RESUMEN
Interleukin 12 (IL-12) is a potent enhancer of interferon gamma production by activated T cells. The high-affinity IL-12 receptor (IL-12R) is a heterodimer of a beta1 and a beta2 subunit. Expression of the signaling IL-12Rbeta2 chain is usually low, as compared with the more abundant beta1 chain, and may be rate-limiting for IL-12 sensitivity. Little is known about the mechanisms controlling IL-12Rbeta2 gene expression. Reporter gene assays in IL-12Rbeta2-expressing Jurkat cells showed that truncation of the region from -151 to -61 abrogated promoter activity. The proximal promoter region does not contain a typical TATA box, suggesting a role for SP-1. Indeed, mutagenesis of the -63 SP-1 consensus site decreased transcription by 50%. Electrophoretic mobility shift experiments confirmed the binding of SP-1 and SP-3 at this site. In contrast, truncation of -252 to -192 increased promoter activity. Likewise, mutagenesis of the consensus nuclear factor of activated T cells site at -206 increased promoter activity by 70%, suggesting silencer activity of this element. Electrophoretic mobility shift experiments with primary Th (T helper) cells showed the formation of a specific, T-cell receptor-inducible complex at this site that is sensitive to cyclosporin A and supershifted with anti-NFATc2 in both Th1 and Th2 cells. Accordingly, cyclosporin A dose-dependently increased IL-12Rbeta2 mRNA expression. These first data on IL-12Rbeta2 gene regulation indicate a TATA-less promoter, depending on SP-1/SP-3 transcription factors, and a negative regulatory NFAT element at -206. This element may contribute to the overall low level of IL-12Rbeta2 expression on Th cells.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Silenciador del Gen , Proteínas Nucleares , Podofilino/análogos & derivados , Regiones Promotoras Genéticas , Receptores de Interleucina/genética , Linfocitos T Colaboradores-Inductores/metabolismo , Factores de Transcripción/fisiología , Secuencia de Bases , Sitios de Unión , Regulación de la Expresión Génica , Genes Reguladores , Humanos , Células Jurkat , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Podofilino/metabolismo , Podofilotoxina/análogos & derivados , Subunidades de Proteína , ARN Mensajero/análisis , Receptores de Interleucina-12RESUMEN
The interleukin-12 receptor (IL-12R) is composed of two subunits, referred to as beta1 and beta2. Both chains are necessary for high-affinity IL-12 binding and signalling, although only the IL-12Rbeta2 chain contains the intracellular tyrosine residues responsible for STAT4 activation. This study presents the intron-exon organization of the human IL-12Rbeta2-chain gene. Polymerase chain reaction (PCR) primers designed across the cDNA (U46198) were used to trace introns, by comparing PCR product sizes obtained using cDNA and genomic DNA as templates. PCR products spanning introns were sequenced to determine the exact splice sites and flanking regions. The coding region of the gene was found to consist of 15 exons and 14 introns. All intron-exon boundaries are consistent with the consensus sequence for splice junctions (5' GT/AG 3'). Comparison of the intron-exon organization with the human GCSFR gene indicated a remarkably well conserved genomic organization between these two class I cytokine receptors. Interestingly, we identified an alternatively spliced mRNA, encoding a putative, truncated protein, lacking all signalling potential.
Asunto(s)
Exones/genética , Intrones/genética , Receptores de Interleucina/genética , Empalme Alternativo/genética , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Codón de Terminación/genética , Secuencia Conservada/genética , Cartilla de ADN/genética , ADN Complementario/genética , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores de Interleucina/química , Receptores de Interleucina/fisiología , Receptores de Interleucina-12 , Eliminación de Secuencia/genética , Linfocitos T/metabolismo , Tirosina/genéticaRESUMEN
The synthesis is described of an N-terminal thyroglobulin (Tg) polypeptide of 27 kDa, which is capable of hormonogenesis, in a baculovirus system. This polypeptide was made using a 657 bp Tg cDNA cloned from human thyroid RNA by a polymerase chain reaction method. The cDNA contained the information for the Tg signal peptide, the N-terminally located site for thyroid hormone formation and, at the 3' end, a sequence coding for six histidine residues. The fragments produced were purified using a nickel-nitrilotriacetic acid column using these six histidine residues. The products were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and showed two glycosylated fragments of 32 and 34 kDa, both of which started with asparagine. Iodination of the fragments with lactoperoxidase in vitro resulted in the formation of thyroxine (T4). The formation rate of T4 in the fragments was about five times lower than that of the mature Tg dimer of 660 kDa, but ten times more rapid than in bovine serum albumin under the same conditions.