* Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada; Institut de Génétique et Biologie Moléculaire et Cellulaire, Department of Developmental Biology, CU de Strasbourg, France; and
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
Correspondence: E-mail: jmwright{at}dal.ca.
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Abstract |
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Key Words: gene duplication gene structure phylogeny genome evolution gene expression subfunctionalization
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Introduction |
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Retinoids are hydrophobic molecules, and their transport in the cytoplasm is thought to be facilitated by a group of intracellular carrier proteins, which specifically bind different isomeric retinoids and target them to various intracellular enzymes or receptors for retinoid metabolism and eventual actualization of their physiological activities. During the past decade, increasing evidence has suggested the involvement of intracellular retinoid-binding proteins in retinoid biological action and metabolism (reviewed by Napoli [1999], Noy [2000] and Adida and Spener 2002). Cellular retinoid-binding proteins belong to the large family of low-molecular-mass (15 kDa) intracellular lipid-binding proteins (iLBP) that bind fatty acids, retinoids, and steroids (reviewed by Ong, Newcomer, and Chytil [1994]. Glatz and Vusse [1996], and Bernlohr et al. [1997]). In mammals, two different types of cellular retinoid-binding proteins with distinct ligand-binding properties have been identified, the cellular retinol-binding proteins (CRBPs) and the cellular retinoic acidbinding proteins (CRABPs). Four types of CRBP, including CRBPI, CRBPII, CRBPIII and CRBPIV, are encoded by different genes in mammals (Bashor, Toft, and Chytil 1973; Ong, Newcomer, and Chytil 1984; Folli et al. 2001; Vogel et al. 2001; Folli et al. 2002). Although ligand-binding affinity (Kd) differs among different CRBP types, all CRBPs have affinity for retinol and retinal but do not bind retinoic acid or retinyl esters (Ong 1984; Folli et al. 2001; Vogel et al. 2001; Folli et al. 2002).
To date, only a single copy of the gene coding for either CRBPI or CRBPII has been identified in mammalian genomes. In previous studies, the gene structure, cDNA sequence, linkage relationship, and expression of the genes coding for cellular retinol-binding protein type I (hereafter referred to as rbp1a) and type II (rbp2a) from zebrafish has been determined (Cameron et al. 2002; Liu et al. 2004). Here, we report the discovery of a pair of duplicate genes coding for CRBPI (rbp1b) and CRBPII (rbp2b) in the zebrafish genome. Phylogenetic analysis, conserved gene structure, and linkage relationship suggest that the zebrafish rbp1b and rbp2b are orthologs of the mammalian genes for CRBPI and CRBPII, respectively, and arose by genome-wide duplication in the euteleost lineage some 250 to 400 MYA (Van de Peer, Taylor, and Meyer 2003). Comparative analysis of the mRNA distribution patterns for the gene duplicates of rbp1 and rbp2 in developing and adult zebrafish indicated that the closely related rbp paralogs have different subfunctions, and that "subfunction shuffling" among duplicates of paralogous genes might be an important mechanism for preservation of duplicated genes.
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Materials and Methods |
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Phylogenetic Analysis
Phylogenetic analysis of the zebrafish rbp1b, rbp2b, and the other fish and mammalian cellular retinoid-binding protein genes was performed with ClustalX (Thompson et al. 1997), and the bootstrap neighbor-joining phylogenetic tree was constructed using the zebrafish intestinal-type fatty acidbinding protein sequence (I-FABP) as an outgroup.
Radiation Hybrid Mapping
Radiation hybrids of the LN54 panel (Hukriede et al. 1999) were used to assign the rbp1b and rbp2b genes to a specific zebrafish linkage group. The sequences of the primers employed to amplify the genomic DNA from cell hybrids of LN54 panel are shown in figure 1 (s3 and as2 for rbp1b and s4 and as3 for rbp2b), and the PCR conditions were the same as previously described (Liu et al. 2003a).
Reverse TranscriptionPolymerase Chain Reaction (RT-PCR)
Conditions for RT-PCR used to determine the tissue-specific distribution of the rbp1b and rbp2b transcripts in adult zebrafish were the same as those previously described (Liu et al. 2003a). Gene-specific primers used for RT-PCR are shown in figure1 (s3 and as1 for rbp1b and s4 and as4 for rbp2b), and reaction products were size-fractionated by agarose gel electrophoresis. The tissue-specific mRNA distribution patterns of rbp1b and rbp2b were compared with that of rbp1a and rbp2a (Liu et al. 2004).
Tissue Section in situ Hybridization and Emulsion Autoradiography
Zebrafish tissue section in situ hybridization and emulsion autoradiography were performed as previously described (Liu et al. 2003b). The antisense oligonucleotide was complementary to the zebrafish rbp1b cDNA sequence (p in figure 1).
Whole-Mount in situ Hybridization to Embryos
To reveal the spatio-temporal distribution of the zebrafish rbp1b and rbp2b transcripts in developing zebrafish, whole-mount in situ hybridization was performed as described by C. Thisse and B. Thisse at the Web site: http://zfin.org/zf_info/zfbook/chapt9/9.82. Antisense RNA probes were prepared from the zebrafish rbp1b and rbp2b cDNA generated by 3' RACE.
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Results |
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The transcription start site of the zebrafish rbp1b gene was defined by 5' RLM-RACE, which generated a single product of the intact 5' cDNA end with the 7-methyl G cap. A negative control in which tobacco acid pyrophosphatase (TAP) treatment was omitted yielded no RACE product (data not shown). The 5' RLM-RACE product was cloned and sequenced, and the transcription start site was mapped to the nucleotide 92 bp upstream of the rbp1b ATG initiation codon (fig. 1). In the same way, the transcription start site of rbp2b was shown to be located 36 bp upstream of the rbp2b initiation codon (fig. 1).
Inspection of the 5' upstream sequence of the zebrafish rbp1b and rbp2b genes with MatInspector Professional version 6.2 (Quandt et al. 1995) revealed a TATA box at position 25 of the rbp2b gene sequence, whereas no TATA box was found within the proximal promoter region of rbp1b gene (fig. 1). A TATA box is present in the promoter of the zebrafish rbp2a gene and the mammalian rbp2 gene but not in the rbp1a gene of zebrafish or the single-copy rbp1 genes from other vertebrate species (Liu et al. 2004; Ong, Newcomer, and Chytil 1994).
Characterization of the Zebrafish rbp1b and rbp2b cDNAs
To further characterize the transcripts of the zebrafish rbp1b and rbp2b genes, we isolated their cDNAs. In addition to 5' RLM-RACE, we performed 3' RACE using gene-specific sense primers corresponding to a sequence in the 5' UTR (fig. 1). A single 3' RACE product of expected size was generated, cloned and sequenced for each gene. The complete nucleotide sequence of the zebrafish rbp1b cDNA (GenBank accession number AY395732), determined by combining the 5' RLM-RACE and 3' RACE sequences, was 971 nt in length, excluding the poly(A) tail. The rbp2b cDNA (GenBank accession number AY619686) was 575 nt, excluding the poly(A) sequence. Putative polyadenylation signals were identified 15 and 16 nt upstream of the poly(A) initiation site of the zebrafish rbp1b and rbp2b cDNA sequences, respectively. Alignment of the zebrafish rbp1b cDNA sequence with the gene sequence revealed a GAG insertion in the cDNA sequence at position 144 to 146. The extra GAG in the cDNA sequence resulted in an insertion of a glutamic acid in the CRBPIb amino acid sequence relative to the amino acid sequence deduced from the rbp1b genomic sequence. This discrepancy between the cDNA and genomic sequence is not likely to be a PCR artifact or a sequencing error but rather an intraspecies genetic variation, because independent clones from separate 3' RACE and 5' RLM-RACE reactions contained this extra GAG. The rbp1b gene sequence and an expressed sequence tag (EST) from GenBank (accession number BI886241) did not contain these three nucleotides. At the present time, we do not know whether this variation has any functional implications. Except for the difference of the GAG triplet, the rest of the coding sequence of the zebrafish rbp1b gene and the cloned cDNA was identical. The nucleotide sequence of the zebrafish rbp2b cDNA is identical to its genomic coding sequence.
The deduced amino acid sequence from the zebrafish rbp1b cDNA sequence consisted of 133 amino acids and the CRBPIb protein has a molecular mass of 15.2 kDa and theoretical isoelectric point of 4.94. Alignment of the zebrafish CRBPIb sequence with mammalian CRBPs and CRABPs showed highest sequence identity with that of the mammalian CRBPIs (74%) and the zebrafish CRBPIa (66%), followed by mammalian and zebrafish CRBPIIs (
56%), human CRBPIII (54%), human CRBPIV (51%), mammalian CRABPIs (
33%), and mammalian CRABPIIs (29% to 33%). The deduced amino acid sequence from the zebrafish rbp2b cDNA sequence consisted of 135 amino acids, with an estimated molecular mass of 15.6 kDa and a theoretical isoelectric point of 7.18. The deduced zebrafish CRBPIIb protein had sequence identity of 67% to 70% with mammalian CRBPIIs, 64% with the zebrafish CRBPIIa, 52% to 53% with mammalian CRBPIs, 53% with human CRBPIV, 50% with human CRBPIII, approximate 30% with mammalian CRABPIIs and approximately 27% with mammalian CRABPIs. The divergence of the amino acid sequences between the zebrafish CRBPIa and CRBPIb as well as between CRBPIIa and CRBPIIb is shown in figure 2.
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Discussion |
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In humans, the RBP1 and RBP2 genes are closely linked and both genes have been physically mapped on human chromosome 3 at 3q23 (De Baere et al. 1998a, 1998b). By examining the human genomic DNA sequence, we found these two genes reside on the same contig sequence (GenBank accession number NT005832) and they are approximately 60 kb apart. In the zebrafish, we also identified a DNA assembly sequence from a single clone (GenBank accession number AL953896) that harbors both rbp1b and rbp2b genes separated by approximately 10 kb of DNA. As expected, these two genes were assigned to the same linkage group, LG 2, and the distance between them is 6.7 cR. The zebrafish rbp1a and rbp2a genes were assigned to LG 15 (Cameron et al. 2002) and LG 16 (Liu et al. 2004), respectively, and each of them has conserved syntenies with the human and mouse RBPI and RBPII genes. In the zebrafish genome, another pair of duplicate sister genes, atp1b3a and atp1b3b, also located on LG 2 and LG 15, syntenic with the zebrafish rbp2b and rbp2a gene, respectively. In humans and mouse, the orthologous gene for ATP1B3 (located at 3q22-q23 in humans and LG 9 51cM in mouse) is also closely linked to the RBPII gene (3q23 in humans and LG 9 57 cM in mouse) (Malik et al. 1998; Besirli, Gong, and Lomax 1998). This finding indicates that these two zebrafish rbp2 genes arose and were retained in the zebrafish genome after chromosomal duplication or whole-genome duplication in the euteleost lineage some 250 to 400 MYA (Van de Peer, Taylor, and Meyer 2003; Taylor et al. 2003). It is likely that after the chromosome or genome duplication in the fish lineage, the syntenic relationship of rbp1 and rbp2 genes in fishes was preserved on one of the duplicate chromosomes (rbp1b and rbp2b) but was not retained on the other (rbp1a and rbp2a), presumably because of translocation or chromosomal fission (fig. 4).
Currently, three theories have been proposed for the evolutionary fate of duplicated genes. First, the nonfunctionalization theory states that one of the gene duplicates becomes silenced because of degenerative mutations and becomes a pseudogene or is eventually lost from the genome. Second, the neofunctionalization theory states that one of the gene duplicates acquires a new and beneficial function, while the other duplicate retains the function of the ancestral gene, such that both genes are preserved. Third, a duplication-degeneration-complementation (DDC) model was recently proposed (Force et al. 1999, and references therein), which states that, in addition to the nonfunctionalization and neofunctionalization fate of duplicate genes, subfunctionalization might be an important mechanism for preservation of duplicated genes. After duplication, degenerative mutations in the cis regulatory elements results in partitioning of the original functions into each duplicate gene. As such, the ancestral function is divided between the duplicates, which functionally complement each other and, thus, the duplicate genes are retained in the host genome (Force et al. 1999).
The mammalian rbp1 genes are expressed in a number of adult tissues. In rats, CRBPI is abundantly distributed in the adult liver and kidney and, to a lesser extent, in the lung, testis, spleen, eye, ovary, uterus, and intestine (Ong, Newcomer, and Chytil 1994), whereas in humans, the ovary contains the highest amount of rbp1 gene transcripts and protein, but the levels of CRBPI are relatively low in human kidney and lung (Fex and Johannesson 1984; Ong and Page 1986; Folli et al. 2001). The relative levels of CRBPI in mammalian adult tissues, therefore, vary among species. Expression of the mammalian and the zebrafish rbp1 genes supports the hypothesis that the ancestral rbp1 gene was expressed in multiple adult tissues, including ovary, liver and in the early developing CNS, and retina. It is clear that the duplicate zebrafish rbp1 genes complement each other to retain some of the ancestral rbp1 functions, where rbp1b transcripts have a specialized distribution in the oocytes and rbp1a transcripts are expressed in the developing CNS and retina (Liu et al. 2004). One feature of the zebrafish rbp1 duplicated genes is that they were subfunctionalized both spatially and temporally, which is not stated by the DDC model of duplicate gene retention (Force et al. 1999). Temporal subfunctionalization might be a common feature for developmentally regulated gene duplicates. Thummel et al. (2004) have noted temporal subfunctionalization of the hoxc13a and hoxc13b genes in zebrafish, where hoxc13a is maternally expressed, and hoxc13b is expressed later in development. Neither rbp1a nor rbp1b is abundantly expressed in the adult liver and do not, therefore, complement each other to retain this ancestral subfunction after duplication. By contrast, both zebrafish rbp2a (Cameron et al. 2002, Liu et al. 2004) and rbp2b are expressed in the adult liver, despite the fact that none of the mammalian rbp2 genes studied to date are expressed in the liver. As such, our data suggests that the ancestral subfunction in the liver for rbp1 has been acquired by the duplicates of a closely related paralogous gene, rbp2, after duplication. We propose that "subfunction shuffling" between duplicates of paralogous genes might be a mechanism for preservation of the duplicates from unifunctional genes, such as the zebrafish rbp2 genes (fig. 8). At present, it is not possible to determine how cis-acting elements are shuffled. However, we speculate that mutation or unequal crossing-over between paralogs could result in the loss of cis-element function. Alternatively, a new subfunction to one of the duplicate gene pair may be acquired by "gain-of-function," such as transposon insertion of a new cis-acting element into the regulatory region of one of the duplicate genes .
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Acknowledgements |
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Footnotes |
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