RESUMO
Bovine spongiform encephalopathy (BSE) is a fatal disorder in cattle characterized by progressive neurodegeneration of the central nervous system. We investigated the molecular mechanisms involved in neurodegeneration during prion infection through the identification of genes that are differentially expressed (DE) between experimentally infected and non-challenged cattle. Gene expression of caudal medulla from control and orally infected animals was compared by microarray analysis using 24,000 bovine oligonucleotides representing 16,846 different genes to identify DE genes associated with BSE disease. In total, 182 DE genes were identified between normal and BSE-infected tissues (>2.0-fold change, P < 0.01); 81 DE genes had gene ontology functions, which included synapse function, calcium ion regulation, immune and inflammatory response, apoptosis, and cytoskeleton organization; 13 of these genes were found to be involved in 26 different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression of five DE genes associated with synapse function (tachykinin, synuclein, neuropeptide Y, cocaine, amphetamine-responsive transcript, and synaptosomal-associated protein 25 kDa) and three DE genes associated with calcium ion regulation (parvalbumin, visinin-like, and cadherin) was further validated in the medulla tissue of cattle at different infection times (6, 12, 42, and 45 months post-infection) by qRT-PCR. These data will contribute to a better understanding of the molecular mechanisms of neuropathology in bovine species.
Assuntos
Encefalopatia Espongiforme Bovina/genética , Bulbo/metabolismo , Animais , Bovinos , Bases de Dados Genéticas , Encefalopatia Espongiforme Bovina/metabolismo , Encefalopatia Espongiforme Bovina/patologia , Expressão Gênica , Perfilação da Expressão Gênica/veterinária , Genoma , Bulbo/patologia , Análise em Microsséries/veterináriaRESUMO
The Mx (myxovirus resistance) gene codes for a protein with antiviral activity. Non-synonymous G/A polymorphism at position 2032 of chicken Mx cDNA results in a change at amino acid 631 of the Mx protein. This mutation has been shown to affect the antiviral activity of the Mx molecule, although recent studies have not confirmed this effect in response to some influenza strains. Nevertheless, the G/A polymorphism could be important for the chicken's response to other viruses. A robust PCR-RFLP protocol for genotyping chicken Mx gene polymorphism associated with the S631N mutation was developed. The F primer anneals to the last intron of the Mx gene, and the R primer anneals to the last exon of the gene, with an expected PCR product of 299 bp. PCR products were digested with Hpy8I. This enzyme cuts the sequence 5'-GTN|NAC-3', 2 bp downstream of the Mx polymorphism for the G allele, whereas the fragment containing the A allele is not cleaved. One hundred and twenty-seven chickens (commercial broilers, White Leghorn and New Hampshire) were genotyped using this protocol, and genotyping data were validated by sequencing. Full identity of results between the two genotyping methods was observed for all 127 samples, proving the reliability and robustness of this PCR-RFLP protocol.