RESUMEN
The presence of damage in the transcribed strand (TS) of active genes and its position in relation to nucleosomes influence nucleotide excision repair (NER) efficiency. We examined chromatin structure, transcription and repair at the MET16 gene of wild-type and cbf1Delta Saccharomyces cerevisiae cells under repressing or derepressing conditions. Cbf1p is a sequence-specific DNA binding protein required for MET16 chromatin remodelling. Irrespective of the level of transcription, repair at the MspI restriction fragment of MET16 exhibits periodicity in line with nucleosome positions in both strands of the regulatory region and the non-transcribed strand of the coding region. However, repair in the coding region of the TS is always faster, but exhibits periodicity only when MET16 is repressed. In general, absence of Cbf1p decreased repair in the sequences examined, although the effects were more dramatic in the Cbf1p remodelled area, with repair being reduced to the lowest levels within the nucleosome cores of this region. Our results indicate that repair at the promoter and coding regions of this lowly transcribed gene are dependent on both chromatin structure and the level of transcription. The data are discussed in light of current models relating NER and chromatin structure.
Asunto(s)
Cromatina/ultraestructura , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Transcripción Genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Proteínas de Unión al ADN/genética , Desoxirribonucleasa HpaII/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Mutación , Nucleosomas/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/biosíntesis , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Dímeros de Pirimidina/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Isw1p and Isw2p are budding yeast homologs of the Drosophila ISWI chromatin-remodeling ATPase. Using indirect-end-label and chromatin immunoprecipitation analysis, we show both independent and cooperative Isw1p- and Isw2p-mediated positioning of short nucleosome arrays in gene-regulatory elements at a variety of transcription units in vivo. We present evidence that both yeast ISWI complexes regulate developmental responses to starvation and that for Isw2p, recruitment by different DNA-binding proteins controls meiosis and haploid invasive growth.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cromatina/metabolismo , Proteínas Fúngicas/metabolismo , Saccharomyces cerevisiae/citología , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/química , Northern Blotting , Cromatina/química , Huella de ADN , ADN de Hongos/análisis , Proteínas Fúngicas/química , Meiosis , Nucleasa Microcócica , Hibridación de Ácido Nucleico , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de Transcripción/químicaRESUMEN
Glutathione (gamma-l-glutamyl-l-cysteinylglycine) is an important antioxidant molecule, helping to buffer the cell against free radicals and toxic electrophiles. Expression of the yeast GSH1 gene, encoding the first enzyme involved in glutathione biosynthesis, gamma-glutamylcysteine synthetase, is regulated by oxidants and the heavy metal cadmium at the level of transcription. We present evidence that the transcription factors involved in controlling the network of sulfur amino acid metabolism genes are also responsible for regulating GSH1 expression in response to cadmium. In particular the transcription factors Met-4, Met-31, and Met-32 are essential for cadmium-mediated regulation of gene expression, whereas the DNA-binding protein Cbf1 appears to play a negative role in controlling GSH1 expression.
Asunto(s)
Cadmio/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/genética , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Cromatina/genética , Cromatina/ultraestructura , Proteínas de Unión al ADN/genética , Farmacorresistencia Microbiana , Proteínas Fúngicas/genética , Genotipo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Eliminación de Secuencia , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
Many regions of chromatin are subject to dynamic changes. We have developed a rapid method for isolation of small chromatin templates from yeast which will facilitate biochemical analysis of chromatin composition. Using the PHO5 promoter we show that templates prepared from cells grown in inducing or repressing conditions show native chromatin structures. This method may be widely applicable as the chromatin structures at a centromere, at ARS1 and at part of the lacZ region on two other plasmids are preserved after chromatin isolation.
Asunto(s)
Cromatina/genética , Proteínas de Transporte de Fosfato , Plásmidos/aislamiento & purificación , Saccharomyces cerevisiae/genética , Proteínas de Transporte de Membrana/genética , Regiones Promotoras GenéticasAsunto(s)
Regulación Fúngica de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Proteínas de Transporte de Fosfato , Fosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Sulfatos/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Genes Fúngicos , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Metionina/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloAsunto(s)
Isomerasas Aldosa-Cetosa , Cromatina/metabolismo , Desoxirribonucleasa I/metabolismo , Nucleasa Microcócica/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Ciclo Celular , ADN de Hongos/análisis , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Esferoplastos/metabolismoRESUMEN
CPF1 is an abundant basic-helix-loop-helix-ZIP protein that binds to the CDEI motif in Saccharomyces cerevisiae centromeres and in the promoters of numerous genes, including those encoding enzymes of the methionine biosynthetic pathway. Strains lacking CPF1 are methionine auxotrophs, and it has been proposed that CPF1 might positively influence transcription at the MET25 and MET16 genes by modulating promoter chromatin structure. We test this hypothesis and show that the regions surrounding the CDEI motifs in the MET25 and MET16 promoters are maintained in a nucleosome-free state and that this requires the entire CPF1 protein. However, the chromatin structure around the CDEI motifs does not change on derepression of transcription and does not correlate with the methionine phenotype of the cell. An intact CDEI motif but not CPF1 is required for transcriptional activation from a region of the MET25 upstream activation sequence. Our results suggest that CPF1 functions to modulate chromatin structure around the CDEI motif but that these changes at the MET25 and MET16 promoters do not explain how CPF1 functions to maintain methionine-independent growth. The presence of CPF1-dependent chromatin structures at these promoters leads to a weak repression of transcription.
Asunto(s)
Centrómero/ultraestructura , Cromatina/ultraestructura , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/fisiología , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/ultraestructura , Secuencia de Aminoácidos , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Secuencias Hélice-Asa-Hélice , Leucina Zippers , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
In Saccharomyces cerevisiae, the CPF1 gene encodes a centromere binding protein that also plays a role in transcription; cpf1 strains are methionine auxotrophs. In this paper we describe four strains that are methionine prototrophs despite containing a defective CPF1 gene. These strains, which contain mutations at either the SPT21, RPD1 (SIN3), RPD3 or CCR4 loci, have defective centromere function and a chromatin structure around the CDEI elements in the MET25 promoter characteristic of strains lacking CPF1. This indicates that the roles of CPF1 in transcription, centromere function and chromatin modulation around CDEI sites are different. We propose that CPF1 functions to overcome the repressing action, mediated via inactive chromatin, of proteins such as SPT21 or RPD1 (SIN3) on gene expression. The absence of proteins such as SPT21 or RPD1 (SIN3) relieves this repression and explains how methionine prototrophy is restored in the absence of CPF1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Centrómero , Proteínas Fúngicas/genética , Prueba de Complementación Genética , Metionina/metabolismo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Regiones Promotoras Genéticas , ARN de Hongos/genética , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
Using the polymerase chain reaction (PCR) we have amplified and cloned genomic DNA encoding the secreted group I allergen proteins from the house dust mite species Euroglyphus maynei, Dermatophagoides pteronyssinus and D. farinae. Affinity chromatography using a monoclonal antibody to the allergen Der p I was used to purify the group I protein from E. maynei. We present the deduced amino acid sequence of a new member of the group I house dust mite allergen family Eur m I. The three proteins show a high level of primary structure similarity: Eur m I and Der p I show 85% amino acid identity, and the three allergen amino acid sequences taken together show 78% identity. A potential N-glycosylation site and residues of the cysteine protease active site are also conserved between the three proteins.
Asunto(s)
Alérgenos/genética , Hormonas de Insectos/genética , Proteínas de Insectos , Ácaros/genética , Secuencia de Aminoácidos , Animales , Antígenos Dermatofagoides , Proteínas de Artrópodos , Secuencia de Bases , Clonación Molecular , ADN/análisis , Hormonas de Insectos/aislamiento & purificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
Because first-year graduate students at the University of Rochester School of Nursing have a widely varying competency in basic physiology, the faculty devised two courses designed not only to provide these students with a review of basic physiology, but also to encourage them to correlate this science with the science of nursing. This prepares the students to function as nurse clinicians who deal with nursing issues, not medical issues.