Alain Bernot

Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype

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.

A candidate gene for familial Mediterranean fever

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 butyrophilin. 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.

Estimate of human gene number provided by genome- wide analysis using Tetraodon nigroviridis DNA sequence

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genome is unknown, with estimates ranging from 50,000 to 90,000 (refs 1, 2), and to more than 140,000 according to unpublished sources.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.

Phenotype-genotype correlation in Jewish patients suffering from familial Mediterranean fever (FMF)

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.

Karyotype and chromosome location of characteristic tandem repeats in the pufferfish Tetraodon nigroviridis

Karyotype analysis of Tetraodon nigroviridis, a pufferfish of the family Tetraodontidae with a small compact genome (385 Mb) which is currently being investigated in our laboratory, indicates that this species has 2n = 42 chromosomes. The small chromosome size (the largest pair measuring less than 3 μm) has complicated accurate chromosome pairing based on morphology alone. DAPI staining, however, provides a banding-like pattern. Because of quantitative variations of some heterochromatin classes, the chromosome formula can not be established precisely, but is estimated to include approximately 20 meta- or submetacentric chromosomes and 22 subtelocentric chromosomes. A centromeric satellite, telomeric repeats, and the major and minor rRNA clusters have been localized unequivocally by FISH. As a result, the 28S and 5S rDNA sequences can be used as chromosome-specific probes.

Linkage of a new member of the lectin supergene family to chicken Mhc genes

With the use of tissue-specific cDNA probes, several genes, which do not correspond to the class I (B-F), class II (B-L), or class IV (B-G) genes, were detected within the cosmid clusters containing the chicken major histocompatibility genes. We isolated cDNA clones with a probe corresponding to one of them, the 17.5 gene, located between two class I genes. The 17.5.3 cDNA, isolated from a chicken spleen cDNA library, encodes a 257-residue-long protein. This sequence shows significant similarity with several members of the C-type animal lectin superfamily and is probably a type II transmembrane protein. Analysis of several cDNA clones, together with Southern blot experiments, strongly suggest that this gene belongs to a multigene family, with at least some of its members being polymorphic. Several arguments lend support to the possibility that, together with the linked Mhc genes, the 17.5 gene is part of the recently described Rfp-Y system.

Analysis of 148 kb of genomic DNA of Tetraodon nigroviridis covering an amylase gene family.

We have sequenced and analysed a 148 kb genomic region of Tetraodon nigroviridis, a teleost fish with a compact genome. Several genes were identified by comparison with genomic or transcript sequences of other species, informatic prediction and screening of a cDNA library. As expected for a compact genome, sizes of the identified genes and introns are very small, and intergenic distances are short. Among identified genes, three code for amylases. As in mammals, these genes are linked, but they are found in a small region of less than 11 kb. These results represent the first description of a genomic sequence larger than 100 kb in this species. Synteny with the human genome is restricted to three regions corresponding to human 1p32.3, 1p13.3 and 1p21.1.

Estimation of the Extent of Synteny Between Tetraodon nigroviridis and Homo sapiens Genomes

This paper presents a genomic comparison between 20 sequenced BACs (or fragments of BACs) from Tetraodon nigroviridis and the human genome. A total of 199 fish genes were identified by informatics resources, together with their putative human orthologues. Comparisons of the localizations in both species led to the identification of 32 syntenic regions and a minimum of 131 rearrangements in these regions that occurred during independent evolution of these species. This made it possible to estimate the rate of genomic rearrangements that occurred per million years (and per megabase). This rate is comparable to that obtained by comparison of the Fugu rubripes shotgun sequence data to human data but is significantly higher that those obtained by comparing the human genome to mammalian genomes. Overall, it suggests that genomic evolution by rearrangement is not uniform within the vertebrate group.