Evolution of Hox gene clusters in gnathostomes: insights from a survey of a shark (Scyliorhinus canicula) transcriptome.

It is now well established that there were four Hox gene clusters in the genome of the last common ancestor of extant gnathostomes. To better understand the evolution of the organization and expression of these genomic regions, we have studied the Hox gene clusters of a shark (Scyliorhinus canicula). We sequenced 225,580 expressed sequence tags from several embryonic cDNA libraries. Blast searches identified corresponding transcripts to almost all the HoxA, HoxB, and HoxD cluster genes. No HoxC transcript was identified, suggesting that this cluster is absent or highly degenerate. Using Hox gene sequences as probes, we selected and sequenced seven clones from a bacterial artificial chromosome library covering the complete region of the three gene clusters. Mapping of cDNAs to these genomic sequences showed extensive alternative splicing and untranslated exon sharing between neighboring Hox genes. Homologous noncoding exons could not be identified in transcripts from other species using sequence similarity. However, by comparing conserved noncoding sequences upstream of these exons in different species, we were able to identify homology between some exons. Some alternative splicing variants are probably very ancient and were already coded for by the ancestral Hox gene cluster. We also identified several transcripts that do not code for Hox proteins, are probably not translated, and all but one are in the reverse orientation to the Hox genes. This survey of the transcriptome of the Hox gene clusters of a shark shows that the high complexity observed in mammals is a gnathostome ancestral feature.

Gene amplification and functional diversification of melanocortin 4 receptor at an extremely polymorphic locus controlling sexual maturation in the platyfish.

In two swordtail species of the genus Xiphophorus, the onset of puberty has been shown to be modulated at the P locus by sequence polymorphism and gene copy-number variation affecting the type 4 melanocortin hormone receptor Mc4r. The system works through the interaction of two allelic types, one encoding wild type and the other dominant-negative receptors. We have analyzed the structure and evolution of the P locus in the platyfish Xiphophorus maculatus, where as many as nine alleles of P determining the onset of sexual maturity in males and females, fecundity in females, and adult size in males are located on both the X and Y chromosomes in a region linked to the master sex-determining locus. In this species, mc4r has been amplified to up to 10 copies on both the X and Y chromosomes through recent large serial duplications. Subsequently, mc4r paralogues have diverged considerably into many different subtypes. Certain copies have acquired new untranslated regions through genomic rearrangements, and transposable element insertions and other mutations have accumulated in promoter regions, possibly explaining observed deviations from the classical mc4r transcriptional pattern. In the mc4r-coding sequence, in-frame insertions and deletions as well as nonsense and missense mutations have generated a high diversity of Mc4r-predicted proteins. Most of these variants are expressed in embryos, adults, and/or tumors. Functional receptor characterization demonstrated major divergence in pharmacological behavior for Mc4r receptors encoded by different copies of platyfish mc4r, with differences in constitutive activity as well as binding and stimulation by hormones. The high degree of allelic and copy-number variation observed between individuals can explain the high level of polymorphism for sexual maturation, fecundity, and body size in the platyfish: multiple combinations of Mc4r variants with different biochemical properties might interact to modulate the melanocortin signaling that regulates the hypothalamus-pituitary-gonadal axis.

Polydnaviruses of braconid wasps derive from an ancestral nudivirus.

Many species of parasitoid wasps inject polydnavirus particles in order to manipulate host defenses and development. Because the DNA packaged in these particles encodes almost no viral structural proteins, their relation to viruses has been debated. Characterization of complementary DNAs derived from braconid wasp ovaries identified genes encoding subunits of a viral RNA polymerase and structural components of polydnavirus particles related most closely to those of nudiviruses--a sister group of baculoviruses. The conservation of this viral machinery in different braconid wasp lineages sharing polydnaviruses suggests that parasitoid wasps incorporated a nudivirus-related genome into their own genetic material. We found that the nudiviral genes themselves are no longer packaged but are actively transcribed and produce particles used to deliver genes essential for successful parasitism in lepidopteran hosts.

Molecular analysis of the sex chromosomes of the platyfish Xiphophorus maculatus: Towards the identification of a new type of master sexual regulator in vertebrates.

In contrast to mammals and birds, fish display an amazing diversity of genetic sex determination systems, with frequent changes during evolution possibly associated with the emergence of new sex chromosomes and sex-determining genes. To better understand the molecular and evolutionary mechanisms driving this diversity, several fish models are studied in parallel. Besides the medaka (Oryzias latipes Temminck and Schlegel, 1846) for which the master sex-determination gene has been identified, one of the most advanced models for studying sex determination is the Southern platyfish (Xiphophorus maculatus, Günther 1966). Xiphophorus maculatus belongs to the Poeciliids, a family of live-bearing freshwater fish, including platyfish, swordtails and guppies that perfectly illustrates the diversity of genetic sex-determination mechanisms observed in teleosts. For X. maculatus, bacterial artificial chromosome contigs covering the sex-determination region of the X and Y sex chromosomes have been constructed. Initial molecular analysis demonstrated that the sex-determination region is very unstable and frequently undergoes duplications, deletions, inversions and other rearrangements. Eleven gene candidates linked to the master sex-determining gene have been identified, some of them corresponding to pseudogenes. All putative genes are present on both the X and the Y chromosomes, suggesting a poor degree of differentiation and a young evolutionary age for platyfish sex chromosomes. When compared with other fish and tetrapod genomes, syntenies were detected only with autosomes. This observation supports an independent origin of sex chromosomes, not only in different vertebrate lineages but also between different fish species.

Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of the plant pathogen Leptosphaeria maculans.

Leptosphaeria maculans, a dothideomycete fungus causing stem canker on oilseed rape, develops gene-for-gene interactions with its host plants. It has the ability to rapidly adapt to selection pressure exerted by cultivars harbouring novel resistance genes as exemplified recently by the 3-year evolution towards virulence at the AvrLm1 locus in French populations. The AvrLm1 avirulence gene was recently cloned and shown to be a solo gene within a 269 kb non-coding, heterochromatin-like region. Here we describe the sequencing of the AvrLm1 genomic region in one avirulent and two virulent isolates to investigate the molecular basis of evolution towards virulence at the AvrLm1 locus. For these virulent isolates, the gain of virulence was linked to a 260 kb deletion of a chromosomal segment spanning AvrLm1 and deletion breakpoints were identical or similar. Among the 460 isolates analysed from France, Australia and Mexico, a similar large deletion was apparent in > 90% of the virulent isolates. Deletion breakpoints were also strongly conserved in most of the virulent isolates, which led to the hypothesis that a unique deletion event leading to the avrLm1 virulence has diffused in pathogen populations. These data finally suggest that retrotransposons are key drivers in genome evolution and adaptation to novel selection pressure in L. maculans.

Molecular analysis of the sex-determining region of the platyfish Xiphophorus maculatus.

Due to the presence of genetically well-defined sex chromosomes, with a relatively restricted sex-determination region containing markers identified at the molecular level, the platyfish Xiphophorus maculatus is one of the best models for the positional cloning of a master sex-determining gene in fish. Both male and female heterogametes and three different types of sex chromosomes have been described in the platyfish, with several loci involved in pigmentation, melanoma formation, and sexual maturity closely linked to the master sex-determining locus. Using the melanoma-inducing oncogene Xmrk, its protooncogenic counterpart egfrb, as well as other X- and Y-linked molecular markers, bacterial artificial chromosome (BAC) contigs have been assembled for the sex-determining region of X. maculatus, which was mapped by fluorescent in situ hybridization to the subtelomeric region of the sex chromosomes. Initial sequence analysis of these contigs revealed several gene candidates and uncovered syntenies with different mammalian and chicken autosomes, supporting an independent origin of sex chromosomes in platyfish and tetrapods. Strikingly, the sex determination region of the platyfish is very instable and frequently undergoes duplications, deletions, and transpositions. This instability might be linked to the high genetic variability affecting sex determination and other sex-linked traits in Xiphophorus.