Seabream ghrelin: cDNA cloning, genomic organization and promoter studies.
Recent studies have indicated that ghrelin stimulates growth hormone release from the pituitary via the growth hormone secretagogue receptor (GHSR). We have previously isolated two GHSR subtypes from the pituitary of the black seabream Acanthopagrus schlegeli. In the present study, we have cloned and characterized ghrelin from the same fish species at both the cDNA and gene levels. The full-length seabream ghrelin cDNA, isolated from sea-bream stomach using a novel approach by exploiting a single conserved region in the coding region, was found to encode a prepropeptide of 107 amino acids, with the predicted mature ghrelin peptide consisting of 20 amino acids (GSSFLSPSQKPQNRGKSSRV). Embedded in this full-length cDNA is a putative fish orthologue of the recently reported mammalian obestatin peptide. The ghrelin gene in black seabream, obtained by genomic PCR, was found to encompass four exons and three introns, possessing the same structural organization as in tilapia and goldfish, but different from that in rainbow trout. In addition, a 2230-bp 5'-flanking region of the seabream ghrelin gene was obtained by genome walking. Sequence analysis revealed that, as in the case of the human ghrelin gene, there is neither a GC box nor a CAAT box present in the isolated 5'-flanking region. However, a number of putative transcription factor-binding sites different from the human counterpart were found in the 5'-flanking region of the seabream ghrelin gene, suggesting that different cis- and trans-acting elements are involved in controlling their gene expression. Functional activity of this 5'-flanking region was examined by cloning it into the pGL3-Basic vector upstream of the luciferase reporter gene and transfected into various cell lines. Positive promoter activity could only be recorded in the colon-derived Caco-2 cells, suggesting that the cloned 5'-flanking region represents the functional promoter of the seabream ghrelin gene, which exhibits tissue-specific promoter activity. Using reverse transcriptase PCR analysis, expression of ghrelin was detected only in the seabream stomach, but not in the other tissues examined, including the brain, gill, intestine, kidney, liver and spleen. This stomach-specific expression of ghrelin in seabream is subject to regulation, as administration of growth hormone or ipamorelin to the fish in vivo was demonstrated to enhance its expression. Reminiscent of the homologous upregulation found in the transcriptional control of the seabream GHSR gene, a similar homologous regulatory mechanism might also exist in controlling the expression of seabream ghrelin. The identification of both GHSR and ghrelin from a single fish species would facilitate our subsequent studies on the elucidation of the physiological functions of the ghrelin/GHSR system in teleost. The possible existence of obestatin in teleost opens up new research avenues on the somatotropic axis in fish.
Yeung CM
，Chan CB
，Woo NY
，Cheng CH
... -
《JOURNAL OF ENDOCRINOLOGY》
Cloning, characterization, and tissue distribution of prolactin receptor in the sea bream (Sparus aurata).
The prolactin receptor (PRLR) was cloned and its tissue distribution characterized in adults of the protandrous hermaphrodite marine teleost, the sea bream (Sparus aurata). An homologous cDNA probe for sea bream PRLR (sbPRLR) was obtained by RT-PCR using gill mRNA. This probe was used to screen intestine and kidney cDNA libraries from which two overlapping clones (1100 and 2425 bp, respectively) were obtained. These clones had 100% sequence identity in the overlapping region (893 bp) and were used to deduce the complete amino acid sequence of sbPRLR. The receptor spans 2640 bp and encodes a protein of 537 amino acids. Features characteristic of PRLR, two pairs of cysteines, WS box, hydrophobic transmembrane domain, box 1, and box 2, were identified and showed a high degree of sequence identity to PRLRs from other vertebrate species. SbPRLR is 29 and 32% identical to tilapia (Oreochromis niloticus) and goldfish (Carassius auratus) PRLRs, respectively. In the sea bream two PRLR transcripts of 2.8 and 3.2 kb were detected in the intestine, kidney, and gills and a single transcript of 2.8 kb was detected in skin and pituitary by Northern blot. Spermiating gonads (more than 95% male tissue; gonado-somatic index of 0.6) contained, in addition to the 2.8-kb transcript, three more transcripts of 1.9, 1.3, and 1.1 kb. RT-PCR, which is a far more sensitive method than Northern blot, detected PRLR mRNA in gills, intestine, brain, pituitary, kidney, liver, gonads, spleen, head-kidney, heart, muscle, and bone. Immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR detected PRLR in several epithelial tissues of juvenile sea bream, including the anterior gut, renal tubule, choroid membrane of the third ventricle, saccus vasculosus, branchial chloride cells, and branchial cartilage.
Santos CR
，Ingleton PM
，Cavaco JE
，Kelly PA
，Edery M
，Power DM
... -
《GENERAL AND COMPARATIVE ENDOCRINOLOGY》
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