Publicação
Molecular biology of prolactin and prolactin receptor in a marine teleost: the sea bream(Sparus aurata)
| Resumo: | Prolactin (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. |
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| Autores principais: | Santos, Cecilia |
| Assunto: | Biologia molecular Prolactina Sparus aurata |
| Ano: | 1999 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Algarve |
| Idioma: | inglês |
| Origem: | Sapientia - Universidade do Algarve |
| Resumo: | Prolactin (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. |
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