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Clonagem do cDNA do receptor de estrogénios na dourada Sparus aurata (Linnaeus, 1758)
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Advisor(s)
Abstract(s)
O receptor de estrogénios (ER), um factor de transcrição que regula a transactivação
de vários genes, pertence à superfamília de receptores nucleares que inclui receptores
para as hormonas esteróides, tiróidea, vitaminas e para vários ligandos ainda não
identificados. O ER tem sido dividido em 6 domínios funcionais (A-F), com os domínios C (ligação ao DNA) e E (ligação à hormona) altamente conservados entre várias espécies, dos peixes aos mamíferos. O objectivo deste estudo foi determinar a sequência do cDNA do ER da dourada, Sparus aurata (dER). Um banco de cDNA de
figado de dourada com 9,1x108 clones, construido em ?LZapII, foi pesquisado com um
fragmento do receptor de estrogénios de Salmo salar (Dr. Glenn Sweeney, Universidade de Cardiff). Foram identificados vários positivos putativos, os quais foram isolados e analisados por Southem blotting, usando a sonda do salmão, com o intuito de seleccionar os clones de maior tamanho e os que apresentavam um sinal mais intenso. Ao mesmo tempo, tentou-se obter por PCR uma sonda homóloga para detecção do cDNA do dER. Os iniciadores foram desenhados através da comparação
das sequências do ER em várias espécies e usados no PCR com cDNA de fígado de
animais previamente estimulados com estradiol-17B. Os fragmentos obtidos foram
clonados e sequenciados para confirmar a respectiva identidade.
The estrogen receptor (ER), a transcriptional factor that regulates the transactivation of target genes, belongs to the nuclear receptor superfarnily that includes receptors for steroid hormones, thyroid horrnones, vitamins and several whose ligands have yet to be identified. The ER has been divided in 6 functional domains (A-F), with domains C (DNA binding) and E (ligand binding) highly conserved among various species from fish to mammals. The aim of this study was to determine the cDNA sequence of ER in seabream, Spams aurata (dER). A seabrearn cDNA liver library of 9,1x1O8 clones, which was constructed in ?\.ZapII, was screened with a fragment of the Salmo salar ER (Dr. Glenn Sweeney, University of Cardifi). Several putative positives were identified, isolated and subjected to Southem blotting, using the salmon probe, in order to select the clones with the largest inserts and which gave the strongest sinal with the probe. At the same time, we used PCR in order to produce a homologous probe for detection of the dER. Primers were designed by comparison of the sequence of ER from different species and used in PCR with cDNA prepared from the liver of animals previously stimulated with estradiol-l7B. The fragments obtained were cloned and sequenced to confirm its identity.
The estrogen receptor (ER), a transcriptional factor that regulates the transactivation of target genes, belongs to the nuclear receptor superfarnily that includes receptors for steroid hormones, thyroid horrnones, vitamins and several whose ligands have yet to be identified. The ER has been divided in 6 functional domains (A-F), with domains C (DNA binding) and E (ligand binding) highly conserved among various species from fish to mammals. The aim of this study was to determine the cDNA sequence of ER in seabream, Spams aurata (dER). A seabrearn cDNA liver library of 9,1x1O8 clones, which was constructed in ?\.ZapII, was screened with a fragment of the Salmo salar ER (Dr. Glenn Sweeney, University of Cardifi). Several putative positives were identified, isolated and subjected to Southem blotting, using the salmon probe, in order to select the clones with the largest inserts and which gave the strongest sinal with the probe. At the same time, we used PCR in order to produce a homologous probe for detection of the dER. Primers were designed by comparison of the sequence of ER from different species and used in PCR with cDNA prepared from the liver of animals previously stimulated with estradiol-l7B. The fragments obtained were cloned and sequenced to confirm its identity.