Tetraodon nigroviridis is a freshwater puffer fish with the smallest known vertebrate genome. Here, we report a draft genome sequence with long-range linkage and substantial anchoring to the 21 Tetraodon chromosomes. Genome analysis provides a greatly improved fish gene catalogue, including identifying key genes previously thought to be absent in fish. Comparison with other vertebrates and a urochordate indicates that fish proteins have diverged markedly faster than their mammalian homologues. Comparison with the human genome suggests ,900 previously unannotated human genes. Analysis of the Tetraodon and human genomes shows that whole-genome duplication occurred in the teleost fish lineage, subsequent to its divergence from mammals. The analysis also makes it possible to infer the basic structure of the ancestral bony vertebrate genome, which was composed of 12 chromosomes, and to reconstruct much of the evolutionary history of ancient and recent chromosome rearrangements leading to the modern human karyotype.Access to entire genome sequences is revolutionizing our understanding of how genetic information is stored and organized in DNA, and how it has evolved over time. The sequence of a genome provides exquisite detail of the gene catalogue within a species, and the recent analysis of near-complete genome sequences of three mammals (human 1 , mouse 2 and rat 3 ) shows the acceleration in the search for causal links between genotype and phenotype, which can then be related to physiological, ecological and evolutionary observations. The partial sequence of the compact puffer fish Takifugu rubripes genome was obtained recently and this survey provided a preliminary catalogue of fish genes 4 . However, the Takifugu assembly is highly fragmented and as a result important questions could not be addressed.Here, we describe and analyse the genome sequence of the freshwater puffer fish Tetraodon nigroviridis with long-range linkage and extensive anchoring to chromosomes. Tetraodon resembles Takifugu in that it possesses one of the smallest known vertebrate genomes, but as a popular aquarium fish it is readily available and is easily maintained in tap water (see Supplementary Notes for naming conventions, natural habitat and phylogeny). The two puffer fish diverged from a common ancestor between 18-30 million years (Myr) ago and from the common ancestor with mammals about 450 Myr ago 5 . This long evolutionary distance provides a good contrast to distinguish conserved features from neutrally evolving DNA by sequence comparison. Tetraodon sequences in fact had an important role in providing a reliable estimate of the number of genes in the human genome 6 . There has been a vigorous and unresolved debate as to whether a whole-genome duplication (WGD) occurred in the ray-finned fish (actinopterygians) lineage after its separation from tetrapods [7][8][9] . By exploiting the extensive anchoring of the Tetraodon sequence to chromosomes, we provide a definitive answer to this question. The distribution of duplicated genes in t...

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by attacks of fever and serositis. In this paper, we define a minimal co-segregating region of 60 kb containing the FMF gene (MEFV) and identify four different transcript units within this region. One of these transcripts encodes a new protein (marenostrin) related to the ret-finger protein and to butyrophllin. Four conservative missense variations co-segregating with FMF have been found within the MEFV candidate gene in 85% of the carrier chromosomes. These variations, which cluster at the carboxy terminal domain of the protein, were not present in 308 control chromosomes, including 162 validated non-carriers. We therefore propose that the sequence alterations in the marenostrin protein are responsible for the FMF disease.

No abstract

The number of genes in the human genome is unknown, with estimates ranging from 50,000 to 90,000 (refs 1, 2), and to more than 140,000 according to unpublished sources. We have developed 'Exofish', a procedure based on homology searches, to identify human genes quickly and reliably. This method relies on the sequence of another vertebrate, the pufferfish Tetraodon nigroviridis, to detect conserved sequences with a very low background. Similar to Fugu rubripes, a marine pufferfish proposed by Brenner et al. as a model for genomic studies, T. nigroviridis is a more practical alternative with a genome also eight times more compact than that of human. Many comparisons have been made between F. rubripes and human DNA that demonstrate the potential of comparative genomics using the pufferfish genome. Application of Exofish to the December version of the working draft sequence of the human genome and to Unigene showed that the human genome contains 28,000-34,000 genes, and that Unigene contains less than 40% of the protein-coding fraction of the human genome.

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurring attacks of fever and serositis. It affects primarily North African Jews, Armenians, Turks and Arabs, in which a founder effect has been demonstrated. The marenostrin-pyrin-encoding gene has been proposed as a candidate gene for the disease ( MEFV ), on the basis of the identification of putative mutations clustered in exon 10 (M680V, M694I, M694V and V726A), each segregating with one ancestral haplotype. In a search for additional MEFV mutations in 120 apparently non-founder FMF chromosomes, we observed eight novel mutations in exon 2 (E148Q, E167D and T267I), exon 5 (F479L) and exon 10 (I692del K695R, A744S and R761H). Except for E148Q and K695R, all mutations were found in a single chromosome. Mutation E148Q was found in all ethnic groups studied and in association with a novel ancestral haplotype in non-Ashkenazi Jews (S2). Altogether, these new findings definitively establish the marenostrin/pyrin-encoding gene as the MEFV locus.

Familial Mediterranean Fever is one of the most frequent recessive disease in non-Ashkenazi Jews. The gene responsible for the disease (MEFV) has very recently been identified. The M694V ('MED') mutation was found in about 80% of the FMF Jewish (Iraqi and North African) chromosomes. To see if the presence of this mutation could be correlated with particular traits of the disease, we examined a number of clinical features in a panel of 109 Jewish FMF patients with 0, 1 or 2 MED mutations. We showed that homozygosity for this mutation was significantly associated with a more severe form of the disease. In homozygous patients, the disease started earlier (mean age 6.4 + /-5 vs 13.6 + /-8.9) and both arthritis and pleuritis were twice as frequent as in patients with one or no M694V mutation. Moreover, 3/3 patients with amyloidosis displayed two MED mutations. No association was found with fever, peritonitis, response to colchicine and erysipeloid eruption. The present result strongly suggests the potential prognostic value of the presence of this mutation.

The major histocompatibility complex (Mhc) of the ring-necked pheasant contains two polymorphic Mhc class II B genes. We show here, by screening of a cDNA library and RT-PCR from RNA, that both of these loci, Phco-DAB1 and Phco-DAB2, normally are transcribed in the spleen. They differ mainly in the 3' untranslated (UT) region, with the transcript lengths, not including the poly(A) tails, being 1,100 nt for DAB1 and 955 nt for DAB2. These two loci are orthologous to the B-LBI and B-LBII loci of the domestic chicken, respectively. DAB1 and DAB2 therefore seem to have evolved from a duplication before the split of the evolutionary lineages leading to the pheasant and the domestic chicken ca. 20 MYA. This is the first report of an orthologous relationship between avian Mhc genes. Yet, the third exons of DAB1 and DAB2 were identical in all available sequences and differed at 10 positions from the exon 3 sequences of B-LBI/B-LBII. The species-specific exon 3 suggests that DAB1 and DAB2 are subject to concerted evolution, i.e., interlocus genetic exchange. The exon 2 sequences show characteristic polymorphism, with hypervariable segments occurring in different combinations in different alleles. Given the divergence in the 3'UT region, the finding of the same exon 2 sequence at both the DAB1 and the DAB2 loci in one of the pheasant haplotypes also suggests that interlocus genetic exchange does occur. Accordingly, the exon 2 sequences tended to cluster irrespective of locus in the phylogenetic analyses. Genetic exchange simultaneously involving both exon 2 and exon 3 may be facilitated by the short length of the intervening intron (< 100 bp) in pheasants and domestic chickens compared with, e.g., humans (about 3 kb).

Five different chicken B-LB genes were cloned and sequenced. The comparison of these sequences shows that they can be classified as members of two different families, the B-LBII family (containing the B-LBI and B-LBII genes) and the B-LBIII family (containing the B-LBIII, B-LBIV, and B-LBV genes). The extent of polymorphism within each of these families was assessed by in vitro amplification of DNA fragments encompassing exon 2 in several haplotypes. The nucleotide sequences were determined, and pairwise relationships were evaluated. In the course of this work, a sixth gene termed B-LBVI was identified, defining a third family (B-LBVI family). Polymorphism of the B-LBIII or B-LBVI families is far less extensive than that of the B-LBII family. In this latter, the distribution of conserved and polymorphic residues is similar to what has been described in mammals. These families seem to have been generated by gene duplication events giving rise to several isotypes, as observed in mammals. However, phylogenetic analyses indicate that these families are not homologous to their mammalian counterparts. Evaluation of the level of transcription of these different genes showed that genes from the B-LBII family are predominantly transcribed over those of the other families.