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Isolation and characterisation of the corticotropin releasing factor receptor 1 (CRFR1) gene in a teleost fish, Fugu rubripes.

Corticotropin releasing factor receptor (CRF) is a member of the secretin family of the G-protein coupled receptor superfamily. These are characterised by the presence of seven transmembrane domains and six conserved cysteines that are important for receptor conformation and ligand binding. IN vertebrates two CRF receptors (CRF1 and CRF2) have been isolated and characterised. In this study the complete structure of the CRF1 receptor was isolated and partially characterised for the first time in a vertebrate using the compact genome of the Japanese pufferfish, Fugu rubripes as a model. The Fugu CRF1 receptor gene is composed of 14 exons is approximately 27 kb in length. A tissue distribution of this receptor in Fugu reveals that it is expressed mainly in liver, gonads, heart and brain, however, expression in the kidney, gut and gills was also detected. In vertebrates this receptor appears to have a different tissue distribution and its presence in the gills may indicate a new role in osmoregulatory processes.

Animals↗

Prelude and Fugue, predicting local protein structure, early folding regions and structural weaknesses.

UNLABELLED: Prelude&Fugue are bioinformatics tools aiming at predicting the local 3D structure of a protein from its amino acid sequence in terms of seven backbone torsion angle domains, using database-derived potentials. Prelude(&Fugue) computes all lowest free energy conformations of a protein or protein region, ranked by increasing energy, and possibly satisfying some interresidue distance constraints specified by the user. (Prelude&)Fugue detects sequence regions whose predicted structure is significantly preferred relative to other conformations in the absence of tertiary interactions. These programs can be used for predicting secondary structure, tertiary structure of short peptides, flickering early folding sequences and peptides that adopt a preferred conformation in solution. They can also be used for detecting structural weaknesses, i.e. sequence regions that are not optimal with respect to the tertiary fold. AVAILABILITY: http://babylone.ulb.ac.be/Prelude_and_Fugue.

Algorithms↗

Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes.

The compact genome of Fugu rubripes has been sequenced to over 95% coverage, and more than 80% of the assembly is in multigene-sized scaffolds. In this 365-megabase vertebrate genome, repetitive DNA accounts for less than one-sixth of the sequence, and gene loci occupy about one-third of the genome. As with the human genome, gene loci are not evenly distributed, but are clustered into sparse and dense regions. Some "giant" genes were observed that had average coding sequence sizes but were spread over genomic lengths significantly larger than those of their human orthologs. Although three-quarters of predicted human proteins have a strong match to Fugu, approximately a quarter of the human proteins had highly diverged from or had no pufferfish homologs, highlighting the extent of protein evolution in the 450 million years since teleosts and mammals diverged. Conserved linkages between Fugu and human genes indicate the preservation of chromosomal segments from the common vertebrate ancestor, but with considerable scrambling of gene order.

Animals↗

Comparative mapping of the human 9q34 region in Fugu rubripes.

Twenty-seven genes have been cloned and mapped in Fugu which have orthologues within the human chromosome 9q34 region. The genes are arranged into five cosmid and BAC contigs which physically map to two different Fugu chromosomes. Considering the gene content of these contigs, it is more probable that a translocation event took place early in the Fugu lineage to split the ancestral 9q34 region onto two chromosomes rather than the alternative hypothesis of a large-scale duplication of the region into two chromosomes with subsequent rapid and dramatic gene loss. There are considerable differences in gene order between the two species, which would appear to be the result of a series of complex chromosome inversions; thus suggesting that there have been no positional constraints on this particular gene set.

Animals↗

Identification of a parathyroid hormone in the fish Fugu rubripes.

UNLABELLED: A PTH gene has been isolated from the fish Fugu rubripes. The encoded protein of 80 amino acid has the lowest homology with any of the PTH family members. Fugu PTH(1-34) had 5-fold lower potency than human PTH(1-34) in a mammalian cell system. INTRODUCTION: Parathyroid hormone (PTH) is the major hypercalcemic hormone in higher vertebrates. Fish lack parathyroid glands, but there have numerous attempts to identify and isolate PTH from fish. MATERIALS AND METHODS: Polymerase chain reaction (PCR) was performed with primers based on preliminary data from the Joint Genome Institute database. PCR amplification was performed on genomic DNA isolated from Fugu rubripes. PCR products were purified and DNA was sequenced. All sequence was confirmed from more than one independently amplified PCR product. Multiple sequence alignments were carried out, and the percentage of identities and similarities were calculated. An unrooted phylogenetic tree, using all the known PTH and PTH-related protein (PTHrP) amino acid sequences, was determined. Synthetic peptides were tested in a biological assay that measured cyclic adenosine 3',5'-monophosphate formation in UMR106.1 cells. Rabbit polyclonal antisera specific for N-terminal human PTHrP and one rabbit polyclonal antiserum specific for N terminus hPTH were used to test the cross-reactivity with fPTH(1-34) in immunoblots.

Amino Acid Sequence↗

Calcitonin: characterisation and expression in a teleost fish, Fugu rubripes.

The present report describes the structure and expression of the calcitonin gene in Fugu rubripes. It is composed of 4 exons and 3 introns. Splicing of exons 1, 2 and 3 generates the calcitonin pre-proprotein, while splicing of exons 1, 2 and 4 generates calcitonin gene-related protein (CGRP). Exons 1 and 2 encoding the signal sequence and the N-terminal peptide are common in both the gene products and this gene organisation has been conserved in human, rat, chicken and salmon. The gene environment around calcitonin in Fugu has been poorly conserved when compared with human, apart from a small gene cluster. The calcitonin gene in Fugu has a widespread tissue distribution but it is most highly expressed in the brain. The abundance of gene expression in the ultimobranchial gland and the pituitary indicates that these are important sites of production and that the peptide is probably secreted into the circulation and/or acts as a paracrine or autocrine controlling factor. Whilst the function of calcitonin in fish is still largely unknown, the distribution described here suggests that one of the potential functions may be as a neuropeptide.

Amino Acid Sequence↗

Identification and characterization of a beta proteasome subunit cluster in the Japanese pufferfish (Fugu rubripes).

The low molecular mass polypeptide (LMP2, LMP7, and MECL-1) genes code for beta-type subunits of the proteasome, a multimeric complex that degrades proteins into peptides as part of the MHC class I-mediated Ag-presenting pathway. These gene products are up-regulated in response to infection by IFN-gamma and replace the corresponding constitutively expressed subunits (X, Y, and Z) during the immune response. In humans, the LMP2 and LMP7 genes both reside within the class II region of the MHC (6p21.3), while MECL-1 is located at 16q22.1. In the present study, we have identified all three IFN-gamma-regulated beta-type proteasome subunits in Fugu, which are present as a cluster within the Fugu MHC class I region. We show that in this species, LMP7, LMP2, and MECL-1 are linked. Also within this cluster is an LMP2-like subunit (which seems specific to all teleosts tested to date) and a closely linked LMP7 pseudogene, indicating that within Fugu and potentially other teleosts, there has been an additional regional duplication involving these genes.

Animals↗

Analysis of 148 kb of genomic DNA around the wnt1 locus of Fugu rubripes.

The analysis of the sequence of approximately 150 kb of a genomic region corresponding to the wnt1 gene of the Japanese pufferfish Fugu rubripes confirms the compact structure of the genome. Fifteen genes were found in this region, and 26.6% of the analyzed sequence is coding sequence. With an average intergenic distance of <5 kb, this gene density is comparable to that of Caenorhabditis elegans. The compactness of this region corresponds to the reduction of the overall size of the genome, consistent with the conclusion that the gene number in Fugu and human genomes is approximately the same. Eight of the genes have been mapped in the human genome and all of them are found in the chromosomal band 12q13, indicating a high degree of synteny in both species, Fugu and human. Comparative sequence analysis allows us to identify potential regulatory elements for wnt1 and ARF3, which are common to fish and mammals.

3' Untranslated Regions↗

A fugue-like state associated with diazepam use.

Diazepam is a long-acting benzodiazepine. Although diazepam is commonly associated with a variety of side effects, it is generally not believed to cause fugue-like states or retrograde amnesia. This report presents the case of an active duty patient who developed a brief fugue-like state with retrograde amnesia. This was associated with the short-term oral use of diazepam. There was no other apparent cause for his symptoms, which resolved within 24 hours after the diazepam was discontinued. This case suggests that short-term use of diazepam can lead to a brief fugue-like state with retrograde amnesia that has not been reported previously.

Administration, Oral↗

[Studies on Fugu (globefish) tetrodotoxin over one century].

One century has passed since fugu toxin was first named tetrodotoxin by Tahara. Chemical problems such as crystallization of tetrodotoxin, followed by structure determination by the research groups headed by Tsuda, Hirata, Woodward, and Mosher, and its subsequent synthesis by Kishi have been solved. In the last 10 years, research on the distribution of tetrodotoxin in nature has shown that tetrodotoxin is not produced by fugu fish, but rather is formed by sea bacteria such as Vibrio, Alteromonas, Photobacterium, and Aeomonas. However, it remains unclear why tetrodotoxin accumulates at a high concentration only in fugu.

Animals↗

The tuberin (TSC2), autosomal dominant polycystic kidney disease (PKD1), and somatostatin type V receptor (SSTR5) genes form a synteny group in the Fugu genome.

The tuberous sclerosis 2 (TSC2) and polycystic kidney disease 1 (PKD1) genes are adjacent on human chromosome 16p13.3 and form part of a conserved synteny group with mouse chromosome 17. We have determined that the PKD1 gene is evolutionarily conserved, single copy, and linked to TSC2 in the Fugu genome. A short cosmid contig has been identified containing both genes based on hybridization, exon trapping, and random sequence data. In addition sequences homologous to the somatostatin type V receptor (SSTR5) were identified 5' to PKD1, defining a larger syntenic region, as this gene has also been mapped to human chromosome 16p13.3. As in mammalian genomes, the Fugu TSC2 and PKD1 genes are adjacent in a tail-to-tail orientation.

Animals↗

Fugu orthologues of human major histocompatibility complex genes: a genome survey.

The major histocompatibility complex (MHC) region in fish has been subjected to piecemeal analysis centering on the in-depth characterization of single genes. The emphasis has been on those genes proven to be involved in the immune response such as the class I and class II antigen presenting genes and the complement genes. The Fugu genome data presents the opportunity to examine the short-range linkage of potentially all the human MHC orthologues and examine conserved synteny with the human and, to a more limited extent, zebrafish genomes. Analysis confirms the existence of a limited MHC locus in Fugu comprising the MHC class Ia genes and associated class II region genes involved in class I antigen presentation. Identification of additional human MHC orthologues indicates the completely dispersed nature of this region in fish, with a maximum of six MHC genes maintained within close proximity in any one contig. The majority of the other genes are present in the genome data as either singletons or pairs. Comparison with zebrafish substantiates previously observed linkages between class III region orthologues and hints at an ancient conserved class III region.

Animals↗

Short-range linkage relationships of the valyl-tRNA synthetase gene in Fugu rubripes.

The class III region of the human major histocompatibility complex (MHC) is gene-dense, averaging one gene every 10-20 kilobases (kb). Its gene order has been compared with other organisms. To extend this analysis further in another non-mammalian vertebrate, the compact genome of Fugu rubripes was investigated for the existence of orthologues of these class III genes and their relative arrangements. Orthologues of the Mr 70000 heat shock protein (HSP70) and valyl-tRNA synthetase genes have been isolated. They do not seem to be closely physically linked as compared with mammals (supported by longer-range analysis using pulsed field gel electrophoresis). Random shotgun sequencing of the two Fugu cosmids containing the gene encoding valyl-tRNA synthetase revealed sequences resembling genes encoding tenascin-X, the nuclear antigen A/Ro of Sjogren's syndrome, and the Landsteiner-Wiener blood group glycoprotein. These linkage relationships recapitulate some mammalian data, albeit imperfectly. Tenascin-X has been located both in the human and mouse Mhc class III regions. Three copies of a sequence found in the gene encoding Sjogren's syndrome nuclear antigen A/Ro have been identified in the human Mhc class I region; the mouse Mhc class I region contains one copy. It is postulated that a fragmented gene pattern had existed prior to convergence in the ancestral mammalian immune response-related Mhc region, and that some of these genes had belonged to the same linkage group.

Animals↗

Cloning and sequencing of the chaperonin-encoding Cctd gene from Fugu rubripes rubripes.

CCT, a chaperonin containing t-complex polypeptide 1 (TCP-1), is a cytosolic molecular chaperone involved in the folding of proteins. We have isolated the Cctd gene from a Fugu rubripes rubripes (Frr) genomic library using a rat Ccta cDNA as a probe, and cloned its cDNA by reverse transcription-polymerase chain reaction (RT-PCR) using a pair of oligodeoxyribonucleotides corresponding to the 5' and 3' non-coding regions of Frr Cctd. Cctd spans a region of 4.7 kb and consists of at least 13 exons with small introns of about 144 bp on average. The Cctd cDNA sequence revealed a deduced polypeptide of 536 amino acids sharing a high degree of homology with that of the mouse Cctd cDNA (88%). Cctd is present as a single-copy gene, as shown by genomic Southern blot analysis, and can be used for evolutionary and classification analyses of Fugu species.

Amino Acid Sequence↗

Demonstration of the mucosal lectins in the epithelial cells of internal and external body surface tissues in pufferfish (Fugu rubripes).

We have previously identified two novel mannose-specific lectins, skin- and intestine-type pufflectins, in the pufferfish, Fugu rubripes [J. Biol. Chem. 278 (2003) 20882]. In the present study, the localization of the lectins and their producing cells were analyzed with antibody and anti-sense probe that recognize both types of pufflectin. Using immunohistochemistry, pufflectins were detected exclusively in epithelial cells in the skin, gills, oral cavity wall and esophagus, whereas in both mucous and epithelial cells in the intestine. Messenger RNAs for pufflectins were detected only in epithelial cells of these tissues with in situ hybridization, suggesting that epithelial cells are able to produce the lectins. Pufflectins are produced and distributed in cells that cover the external and internal body surfaces, which might mean that Fugu have a common immunological system on both surfaces.

Animals↗

Characterization of snRNA and snRNA-type genes in the pufferfish Fugu rubripes.

Vertebrate snRNA and snRNA-type genes occur in independent transcription units with external promoters. The transcription level from the basal promoter is enhanced by the distal sequence element DSE. This element contains almost invariably two activator submotifs, the Staf binding site and the octamer motif, recruiting the Staf and Oct-1 transcriptional activators. In the present work, database search identified 35 snRNA and snRNA-type genes in the genome sequence of the pufferfish Fugu rubripes. Sequence comparisons of promoter elements, determination of template activities by microinjection into Xenopus oocytes and DNA binding assays of the transcriptional activators led to the surprising finding that only two Fugu genes conform to the general scheme with the expected two submotifs in the DSE. Distinctively, all the other DSEs harbor a unique Staf binding site. Also striking was the observation that the tRNA(Sec), and the snRNA genes that are tandemly repeated, are transcribed from promoter-less DSEs. Evolutionary implications of these results are discussed.

Amino Acid Sequence↗

Flounder and fugu have a single lefty gene that covers the functions of lefty1 and lefty2 of zebrafish during L-R patterning.

The lefty gene encodes a member of the TGF-beta superfamily that regulates L-R axis formation during embryogenesis via antagonistic activity against Nodal, another TGF-beta superfamily member. Both mouse and zebrafish have two lefty genes, lefty1 and lefty2. Interestingly, the expression domains of mouse and zebrafish lefty are different from one another. At present, the orthology and functional diversity of the mouse and zebrafish lefty genes are not clear. Here, we report that flounder and two fugu species, Takifugu and Tetraodon, have a single lefty gene in their genomes. In addition, we provide evidence that the mouse lefty genes were duplicated on a single chromosome but the zebrafish lefty genes arose from a whole-genome duplication that occurred early in the divergence of ray-finned fishes. These independent origins likely explain the difference in the expression domains of the mouse and zebrafish lefty gene pairs. Furthermore, we found that the duplication corresponding to the zebrafish lefty2 gene was lost from the fugu genome, suggesting that loss of lefty2 in the fugu/flounder lineage occurred after its divergence from the zebrafish lineage. During L-R patterning, the single lefty gene of flounder covers two expression domains, the left side of the dorsal diencephalon and the left LPM, which are regulated separately by lefty1 and lefty2 in zebrafish. We infer that the lefty genes of the ray-finned fishes and mammals underwent independent gene duplication events that resulted in independent regulation of lefty expression.

Amino Acid Sequence↗

Accumulation of tetrodotoxin (TTX) in Pseudocaligus fugu, a parasitic copepod from panther puffer Takifugu pardalis, but without vertical transmission--using an immunoenzymatic technique.

The caligid copepod Pseudocaligus fugu, a common parasite on the body surface of both toxic (Takifugu pardalis) and cultured, non-toxic (Takifugu rubripes) puffer fishes, was isolated and analyzed for the presence of tetrodotoxin (TTX) using a monoclonal anti-TTX antibody-based immunohistochemical technique. Histological sections of female P. fugu from Takifugu pardalis explicitly revealed that positive brown staining was visible in regions of gut and appendages, and also in the general body tissues from the prosome to the urosome. It is absent from the epicuticle, ovary, oviduct, uterus and egg sacs, where clear negative blue color reaction was obtained. In contrast, the caligids on cultured, non-toxic Takifugu rubripes, considered as negative control, had no sign of TTX. The results indicate that there is no vertical transmission of TTX in the parasitic caligids, which could acquire TTX by feeding on the toxic mucus and skin tissues of host puffer fish.

Animals↗