Cloning of the dmrt1 gene of Xiphophorus maculatus: dmY/dmrt1Y is not the master sex-determining gene in the platyfish.

In contrast to the situation observed in mammals and birds, a switching between different sex determination systems frequently occurred during the evolution of the teleost fish lineage. This might be due to a frequent turnover of sex-determining signals at the top of the sex determination cascade (master sex-determining genes in the case of genetic sex determination). Alternatively, different variations of a same master gene might decide the sex of individuals in different sex determination systems. In the medaka Oryzias latipes, a Y-specific copy of the putative transcription factor gene dmrt1 very likely corresponds to the master sex-determining gene inducing male formation [Nature 417 (2002) 559; Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 11778]. This gene, alternately called dmY and dmrt1Y, has been formed by duplication of the autosomal dmrt1. In order to determine if an orthologue of dmrt1Y was also located in the sex determination region of the related platyfish Xiphophorus maculatus, Southern blot analysis was performed on genomic DNA from XY and YY males and WY and XX females using both medaka and human dmrt1 cDNA as probes. Using different restriction enzymes, no evidence for sex-specific dmrt-containing genomic fragments could be found in the platyfish. No dmrt gene could be detected by low-stringency Southern blot analysis of genomic inserts from 60 bacterial artificial chromosome (BAC) clones linked to the sex-determining locus on the X and Y chromosomes. Six different groups of BAC clones containing dmrt genes were isolated from a platyfish genomic library. X. maculatus dmrt1 gene and cDNA were characterised. In adult, expression of dmrt1 was detected only in testis. Phylogenetic analysis indicated that the duplication of dmrt1 that led to the formation of dmY/dmrt1Y within the medaka lineage clearly occurred after its divergence from the platyfish lineage. Hence, a dmY/dmrt1Y orthologue generated by this particular event of duplication does not correspond to the master sex-determining gene in the platyfish.

The Xmrk oncogene can escape nonfunctionalization in a highly unstable subtelomeric region of the genome of the fish Xiphophorus.

The Xmrk oncogene involved in melanoma formation in the fish Xiphophorus was formed relatively recently by duplication of the epidermal growth factor co-orthologue egfrb. In the platyfish X. maculatus, Xmrk is located close to the major sex-determining locus in a subtelomeric region of the X and Y sex chromosomes that frequently undergoes duplications and other rearrangements. This region accumulates repetitive sequences: more than 80% of the 33-kb region 3' of Xmrk is constituted by retrotransposable elements. The high degree of nucleotide identity between X- and Y-linked sequences and the rarity of gonosome-specific rearrangements indicated that the instability observed was not a manifestation of gonosome-specific degeneration. Seven other duplicated genes were found, all corresponding, in contrast to Xmrk, to pseudogenes (nonfunctionalization). Functional persistence of Xmrk in a highly unstable region in divergent Xiphophorus species suggests a beneficial function under certain conditions for this dispensable and potentially injurious gene.

An active non-LTR retrotransposon with tandem structure in the compact genome of the pufferfish Tetraodon nigroviridis.

The fish retrotransposable element Zebulon encodes a reverse transcriptase and a carboxy-terminal restriction enzyme-like endonuclease, and is related phylogenetically to site-specific non-LTR retrotransposons from nematodes. Zebulon was detected in the pufferfishes Tetraodon nigroviridis and Takifugu rubripes, as well as in the zebrafish Danio rerio. Structural analysis suggested that Zebulon, in contrast to most non-LTR retrotransposons, might be able to retrotranspose as a partial tandem array. Zebulon was active relatively recently in the compact genome of T. nigroviridis, in which it contributed to the extension of intergenic and intronic sequences, and possibly to the formation of genomic rearrangements. Accumulation of Zebulon together with other retrotransposons was observed in some heterochromatic chromosomal regions of the genome of T. nigroviridis that might serve as reservoirs for active elements. Hence, pufferfish compact genomes are not evolutionarily inert and contain active retrotransposons, suggesting the presence of mechanisms allowing accumulation of retrotransposable elements in heterochromatin, but minimizing their impact on euchromatic regions. Homologous recombination between partial tandem sequences eliminating active copies of Zebulon and reducing the size of insertions in intronic and intragenic regions might represent such a mechanism.

Construction and initial analysis of bacterial artificial chromosome (BAC) contigs from the sex-determining region of the platyfish Xiphophorus maculatus.

Despite the major importance of sex determination in aquaculture, no master sex-determining gene has been identified so far in teleost fish. In the platyfish Xiphophorus maculatus, this master gene is flanked by two receptor tyrosine kinase genes, the Xmrk oncogene responsible for melanoma formation in some Xiphophorus interspecific hybrids, and its proto-oncogenic counterpart. Both Xmrk genes, which have already been characterised at the molecular level, delimit a region of about 1 Mb that contains other gene loci involved in sexual maturity, pigmentation and melanoma formation. We have constructed a genomic bacterial artificial chromosome (BAC) library of X. maculatus with a tenfold coverage of the haploid genome and walked on both X and Y sex chromosomes starting from both Xmrk genes. This led to the assembly of BAC contigs from the sex-determining region covering approximately 950 kb of the X and 750 kb of the Y chromosome. To our knowledge, these are the largest contigs reported so far for sex chromosomes in fish. Molecular analysis suggests that the sex-determining region of X. maculatus frequently undergoes retrotranspositions and other kinds of rearrangements. This genomic plasticity might be related to the high genetic variability observed in Xiphophorus for sex determination, sexual maturity, pigmentation and melanoma formation, which are encoded by gene loci located in the sex-determining region.