RESUMEN

Reactive oxygen species are the most important source of DNA lesions in aerobic organisms, but little is known about the activation of the DNA checkpoints in response to oxidative stress. We show that treatment of yeast cells with sublethal concentrations of hydrogen peroxide induces a Mec1-dependent phosphorylation of Rad53 and a Rad53-dependent cell cycle delay specifically during S phase. The lack of Rad53 phosphorylation after hydrogen peroxide treatment in the G1 and G2 phases is due to the silent repair of oxidative DNA lesions produced at these stages by the base excision repair (BER) pathway. Only the disruption of the BER pathway and the accumulation and/or treatment of DNA intermediates by alternative repair pathways reveal the existence of primary DNA lesions induced at all phases of the cell cycle by hydrogen peroxide. Our data illustrate both the concept of silent repair of DNA damage and the high sensitivity of S-phase cells to hydrogen peroxide.

Asunto(s)

Proteínas de Ciclo Celular , Ciclo Celular/fisiología , Daño del ADN , Reparación del ADN/fisiología , Estrés Oxidativo/fisiología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Secuencia de Bases , Quinasa de Punto de Control 2 , ADN de Hongos/efectos de los fármacos , ADN de Hongos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genotipo , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Cinética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Oxidación-Reducción , Fosforilación , Proteínas Quinasas/genética , Especies Reactivas de Oxígeno/fisiología , Fase S , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Moldes Genéticos

RESUMEN

Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC. Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes. Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro. The transcripts of wild-type and promoter-defective SNR6 genes decreased or became undetectable in an nhp6ADelta nhp6BDelta double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost in nhp6ADelta nhp6BDelta cells at 37 degrees C. In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6ADelta nhp6BDelta cells or reconstituted transcription systems. Finally, NHP6B activated SNR6 transcription in a TFIIIC-independent assay. These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxillary factors are required for the optimal transcription of at least some specific Pol III genes.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas Nucleares/metabolismo , ARN Polimerasa III/genética , ARN de Hongos , ARN Nuclear Pequeño , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas HMGN , Proteínas del Grupo de Alta Movilidad/genética , Mutagénesis , Proteínas Nucleares/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , TATA Box , Transactivadores/genética , Transcripción Genética , Activación Transcripcional

RESUMEN

RAD53 encodes a conserved protein kinase that acts as a central transducer in the DNA damage and the DNA replication checkpoint pathways in Saccharomyces cerevisiae. To identify new elements of these pathways acting with or downstream of RAD53, we searched for genes whose overexpression suppressed the toxicity of a dominant-lethal form of RAD53 and identified PTC2, which encodes a protein phosphatase of the PP2C family. PTC2 overexpression induces hypersensitivity to genotoxic agents in wild-type cells and is lethal to rad53, mec1, and dun1 mutants with low ribonucleotide reductase activity. Deleting PTC2 specifically suppresses the hydroxyurea hypersensitivity of mec1 mutants and the lethality of mec1Delta. PTC2 is thus implicated in one or several functions related to RAD53, MEC1, and the DNA checkpoint pathways.

Asunto(s)

Proteínas de Ciclo Celular , ADN de Hongos/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alelos , Secuencia de Bases , Quinasa de Punto de Control 2 , Cartilla de ADN , Genes Letales , Proteínas Quinasas/genética , Proteína Fosfatasa 2 , Proteína Fosfatasa 2C , Ribonucleótido Reductasas/metabolismo , Saccharomyces cerevisiae/enzimología , Especificidad por Sustrato

Asunto(s)

ARN Polimerasa III/metabolismo , Transcripción Genética , Secuencia de Bases , Enzimas de Restricción del ADN , ADN Complementario/metabolismo , Escherichia coli/genética , Genes Reporteros , Vectores Genéticos , Oligodesoxirribonucleótidos , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo/métodos , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo

RESUMEN

Transcription factor IIIC (TFIIIC) (or tau) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, tau91, present in affinity-purified TFIIIC. Tau91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of tau91 was established by the following biochemical and genetic evidence. Antibodies to tau91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to tau91; and a point mutation in TFC6 (tau91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349E mutant affected in the B block binding subunit (tau138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that tau91 cooperates with tau138 for DNA binding. Recombinant tau91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.

Asunto(s)

ADN/metabolismo , Saccharomyces cerevisiae/genética , Factores de Transcripción TFIII , Factores de Transcripción/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Clonación Molecular , Datos de Secuencia Molecular , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia , Factores de Transcripción/metabolismo

RESUMEN

In a previous study, we explored the mechanisms of SNR6 gene activation by grafting a heterologous DNA-binding domain, GAL4-(1-147), to various components of the yeast RNA polymerase III transcription system. Here, we demonstrate that a modified SNR6 gene harboring GAL4-binding sites (UAS(G)-SNR6) can be efficiently activated via an intervening, unrelated protein-protein interaction, thus laying the foundations of a RNA polymerase III-based two-hybrid system. In a model system, the interacting proteins recruiting TFIIIC to DNA were PRP21 and PRP9 or PRP21 and PRP11. Mutations affecting the interaction between PRP21 and PRP9, or PRP21 and PRP11 decreased UAS(G)-SNR6 activation level proportionally. RNA polymerase II transcriptional activators, like GAL4, VP16 or p53, fused to GAL4 DNA-binding domain, did not activate the UAS(G)-SNR6 gene. However, GAL4 strongly activated UAS(G)-SNR6 when GAL80, an interacting protein, was fused to TFIIIC. This result indicates that this two-hybrid system can be used to assess the interactions between RNA polymerase II regulatory proteins and their partners.

Asunto(s)

ARN Polimerasa III/metabolismo , ARN Polimerasa II/metabolismo , ARN de Transferencia/genética , Proteínas de Unión al ARN , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción TFIII , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Unión Proteica , Saccharomyces cerevisiae/genética , Transcripción Genética , Activación Transcripcional

RESUMEN

Recent work has demonstrated a repressive effect of chromatin on the transcription of the yeast SNR6 gene in vitro. Here, we show the relations between chromatin structure and transcriptional activity of this gene in vivo. Analysis of the SNR6 locus by micrococcal nuclease digestion showed a protection of the TATA box, nuclease-sensitive sites around the A and B blocks, and arrays of positioned nucleosomes in the flanking regions. Analysis of a transcriptionally silent SNR6 mutant containing a 2-bp deletion in the B block showed a loss of TATA-protection and rearrangement or destabilization of nucleosomes in the flanking regions. Hence, SNR6 organizes the chromatin structure in the whole region in a manner dependent on its transcriptional state. Transcriptional analysis was performed by use of maxi-gene SNR6 constructs introduced into histone-mutated strains. Chromatin disruption induced by histone H4 depletion stimulated the transcription of promoter-deficient, but not of wild-type SNR6 genes, revealing a competition between the formation of nucleosomes and the assembly of Pol III transcription complexes that was much in favor of transcription factors. On the other hand, amino-terminal mutations in histone H3 or H4 had no effect (H4) or only a moderate stimulatory effect (H3) on the transcription of promoter-deficient SNR6 genes.

Asunto(s)

Cromatina/ultraestructura , Regulación Fúngica de la Expresión Génica , ARN Nuclear Pequeño/biosíntesis , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Bases , Genes Fúngicos/genética , Histonas/genética , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Nucleosomas/ultraestructura , Unión Proteica , ARN Mensajero/biosíntesis , ARN Nuclear Pequeño/genética , Eliminación de Secuencia , TATA Box/genética

RESUMEN

A DNA fragment containing sequences hybridizing to the 5' region of GS15, a gene encoding soybean cytosolic glutamine synthetase, was isolated from a soybean genomic library. Mapping and partial sequence analysis of the genomic clone revealed that it encodes a cytosolic GS gene, GS21, which is different from GS15. In parallel, a number of cDNA clones encoding cytosolic GS were isolated using the coding region of pGS20 as a probe (pGS20 is a cDNA clone which corresponds to a transcript of the GS15 gene). Two new full-length cDNAs designated pGS34 and pGS38 were isolated and sequenced. In the 5' non-coding region a strong homology was found between the two clones and the GS21 gene. However, none of these sequences were identical, which suggests that there are at least three members in this group of genes. In order to determine their relative levels of transcription, specific sequences from pGS34, pGS38 and GS21 were used in an RNAse protection assay. This experiment clearly showed that GS21 and the gene encoding pGS38 are specifically expressed in young or mature nodules, whereas the gene encoding pGS34 is highly transcribed in nodules and constitutively expressed at a lower level in other soybean organs. In order to further analyse the molecular mechanisms controlling GS21 transcription, different fragments of the promoter region were fused to the Escherichia coli reporter gene encoding beta-glucuronidase (GUS) and the constructs were introduced into Lotus corniculatus via Agrobacterium rhizogenes-mediated transformation. Analysis of GUS activity showed that the GS21 promoter-GUS constructs were expressed in the vasculature of all vegetative organs. This result is discussed in relation to species-specific metabolic and developmental characteristics of soybean and Lotus.

Asunto(s)

Compartimento Celular , Regulación de la Expresión Génica de las Plantas , Glutamato-Amoníaco Ligasa/genética , Glycine max/genética , Tumores de Planta , Secuencia de Aminoácidos , Secuencia de Bases , Citosol/enzimología , ADN Complementario/genética , Fabaceae/genética , Glucuronidasa/biosíntesis , Glucuronidasa/genética , Isoenzimas/genética , Datos de Secuencia Molecular , Fijación del Nitrógeno/genética , Plantas Modificadas Genéticamente , Plantas Medicinales , Regiones Promotoras Genéticas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Rhizobium/genética , Glycine max/enzimología , Distribución Tisular

RESUMEN

A yeast chimeric RNA polymerase III transcription system was constructed to explore the ordered, multistep process of gene activation in vivo. A promoter-deficient U6 RNA gene harboring GAL4-binding sites could be reactivated by fusing the GAL4 DNA-binding domain to components of the general transcription factor TFIIIC (tau) or TFIIIB. Expression of chimeric tau 138 or tau 131 (but not tau 95) subunits activated transcription from GAL4-binding sites located at various positions, including upstream of or within the gene. The function(s) of the B block binding domain of TFIIIC was provided by the fused GAL4-(1-147) domain. The GAL4-(1-147)-TFIIIB70 fusion protein acted at a distance like an activator of transcription. In contrast, none of the 10 different GAL4-(1-147)-polymerase subunit fusions was able to induce transcription, suggesting that RNA polymerase recruitment is not sufficient to initiate transcription.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , ARN Polimerasa III/biosíntesis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción , Secuencia de Bases , Sitios de Unión , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Insercional , Regiones Promotoras Genéticas , ARN Polimerasa III/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , TATA Box , Transcripción Genética , Activación Transcripcional

RESUMEN

The yeast U6 snRNA gene, SNR6, transcribed by RNA polymerase III or C, is shown to have a mixed promoter with upstream, intragenic and downstream elements. The distant downstream B block behaves as a typical enhancer element. Required in vivo, and for transcription of chromatin templates in vitro, it was also active in reversed orientation. As shown by footprinting and electron microscopy, the factor TFIIIC, or tau, bound the B block in an oriented manner and was able to induce DNA looping. The factor TFIIIC appeared to act via a weak A block located at position +21. This A block-related motif was essential in vivo and with chromatin templates. When changed into a consensus A block it favored DNA looping by TFIIIC firmly anchored on the B block, and activated a B block lacking gene in vivo and in vitro. The role of the TATA box at -30 was most apparent using a purified transcription system. With the A block, it appeared to contribute to start site selection. The results suggest a model where three weak promoter elements collaborate to assemble the transcription complex by DNA looping and synergistic protein-DNA interactions.

Asunto(s)

Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , ARN Nuclear Pequeño/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción TFIII , Secuencia de Bases , ADN de Hongos/metabolismo , ADN de Hongos/ultraestructura , Genes Fúngicos , Datos de Secuencia Molecular , Mutación , ARN de Hongos/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/ultraestructura , Transcripción Genética

RESUMEN

Glutamine synthetase (GS; EC 6.3.1.2) is present in different subcellular compartments in plants. It is located in the cytoplasm in root and root nodules while generally present in the chloroplasts in leaves. The expression of GS gene(s) is enhanced in root nodules and in soybean roots treated with ammonia. We have isolated four genes encoding subunits of cytosolic GS from soybean (Glycine max L. cv. Prize). Promoter analysis of one of these genes (GS15) showed that it is expressed in a root-specific manner in transgenic tobacco and Lotus corniculatus, but is induced by ammonia only in the legume background. Making the GS15 gene expression constitutive by fusion with the CaMV-35S promoter led to the expression of GS in the leaves of transgenic tobacco plants. The soybean GS was functional and was located in the cytoplasm in tobacco leaves where this enzyme is not normally present. Forcing this change in the location of GS caused concomitant induction of the mRNA for a native cytosolic GS in the leaves of transgenic tobacco. Shifting the subcellular location of GS in transgenic plants apparently altered the nitrogen metabolism and forced the induction in leaves of a native GS gene encoding a cytosolic enzyme. The latter is normally expressed only in the root tissue of tobacco. This phenomenon may suggest a hitherto uncharacterized metabolic control on the expression of certain genes in plants.

Asunto(s)

Citosol/enzimología , Glutamato-Amoníaco Ligasa/genética , Glycine max/genética , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Cromatografía en Gel , ADN , Regulación Enzimológica de la Expresión Génica , Glutamato-Amoníaco Ligasa/metabolismo , Inmunohistoquímica , Focalización Isoeléctrica , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Plantas Modificadas Genéticamente , Plantas Tóxicas , Regiones Promotoras Genéticas , Mapeo Restrictivo , Homología de Secuencia , Glycine max/enzimología , Nicotiana

RESUMEN

A full-length cDNA clone encoding cytosolic glutamine synthetase (GS), expressed in roots and root nodules of soybean, was isolated by direct complementation of an Escherichia coli gln A- mutant. This sequence is induced in roots by the availability of ammonia. A 3.5-kilobase promoter fragment of a genomic clone (lambda GS15) corresponding to this cDNA was isolated and fused with a reporter [beta-glucuronidase (GUS)] gene. The GS-GUS fusion was introduced into a legume (Lotus corniculatus) and a nonlegume (tobacco) plant by way of Agrobacterium-mediated transformations. This chimeric gene was found to be expressed in a root-specific manner in both tobacco and L. corniculatus, the expression being restricted to the growing root apices and the vascular bundles of the mature root. Treatment with ammonia increased the expression of this chimeric gene in the legume background (i.e., L. corniculatus); however, no induction was observed in tobacco roots. Histochemical localization of GUS activity in ammonia-treated transgenic L. corniculatus roots showed a uniform distribution across all cell types. These data suggest that the tissue specificity of the soybean cytosolic GS gene is conserved in both tobacco and L. corniculatus; however, in the latter case, this gene is ammonia inducible. Furthermore, the ammonia-enhanced GS gene expression in L. corniculatus is due to an increase in transcription. That this gene is directly regulated by externally supplied or symbiotically fixed nitrogen is also evident from the expression of GS-GUS in the infection zone, including the uninfected cells, and the inner cortex of transgenic L. corniculatus nodules, where a flux of ammonia is encountered by this tissue. The lack of expression of GS-GUS in the outer cortex of the nodules suggests that ammonia may not be able to diffuse outside the endodermis.

Asunto(s)

Amoníaco/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutamato-Amoníaco Ligasa/genética , Glycine max/genética , Plantas Modificadas Genéticamente/genética , Secuencia de Aminoácidos , Secuencia de Bases , Citosol/enzimología , Fabaceae/genética , Glutamato-Amoníaco Ligasa/biosíntesis , Datos de Secuencia Molecular , Plantas Medicinales , Plantas Tóxicas , Glycine max/enzimología , Nicotiana/genética