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- Cloning, expression, and tissue localisation of prolactin in adult sea bream (Sparus aurata)Publication . Santos, Cecilia; Brinca, Lilia; Ingleton, P. M.; Power, DeborahA major action of prolactin (PRL) in teleost fish is the maintenance of hydromineral balance in euryhaline species in fresh water. The function of PRL in marine teleosts is less certain and unlike euryhaline teleosts, such as tilapia and salmon, there is relatively little information about protein or gene structure. Associated with studies to determine potential functions of PRL, pituitary prolactin cDNA has been cloned and sequenced from sea bream (Sparus aurata), a marine teleost. The sequence obtained spanned 1349 bp and contained an open reading frame encoding a protein of 212 amino acids composed of a putative signal peptide of 24 residues and a mature protein of 188 amino acids. N-terminal sequencing of the native protein confirmed unambiguously the cleavage site, Ala24, Val25, predicted from alignments of the sea bream PRL cDNA with that of other teleosts. The presence of only one form of PRL in sea bream was supported by identification using Northern blots of only a single transcript of 1.35 kb. Reverse transcription and polymerase chain reaction techniques coupled with Southern blot analysis resulted in the detection of PRL in the pituitary but also in the intestine, liver, ovary, and testes.
- Cloning, characterisation and tissue distribution of an aquaporin-3 cDNA from fish (Sparus aurata)Publication . Santos, CRA; Fuentes, Juan; Cardoso, João CR; Estêvão, Dulce; Power, DeborahThe major intrinsic protein (MIP) family consists of several transmembrane channel proteins specific for water and neutral solutes. All proteins belonging to the MIP family evolved from two divergent bacterial paralogues, one giving rise to the CHIP group, functionally characterised as water channels and the other to the GLP group, specialised in glycerol transport. Three forms of MIP proteins belonging to the GLP group have been identified in mammals: aquaporin-3 (AQP-3), aquaporin-7 (AQP-7) and aquaporin-9 (AQP-9). We have recently cloned and characterised a GLP cDNA from the marine teleost sea bream (Sparus aurata) and studied its tissue distribution. Phylogenetic analysis revealed it was most like AQP-3 and further studies are now underway to determine its role in hydromineral balance.
- Cloning, characterization, and tissue distribution of prolactin receptor in the sea bream (Sparus aurata)Publication . Santos, Cecilia; Ingleton, P. M.; Cavaco, J. E. B.; Kelly, P. A.; Edery, M.; Power, DeborahThe 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.
- Cloning and characterisation of a fish aldolase B genePublication . Llewellyn, Lynda; Ramsurn, Vimi P.; Sweeney, Glen E.; Wigham, Trevor; Santos, Cecilia; Power, DeborahA full length cDNA clone representing an aldolase mRNA was isolated from a sea bream (Sparus aurutu) liver cDNA library. Sequencing of this clone revealed it to encode a 364 amino acid protein with 74% amino acid identity to human aldolase B and slightly lower similarity to human aldolase A and C. In view of the sequence data and of Northern blot analysis showing strong expression of a 1.6 kb transcript in liver it was concluded that the cloned gene represents aldolase B. This clone represents the first aldolase gene to be sequenced from any fish species thus providing new data on the evolution of the vertebrate aldolase gene family.
- High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxinePublication . Eneqvist, Therese; Lundberg, Erik; Karlsson, Anders; Huang, Shenghua; Santos, Cecilia; Power, Deborah; Sauer-Eriksson, A. ElisabethTransthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3 -triiodo- L-thyronine (T3) and 3,5,3 ,5 -tetraiodo-L-thyronine (thyroxine, T4). Human TTR has higher affinity for T4 than T3, whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 Å resolution and bound to ligands T3 and T4, both at 1.9 Å resolution. The apo structure is similar to human TTR with structural changes only at -strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR T4 complex shows the T4-binding site to be similar but not identical to human TTR, whereas the TTR T3 complex shows the I3 halogen situated at the site normally occupied by the hydroxyl group of T4. The significantly wider entrance of the hormone- binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T3 and T4.
- Piscine (Sparus aurata) transthyretin cDNA cloning and characterizationPublication . Santos, Cecilia; Power, DeborahTransthyretin (TTR) is one of the three plasma proteins that participate in the transport of thyroid hormones, thyroxine (T4) and triiodothyronine (T3); it is also involved in the carriage of retinol through the mediation of retinol-binding protein.1 The liver and choroid plexus are the major sites of TTR synthesis in mammals, birds, and diprotodont marsupials; in reptiles, TTR is only synthesized in the choroid plexus. 2 cDNA coding for TTR has been cloned from several mammalian, reptilian, and avian species and is highly conserved especially in the domains involved in binding to thyroid hormones.3 TTR expression has never been reported in fish, in which albumin is considered to be the main carrier for T3 and T4.2 We report the cloning of a TTR cDNA from sea bream (Sparus aurata) and demonstrate the presence of TTR mRNA in the liver of this fish.
- Cloning and sequencing of a full-length sea bream (Sparus aurata) beta-actin cDNAPublication . Santos, Cecilia; Power, Deborah; Kille, Peter; Llewellyn, Lynda; Ramsurn, Vimi P.; Wigham, Trevor; Sweeney, Glen E.A full-length cDNA clone encoding beta-actin (b-actin) was isolated from a sea bream (Sparus aurata) liver cDNA library. Sequencing of this clone reveals an open reading frame encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The sea breamb-actin sequence showed 98% identity to carp and human b-actin and 95% and 94% identity to sea squirt and Dictyostelium cytoplasmic actins, respectively.
- Identification of transthyretin in fish (Sparus aurata) cDNA cloning and characterisationPublication . Santos, Cecilia; Power, DeborahTransthyretin (TTR) has been proposed to have first evolved in reptiles and is one of the three plasma proteins important in the transport of thyroid hormones in higher vertebrates.
- Molecular biology of prolactin and prolactin receptor in a marine teleost: the sea bream(Sparus aurata)Publication . Santos, Cecilia; Power, DeborahProlactin (PRL) is a versatile hormone principally secreted by the pituitary gland, which is involved in up to 300 physiological functions in vertebrates. PRL interacts with target cells by binding to specific membrane located receptors, and modulates gene expression via a cascade of intracellular signalling mechamsms mvolving tyrosine kinases and signal transducers and activators of transcription. The present thesis aimed at isolating and characterising the cDNA for PRL and its receptor in sea bream, providing the essential molecular tools for studying the role of PRL in osmoregulation, development, growth and reproduction in the sea bream. In this conlext sbPRL and PRL receptor (PRLR) were cloned and the expression of both the receptor and the ligand was determined in adult tissues and during the early stages of development of this species. In an initial step, with the objective of establishing most of the molecular biology íechniques required to accomplish the aims of the project, and so that a cDNA sequence of a sea bream housekeeping gene became available, [3-actin was cloned from a sea bream liver cDNA. Sea bream PRL (sbPRL) cDNA was cloned from a sea bream pituitary cDNA library using a homologous probe generated by RT-PCR, and its pnmary structure was compared with its tetrapod counterparts and also with the available sequences from other teleosts. A companson among PRL. growth hormone (GH) and somatolactin (SL) from the sea bream and other fish was also carried out. Using sbPRL cDNA as a probe the expression of PRL In several sea bream tissues was investigated by northem blot and RTPCR, The molecular weight size of the native hormone was determined in pituitary protein extracts and compared to the in vitro translation product of the cloned cDNA. The spatial location of PRL transcripts in the pituitary gland was determined by In situ hybndisation and compared to the protein distribution. So that the complete nucleotide sequence of the sea bream PRL receptor (sbPRLR) could be determined, a sbPRLR homologous probe v/as obtained by RT-PCR using degenerate primers designed from highly consen/ed regions among mammalian and avian PRL receptors. This probe was used to screen intestine and kidney cDNA libraries from which it was possible to isolate three clones. Sequence analysis of these clones provided the complete nucleotide and deduced amino acid sequence of the sbPRLR. The amino acid sequence of sbPRLR was compared with tilapia PRLR and tetrapod counterparts. The expression of sbPRLR was determined in several tissues by northern blot and RT-PCR and also in embryos and in larvae at the early stages of development. Moreover, part of the genomic sequence of sbPRLR was determined and compared to mouse PRLR gene. Aiming at carrying out an evolutionary approach of PRLR either at the functional and structural levei, part of the sequence of an amphibian PRLR {Xenopus laevis) was determined and its expression analysed. The sea bream (3-actin clone spanned 1806 nucleotides (nt) and contained an open reading frame encoding a protein of 375 amino acids (aa), sbPRL spanned 1349 nt and contained an open reading frame encoding a protein of 212 aa including a signal peptide comprising 24 residues and the mature protein of 188 amino acids. sbPRL was found to be more closely related to other piscine PRL, particularly to other perciformes (72-78%). Homology to tetrapod species decreases dramatically to 25- 28%. Multiple sequence alignment of sbPRL with available protein sequences of GH. SL and PRL from other piscine species showed that sbPRL is more closely related to other PRLs than to SL or GH. PRL expression was detected in the pituitary where it is expressed at very high leveis but also in the intestine and in the gonads. In vitro translation of sbPRL cDNA gave ongin to a protein of 26Kda which had the same molecular weight as PRL extracted from the pituitary. In situ hybndisation carried out with sbPRL probe allowed the detection of PRL transcripts in the rostral pars distallis of the sea bream pituitary. sbPRLR comprises 542 amino acids, the extracellular domain is composed by 208 aa and is followed by the transmembrane domain comprising 24 residues and an intracellular domain composed of 542 aa. Within the extracellular domain the characteristic motifs of PRLRs are present: two pairs of cysteine residues and the WS box (WSEWT). The transmembrane domain is composed largely of hydrophobic residues. Within the intracellular domain, box 1 (PPVPGPKI) is 100%conserved in relation to other PRLRs and box 2 and several tyrosine residues were also identified. Northem blot of mRNA samples from several tissues showed that the pituitary, intestine, kidney. gills, and to a lower extent, skin were the tissues expressing PRLR at higher leveis. In intestine, kidney and gills, two transcnpts of 2,8 and 3.2Kb were identified whereas In pituitary and skin only the smaller transchpt was present, In brain, spleen and in the gonads no message was detected. Nevertheless, by RT-PCR it was possible to detect PRLR transcnpts in ali the tissues above and also in embryos and in larvae at early stages of development. Partial sequencing of sbPRLR gene showed that in this species, within the identified region, sbPRLR gene shares the same organisation as mouse PRLR gene. The partial sequence obtamed for Xenopus laevis PRLR comprised 693bp and exhibited the charactenstic motifs of PRLRs. The analysis of the expression of Xenopus PRLR in several tissues demonstrated that liver, testis. kidney. brain, muscle, intestine, stomach, boné and lungs do express PRLR. The results obtained are discussed and compared to the most relevant literature on the subject and in a functional, structural and evolutionary perspective.
- Identification of transthyretin in fish (Sparus aurata): cDNA cloning and characterisationPublication . Santos, CRA; Power, DMTransthyretin (TTR) has been proposed to have first evolved in reptiles and is one of the three plasma proteins important in the transport of thyroid hormones in higher vertebrates. A full-length cDNA encoding TTR was isolated from a sea bream (Sparus aurata) liver cDNA library using a homologous TTR cDNA probe generated by RT-PCR. Comparison of the deduced amino acid sequence of sea bream TTR with other published sequences, revealed an overall identity of 47-54%, although the amino acids in the active binding site were almost 100% conserved. Distribution of TTR was studied in sea bream adult tissue by RT-PCR and was detected in liver, brain, pituitary, gills, kidney, intestine and testis although northern blot analysis only revealed TTR in the liver, suggesting that in sea bream, liver is the main source of this protein. TTR was also expressed in larvae from the first day post-hatch (48h post-fertilisation), Analysis of thyroxine (T-4) and triiodo-L-thyronine (T-3) binding to sea bream serum proteins demonstrated that both T-4 and T-3 bind to albumin and TTR. By demonstrating the existence of TTR in teleost fish this study indicates TTR must have evolved in a common fish ancestor of the tetrapod evolutionary line.