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.     

Characterisation of two topoisomerase 1 genes in the pufferfish (Fugu rubripes)

Eukaryotic DNA topoisomerase I manipulates the higher order structures of DNA. Only one functional topoisomerase 1 (top1) gene has previously been identified in any individual eukaryotic species. Here we report the identification and characterisation of two top1 genes in the pufferfish, Fugu rubripes. This shows that the copy number of top1, like that of other topoisomerases, may vary between eukaryotes. Both Fugu genes have 21 exons; a gene structure similar to that of human TOP1. Despite this conservation of structure, and some non-coding elements, both genes are less than a tenth of the size of the human gene. Sequence and phylogenetic analyses have shown that this duplication is ancient and also affects other species in the fish lineage.     

Isolation and genomic characterization of the TUPLE1/HIRA gene of the pufferfish Fugu rubripes

In an effort to obtain a small genomic construct for the generation of a HIRA transgenic mouse, we have isolated and sequenced the Fugu TUPLE1/HIRA gene. We have compared the gene organization and the proteins encoded in pufferfish and human and also searched for conserved DNA sequences that might be important in gene regulation. The pufferfish gene spans approx. 9 kb, which is approx. 11 times smaller than the human gene, owing to the reduced size of the introns. Like its human counterpart, it is organized into 25 exons. The majority of the splice sites are in identical positions to those found in the human gene, however, for three internal exons the positions of the splice sites are not directly comparable. The coding regions are almost identical in size and show a high degree of similarity, especially at the amino and carboxy termini. Comparisons of 5′ and 3′ sequences failed to detect similarities or sequences involved in regulation.     

Identification and analysis of cis-regulatory elements in development using comparative genomics with the pufferfish, Fugu rubripes

The control of vertebrate development is facilitated by cis-regulatory sequences hardwired into the genome. Given that many developmental processes are strikingly similar across all backboned animals, it is reasonable to expect these sequences to be conserved at the nucleotide level, their potential for mutation being constrained by their function. Comparison between the genomes of highly divergent organisms allows such sequences to be identified and some of the most successful approaches have compared regions from the pufferfish, Fugu rubripes, with its distant mammalian relatives, rodents and humans. This review describes progress made in this kind of comparison, from small regions of individual genes, to whole genome alignments.     

Identification of novel tropomyosin 1 genes of pufferfish (Fugu rubripes) on genomic sequences and tissue distribution of their transcripts

Fugu genome database enabled us to identify two novel tropomyosin 1 (TPM1) genes through in silico data mining and isolation of their corresponding cDNAs in vivo. The duplicate TPM1 genes in Japanese pufferfish Fugu rubripes suggest that additional an ancient segmental duplication or whole genome duplication occurred in fish lineage, which, like many other reported Fugu genes, showed reduction in genomic size in comparison with their human homologue. Computer analysis predicted that the coiled-coil probabilities, that were thought to be the most major function of TPM, were the same between the two TPM1 isoforms. We confirmed that the tissue expression profiles of the two TPM1 genes differed from each other, which implied that changes in expression pattern could fix duplicated TPM1 genes although the two TPM1 isoforms appear to have similar function.     

The nicotinic acetylcholine receptor gene family of the pufferfish,Fugu rubripes

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission at nerve-muscle junctions and in the brain. However, the complete gene family of nAChRs has not so far been reported for any vertebrate organism. We have identified the complete nAChR gene family from the reference genome of the pufferfish, Fugu rubripes. It consists of 16 alpha and 12 non-alpha candidate subunits, making it the largest vertebrate nAChR gene family known to date. The gene family includes an unusual set of muscle-like nAChR subunits comprising two alpha1s, two beta1s, one delta, one epsilon, and one gamma. One of the beta1 subunits possesses an aspartate residue and N-glycosylation sites hitherto shown to be necessary for delta-subunit function. Potential Fugu orthologs of neuronal nAChR subunits alpha2-4, alpha6, and beta2-4 have been identified. Interestingly, the Fugu alpha5 counterpart appears to be a non-alpha subunit. Fugu possesses an expanded set of alpha7-9-like subunits and no alpha10 ortholog has been found. Two new candidate beta subtypes, designated beta5 and beta6, may represent subunits yet to be found in the human genome. The Fugu nAChR gene structures are considerably more diverse than those of higher vertebrates, with evidence of "intron gain" in many cases. We show, using RT-PCR, that the Fugu nAChR subunits are expressed in a variety of tissues.     

Molecular characterization and expressional affirmation of the beta proteasome subunit cluster in rock bream immune defense

Immunoproteasomes are primarily induced upon infection and formed by replacing constitutive beta subunits with inducible beta subunits which possess specific cleavage properties that aid in the release of peptides necessary for MHC class I antigen presentation. In this study, we report the molecular characterization and expression analysis of the inducible immunosubunits PSMB8, PSMB9, PSMB9-L, and PSMB10 from rock bream, Oplegnathus fasciatus. The three subunits shared common active site residues and were placed in close proximity to fish homologues in the reconstructed phylogenetic tree, in which the mammalian homologues formed separate clades, indicating a common ancestral origin. The rock bream immunosubunits possessed higher identity and similarity with the fish homologues. RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 were multi-exonic genes with 6, 6, 7 and 8 exons, respectively. These four genes were constitutively expressed in all the examined tissues. Immunostimulants such as lipopolysaccharide and poly I:C induced RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 in liver and head kidney, suggesting their possible involvement in immune defense in rock bream.     

Sushi gets serious: the draft genome sequence of the pufferfish Fugu rubripes

The publication of the Fugu rubripes draft genome sequence will take this fish from culinary delicacy to potent tool in deciphering the mysteries of human genome function.     

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.     

The mitochondrial genome of the pufferfish,Fugu rubripes,and ordinal teleostean relationships

The small nuclear genome of the pufferfish, Fugu rubripes (order Tetraodontiformes), makes this species highly interesting for genome research. In order to establish the phylogenetic position of the Tetraodontiformes relative to other teleostean orders that might also have a reduced nuclear genome size, we have sequenced the mitochondrial (mt) genome of the pufferfish. The gene order, nucleotide composition and evolutionary rate of the mt genome of the fugu correspond to those of other teleosts. This suggests that the evolution of this genome has not been affected by the processes that led to the dramatic reduction of the size of the nuclear genome of the fugu. The phylogenetic analyses, which were based on the concatenated amino acid sequences of twelve protein-coding mt genes, placed the fugu among the percomorphs. The affinities between the Tetraodontiformes and either the Perciformes or the Zeiformes were limited, however. The common notion of a separate euteleostean clade remained unsupported. The analyses did not support the traditional systematic understanding that the Clupeiformes constitute a basal teleostean lineage. In addition the findings strongly suggest that three teleostean orders, the Perciformes, Zeiformes and Scorpaeniformes, are paraphyletic.