J. Biol. Chem. 279, 48477–48485. Friedman, R., Hughes, A.L., 2001. Pattern and timing of gene duplication in animal genomes. Genome Res. 11, 1842–1847. Holland, P.W., Garcia-Fernandez, J., Williams, N.A., Sidow, A., 1994. Gene duplications and the origins of vertebrate development. Dev. Suppl. 125–133. Huang, X., Madan, A., 1999. CAP3: A DNA sequence assembly program. Genome Res. 9, 868–877.

G. Li et al. / Gene 405 (2007) 88–95 Hughes, A.L., 1998. Phylogenetic tests of the hypothesis of block duplication of homologous genes on human chromosomes 6, 9, and 1. Mol. Biol. Evol. 15, 854–870. Hughes, A.L., 1999. Phylogenies of developmentally important proteins do not support the hypothesis of two rounds of genome duplication early in vertebrate history. J. Mol. Evol. 48, 565–576. Illing, M., Molday, L.L., Molday, R.S., 1997. The 220-kDa rim protein of retinal rod outer segments is a member of the ABC transporter superfamily. J. Biol. Chem. 272, 10303–10310. Kaminski, W.E., Orsó, E., 2000. Identification of a Novel Human SterolSensitive ATP-Binding Cassette Transporter (ABCA7). Biochem. Biophys. Res. Commun. 273, 532–538. Kent, W.J., 2002. BLAT—the BLAST-like alignment tool. Genome Res. 12, 656–664. Klein, I., Sarkadi, B., Varadi, A., 1999. An inventory of the human ABC proteins. Biochim. Biophys. Acta 1461, 237–262. Langmann, T., Aslanidis, C., 1999. Molecular cloning of the human ATPbinding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages. Biochem. Biophys. Res. Commun. 257, 29–33. Li, G., Shi, P., Wang, Y., 2007. Evolutionary dynamics of the ABCA chromosome 17q24 cluster genes in vertebrates. Genomics 89, 385–391. Luke, G.N., Castro, L.F., McLay, K., Bird, C., Coulson, A., Holland, P.W., 2003. Dispersal of NK homeobox gene clusters in amphioxus and humans. Proc. Natl. Acad. Sci. U. S. A. 100, 5292–5295. Martin, A., 2001. Is tetralogy true? Lack of support for the “one-to-four rule”. Mol. Biol. Evol. 18, 89–93. Minguillon, C., et al., 2005. No more than 14: the end of the amphioxus Hox cluster. Int. J. Biol. Sci. 1, 19–23. Nei, M., Kumar, S., 2000. Molecular Evolution and Phylogenetics. Oxford University Press, New York. Ohno, S., 1970. Evolution by gene duplication. Springer-Verlag, New York. Rosenberg, M.F., Kamis, A.B., Callaghan, R., Higgins, C.F., Ford, R.C., 2003. Three-dimensional structures of the mammalian multidrug resistance Pglycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding. J. Biol. Chem. 278, 8294–8299.

95

Rust, S., Rosier, M., 1999. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat. Genet. 22, 352–355. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Sheps, J.A., Ralph, S., Zhao, Z., Baillie, D.L., Ling, V., 2004. The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes. Genome Biol. 5, R15. Shi, P., Zhang, J., 2006. Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor genes. Mol. Biol. Evol. 23, 292–300. Shulenin, S., Nogee, L.M., Annilo, T., Wert, S.E., Whitsett, J.A., Dean, M., 2004. ABCA3 gene mutations in newborns with fatal surfactant deficiency. N. Engl. J. Med. 350, 1296–1303. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Wang, N., et al., 2003. ATP-binding cassette transporter A7 (ABCA7) binds apolipoprotein A-I and mediates cellular phospholipid but not cholesterol efflux. J. Biol. Chem. 278, 42906–42912. Wotton, K.R., Shimeld, S.M., 2006. Comparative genomics of vertebrate Fox cluster loci. BMC Genomics 7, 271. Yang, H., Shi, P., Zhang, Y.P., Zhang, J., 2005. Composition and evolution of the V2r vomeronasal receptor gene repertoire in mice and rats. Genomics 86, 306–315. Zhang, J.Z., 2003. Evolution by gene duplication: an update. Trends Ecol. Evol. 18, 292–298. Zhang, Q.J., Zhong, J., Fang, S.H., Wang, Y.Q., 2006. Branchiostoma japonicum and B. belcheri are distinct lancelets (Cephalochordata) in Xiamen Waters in China. Zool. Sci. 23, 573–579.