Identification of three isoforms for mitochondrial adenine nucleotide translocator in the pufferfish Takifugu rubripes

Three adenine nucleotide translocator (ANT) genes were identified through in silico data mining of the Fugu genome database along with isolation of their corresponding cDNAs in vivo from the pufferfish (Takifugu rubripes). As a result of phylogenetic analysis, the ANT gene on scaffold_254 corresponded to mammalian ANT1, whereas both of those on scaffold_6 and scaffold_598 to mammalian ANT3. The ANT gene encoded by scaffold_6 was expressed ubiquitously in various tissues, whereas the ANT genes encoded by scaffold_254 and scaffold_598 were predominantly expressed in skeletal muscle and heart, respectively.     

Functional characterisation and genomic analysis of an epithelial calcium channel (ECaC) from pufferfish, Fugu rubripes

An orthologue to the mammalian epithelial calcium channels, ECaC1 (TRPV5) and ECaC2 (TRPV6), was cloned from gill of pufferfish (Fugu rubripes) and characterised, demonstrating that this gene predates the evolution of land-living vertebrates. The F. rubripes ECaC (FrECaC) protein displays all structural features typical for mammalian ECaCs including three ankyrin repeats, six transmembrane domains, and a putative pore region between TM V and TM VI. Functional expression of FrECaC in Madin-Darby canine kidney (MDCK) cells confirmed that the channel mediates Ca(2+) influx. FrECaC was also permeable to Zn(2+) and, to a small extent, to the Fe(2+) ion. Thus, in addition to a role in Ca(2+) uptake FrECaC might serve as a pathway for zinc and iron acquisition. FrECaC mRNA was highly abundant in the gill, but sparsely present in the intestine. Calcium absorption via FrECaC in pufferfish may be subject to the regulation of 1.25(OH)(2)D(3), estrogen and progesterone as consensus cis regulatory elements for the respective steroid hormone receptors were found in the upstream regulatory region of the FrECaC gene. FrECaC gene organisation is very conserved when compared with mammalian ECaCs. Only one ECaC gene seems to exist in the F. rubripes genome, and the corresponding protein clusters together with ECaC2 from mammals upon phylogenetic analysis. Thus, the two mammalian ECaC genes may originate from a single ancestral ECaC2 gene in vertebrates appearing early in evolution.     

Pattern of divergence of amino acid sequences encoded by paralogous genes in human and pufferfish

We used phylogenetic analyses of protein families containing two or more pairs of orthologues in the genomes of human and pufferfish (Takifugu rubripes) to test the hypothesis that these sequences show a strong signal of polyploidization events hypothesized to have occurred early in vertebrate history. In order to test for evidence of two distinct rounds of polyploidization (the 2R hypothesis), we compared the pattern of amino acid sequence divergence of proteins encoded by genes duplicated just prior to the most recent common ancestor of human and pufferfish with that of proteins encoded genes duplicated earlier. These sequence divergences were statistically indistinguishable, contrary to the prediction of the 2R hypothesis. The variance of amino acid sequence divergences between paralogues was significantly greater than expected from that of orthologues in the same families. Estimation of gene duplication times assuming a molecular clock provided earlier estimates than expected, suggesting that it may not be appropriate to time the duplication of paralogues using rate estimates derived from orthologous comparisons. Overall, the results indicate that amino acid sequences do not provide a strong signal supporting the hypothesis that gene duplications early in vertebrate history occurred by polyploidization. On the other hand, the data are easily explained under an alternative model that gene duplications occurred at different times in different vertebrate gene families.     

Cloning and sequencing of the CRABP-I locus from chicken and pufferfish: analysis of the promoter regions in transgenic mice

Retinoic acid (RA), a derivative of vitamin A, is an important molecule for development and homeostasis of vertebrate organisms. The intracellular retinoic acid binding protein CRABP-I has a high affinity for RA, and is thought to be involved in the mechanism of RA signalling. CRABP-I is well conserved in evolution and shows a specific expression pattern during development, but mice made deficient for the protein by gene targeting appear normal. However, the high degree of homology with CRABP-I from other species indicates that the protein has been subject to strong selective conservation, indicative of an important biological function. In this paper we have compared the conservation in the expression pattern of the mouse, chicken and pufferfish CRABP-I genes to substantiate this argument further. First we cloned and sequenced genes and promoter regions of the CRABP-I genes from chicken and the Japanese pufferfish, Fugu rubripes. Sequence comparison with the mouse gene did not show any large blocks of homology in the promoter regions. Nevertheless, the promoter of the chicken gene directed expression to a subset of the tissues that show expression with the promoter from the mouse gene. The pattern observed with the pufferfish promoter is even more restricted, essentially to rhombomere 4 only, indicating that this region may be functionally the most important for CRABP-I expression in the developing embryo     

Genomic structure and sequence of the leukocyte common antigen (CD45) from the pufferfish Fugu rubripes and comparison with its mammalian homologue

The leukocyte common antigen (CD45) is a transmembrane protein tyrosine phosphatase expressed only in nucleated hematopoietic cells. It can be expressed as different isoforms depending on the cell type and the state of activation or differentiation and it is known to play a crucial role in the maturation and differentiation of B and T lymphocytes. However, the regulation of CD45 expression and function has been difficult to study due to the complexity of the gene in mammals. In this paper, we report the isolation and characterization of a CD45 orthologue gene from the Japanese pufferfish Fugu rubripes (Fugu). The Fugu CD45 cDNA sequence contains an open reading frame of 1,246 amino acids with a variable extracellular region as a result of the alternative splicing of two exons. The intracellular region is organized into two highly conserved tyrosine phosphatase domains. The extracellular region is not conserved except in some structural domains. The Fugu CD45 gene has a similar exon/intron organization to that of mammals except in the 5' end where some exons are missing or fused together. By contrast, the gene is ten times smaller in Fugu due to the small size of the introns. These studies show a greater flexibility to evolve at the 5' end of the gene and provide clues to the functionally important domains of the molecule. In addition, the lower complexity of this gene in Fugu should allow easier mapping of its regulatory sequences.     

Identification of pepsinogen gene in the genome of stomachless fish, Takifugu rubripes

Pepsinogen is a precursor of pepsin, a gastric specific protease belonging to the aspartic proteinase family. In teleosts, several species, such as zebrafish and puffer, have independently lost gastric glands. So whether puffer have pepsinogen gene or not is an interesting issue. A search of GSS database for pufferfish, Takifugu rubripes, revealed five different aspartic proteinase genes in its genome. One of them (pPep) has typical pepsinogen structure and belongs to the fish pepsinogen cluster by phylogenic analysis. The pPep antisense probe hybridized to the gastric glands of flounder stomach. Therefore, we concluded that pPep is a pufferfish pepsinogen. The pufferfish pepsinogen mRNA was not expressed in the digestive organs but specifically in the skin. We speculated that while Tetraodontiformes evolutionarily lost gastric glands, pufferfish pepsinogen acquired an alternative function to food digestion.     

Characterization of the agr2 gene, a homologue of X. laevis anterior gradient 2, from the zebrafish, Danio rerio

We characterized a zebrafish (Danio rerio) anterior gradient 2 homologue (agr2) gene. agr2 contains an open reading frame of 513bp encoding 171 amino acids. Deduced amino acid sequence comparison showed that the zebrafish agr2 protein shares high (80-89%) amino acid sequence similarity with those homologues of anterior gradient 2 (HAGR2, MAgr2, Tagr2, and Sagr2) from the human, mouse, pufferfish, and Atlantic salmon, while sharing less (67-71%) sequence similarity with those anterior gradient 2 genes (XAG-2, XAG-1, XAgr2, MAgr3, and HAGR3) from Xenopus laevis, mouse, and human. Both phylogenetic and syntenic analyses indicate that zebrafish agr2 is the orthologue of human AGR2 and mouse Agr2 genes. Whole-mount in situ hybridization indicated that zebrafish agr2 is expressed in most organs, such as epidermis, olfactory bulbs, otic vesicles, pharynx, esophagus, pneumatic duct, swim bladder, and intestine, which contain mucus-secreting cells. Moreover, semi-quantitative RT-PCR demonstrated agr2 is expressed in the gill, pharynx/esophagus, swim bladder/pneumatic duct, and intestine in the adult fish. In contrast, Xenopus anterior gradient 2 homologues are mainly expressed in ectoderm-derived organs including the cement gland and otic vesicles, while human and mouse anterior gradient 2 orthologues are mainly distributed in endoderm-derived organs including the trachea, lungs, stomach, intestines, and colon.     

Genomic structure and evolutionary conservation of the tyrosinase gene family from Fugu

The tyrosinase gene family encompasses three members, tyrosinase, tyrosinase-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct), which encode for proteins implicated in melanin synthesis. In human and mouse, genomic organization is known for all three genes, revealing common features of regulatory elements and of exon/intron structure. We have set out to identify the complete family from a more primitive vertebrate, the pufferfish Fugu (Takifugu rubripes), which is characterized by a compact genome. We had recently isolated and characterized the Fugu tyrosinase gene (Genesis 28 (2000) 99-105). We now report the isolation and characterization of the two other members of the family, Tyrp1 and Dct. Regulatory sequences from these genes function in mouse pigment cells and are able to mediate reporter gene expression. Our results demonstrate the existence of all three tyrosinase family members in teleosts and underline the evolutionary conservation of the pigmentary system.     

Compact intergenic regions of the pufferfish genome facilitate isolation of gene promoters: characterization of Fugu 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (fPapss2) gene promoter function in transgenic Xenopus

The highly compact nature of the pufferfish (Fugu rubripes) genome renders it a useful tool not only for annotating coding regions within vertebrate genomes, but also for the identification of sequences important to gene regulation. Indeed, owing to this compaction it will be feasible in many instances to initiate analyses using entire intergenic regions when mapping gene promoters; a strategy that is very rarely feasible with the expanded genomes of other species. Stemming from our interest in studying promoters expressed in chondrocytes, we selected for study the intergenic region upstream of Fugu 3'-phosphoadenosine 5'-phosphosulfate synthase 2, fPapss2, a gene required for the normal development of cartilage extracellular matrix. Functional characterization of the entire fPapss2 5' intergenic region was carried out by monitoring expression of the enhanced green fluorescent protein (EGFP) gene reporter in the developing cartilage of transgenic Xenopus laevis. By evaluating a series of 5' intergenic region deletions we defined a minimal fPapss2 sequence of approximately 300 bp that was essential for EGFP expression in tadpole cartilage. This functional analysis of an entire Fugu intergenic region, combined with the efficiency of Xenopus transgenesis, serves as a model for the rapid characterization of evolutionarily-conserved regulatory regions of other pufferfish genes.     

Identification of Cis-regulatory elements in the mouse Pax9/Nkx2-9 genomic region: implication for evolutionary conserved synteny

We previously reported close physical linkage between Pax9 and Nkx2-9 in the human, mouse, and pufferfish (Fugu rubripes) genomes. In this study, we analyzed cis-regulatory elements of the two genes by comparative sequencing in the three species and by transgenesis in the mouse. We identified two regions including conserved noncoding sequences that possessed specific enhancer activities for expression of Pax9 in the medial nasal process and of Nkx2-9 in the ventral neural tube. Remarkably, the latter contained the consensus Gli-binding motif. Interestingly, the identified Pax9 cis-regulatory sequences were located in an intron of the neighboring gene Slc25a21. Close examination of an extended genomic interval around Pax9 revealed the presence of strong synteny conservation in the human, mouse, and Fugu genomes. We propose such an intersecting organization of cis-regulatory sequences in multigenic regions as a possible mechanism that maintains evolutionary conserved synteny.