Browsing by Author "Deloffre, Laurence A. M."
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- Alternative transcripts of DMRT1 in the European sea bass: expression during gonadal differentiationPublication . Deloffre, Laurence A. M.; Martins, Rute S. T.; Mylonas, Constantinos C.; Canario, Adelino V. M.DMRT1 is conserved from invertebrates to human and has been implicated in sex differentiation and testis function in many organisms.We report the cloning of two DMRT1 transcripts, DMRT1a and DMRT1b, encoded by a single gene in the European sea bass, Dicentrarchus labrax, a teleost fish with polygenic sex determination influenced by temperature. DMRT1a and DMRT1b are specific to the testis and are present as transcripts of 2.7 and 4.6 kb, as determined by Northern blot, although the cloned cDNAs are 1.2 and 1.9 kb. DMRT1a, encodes a protein of 306-amino acids, of similar size to what has been found in other teleosts, while the DMRT1b encodes a unique protein which differ from DMRT1a by having a 26 amino acids insertion which separates the S and Y domains. This insertion is the result of an extra exon (exon 4), present in the genomes of teleosts but not in other vertebrate genomes. Furthermore, unlike in the European sea bass, the putative product of DMRT1b in other teleost species is a truncated peptide. In European sea bass, the two transcripts are expressed at a similar level and have a similar expression profile in developing gonads. They are first detected by reverse transcription-polymerase chain reaction at 150 days post-hatch coinciding with the start of sex differentiation. After 200 days, expression increases in testis and decreases in ovary. Although it is not clear whether the two transcripts have differential roles it is suggested that both are required for testicular function.
- A cDNA for European sea bass (Dicentrachus labrax) 11 beta-hydroxylase: Gene expression during the thermosensitive period and gonadogenesisPublication . Socorro, S.; Martins, Rute S. T.; Deloffre, Laurence A. M.; Mylonas, Constantinos C.; Canario, Adelino V. M.Steroid P450 11 -hydroxylase, encoded by the CYP11B gene, is a key mitochondrial enzyme for the production of 11-oxygenated androgens, which have been shown to be potent masculinising steroids in several Wsh species. In this study we have isolated a CYP11B cDNA of 1903 base pairs from the testis of European sea bass (Dicentrarchus labrax) encoding a predicted protein of 552 amino acids. The amino acid identities to other vertebrate 11 -hydroxylase proteins ranged from 66% to 72% to other Wsh; 45% to amphibian and 35–39% to mammalian. Southern blot indicated that a single CYP11B gene is present. Northern blot analysis detected two transcripts in testis and head kidney, one of the same size of the isolated clone and the other of 3.9 kb. Reverse transcriptase-polymerase chain reaction showed abundant mRNA expression only in testis and head kidney, being residual in a range of other tissues. Expression of CYP11B and CYP19A (which encodes for ovarian aromatase) was detected from at least 4 days post-hatching and did not appear to be aVected by rearing temperature (15 and 20 °C) during the Wrst 60 days, a period in which high temperatures promote masculinisation in European sea bass. Throughout, gonadogenesis (60–300 dph), a highly dimorphic pattern of CYP11B expression was consistent with a role of this gene in testicular development.
- DAX1 regulatory networks unveil conserved and potentially new functionsPublication . Martins, Rute S. T.; Power, Deborah; Fuentes, J.; Deloffre, Laurence A. M.; Canario, Adelino V. M.DAX1 is an orphan nuclear receptorwith actions in mammalian sex determination, regulation of steroidogenesis, embryonic development and neural differentiation. Conserved patterns of DAX1 gene expression frommammals to fish have been taken to suggest conserved function. In the present study, the European sea bass, Dicentrarchus labrax, DAX1 promoter was isolated and its conserved features compared to other fish and mammalian DAX1 promoters in order to derive common regulators and functional gene networks. Fish andmammalian DAX1 promoters share common sets of transcription factor frameworkswhichwere also present in the promoter region of another 127 genes. Pathway analysis clustered these into candidate gene networks associated with the fish and mammalian DAX1. The networks identified are concordantwith described functions for DAX1 in embryogenesis, regulation of transcription, endocrine development and steroid production. Novel candidate gene network partners were also identified, which implicate DAX1 in ion homeostasis and transport, lipid transport and skeletal development. Experimental evidence is provided supporting roles for DAX1 in steroid signalling and osmoregulation in fish. These results highlight the usefulness of the in silico comparative approach to analyse gene regulation for hypothesis generation. Conserved promoter architecture can be used also to predict potentially newgene functions. The approach reported can be applied to genes from model and non-model species.
- Developmental expression of DAX1 in the European sea bass, Dicentrarchus labrax: lack of evidence for sexual dimorphism during sex differentiationPublication . Martins, Rute S. T.; Deloffre, Laurence A. M.; Mylonas, Constantinos C.; Power, Deborah; Canario, Adelino V. M.DAX1 (NR0B1), a member of the nuclear receptors super family, has been shown to be involved in the genetic sex determination and in gonadal differentiation in several vertebrate species. In the aquaculture fish European sea bass, Dicentrarchus labrax, and in the generality of fish species, the mechanisms of sex determination and differentiation have not been elucidated. The present study aimed at characterizing the European DAX1 gene and its developmental expression at the mRNA level.