Browsing by Author "Bastos, Estela"
Now showing 1 - 5 of 5
Results Per Page
Sort Options
- Identification and characterization of four splicing variants of ovine POU1F1 genePublication . Bastos, Estela; Ávila, Sílvia; Cravador, A.; Renaville, Robert; Guedes-Pinto, Henrique; Castrillo, José LuísExpression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone (GH ), prolactin (PRL), thyroid-stimulating hormone (TSH ) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-β, with a 78 bp insert in the trans-activation domain; POU1F1-γ that lacks exon 3 and POU1F1-δ that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the β variant presented only 12% of the Wild-type trans-activation capacity. Variants γ and δ showed no capacity to trans-activate PRL promoter. Both γ and δ variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone ( p<0.05). Concerning the GH promoter, the β variant presented a trans-activation capacity 10% higher than Wt. Wt and β variants strongly interact in the activation of GH promoter doubling the trans-activation potential of Wt. Variants γ and δ showed no capacity to trans-activate the GH promoter and both acted as repressors, reducing significantly ( p<0.001) the trans-activation performed by Wt. This work presents, for the first time, the characterization of four splicing forms of Ovis aries POU1F1 gene.
- Ovis aries POU1F1 gene: cloning, characterization and polymorphism analysisPublication . Bastos, Estela; Santos, Ingrid; Parmentier, I.; Castrillo, José Luís; Cravador, A.; Guedes-Pinto, Henrique; Renaville, RobertPOU1F1 (PIT-1/GHF-1) is a transcription factor with critical role in the transcriptional regulation of multiple genes in the pituitary and also important for the survival, differentiation and proliferation of three pituitary cell types. To understand the regulation of POU1F1 gene in Ovis aries we report its cloning, sequencing and characterization. The sequenced 5787 bp included six exons and two complete introns. Ovine POU1F1 gene has a high level of conservation with its bovine, human and rat counterparts showing 98.2%, 91.2% and 86.2% of similarity at the coding level, respectively. All six exons were analyzed for polymorphism detection in 100 animals of the Portuguese indigenous ovine breed ‘Churra da Terra Quente’. One polymorphism was found at codon 58 in exon 2, in one allele of 4 animals leading to a change from cysteine to tyrosine (2% allelic frequency). In exon 3 two polymorphisms were detected: a G to A transition altering a glycine to an asparagine at codon 89 in one allele of one animal (0.5% allelic frequency) and another G to A transition at codon 105 converting an alanine into a threonine in one allele of 3 animals (1.5% allelic frequency). These polymorphisms might change the structure of the POU1F1 protein and modify gene-expression. In intron 4, an A to G transition was detected in one allele of six animals (3% allelic frequency). Exons 1, 4 and 6 showed no polymorphisms.
- Single strand conformation polymorphism (SSCP) detection in six genes in the Portuguese indigenous sheep breed "Churra da Terra Quente"Publication . Bastos, Estela; Cravador, A.; Azevedo, Jorge; Guedes-Pinto, HenriqueEvaluation of the genetic diversity for six genes in forty animals of the Portuguese indigenous sheep breed (Ovis aries) “Churra da Terra Quente” was done. A non-radioactive method to allow single-strand conformation polymorphism (SSCP) detection was optimised, starting from genomic DNAand PCR amplification of seven fragments: exon 1 of the a-lactalbumin gene; exons 10 and 11 of the as1-casein gene; exon 7 of the b-casein gene; exon 4 of the k-casein gene; exons 4 and 5 of the growth hormone gene and exon 6 of the growth hormone receptor gene. Polymorphisms were detected in five of the PCR products. Only k-casein and growth hormone receptor were monomorphic. a-lactalbumin and as1-casein exons showed three conformational patterns, b-casein and growth hormone exon 4 showed two electrophoretic patterns and growth hormone exon 5 showed five conformational patterns. These data provide evidence that “Churra da Terra Quente” has a high genetic variability, which opens interesting prospects for future selection programs and also for preservation strategies. Also, our data show that PCR-SSCP is an appropriate tool for evaluating genetic variability.
- Single-strand conformation polymorphism (SSCP) analysis of exon 4 and exon 5 of growth hormone gene in “Serrana Transmontana” goat breedPublication . Varejão, M. C.; Bastos, Estela; Chaves, R.; Cravador, A.; Azevedo, Jorge; Guedes-Pinto, HenriqueThe origin of Serrana goat breed is lost in time: however the wild goat Capra pyrenaica, from the Serra da Estrela, is probably its ancestor. The Serrana is the most representative of Portuguese breeds and is considered to have great productive potentialities and considerable expansion possibilities due to its productive and reproductive indices, high degree of ruggedness and high quality products: cheese and meat.
- Single-strand conformation polymorphism detection of exon 7 of beta-casein gene in “Churra da Terra Quente” Portuguese indigenous ovine breedPublication . Bastos, Estela; Cravador, A.; Varejão, M. C.; Chaves, R.; Guedes-Pinto, HenriqueMilk protein polymorphisms provide a powerful tool for the knowledge o their molecular biology and physichochemical properties. It is becoming increasingly recognised that genetic variation may offer a source of differentiation that can improve milk technology, including cheesemaking.