Asunto(s)
Linfadenitis , Faringitis , Estomatitis Aftosa , Fiebre , Humanos , Faringitis/diagnóstico , Faringitis/tratamiento farmacológicoRESUMEN
Infection of Nicotiana tabacum Samsun NN with tobacco mosaic virus (TMV) results in a hypersensitive plant response and leads to systemic acquired resistance (SAR). The induction of SAR is mediated by the plant hormone salicylic acid (SA) and is accompanied by the induced expression of a number of genes including the pathogenesis-related (PR) gene 1a. Previously, it has been found that TMV infection and SA treatment resulted in a reduction of binding of nuclear protein GT-1 to far-upstream regions (-902 to -656) of the PR-1a gene. To test if GT-1 is a negative regulator of PR-1a gene expression, the effects of mutations in the seven putative GT-1 binding sites in this region were studied in vitro using dimethyl sulfate interference footprinting and band shift assays. This showed that at least one of the seven sites is indeed a GT-1 binding site. However, when tested in transgenic plants, the mutations did not result in constitutive expression of the chimeric PR-1a/GUS transgene, while inducible expression after SA treatment was decreased. The results suggest that binding of GT-1-like proteins to far-upstream PR-1a promoter regions indeed influences gene expression. A possible model for GT-1's mode of action in PR-1a gene expression is discussed.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Secuencia de Bases , Sitios de Unión/genética , ADN de Plantas/genética , ADN de Plantas/metabolismo , Expresión Génica/efectos de los fármacos , Genes de Plantas , Datos de Secuencia Molecular , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Plantas Modificadas Genéticamente , Plantas Tóxicas , Mutación Puntual , Regiones Promotoras Genéticas , Ácido Salicílico/farmacología , Nicotiana/genética , Nicotiana/metabolismo , Virus del Mosaico del Tabaco/patogenicidadRESUMEN
The 900 bp promoter region of the tobacco PR-1a gene was divided into eight fragments using PCR. The fragments were tested for their ability to bind to nuclear factors isolated from tobacco leaf. Band shift assays demonstrated that all but one of the fragments specifically interacted with nuclear proteins. From competition experiments it was determined that the same nuclear factors bind various promoter fragments with different affinity. Moreover, efficient competition with a synthetic tetramer of box II of the rbcS promoter indicated that GT-1-like nuclear factors are involved in these interactions. Furthermore, in comparison to extracts from untreated plants, nuclear protein preparations from tobacco mosaic virus-infected tobacco showed a reduced GT-1 binding activity. These results will be discussed in relation to induced PR-1a gene expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Unión Competitiva , ADN de Plantas/genética , Datos de Secuencia Molecular , Plantas Tóxicas , Reacción en Cadena de la Polimerasa , Unión Proteica , Nicotiana/genética , Factores de TranscripciónRESUMEN
To study regulation of the (Ds) transposition process in heterologous plant species, the transposase gene of Ac was fused to several promoters that are active late during plant development. These promoters are the flower-specific chalcone synthase A promoter (CHS A), the anther-specific chalcone isomerase B promoter CHI B and the pollen-specific chalcone isomerase A2 promoter CHI A2. The modified transposase genes were introduced into a tobacco tester plant. This plant contains Ds stably inserted within the leader sequence of the hygromycin resistance (HPT II) gene. As confirmed with positive control elements, excision of Ds leads to the restoration of a functional HPT II gene and to a hygromycin resistant phenotype. No hygromycin resistance was observed in negative control experiments with Ac derivatives lacking 5' regulatory sequences. Although transactivation of Ds was observed after the introduction of transposase gene fusions in calli, excision in regenerated plants was observed only for the CHS A- or CHI B-transposase gene fusions. With these modified transposase genes, somatic excision frequencies were increased (68%) and decreased (22%), respectively, compared to the situation with the Ac element itself (38%). The shifts in transactivation frequencies were not associated with significant differences in the frequencies of germinally transmitted excision events (approximately 5%). The relative somatic stability of Ds insertions bearing the CHI B-transposase gene fusion suggests the usefulness of this activator element for transposon tagging experiments.