RESUMEN
The genotypic characterization of Coxiella burnetii provides useful information about the strains circulating at the farm, region, or country level and may be used to identify the source of infection for animals and humans. The aim of the present study was to investigate the strains of C. burnetii circulating in caprine and bovine Belgian farms using a single nucleotide polymorphism (SNP) technique. Direct genotyping was applied to different samples (bulk tank milk, individual milk, vaginal swab, fetal product, and air sample). Besides the well-known SNP genotypes, unreported ones were found in bovine and caprine samples, increasing the variability of the strains found in the two species in Belgium. Moreover, multiple genotypes were detected contemporarily in caprine farms at different years of sampling and by using different samples. Interestingly, certain SNP genotypes were detected in both bovine and caprine samples, raising the question of interspecies transmission of the pathogen.
Asunto(s)
Enfermedades de los Bovinos/microbiología , Coxiella burnetii/genética , Coxiella burnetii/aislamiento & purificación , Enfermedades de las Cabras/microbiología , Polimorfismo de Nucleótido Simple , Fiebre Q/veterinaria , Animales , Bélgica , Bovinos , Coxiella burnetii/clasificación , Genotipo , Cabras , Humanos , Filogenia , Fiebre Q/microbiologíaRESUMEN
The growth hormone secretagogue receptor (GHSR) is involved in the regulation of energetic homeostasis and GH secretion. In this study, the bovine GHSR gene was mapped to BTA1 between BL26 and BMS4004. Two different bovine GHSR CDS (GHSR1a and GHSR1b) were sequenced. Six polymorphisms (five SNPs and one 3-bp indel) were also identified, three of them leading to amino acid variations L24V, D194N, and Del R242. These variations are located in the extracellular N-terminal end, the exoloop 2, and the cytoloop 3 of the receptor, respectively.
Asunto(s)
Bovinos/genética , Mapeo Cromosómico , Polimorfismo Genético , Receptores de Ghrelina/genética , Animales , Genómica , MasculinoRESUMEN
Expression 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-beta, with a 78 bp insert in the trans-activation domain; POU1F1-gamma that lacks exon 3 and POU1F1-delta 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 beta variant presented only 12% of the Wild-type trans-activation capacity. Variants gamma and delta showed no capacity to trans-activate PRL promoter. Both gamma and delta variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone (p<0.05). Concerning the GH promoter, the beta variant presented a trans-activation capacity 10% higher than Wt. Wt and beta variants strongly interact in the activation of GH promoter doubling the trans-activation potential of Wt. Variants gamma and delta 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.
Asunto(s)
Ovinos/genética , Factor de Transcripción Pit-1/genética , Empalme Alternativo , Animales , Secuencia de Bases , Línea Celular , ADN/genética , Variación Genética , Hormona del Crecimiento/genética , Células HeLa , Humanos , Hipófisis/metabolismo , Prolactina/genética , Regiones Promotoras Genéticas , Ratas , Proteínas Recombinantes/genética , Activación TranscripcionalRESUMEN
The five exons and the 5' and 3'-untranslated regions (5'-UTR and 3'-UTR) of the oGH gene were screened for mutations using PCR-single strand conformation polymorphism (PCR-SSCP) procedures in 523 Serra da Estrela ewes and were found to be highly polymorphic. The region extending across and between the GH2-N and GH2-Z copies was sequenced allowing the design of primers for the specific PCR amplification of each copy. These were cloned and sequenced in 20 animals representative of all SSCP patterns. The corresponding genotypes were established for each copy following nucleotide sequencing of SSCP alleles. Twenty-four polymorphic sites were found at the GH2-N (or GH1) and fourteen at the GH2-Z copies. Eight amino acid substitutions were predicted at the GH2-N and six at the GH2-Z copies. Milk yield adjusted to 150 lactation days was analysed for the genotype of each oGH gene copy taken separately or together (associated genotypes) by restricted maximum likelihood (REML) through a univariate best linear unbiased prediction (BLUP) animal model with repeated measures. Significant associations between genotypes and milk yield were observed. Within GH2-N genotypes there was a milk yield differential of 21.4+/-0.2 l/150 d between the most (N7) and the least (N5) productive ones. Within GH2-Z genotypes there was a differential of 21.6+/-0.2 l/150 d between the most (Z8) and the least (Z1) productive ones. The effect of associated GH2-N and GH2-Z genotypes revealed a differential of 39.6+/-0.3 l/150 d between the most (N1+Z7) and the least (N3+Z2) productive associated genotypes. The results show that GH2-N and GH2-Z genotypes significantly affect milk yield in Serra da Estrela ewes. Moreover, the apparent joint effect of GH2-N and GH2-Z genotype could improve milk yield in 25% as compared with the mean milk production of the analysed population.
Asunto(s)
Hormona del Crecimiento/genética , Lactancia/genética , Leche/metabolismo , Polimorfismo Genético , Ovinos/fisiología , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Genotipo , Lactancia/metabolismo , Funciones de Verosimilitud , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa/métodos , Ovinos/genéticaRESUMEN
POU1F1 (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.