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A multicopy Y-chromosomal SGNH hydrolase gene expressed in the testis of the platyfish has been captured and mobilized by a Helitron transposon.
Tomaszkiewicz M
,
Chalopin D
,
Schartl M
,
Galiana D
,
Volff JN
.
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BACKGROUND: Teleost fish present a high diversity of sex determination systems, with possible frequent evolutionary turnover of sex chromosomes and sex-determining genes. In order to identify genes involved in male sex determination and differentiation in the platyfish Xiphophorus maculatus, bacterial artificial chromosome contigs from the sex-determining region differentiating the Y from the X chromosome have been assembled and analyzed.
RESULTS: A novel three-copy gene called teximY (for testis-expressed in Xiphophorus maculatus on the Y) was identified on the Y but not on the X chromosome. A highly related sequence called texim1, probably at the origin of the Y-linked genes, as well as three more divergent texim genes were detected in (pseudo)autosomal regions of the platyfish genome. Texim genes, for which no functional data are available so far in any organism, encode predicted esterases/lipases with a SGNH hydrolase domain. Texim proteins are related to proteins from very different origins, including proteins encoded by animal CR1 retrotransposons, animal platelet-activating factor acetylhydrolases (PAFah) and bacterial hydrolases. Texim gene distribution is patchy in animals. Texim sequences were detected in several fish species including killifish, medaka, pufferfish, sea bass, cod and gar, but not in zebrafish. Texim-like genes are also present in Oikopleura (urochordate), Amphioxus (cephalochordate) and sea urchin (echinoderm) but absent from mammals and other tetrapods. Interestingly, texim genes are associated with a Helitron transposon in different fish species but not in urochordates, cephalochordates and echinoderms, suggesting capture and mobilization of an ancestral texim gene in the bony fish lineage. RT-qPCR analyses showed that Y-linked teximY genes are preferentially expressed in testis, with expression at late stages of spermatogenesis (late spermatids and spermatozeugmata).
CONCLUSIONS: These observations suggest either that TeximY proteins play a role in Helitron transposition in the male germ line in fish, or that texim genes are spermatogenesis genes mobilized and spread by transposable elements in fish genomes.
Figure 1. Structure of the teximY gene cluster in the sex-determining region of the Y chromosome of Xiphophorus maculatus. Red arrowheads show the position and predicted transcription orientation of teximY copies. The four sequenced BAC clones (B14, B29, B17 and N20) are shown as black lines. The distance between teximY copies is given. Locations of the Y-specific Y-cript pseudogene and of two copies of the type 4 melanocortin receptor gene mc4r[14] are indicated.
Figure 2. Exon/intron structure of texim genes in the platyfish genome. The SGNH hydrolase domain(-coding region) is marked in light blue. Introns are represented by broken lines. Predicted start codons are in green and stop codons in red. cDNA and predicted protein sizes are provided. Helitron transposon positions are indicated. Question marks indicate lack of information due to absence of sequence data. Both coding and protein sequences of texim4 are partial.
Figure 3. Conserved amino-acid blocks in the SGNH domain of predicted Texim proteins. Critical residues important for catalytic activity and oxyanion hole residues are indicated, as well as minimum-maximum amino-acid distances between the domains.
Figure 4. Maximum likelihood phylogenetic tree of Texim-related SGNH hydrolase protein domains found in different organisms. The Maximum Likelihood phylogenetic tree is based on an alignment of 112 amino-acids from the SGNH hydrolase domains. Bootstrap values are indicated (1000 repetitions). Accession numbers: Fish Texim: Lipase_GDSL2_Sphoeroides: Sphoeroides nephelus, protein prediction, AF094698.1; Lipase_GDSL2_Dicentrarchus: Dicentrarchus labrax, protein prediction, LG1, FQ310506.3; Lipase_GDSL2_Oryzias: Oryzias latipes, protein prediction, ENSEMBL, LG1:scaffold 395; Lipase_GDSL2_Fundulus-2: Fundulus grandis, JW608837.1; Lipase_GDSL2_Fundulus: Fundulus grandis, JW617431.1; Lipase_GDSL2_Gadus: Gadus morhua, consensus sequence from ESTs EL616197.1, ES481574.1 and ES474748; Sialate_O-acetylesterase_like2_Lepisosteus: Lepisosteus oculatus, protein prediction, LG7. Invertebrate deuterostome Texim: Hydrolase_like1_Amphioxus: Branchiostoma floridae, XP_002590490.1; Hydrolase_like1_Amphioxus-2: Branchiostoma floridae, protein prediction, scaffold 113; Hydrolase_like1_Strongylocentrotus: Strongylocentrotus purpuratus, protein prediction, scaffold 80934; Lipase_GDSL2_Lytechinus: Lytechinus variegatus, JI442514.1; Sialate_O-acetylesterase_like2_Strongylocentrotus: Strongylocentrotus purpuratus, XP_003726742.1; Hydrolase_like_Amphioxus: Branchiostoma floridae, XP_002586334.1; Hydrolase_like_Oikopleura: Oikopleura dioica, CBY22153.1. Accession numbers for PAF acetylhydrolases, prokaryotic hydrolases, CR1 retrotransposons and outgroups are available upon request.
Figure 5. Association of texim genes with Helitron transposons in different fish species, plotted on a Texim molecular phylogeny.Texim phylogeny was performed on a 112 amino-acid alignment using the Maximum Likelihood method with 100 bootstraps (values are provided). Blue boxes indicate texim exons, light blue regions encode the SGNH domain. Grey boxes show Helitron transposons, broken boxes indicate that the coding region is corrupted by point mutations. Nucleotide distance between texim and Helitron is provided. Accession numbers are given in the legend of Figure 4.
Figure 6. RT-qPCR expression analysis of texim genes in adult tissues of Xiphophorus maculatus population Rio Jamapa. All qPCR results were normalized against the ribosomal protein L7 housekeeping gene rpl7, the value 1 was assigned to the tissue/organ with the highest level of expression. M: males (in blue); F: females (in red). TeximYa, teximYb, texim1 and texim4 are preferentially expressed in testis, with teximYb being almost exclusively expressed in the male gonad. Texim1 is also significantly expressed in ovaries. Texim2 and texim3 show predominant expression in brain.
Figure 7. RT-qPCR expression analysis of texim genes in adult tissues of Xiphophorus maculatus population Rio Usumacinta. All qPCR results were normalized against the ribosomal protein L7 housekeeping gene rpl7, the value 1 was assigned to the tissue/organ with the highest level of expression. M: males (in blue); F: females (in red). All texim genes are strongly expressed in testis, with teximYb and texim2 being almost exclusively expressed in the male gonad. Texim1 is also strongly expressed in gills, and texim4 in brain.
Figure 8. In situ RNA hybridization analysis of vasa and texim1/Y expression in platyfish adult testis. A. Transversal testis section stained with Masson’s trichrome. B. Vasa sense probe, C. Vasa antisense probe, D. Texim sense probe, E. Texim antisense probe. SG, spermatogonia; SC, spermatocytes; ST, spermatids; SZ, spermatozeugmata. The probe used does not allow to differentiate between teximY and texim1. Scale bars: 50 μm.
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