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PubMed2000

[Recent advances in tetrodotoxin research].

One century has passed since fugu toxin was named tetrodotoxin (TTX) by Tahara. Chemical problems such as crystallization of tetrodotoxin and subsequent structure determination were solved by research groups headed by Tsuda, Hirata, Woodward, and Mosher. The International Symposium on the Chemistry of Natural Products in Kyoto (1964) was well known as symposium which the structure of TTX was internationally clarified. Since the first isolation of toxin from taricha torosa (imori) as natural source except for fugu fishes, distribution of toxin in nature has been widely investigated. And, it was proved that toxin is not produced by fugu fishes, but rather is formed by sea bacteria (30 sp.) such as Alteromonas sp, Vibrio sp, Shewanella. However, it seems to be difficult to explain the tetrodotoxin accumulation at high concentration in fugu by only toxin production by bacteria. TTX analogues were isolated from natural origins such as crabs, fish, annelids, and algae. Based on the structure of these toxin analogues, the biosynthesis of toxin and the structure-activity relationship (Na+ channel) were proposed by Yasumoto-Yamashita. The findings of wide distribution of toxin in nature may be attributed to development of highly sensitive detection method for toxin. The interesting proposal for the biosynthesis and the structure activity, and the detection method for toxin are outlined in this review.

Alteromonas↗
PubMed2003

Identification and characterization of human TIPARP gene within the CCNL amplicon at human chromosome 3q25.31.

Array CGH combined with mRNA microarray analyses was successfully applied for genome-wide screening of proto-oncogenes as well as tumor suppressor genes in 2002. It has been reported that an uncharacterized gene, corresponding to a 5'-truncated partial cDNA DKFZp434J214, is amplified and up-regulated together with CCNL in head-and-neck squamous cell carcinoma (HNSCC). Here, we identified that the novel gene, corresponding to the 5'-truncated DKFZp434J214 cDNA, is the human ortholog of mouse Tiparp gene, by using bioinformatics. Complete coding sequence of human TIPARP mRNA was determined in silico by assembling nucleotide sequences of BC034397 and DKFZp434J214 cDNAs. Human TIPARP gene, consisting of six exons, was located between SSR3 and CCNL genes at human chromosome 3q25.31. Fugu tiparp gene was located around nucleotide position 14280-23355 of fugu genome draft sequence CAAB01003597.1. Human TIPARP showed 91.8% and 52.7% total amino-acid identity with mouse Tiparp and fugu tiparp, respectively. TPH domain (codon 242-296 of human TIPARP), WWE domain (codon 329-378) and PARP-like domain (codon 465-648) were evolutionarily conserved among TIPARP proteins. CCCH-type zinc finger was located within the N-terminal part of the TPH domain. Human TIPARP showed 27.5% and 26.0% total-amino-acid identity with human FLJ22693 and ZAP, respectively. TIPARP, FLJ22693 and ZAP, sharing the common structure with the TPH, WWE and PARP-like domains, were found to constitute the TIPARP family. This is the first report on human TIPARP and fugu tiparp genes as well as on the TIPARP family.

Amino Acid Sequence↗
PubMed1995

Structure and organization of the isotocin and vasotocin genes from teleosts.

Oxytocin and vasopressin are structurally related mammalian neurohypophysial hormones with distinct physiological activities. Homologues of these two hormones are found in all vertebrate groups except the primitive cyclostomes in which only a vasopressin-like hormone has been reported so far. Genes encoding the mammalian oxytocin and vasopressin hormone precursors exhibit a similar genomic organisation, and are closely linked (3.5 kb to 12 kb apart) in a tail-to-tail manner suggesting a common evolutionary origin. However, no linkage among genes of the vasopressin/oxytocin family has been demonstrated in any of the lower vertebrates. The Japanese pufferfish, Fugu rubripes, has been shown to have a very compact genome of about 400 Mb with very few repetitive sequences. We used this model vertebrate genome to analyse the genomic organisation of vasotocin and isotocin genes, the teleost homologues of vasopressin and oxytocin. We have cloned and mapped the vasotocin/isotocin locus in Fugu and obtained complete nucleotide sequences of the vasotocin and isotocin genes. The coding sequences of the Fugu vasotocin and isotocin genes are interrupted by two introns at identical positions, similar to their homologues in higher vertebrates. The deduced amino acid sequences as well as the coding sequences of the two genes show high homology in the neurophysin region. Furthermore, the two genes are linked in a tandem manner with an intergenic region of about 20 kb. Random sequencing has shown that there are at least two known vertebrate genes in the intergenic region. Genomic organisation of the Fugu vasotocin/isotocin locus suggests that the teleost vasotocin and isotocin genes have evolved from a common ancestor through tandem duplication and that this locus seems to have undergone a localised reorganisation during vertebrate evolution.

Amino Acid Sequence↗
PubMed2007

Comparison of diverged Hoxc8 early enhancer activities reveals modification of regulatory interactions at conserved cis-acting elements.

The Hoxc8 early enhancer that controls the initiation and establishment of Hoxc8 expression in the developing mouse embryo is found in different vertebrate lineages including mammals, birds and fish. Mouse and Fugu Hoxc8 early enhancers (200 bp) have diverged in the composition of elements located towards the 3' region. However, they share cis-acting elements A-E located in the 5' region. Mutations at these elements in the context of the mouse Hoxc8 early enhancer affect reporter gene expression in the posterior neural tube, somites and lateral plate mesoderm of day 9.5 mouse embryos. Here, we demonstrate that mutations introduced at the same elements but in the context of the Fugu Hoxc8 early enhancer had different consequences on the reporter gene expression in transgenic mouse embryos. Furthermore, in contrast to the mouse enhancer the Fugu enhancer does not utilize elements D and E in achieving posterior neural tube and somite expression. These results suggest that the diverged sequences prevent regulatory interactions at conserved cis-acting elements. We propose that divergent sequences modify regulatory interactions at conserved elements by providing a "contextual change". Our finding that the enhancer elements do not act in a unitary fashion but function in the context of the surrounding sequence brings a new dimension to the study of cis-regulatory evolution.

Animals↗
PubMed2003

Comparative analysis of vertebrate Shh genes identifies novel conserved non-coding sequence.

The puffer fish Takifugu rubripes (Fugu), with its compact genome, is an ideal model organism for comparative genomics. Sonic hedgehog (Shh) is a key protein in the patterning of differentiating cells during embryonic development. We have sequenced the Fugu Shh gene and compared it with the mammalian and zebrafish orthologs, identifying a number of novel conserved, non-coding sequences upstream of exon one and within the two introns. Additional conserved sequences serve to delineate activator regions and enhancers previously characterized through functional analysis. Control elements can thus be rapidly and effectively predicted by comparative methodology in its own right as well as complementing other, functional methods. This work demonstrates the value of using Fugu in comparative genomics, which has allowed identification of new putative regulatory elements, as well as corroborating enhancers identified by the more traditional deletion mapping method.

Animals↗
PubMed2005

Fish possess multiple copies of fgfrl1, the gene for a novel FGF receptor.

FGFRL1 is a novel FGF receptor that lacks the intracellular tyrosine kinase domain. While mammals, including man and mouse, possess a single copy of the FGFRL1 gene, fish have at least two copies, fgfrl1a and fgfrl1b. In zebrafish, both genes are located on chromosome 14, separated by about 10 cM. The two genes show a similar expression pattern in several zebrafish tissues, although the expression of fgfrl1b appears to be weaker than that of fgfrl1a. A clear difference is observed in the ovary of Fugu rubripes, which expresses fgfrl1a but not fgfrl1b. It is therefore possible that subfunctionalization has played a role in maintaining the two fgfrl1 genes during the evolution of fish. In human beings, the FGFRL1 gene is located on chromosome 4, adjacent to the SPON2, CTBP1 and MEAEA genes. These genes are also found adjacent to the fgfrl1a gene of Fugu, suggesting that FGFRL1, SPON2, CTBP1 and MEAEA were preserved as a coherent block during the evolution of Fugu and man.

Amino Acid Sequence↗
PubMed2005

The gene repertoire and the common evolutionary history of glutamate, pheromone (V2R), taste(1) and other related G protein-coupled receptors.

Glutamate receptors (also known as clan C) are one of the main groups of GPCRs with many subgroup linked through complex evolutionary relationships. We performed thorough searches for genes coding for proteins belonging to this family in the human, mouse, Fugu, and zebrafish genomes, as well as in four invertebrate species. We assembled over 70 new full-length sequences from protein predictions excluding pseudogenes. This resulted in a total of 22 full-length sequences from the human genome, 79 from the mouse genome, 30 from the Fugu genome, and 32 from the zebrafish genome (pseudogenes are not included in these numbers). We show that the vertebrate Glutamate GPCRs form four main phylogenetic groups with a total of eight subgroups (Group I: V2R, TAS1R, GPRC6A, and CASR, Group II: GRM, Group III: GABA together with previously unpublished GPR158 and GPR158L and Group IV: GPRC5). All eight receptor subgroups are present both in mammals and fish, except for GPRC5 and GPR158. The pheromone (V2R), GPRC6, and sweet taste (TAS1) receptors were not found in invertebrates while GRM, GABA, and CASR were found in both C. elegans and C. intestinalis. The pheromone receptors are found in high numbers in mouse, zebrafish and Fugu but are only found as pseudogenes in the human genome. This report provides a comprehensive overview of the expansion/deletions of the groups within the Glutamate receptor family.

Amino Acid Motifs↗
PubMed2005

Analysis and characterization of the expression of the secretory and membrane forms of IgM heavy chains in the pufferfish, Takifugu rubripes.

We investigated the structure and expression of immunoglobulin genes in the pufferfish, Takifugu rubripes, a highly prized and economically important fish species. The cDNA fragment that partially encodes the constant region of the IgM heavy chain was isolated in these animals by RACE using degenerate primers after which it was used as a probe for screening IgM heavy chains in a fugu splenic cDNA library. The structural feature of the constant region of fugu sIgM was found to consist of four constant domains (CH1 to CH4), while mIgM was shown to contain a deletion of the CH4 domain, and its transmembrane domain was directly spliced to the CH3 domain as found in other teleosts. This feature may be common to all teleosts. In addition, five VH genes isolated in this study fell into two families based on their variability. Analysis of genomic sequences from the fugu genomic database also showed that there are only two VH families in the genome. The IgM gene was preferentially expressed in presumptive lymphoid tissues. Moreover, in situ hybridization revealed that large numbers of IgM positive cells were widely distributed throughout the spleen, head kidney, kidney, and thymus, confirming that these tissues were major sites of antibody production in fish. The expressions of IgM in the mucosal organs such as the skin, gills, and intestine suggest that they, too, contribute to humoral immunity in aquatic animals. The expression of IgM mRNA in the early development stages of this fish suggests that its larval form possesses a protective defense mechanism against foreign invaders.

Animals↗
PubMed2006

Identification and characterization of suppressor of cytokine signaling 3 (SOCS-3) homologues in teleost fish.

The suppressor of cytokine signaling 3 (SOCS-3) is a member of a newly discovered protein family, which have been shown to regulate the responses of many immune cytokines, such as interferon (IFN), interleukin-2 (IL-2) and IL-6, etc., by inhibiting Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling in a negative auto-regulatory manner. Although SOCS-3 was well characterized in several mammal species, there was still no report in fish. In present study, we initially identified and characterized the SOCS-3 genes from three fishes, the Tetraodon nigroviridis, the Danio rerio and the Fugu rubripes. The results showed that Tetraodon SOCS-3 gene located within a 2666 bp genomic fragment of chromosome 3, transcribed into a 1445 bp mRNA including 273 bp 5' UTR (untranslated region), 606 bp ORF (open reading frame) and 566 bp 3' UTR. Tetraodon SOCS-3 with 201aa (amino acid) has a calculated molecular mass of 22.76 kDa and a theoretical pI of 8.99. Danio SOCS-3 gene located within a 3617 bp genomic fragment of chromosome 3, transcribed into a 1927 bp mRNA including 178 bp 5' UTR, 624 bp ORF and 1125 bp 3' UTR. Danio SOCS-3 with 207aa has a calculated molecular mass of 23.68 kDa and a theoretical pI of 9.19. Fugu SOCS-3 gene located within a 2842 bp genomic fragment of Scaffold_1118, transcribed into a 1528 bp mRNA including 209 bp 5' UTR, 606 bp ORF and 713 bp 3' UTR. Fugu SOCS-3 with 201aa has a calculated molecular mass of 22.76 kDa and a theoretical pI of 8.18. The fish SOCS-3-encoding genes with the same organization as the mammalians consist of two exons and a single intron that lies in the 5' UTR of the transcript. The deduced amino acid sequences of the fish SOCS-3s showed: 60.7-61.7% sequence identity to mammalian SOCS-3s; 62.3-63.2% sequence identity to bird SOCS-3s; and 55.3-57.8% sequence identity to amphibian SOCS-3s. Phylogenetic analysis separates the fish SOCS-3s into an exclusive group. Expression study of Tetraodon SOCS-3 mRNA in ten selected tissues showed that it was constitutively expressed and induced by lipopolysaccharide (LPS) strikingly. These results indicated that SOCS-3s in fish may be involved in inflammatory responses. This is the first report of cloning and characterization of SOCS-3 cDNAs and genes in fish.

Amino Acid Sequence↗
PubMed2006

Detection of tetrodotoxin (TTX) from two copepods infecting the grass puffer Takifugu niphobles: TTX attracting the parasites?

In May 2002, two parasitic copepods, Pseudocaligus fugu and Taeniacanthus sp., were collected from the body surface and gill of the grass puffer Takifugu niphobles, respectively, in Takehara city, Hiroshima Prefecture, faced with Seto Inland Sea located in the western part of Japan. To them was added 5 ml of 0.1% acetic acid, then the suspension was subjected to ultrasonic disruption with an ultrasonicator for 10 min. The resulting mixture was heated in a boiling water bath for 10 min, and then centrifuged. The supernatant was concentrated under reduced pressure, and loaded on to a Sep-Pak plus C18 Environmental Cartridge (Waters). The unbound fraction was analyzed by HPLC and gas chromatography-mass spectrometry (GC-MS) for tetrodotoxin (TTX). It was rather unexpectedly revealed from these results that this fraction was comprised of TTX and its analogues. As far as we know, this is the first record to show the existence of TTX in the copepods. In addition, relationships between the more and less than the average number of the two parasites and the toxicity of its skin mucus of the host were examined by student's t-test. In P. fugu, the average number per host was 13.9, and those are 520.7 (n=9) and 269.0 MU/g (n=22), respectively. A highly significant difference between them was detected at p-value 0.0011. In contrast, as for Taeniacanthus sp., the average number was 2.7, and those were 338.0 (n=14) and 345.5 MU/g (n=17), respectively. No significant difference was detected in Taeniacanthus sp. The high host-specificity of P. fugu on the toxic puffer and the present bioassay of its skin mucus suggest a possibility that TTXs may attract the parasite.

Animals↗
PubMed2006

Prolactin and prolactin receptor expressions in a marine teleost, pufferfish Takifugu rubripes.

To investigate the physiological significance of prolactin (PRL) in a marine teleost, pufferfish (or fugu), Takifugu rubripes, we cloned and characterized cDNAs encoding its PRL and PRL receptor (PRLR) from the pituitary and gills, respectively. The fugu PRL cDNA consisted of 995 bp and encoded a protein of 213 amino acids. The PRLR, consisting of 510 amino acids, contained a putative signal peptide, an extracellular domain with two pairs of cysteines, a WSXWS motif, a single transmembrane domain, and a cytoplasmic (intracellular) domain with box 1 and box 2 regions, all of which are characteristic of the cytokine receptor superfamily. Reverse transcription-PCR showed the expression of PRLR mRNA in osmoregulatory organs, such as gills, kidney, and intestine, whereas pufferfish PRL mRNA was detected only in the pituitary. Furthermore, in situ hybridization revealed the expression of pufferfish PRLR in branchial chloride cells, kidney tubule cells, and intestinal epithelia. The PRL-gene expression levels in the pituitary were about five times higher in 25%-diluted seawater than in full-strength seawater. These results suggest that fugu PRL regulates water and electrolyte balances through PRLR expressed in the osmoregulatory organs, as is the case with freshwater-adapted euryhaline species.

Amino Acid Sequence↗
PubMed2005

In vivo characterization of a vertebrate ultraconserved enhancer.

Genomic sequence comparisons among human, mouse, and pufferfish (Takifugu rubripes (Fugu)) have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2, located near the dachshund gene, which displays a human-Fugu identity of 84% over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical among human, mouse, chicken, zebrafish, and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424-bp Dc2-conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144-bp 100% conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16-bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144-bp 100% conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on the Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence.

Animals↗
PubMed2000

Comparative analysis of the RED1 and RED2 A-to-I RNA editing genes from mammals, pufferfish and zebrafish.

One type of RNA editing involves the deamination of adenosine (A) residues to inosines (I) at specific sites in specific pre-mRNAs. These inosines are subsequently read as guanosines by the ribosome, with potentially significant consequences for protein sequence. In mammals, two such A-to-I RNA editases are RED1, which edits some serotonin and glutamate receptors, and RED2, with unidentified substrates. To study the evolutionary conservation among these editases, we have isolated homologous genes from the Japanese pufferfish, Fugu rubripes. Fugu has two genes homologous to Red1 that are similar in size and organization and that show a fivefold compaction relative to the human gene; they differ, however, in their base compositional features. The Fugu gene for RED2 is unusually large, spanning more than 50kb; within the largest intron, there is evidence for a novel gene on the opposite strand. Because of these unusual features, the partial genomic structure was determined for the mouse RED2 gene. A partial cDNA for RED1 was also isolated from zebrafish. Comparisons between fish and between fish and mammals of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA-binding domains vary within a single protein in their levels of conservation. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation.

Adenosine Deaminase↗
PubMed2002

Genomic organization, transcript variants and comparative analysis of the human nucleoporin 155 (NUP155) gene.

Nucleoporin 155 (Nup155) is a major component of the nuclear pore complex (NPC) involved in cellular nucleo-cytoplasmic transport. We have acquired the complete sequence and interpreted the genomic organization of the Nup155 orthologos from human (Homo sapiens) and pufferfish (Fugu rubripes), which are approximately 80 and 8 kb in length, respectively. The human gene is ubiquitously expressed in many tissues analyzed and has two major transcript variants, resulted from an alternative usage of the 5' cryptic or consensus splice donor in intron 1 and two polyadenylation signals. We have also cloned DNA complementary to RNAs of the Nup155 orthologs from Fugu and mouse. Comparative analysis of the Nup155 orthologs in many species, including H. sapiens, Mus musculus, Rattus norvegicus, F. rubripes, Arabidopsis thaliana, Drosophila melanogaster, and Saccharomyces cerevisiae, has revealed two paralogs in S. cerevisiae but only a single gene with increasing number of introns in more complex organisms. The amino acid sequences of the Nup155 orthologos are highly conserved in the evolution of eukaryotes. Different gene orders in the human and Fugu genomic regions harboring the Nup155 orthologs advocate cautious interpretation of synteny in comparative genomic analysis even within the vertebrate lineage.

3T3 Cells↗
PubMed2002

Complete genomic structure of human rpS3: identification of functional U15b snoRNA in the fifth intron.

Analysis of the complete genomic structure of the human ribosomal protein S3 (rpS3) gene revealed the presence of a functional U15b snoRNA gene in its intron. Human ribosomal protein S3 (rpS3) gene of 6115 bp long has been identified to contain six introns and seven exons in this study. The first and fifth introns of human S3 gene contain functional U15 snoRNA genes. Although Xenopus and Fugu counterparts also have six introns and seven exons, S3 gene of Fugu contains two functional U15 snoRNAs in the fourth and sixth introns and two pseudo genes for U15 snoRNAs in the first and fifth introns. In Xenopus S1 gene encoding ribosomal protein S3, however, three of its six introns contain U15 snoRNA gene sequence. Sequence comparison of the U15 genes from Xenopus, Fugu and human revealed that the regions involved in binding to 28S rRNA and the consensus sequence (C, D and D' boxes) for snoRNAs are highly conserved among those genes from these three species. Human U15a and U15b RNAs which are derived from the first and the fifth introns, respectively, have been identified to be functional by microinjection of human U15a and U15b snoRNAs into Xenopus oocyte. Northern blot and primer extension analyses confirm that human U15b snoRNA is expressed in vivo.

Animals↗
PubMed2005

Functional expression of a low-affinity zinc uptake transporter (FrZIP2) from pufferfish (Takifugu rubripes) in MDCK cells.

Zinc is a vital micronutrient to all organisms and it is therefore very important to determine the mechanisms that regulate cellular zinc uptake. Previously, we reported on zinc uptake transporters from zebrafish (Danio rerio; DrZIP1) and Fugu pufferfish (Takifugu rubripes; FrZIP1) that facilitated cellular zinc uptake of high affinity (K(m)<0.5 microM) in both CHSE214 [chinook salmon (Oncorhynchus tshawytscha) embryonic 214] cells and Xenopus laevis oocytes. To investigate additional biochemical pathways of zinc uptake in fish, we molecularly cloned the second fish member (FrZIP2) of the SLC39 subfamily II from Fugu pufferfish gill. Functional characterization suggests that FrZIP2 stimulated zinc uptake in a temperature-, time-, concentration- and pH-dependent manner when overexpressed in MDCK cells (Madin-Darby canine kidney cells). In comparison with FrZIP1 and DrZIP1 (<0.5 microM), FrZIP2 appears to represent a low-affinity zinc uptake transporter (K(m)=13.6 microM) in pufferfish. FrZIP2 protein was selective for zinc, but it might also transport Cu2+, since 20 times excess of Cu2+ completely abolished its zinc uptake activity. The zinc uptake by FrZIP2 was stimulated in a slightly acidic medium (pH 5.5-6.5) and was completely blocked at pH 7.5 and above, suggesting that an inward H+ gradient might provide a driving force for zinc transport by FrZIP2. Furthermore, FrZIP2-mediated zinc uptake activity was slightly inhibited by 0.5 mM HCO3-, indicating that FrZIP2 may employ a different mechanism of zinc translocation from the assumed HCO3--coupled zinc transport used by human SLC39A2. The FrZIP2 gene was expressed in all the tissues studied herein, with especially high levels in the ovary and intestines. Thus FrZIP2 may be a prominent zinc uptake transporter of low affinity in many cell types of Fugu pufferfish.

Amino Acid Sequence↗
PubMed2004

Variation in sequence and organization of splicing regulatory elements in vertebrate genes.

Although core mechanisms and machinery of premRNA splicing are conserved from yeast to human, the details of intron recognition often differ, even between closely related organisms. For example, genes from the pufferfish Fugu rubripes generally contain one or more introns that are not properly spliced in mouse cells. Exploiting available genome sequence data, a battery of sequence analysis techniques was used to reach several conclusions about the organization and evolution of splicing regulatory elements in vertebrate genes. The classical splice site and putative branch site signals are completely conserved across the vertebrates studied (human, mouse, pufferfish, and zebrafish), and exonic splicing enhancers also appear broadly conserved in vertebrates. However, another class of splicing regulatory elements, the intronic splicing enhancers, appears to differ substantially between mammals and fish, with G triples (GGG) very abundant in mammalian introns but comparatively rare in fish. Conversely, short repeats of AC and GT are predicted to function as intronic splicing enhancers in fish but are not enriched in mammalian introns. Consistent with this pattern, exonic splicing enhancer-binding SR proteins are highly conserved across all vertebrates, whereas heterogeneous nuclear ribonucleoproteins, which bind many intronic sequences, vary in domain structure and even presence/absence between mammals and fish. Exploiting differences in intronic sequence composition, a statistical model was developed to predict the splicing phenotype of Fugu introns in mammalian systems and was used to engineer the spliceability of a Fugu intron in human cells by insertion of specific sequences, thereby rescuing splicing in human cells.

Animals↗
PubMed2006

Evolution of cis elements in the differential expression of two Hoxa2 coparalogous genes in pufferfish (Takifugu rubripes).

Sequence divergence in cis-regulatory elements is an important mechanism contributing to functional diversity of genes during evolution. Gene duplication and divergence provide an opportunity for selectively preserving initial functions and evolving new activities. Many vertebrates have 39 Hox genes organized into four clusters (Hoxa-Hoxd); however, some ray-finned fishes have extra Hox clusters. There is a single Hoxa2 gene in most vertebrates, whereas fugu (Takifugu rubripes) and medaka (Oryzias latipes) have two coparalogous genes [Hoxa2(a) and Hoxa2(b)]. In the hindbrain, both genes are expressed in rhombomere (r) 2, but only Hoxa2(b) is expressed in r3, r4, and r5. Multiple regulatory modules directing segmental expression of chicken and mouse Hoxa2 genes have been identified, and each module is composed of a series of discrete elements. We used these modules to investigate the basis of differential expression of duplicated Hoxa2 genes, as a model for understanding the divergence of cis-regulatory elements. Therefore, we cloned putative regulatory regions of the fugu and medaka Hoxa2(a) and -(b) genes and assayed their activity. We found that these modules direct reporter expression in a chicken assay, in a manner corresponding to their endogenous expression pattern in fugu. Although sequence comparisons reveal many differences between the two coparalogous genes, specific subtle changes in seven cis elements of the Hoxa2(a) gene restore segmental regulatory activity. Therefore, drift in subsets of the elements in the regulatory modules is responsible for the differential expression of the two coparalogous genes, thus providing insight into the evolution of cis elements.

Animals↗