RESUMO
In higher plants, as-1-like cis elements mediate auxin- and salicylic acid-inducible transcription. Originally found in viral and T-DNA promoters, they are also functional elements of plant promoters activated during the defence response against pathogens. Tobacco bZIP transcription factor TGA1a was the first recombinant protein shown to bind to as-1. cDNAs for two novel tobacco as-1-binding bZIP proteins (TGA2.1 and TGA2.2) were isolated. Revealing a high degree of amino acid identity in the bZIP domain (89%) and the C-terminus (79%), the two TGA2 factors differ remarkably with respect to the length of the N-terminus (170 amino acids in TGA2.1 versus 43 amino acids in TGA2.2). TGA2.1 and TGA2.2, but not TGA1a, interacted with ankyrin repeat protein NPR1, a central activator of the plant defence response. In contrast, TGA2.1 and TGA1a, but not TGA2.2, functioned as transcriptional activators in yeast. Apart from conferring transcriptional activation, the N-terminal domain of TGA2.1 led to reduced in vitro as-1-binding activity and almost completely abolished binding to one half site of this bifunctional element. When being part of a heterodimer with TGA2.2, TGA2.1 was efficiently recruited to a single half site, though double occupancy of the element was still preferred. In contrast, TGA1a preferred to bind to only one palindrome, a feature that was also maintained in heterodimers between TGA1a and TGA2.1 or TGA2.2.
Assuntos
Proteínas de Arabidopsis , Proteínas de Ligação a DNA/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Plantas Tóxicas , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Northern Blotting , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Dimerização , Regulação da Expressão Gênica de Plantas , Óperon Lac/genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Nicotiana/genética , Fatores de Transcrição/genética , Ativação Transcricional , Técnicas do Sistema de Duplo-HíbridoRESUMO
In higher plants, activating sequence-1 (as-1) of the cauliflower mosaic virus 35 S promoter mediates both salicylic acid (SA)- and auxin-inducible transcriptional activation. Originally found in promoters of several viral and bacterial plant pathogens, as-1-like elements are also functional elements of plant promoters activated in the course of a defense response upon pathogen attack. Nuclear as-1-binding factor (ASF-1) and cellular salicylic acid response protein (SARP) bind specifically to as-1. Four different tobacco bZIP transcription factors (TGA1a, PG13, TGA2.1, and TGA2.2) are potential components of either ASF-1 or SARP. Here we show that ASF-1 and SARP are very similar in their composition. TGA2.2 is a major component of either complex, as shown by supershift analysis and Western blot analysis of DNA affinity-purified SARP. Minor amounts of a protein immunologically related to TGA2.1 were detected, whereas TGA1a was not detectable. Overexpression of either TGA2.2 or a dominant negative TGA2.2 mutant affected both SA and auxin (2, 4D) inducibility of various target promoters encoding as-1-like elements, albeit to different extents. This indicates that TGA2.2 is a component of the enhancosome assembling on these target promoters, both under elevated SA and 2,4D concentrations. However, the effect of altered TGA2.2 levels on gene expression was more pronounced upon SA treatment than upon 2,4D treatment.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Ácidos Indolacéticos/farmacologia , Regiões Promotoras Genéticas , Ácido Salicílico/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/fisiologia , Alelos , Fatores de Transcrição de Zíper de Leucina Básica , Northern Blotting , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes Dominantes , Glucuronidase/metabolismo , Mutação , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Tóxicas , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Nicotiana/química , Nicotiana/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
1. In mature mammalian muscle, the muscular chloride channel ClC-1 contributes about 75% of the sarcolemmal resting conductance (Gm). In mice carrying two defective alleles of the corresponding Clc1 gene, chloride conductance (GCl) is reduced to less than 10% of that of wild-type, and this causes hyperexcitability, the salient feature of the disease myotonia. Potassium conductance (GK) values in myotonic mouse muscle fibres are lowered by about 60% compared with wild-type. 2. The defective Clcadr allele causes loss of the 4.5 kb ClC-1 mRNA. Mice heterozygous for the defective Clc1adr allele contain about 50% functional mRNA in their muscles compared with homozygous wild-type mice. 3. Despite a halved functional gene dosage, heterozygous muscles display an average GCl which is not significantly different from that of homozygous wild-type animals. The GK values in heterozygotes are also indistinguishable from homozygous wild-type animals. 4. These results indicate that a regulatory mechanism acting at the post-transcriptional level limits the density of ClC-1 channels. GK is probably indirectly regulated by muscle activity.
