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PubMed1998

Genomic structure and sequence of the pufferfish (Fugu rubripes) gene encoding an actin-related protein.

Using a polymerase chain reaction (PCR)-generated gene fragment as a probe, we isolated and sequenced a gene encoding an actin-related protein belonging to the Arp3 family from the pufferfish, Fugu rubripes. The Fugu Arp3 gene spans 3.7kb from the transcription start site to the polyadenylation signal. This is the first report of the genomic sequence of a vertebrate Arp3 gene. The Fugu Arp3 gene consists of 12 exons and 11 introns compared to Drosophila homologue, which has six exons and five introns. The protein sequence encoded by the Fugu gene is 97% and 80% identical to bovine and Drosophila homologues, respectively. The Fugu Arp3 gene is expressed in a wide range of tissues, with higher levels detected in the gills, kidney, ovary, skin and testis. Three different size transcripts of the Arp3 gene (1.4kb, 1.8kb and 2.2kb) were identified in various tissues.

Actin-Related Protein 3↗
PubMed1999

Structures, sequence characteristics, and synteny relationships of the transcription factor E4TF1, the splicing factor U2AF35 and the cystathionine beta synthetase genes from Fugu rubripes.

A cosmid containing the beta-amyloid precursor protein (APP) from Fugu rubripes has been completely sequenced. In addition to APP, the cosmid contains the E4TF1-60 transcription factor, the U2AF35 pre-mRNA splicing factor, and the cystathionine beta synthetase (CBS) gene. The human homologues of all four genes map to human chromosome 21 but are not clustered; APP and E4TF1-60 map within 21q21, whereas U2AF35 and CBS map approximately 20Mb distal in 21q22. 3. The protein sequences of the Fugu genes vary in their overall level of similarity to their mammalian homologues, but several regions of functional importance are almost identical. As expected, the intron/exon structures of the homologous pairs of genes are highly conserved, but there are significant differences in the compaction ratios. The introns of APP and E4TF1-60 are 49- and 24-fold smaller in Fugu than in human, and the intergenic distance is compressed at least 100-fold. For U2AF35 and CBS, the introns are compressed only five- to eightfold. These size differences were compared with those for a number of previously reported Fugu genes; in general, levels of compaction of Fugu genes are consistent with the isochore locations of the human homologues.

Amino Acid Sequence↗
PubMed2006

Synuclein proteins of the pufferfish Fugu rubripes: sequences and functional characterization.

In humans, three genes encode the related alpha-, beta-, and gamma-synucleins, which function as lipid-binding proteins in vitro. They are being widely studied, mainly because of the central involvement of alpha-synuclein in a number of neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In these diseases, the normally soluble alpha-synuclein assembles into abnormal filaments. Here, we have identified and characterized the synuclein gene family from the pufferfish Fugu rubripes. It consists of four genes, which encode alpha-, beta-, gamma1-, and gamma2-synucleins. They range from 113 to 127 amino acids in length and share many of the characteristics of human synucleins, including the presence of imperfect amino-terminal repeats of 11 amino acids, a hydrophobic middle region, and a negatively charged carboxy-terminus. All four synucleins are expressed in the Fugu brain. Recombinant Fugu synucleins exhibited differential liposome binding, which was strongest for alpha-synuclein, followed by beta-, gamma2-, and gamma1-synucleins. In assembly experiments, Fugu alpha-, gamma1-, and gamma2-synucleins formed filaments more readily than human alpha-synuclein. Fugu beta-synuclein, by contrast, failed to assemble in bulk. Filament assembly of synucleins was directly proportional to their degree of hydrophobicity and their tendency to form beta-sheet structure, and correlated inversely with their net charge.

Amino Acid Sequence↗
PubMed1997

Transgenic rats reveal functional conservation of regulatory controls between the Fugu isotocin and rat oxytocin genes.

We have asked whether comparative genome analysis and rat transgenesis can be used to identify functional regulatory domains in the gene locus encoding the hypothalamic neuropeptides oxytocin (OT) and vasopressin. Isotocin (IT) and vasotocin (VT) are the teleost homologues of these genes. A contiguous stretch of 46 kb spanning the Fugu IT-VT locus has been sequenced, and nine putative genes were found. Unlike the OT and vasopressin genes, which are closely linked in the mammalian genome in a tail-to-tail orientation, Fugu IT and VT genes are linked head to tail and are separated by five genes. When a cosmid containing the Fugu IT-VT locus was introduced into the rat genome, we found that the Fugu IT gene was specifically expressed in rat hypothalamic oxytocinergic neurons and mimicked the response of the endogenous OT gene to an osmotic stimulus. These data show that cis-acting elements and trans-acting factors mediating the cell-specific and physiological regulation of the OT and IT genes are conserved between mammals and fish. The combination of Fugu genome analysis and transgenesis in a mammal is a powerful tool for identifying and analyzing conserved vertebrate regulatory elements.

Animals↗
PubMed1998

Complete sequencing of the Fugu WAGR region from WT1 to PAX6: dramatic compaction and conservation of synteny with human chromosome 11p13.

The pufferfish Fugu rubripes has a genome approximately 7.5 times smaller than that of mammals but with a similar number of genes. Although conserved synteny has been demonstrated between pufferfish and mammals across some regions of the genome, there is some controversy as to what extent Fugu will be a useful model for the human genome, e.g., [Gilley, J., Armes, N. & Fried, M. (1997) Nature (London) 385, 305-306]. We report extensive conservation of synteny between a 1.5-Mb region of human chromosome 11 and <100 kb of the Fugu genome in three overlapping cosmids. Our findings support the idea that the majority of DNA in the region of human chromosome 11p13 is intergenic. Comparative analysis of three unrelated genes with quite different roles, WT1, RCN1, and PAX6, has revealed differences in their structural evolution. Whereas the human WT1 gene can generate 16 protein isoforms via a combination of alternative splicing, RNA editing, and alternative start site usage, our data predict that Fugu WT1 is capable of generating only two isoforms. This raises the question of the extent to which the evolution of WT1 isoforms is related to the evolution of the mammalian genitourinary system. In addition, this region of the Fugu genome shows a much greater overall compaction than usual but with significant noncoding homology observed at the PAX6 locus, implying that comparative genomics has identified regulatory elements associated with this gene.

Amino Acid Sequence↗
PubMed2003

Faithful expression of a tagged Fugu WT1 protein from a genomic transgene in zebrafish: efficient splicing of pufferfish genes in zebrafish but not mice.

The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal beta-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.

Alternative Splicing↗
PubMed2003

Fugu ESTs: new resources for transcription analysis and genome annotation.

The draft Fugu rubripes genome was released in 2002, at which time relatively few cDNAs were available to aid in the annotation of genes. The data presented here describe the sequencing and analysis of 24,398 expressed sequence tags (ESTs) generated from 15 different adult and juvenile Fugu tissues, 74% of which matched protein database entries. Analysis of the EST data compared with the Fugu genome data predicts that approximately 10,116 gene tags have been generated, covering almost one-third of Fugu predicted genes. This represents a remarkable economy of effort. Comparison with the Washington University zebrafish EST assemblies indicates strong conservation within fish species, but significant differences remain. This potentially represents divergence of sequence in the 5' terminal exons and UTRs between these two fish species, although clearly, complete EST data sets are not available for either species. This project provides new Fugu resources, and the analysis adds significant weight to the argument that EST programs remain an essential resource for genome exploitation and annotation. This is particularly timely with the increasing availability of draft genome sequence from different organisms and the mounting emphasis on gene function and regulation.

Animals↗
PubMed1996

Fugu intron oversize reveals the presence of U15 snoRNA coding sequences in some introns of the ribosomal protein S3 gene.

We present here the analysis of the genomic organization of the Fugu gene coding for ribosomal protein S3 and its intron encoded U15 RNA, and compare it with the homologous human and Xenopus genes. Only two of the six Fugu S3 gene introns do not contain the U15 sequence and are in fact shorter than 100 nucleotides, as most Fugu introns. The other four introns are somewhat longer and contain sequences homologous to U15 RNA; two of these represent functional copies, as shown by microinjections of Fugu transcripts into Xenopus oocytes, whereas the other two appear to be nonfunctional pseudocopies. Thus Fugu turns out to be ideal for the study of intron encoded snoRNAs, partly because of the reduced cloning and sequencing workload, and partly because the intron length per se can be an indication of the presence of a snoRNA coding sequence.

Animals↗
PubMed1997

Characterization of cell cultures derived from Fugu, the Japanese pufferfish.

The Japanese pufferfish (genus Fugu), which possesses a highly compact genome, is becoming a popular model among those interested in sequencing and mapping the genomes of higher vertebrates. Although genomic libraries have been derived and used to study the molecular biology of Fugu, biological material derived from the living organism is difficult to obtain for laboratories distant from the Asian Pacific. We have established cell cultures from two Fugu species: kusafugu, Fugu niphobles, and torafugu, F. rubripes. Cultures derived from F. niphobles fry and F. rubripes eye have been passaged more than 60 times over the course of one year, representing approximately 180 population doublings. Proliferating cultures were also initiated from F. rubripes brain, liver, fin, spleen, kidney, swimbladder, and muscle. Karyotype analyses indicated that F. rubripes eye-derived cells possessed a chromosome number in the diploid range; F. niphobles fry cells were slightly hyperploid. Flow cytometry confirmed that the relative amounts of DNA present in cultured cells from both Fugu species were similar to that measured in blood cells collected from F. rubripes, and approximately one-seventh of that measured in diploid human cells. Telomerase activity was easily detectable in lysates prepared from F. niphobles fry cells and F. rubripes eye cells, consistent with the notion that these cultures are capable of indefinite proliferation.

Animals↗
PubMedDate not supplied

The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse.

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.

Amino Acid Sequence↗
PubMed2005

Comparative genomics using fugu: a tool for the identification of conserved vertebrate cis-regulatory elements.

With the imminent completion of the whole genome sequence of humans, increasing attention is being focused on the annotation of cis-regulatory elements in the human genome. Comparative genomics approaches based on evolutionary conservation have proved useful in the detection of conserved cis-regulatory elements. The pufferfish, Fugu rubripes, is an attractive vertebrate model for comparative genomics, by virtue of its compact genome and maximal phylogenetic distance from mammals. Fugu has lost a large proportion of nonessential DNA, and retained single orthologs for many duplicate genes that arose in the fish lineage. Non-coding sequences conserved between fugu and mammals have been shown to be functional cis-regulatory elements. Thus, fugu is a model fish genome of choice for discovering evolutionarily conserved regulatory elements in the human genome. Such evolutionarily conserved elements are likely to be shared by all vertebrates, and related to regulatory interactions fundamental to all vertebrates. The functions of these conserved vertebrate elements can be rapidly assayed in mammalian cell lines or in transgenic systems such as zebrafish/medaka and Xenopus, followed by validation of crucial elements in transgenic rodents.

Animals↗
PubMed1996

Molecular cloning of two cannabinoid type 1-like receptor genes from the puffer fish Fugu rubripes.

The puffer fish, Fugu rubripes (Fugu), has been proposed as a model vertebrate genome. We have characterized two putative G-protein-coupled receptor encoding genes, FCB1A and FCB1B, obtained by degenerate PCR and low-stringency hybridization of a Fugu genomic library. These two genes show high homology to the human cannabinoid receptor type 1 (HCB1), but very low homology to the type 2 receptor. The amino acid sequences of the FCB1A and FCB1B genes are 66.2% identical, and the homology of each gene to HCB1 is 72.2 and 59.0%, respectively. The transcripts of both the FCB1A and the FCB1B receptors are abundant in the brain. No type 2 receptor could be cloned from Fugu. These data suggest that although two cannabinoid receptor-like genes are found in the puffer fish, both show similarity to the type 1 receptor found in human.

Amino Acid Sequence↗
PubMed1996

Isolation, characterization and evolution of nine pufferfish (Fugu rubripes) actin genes.

The Japanese pufferfish Fugu rubripes (Fugu) has a small genome of about 400 Mb. Nine different actin genes have been isolated and sequenced from a genomic library constructed from this teleost. The six muscle-type actin genes include two alpha-skeletal actins, three alpha-cardiac actins and an alpha-anomalous (testis type) actin, and the three cytoplasmic actins include two beta-cytoplasmic actins and a beta-cytoplasmic (vascular type) actin. The two skeletal muscle actin genes have identical genomic organization, but differ by five amino acid residues. The three cardiac actin genes code for the same protein but differ in their nucleotide sequences and genomic organization. beta-Cytoplasmic actin1 differs by three amino acids from beta-cytoplasmic actin2. The alpha-anomalous (testis type) and beta-cytoplasmic (vascular type) actins are novel vertebrate actins. The amino acid sequence of alpha-anomalous (testis type) actin is the most divergent of all the known vertebrate actins and transcripts of this gene are abundant in the testis. The beta-cytoplasmic (vascular type) actin gene has eight introns, similar to mammalian smooth muscle actins, and is expressed in vascular tissues such as the gills, kidney and skin. Several known regulatory elements are found in the 5' flanking sequences and the first intron of various Fugu actin genes. The intron patterns of the various Fugu actins seem to be the result of loss of certain introns from a common ancestral gene.

Actins↗
PubMed2001

FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties.

FUGUE, a program for recognizing distant homologues by sequence-structure comparison (http://www-cryst.bioc.cam.ac.uk/fugue/), has three key features. (1) Improved environment-specific substitution tables. Substitutions of an amino acid in a protein structure are constrained by its local structural environment, which can be defined in terms of secondary structure, solvent accessibility, and hydrogen bonding status. The environment-specific substitution tables have been derived from structural alignments in the HOMSTRAD database (http://www-cryst.bioc. cam.ac.uk/homstrad/). (2) Automatic selection of alignment algorithm with detailed structure-dependent gap penalties. FUGUE uses the global-local algorithm to align a sequence-structure pair when they greatly differ in length and uses the global algorithm in other cases. The gap penalty at each position of the structure is determined according to its solvent accessibility, its position relative to the secondary structure elements (SSEs) and the conservation of the SSEs. (3) Combined information from both multiple sequences and multiple structures. FUGUE is designed to align multiple sequences against multiple structures to enrich the conservation/variation information. We demonstrate that the combination of these three key features implemented in FUGUE improves both homology recognition performance and alignment accuracy.

Algorithms↗
PubMed2003

Characterisation, expression and promoter analysis of an interleukin 10 homologue in the puffer fish, Fugu rubripes.

Using computer-based tools, an interleukin (IL) 10 homologue has been identified from the puffer fish ( Fugu rubripes) genome database. This is the first report on the existence of an IL-10 homologue in a non-mammalian vertebrate species. The Fugu IL-10 gene is located within a 2790-bp fragment including 549 bp of coding sequence which translates into an 183-amino-acid protein. It is predicted to contain five exons and four introns, sharing the same organization with the mammalian IL-10 genes. The size of the introns in the Fugu IL-10 gene is much smaller compared to mammalian IL-10 genes, whilst the size of the exons is similar. The deduced protein sequence shares 44-50% homology with the mammalian IL-10 sequences, 39-42% with the viral IL-10 sequences and 37-42% with other members of the IL-10 family, IL-20 and IL-22. Southern blot analysis indicates that a single copy of the IL-10 gene is present in the Fugu genome. A very low level of constitutive expression was detected in tissues of healthy fish including liver, kidney, gut and spinal cord, whilst no expression was detectable in spleen, gill, brain, gonad and eye. Analysis of the transcription regulation elements in the promoter region revealed that trans elements are located in the region between 1 bp and 721 bp, cis elements between 934 bp and 1114 bp and a tumour necrosis factor alpha responsive transcription element was located 92 bp upstream of the TATA box.

Amino Acid Sequence↗
PubMed2003

Fugu rubripes possesses genes for the entire set of the ITAM-bearing transmembrane signal subunits.

The transmembrane signaling subunits (TSSs) bearing the immunoreceptor tyrosine-based activation motif (ITAM) play a crucial role in triggering the effector functions of mammalian leukocytes. The involvement in key immune reactions and obvious extension through duplication events make TSSs valuable markers of the evolution of the immune system. We surveyed the genomic sequences of the teleostean fish Fugu rubripes for the presence of genes encoding these accessory molecules. Automatic gene prediction was not efficient because of the poor ability of the programs used to recognize the short exons encoding the intracellular regions of TSSs. However, the unique compactness of the Fugu genome and the conservation of the exon/intron arrangements of the TSS genes facilitated their recognition by visual inspection of the candidate genomic sequences. Evidence for the presence of the CD3epsilon, CD3gamma/delta, CD79a, CD79b, TCRzeta, FcRgamma, DAP12 and DAP10 genes in the Fugu genome was obtained. Furthermore, conserved synteny for the short regions including the TSS genes was revealed by comparison of the Fugu and human genomes. The data demonstrate that the set of TSSs arose before the teleost-tetrapod split and provide a starting point for experimental investigation of the molecular evolution of the leukocyte-activating receptor complexes from fish species to mammals.

Amino Acid Sequence↗
PubMed2004

Characterization and expression of the immunoglobulin light chain in the fugu: evidence of a solitaire type.

In this study, we characterized the immunoglobulin light (IgL) chain gene and examined its expression in the fugu (Takifugu rubripes). The cDNA fragment that partially encodes the IgL chain was isolated by RACE and used as a probe for screening for IgL in a fugu splenic cDNA library. The IgL cDNA sequence that we found consisted of a variable (V(L)) and a constant (C(L)) segment. Its structural features were similar to the IgL isotype commonly found in teleosts. Genomic sequence analysis revealed that the IgL gene was organized as two V(L) gene segments (designed V(L1) and V(L2)) followed by single joining (J(L)) and C(L) segment. In addition, an unusual duplicate V(L1) gene segment was found downstream of the C(L) segment. The transcriptional orientation of the V(L) exons was found to be opposite to that of the J(L) and C(L) segments. Genomic blot hybridizations with V(L) and C(L) probes gave multibands, supporting the contention that the teleost IgL forms a multicluster. Both genomic and cDNA sequences analyses showed that all of the constant segments found in the fugu are identical, suggesting that no other isotypes could be found in this species. Comparison of the deduced amino acid sequence of the fugu C(L) domain with those of other species showed a high degree of identity (from 40 to 77%). IgL mRNAs were found to be expressed primarily in the lymphoid tissues. In situ hybridization revealed the presence of IgL-positive cells widely distributed throughout the spleen, head kidney, kidney, and thymus. These results support the contention that the lymphoid tissues are the major sites of antibody production in fish. Since IgL mRNA was also expressed in the skin and gill that are exposed to external antigens, it is likely that mucosal Ig plays an important role in immune protection.

Amino Acid Sequence↗
PubMed2001

Characterization of the MHC class I region of the Japanese pufferfish (Fugu rubripes).

A BAC map of the Japanese pufferfish (Fugu) MHC class I region was constructed using a mixture of sequence scanning and sequence-tagged site mapping methodologies. The Fugu MHC class Ia genes are linked to genes which are found within the human classical MHC class II and extended class II regions, a situation which has been found in the MHC of all teleosts mapped so far. The 300-kb contig comprises 24 MHC-related genes and is bounded by six non-MHC genes, which are thought to represent an evolutionary breakpoint within the region. Comparative analysis with both human and zebrafish MHC maps indicates two blocks of genes (KNSL2, ZNF297, DAXX, TAPBP, FLOTILLIN; and PSMB8, PSMB10, PSMB9, ABCB3, FABGL, BRD2, COL11A2, RXRB) which have remained linked over 400 million years and may represent an ancestral arrangement of the vertebrate MHC. Zebrafish and Fugu diverged between 100-200 million years ago and differences exist between these two fish species. The position and number of MHC class Ia genes is not conserved between species, there is an inversion of a block of nine genes centering on the PSMB cluster, and additional genes are present in zebrafish coding for a transport-associated protein and a beta proteasome subunit. The extent of these differences has implications for the extrapolation of fish model organism data to commercial aquaculture species. The data presented here represent the most extensive analysis of a fish MHC class Ia region described so far and clearly delimit the extent of this region in Fugu and, potentially, all teleosts.

Animals↗