RESUMO
The availability of sequenced genomes has generated a need for experimental approaches that allow the simultaneous analysis of large, or even complete, sets of genes. To facilitate such analyses, we have developed GST-PRIME, a software package for retrieving and assembling gene sequences, even from complex genomes, using the NCBI public database, and then designing sets of primer pairs for use in gene amplification. Primers were designed by the program for the direct amplification of gene sequence tags (GSTs) from either genomic DNA or cDNA. Test runs of GST-PRIME on 2000 randomly selected Arabidopsis and Drosophila genes demonstrate that 93 and 88% of resulting GSTs, respectively, fulfilled imposed length criteria. GST-PRIME primer pairs were tested on a set of 1900 Arabidopsis genes coding for chloroplast-targeted proteins: 95% of the primer pairs used in PCRs with genomic DNA generated the correct amplicons. GST-PRIME can thus be reliably used for large-scale or specific amplification of intron-containing genes of multicellular eukaryotes.
Assuntos
Proteínas de Arabidopsis/genética , Primers do DNA/genética , Proteínas de Drosophila/genética , Genoma , Reação em Cadeia da Polimerase/métodos , Software , Automação/métodos , Pareamento de Bases , Cloroplastos/metabolismo , Primers do DNA/química , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Genes de Insetos/genética , Genes de Plantas/genética , Íntrons/genética , Conformação de Ácido Nucleico , Transporte Proteico , Edição de RNA/genética , Sítios de Splice de RNA/genéticaRESUMO
Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease of unknown etiology characterized by high-titer anti-mitochondrial antibodies. The major autoantigen has been identified as the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). The fact that PDC-E2 is present in all nucleated cells, but autoimmune damage is confined to biliary epithelial cells, prompted us to investigate the possibility that mucosally-derived IgA may be pathogenic for biliary epithelial cells. Serum IgA was purified from six patients with PBC and its localization and ability to penetrate cells was studied using Madine-Darby canine kidney (MDCK) cells transfected with the human IgA receptor (MDCK-pIgR). The potential of IgA to be transported through the cells was studied by a combination of immunohistochemistry and dual color fluorescent microscopy. Interestingly, IgA from all PBC patients co-localized with PDC-E2 (the major autoantigen of PBC) inside the cells; this was demonstrated by dual staining with anti-human IgA and a mouse monoclonal antibody directed to PDC-E2. In contrast, no co-localization was observed for IgA controls. Furthermore, dual staining of liver sections from PBC patients demonstrated co-localization of IgA and PDC-E2, both cytoplasmically and at the apical surface. We postulate that there may be a direct effect of these autoantibodies on the mitochondrial function of biliary epithelial cells.
