RESUMEN
In hemodialysis patients, vertebral fractures were associated with elevated sclerostin levels, suggesting that sclerostin could reflect bone fragility in these patients. INTRODUCTION: Fragility fractures are common in hemodialysis patients. The aims of our study were to determine the prevalence of vertebral fracture and analyze associations between sclerostin serum levels and vertebral fractures in hemodialysis patients. METHODS: Ninety-two hemodialysis patients and 100 controls matched for age and sex were studied. Bone mineral density was measured by ultrasonography at non-dominant heel. The markers of bone turnover included serum osteocalcin, C-terminal telopeptide, and sclerostin. All participants underwent radiography of the thoracic and lumbar spine to ascertain the presence of vertebral fractures. RESULTS: Bone ultrasound parameters at calcaneus were significantly lower in hemodialysis patients compared with controls; bone turnover markers and parathyroid hormone level were significantly higher, while serum of 25-OH-D3 was significantly lower in hemodialysis group. One or more moderate or severe vertebral fractures were found in 38 hemodialysis patients, whereas in control group, 10 patients had a vertebral fracture. In hemodialysis group, the comparison between patients with and without vertebral fractures showed that the patients with vertebral fractures had the serum sclerostin levels statistically higher than patients without vertebral, while serum levels of 25-OH-D3 was significantly lower in patients with vertebral fractures compared to the patients without vertebral fractures. Multivariate analysis disclosed that sclerostin levels were associated with an increased risk of vertebral fractures in hemodialysis patients after adjusting for multiple variables. CONCLUSIONS: Our data shows high prevalence of vertebral fractures in hemodialysis patients and that it is associated with elevated sclerostin levels, reflecting bone fragility in these patients.
Asunto(s)
Proteínas Morfogenéticas Óseas/sangre , Fracturas Osteoporóticas/etiología , Diálisis Renal/efectos adversos , Fracturas de la Columna Vertebral/etiología , Deficiencia de Vitamina D/complicaciones , Proteínas Adaptadoras Transductoras de Señales , Anciano , Anciano de 80 o más Años , Densidad Ósea/fisiología , Estudios de Casos y Controles , Femenino , Marcadores Genéticos , Talón/diagnóstico por imagen , Humanos , Masculino , Persona de Mediana Edad , Osteoporosis/complicaciones , Osteoporosis/diagnóstico por imagen , Osteoporosis/fisiopatología , Fracturas Osteoporóticas/sangre , Fracturas Osteoporóticas/diagnóstico por imagen , Fracturas Osteoporóticas/fisiopatología , Radiografía , Medición de Riesgo/métodos , Fracturas de la Columna Vertebral/sangre , Fracturas de la Columna Vertebral/diagnóstico por imagen , Fracturas de la Columna Vertebral/fisiopatología , Ultrasonografía , Calcificación Vascular/sangre , Calcificación Vascular/etiología , Deficiencia de Vitamina D/fisiopatologíaRESUMEN
CONTEXT: Percutaneous laser ablation (PLA) may be useful in treating patients with metachronous metastatic lymph nodes in the neck. OBJECTIVE: Our objective was to assess PLA as a treatment of difficult-to-treat metachronous cervical lymph node metastases from papillary thyroid carcinoma. DESIGN AND SETTING: We conducted a retrospective analysis of prospectively collected data at a public hospital. PATIENTS: Fifteen patients with previous resection of papillary thyroid carcinoma with elevated serum levels of thyroglobulin (Tg) or anti-Tg antibodies (TgAbs) and 24 metachronous nodal metastases treated between September 2010 and April 2012 were followed with [¹8F]fluorodeoxyglucose (¹8FDG) positron emission tomography (PET)/computed tomography (CT) and contrast-enhanced ultrasound (CEUS). INTERVENTION: Intervention was PLA. OUTCOME MEASURES: Technique feasibility and technical success were evaluated. Tg/TgAb serum levels and ¹8FDG-PET/CT, and CEUS appearance were assessed at 6 and 12 months and compared with baseline. Complications were recorded. RESULTS: PLA was always feasible, and technical success was achieved in all patients. At 6 months, local control was achieved in 11 of 15 patients (73%), with 6 (40%) having serum Tg/TgAb normalized (P = .017 vs baseline). Whereas 20 of 24 (83%) nodes were negative at ¹8FDG-PET/CT and CEUS (P < .001 vs baseline), 4 were ¹8FDG-PET/CT-positive (3 also CEUS-positive). At the 12-month follow-up, local control was achieved in 10 of 14 patients (71.4%). Sixteen of 20 nodes (80%) were negative at ¹8FDG-PET/CT and CEUS (P < .001 vs baseline), 4 were ¹8FDG-PET/CT-positive (2 also CEUS-positive). Four of 10 (40%) patients had normalization of serum Tg/TgAb (P = .098 vs baseline). No major complications occurred. CONCLUSIONS: PLA is potentially feasible, safe, and effective for the treatment of metachronous cervical nodal metastases from papillary thyroid carcinoma. This procedure may reduce or delay a large number of highly invasive repeat neck dissections.
Asunto(s)
Carcinoma Papilar/cirugía , Carcinoma/cirugía , Ablación por Catéter , Terapia por Láser , Ganglios Linfáticos/cirugía , Neoplasias de la Tiroides/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Autoanticuerpos/análisis , Carcinoma/sangre , Carcinoma Papilar/diagnóstico por imagen , Carcinoma Papilar/secundario , Ablación por Catéter/efectos adversos , Estudios de Factibilidad , Femenino , Estudios de Seguimiento , Humanos , Terapia por Láser/efectos adversos , Ganglios Linfáticos/diagnóstico por imagen , Metástasis Linfática , Masculino , Persona de Mediana Edad , Cuello , Complicaciones Posoperatorias/prevención & control , Cintigrafía , Estudios Retrospectivos , Tiroglobulina/sangre , Tiroglobulina/metabolismo , Cáncer Papilar Tiroideo , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/sangre , UltrasonografíaRESUMEN
We used the cDNA microarray technique to monitor simultaneously possible changes induced by hypergravity in the expression level of thousands of hippocampal genes. We tested the mRNA level of about 5000 genes in the hippocampus of mice subjected to 1.09 g (1g) or to 1.85 g (2g) for five repeated 1-h daily rotations in a centrifuge (g = 9.81 m/s2). Data were compared with those obtained for mice kept stationary (C). The ratios 1g/C and 2g/C identified genes affected by rotation and rotation + hypergravity, respectively, whereas 2g/1g ratio identified those affected by hypergravity. We found that about 200 genes were affected by rotation and/or rotation + hypergravity. Almost all the genes affected by rotation + hypergravity were up-regulated, only five being down-regulated. The modulated genes code for proteins involved in a wide range of cellular functions (DNA/RNA metabolism, protein processing, intermediate metabolism, cytoskeleton and motility, cell cycle and apoptosis, signal transduction, neuronal structure/function), suggesting that rotation + hypergravity may affect several aspects of the hippocampal function in order to compensate for environmental changes. Six genes directly or indirectly involved in synaptic transmission and plasticity (proSAAS, neuroblastoma ras oncogene, ESTs moderately similar to thymosin beta-10, syndet, inhibin beta E and Ngfi-A binding protein 2) were found to be significantly modulated by hypergravity and unaffected or only slightly affected by rotation. The modulation by hypergravity of these genes suggests that this stimulus might induce plastic remodelling of the hippocampal circuits, possibly both at structural and functional level.
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Regulación de la Expresión Génica , Expresión Génica/fisiología , Hipocampo/metabolismo , Hipergravedad , Animales , Centrifugación/métodos , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Whole-genome analysis was performed using DNA microarrays to define the changes in the gene expression patterns occurring in Saccharomyces cerevisiae cells exposed to ionizing radiation. The effects of sublethal dose on wild-type, rad53 (enhanced sensitivity to radiation and impaired in a cell cycle damage checkpoint), and rad6 (enhanced sensitivity to radiation and functional cell cycle block by radiation) mutant backgrounds and of a higher dose on the wild-type and G(2)-phase-arrested cells were analyzed. Several gene pathways were identified as being implicated in the response to radiation. In particular, the cell cycle blockage that occurred in the wild-type strain after a high radiation dose and in the rad6 mutant after a lower dose entailed modifications of defined gene expression patterns, which are described here and are compared with the gene modulation patterns observed in the rad53 strain in the absence of efficient blockage. Loss of the RAD53 function caused a major increase in the number of genes modulated by radiation. Given that Rad53-Sad1p, the protein encoded by RAD53, has functions other than those directly connected to cell cycle arrest, we determined the gene patterns that were modulated upon irradiation of rad53 cells that had been forced to arrest in G(2) phase by nocodazole treatment. These differential whole-genome analyses shed light on the multiplicity of functions of the pivotal Rad53-Sad1p protein. The results obtained describe how the cells respond to different irradiation conditions by modulating important gene classes, including those associated with stress defense, ribosomal proteins, histones, ergosterol and GCR1-controlled sugar metabolism.
Asunto(s)
Proteínas de Ciclo Celular , Daño del ADN , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/efectos de la radiación , Transcripción Genética/efectos de la radiación , Ciclo Celular , Quinasa de Punto de Control 2 , Relación Dosis-Respuesta en la Radiación , Fase G2 , Proteínas Serina-Treonina Quinasas/fisiologíaRESUMEN
The synthesis of prebiotic molecules is a major problem in chemical evolution as well as in any origin-of-life theory. We report here a plausible new prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide under catalytic conditions. In the presence of CaCO(3) and different inorganic oxides, namely silica, alumine, kaolin, and zeolite (Y type), neat formamide undergoes the formation of purine, adenine, cytosine, and 4(3H)-pyrimidinone, from acceptable to good yields. The role of catalysts showed to be not limited to the improvement of the yield but it is also relevant in providing a high selectivity in the products distribution.
Asunto(s)
Citosina/síntesis química , Evolución Química , Formamidas/química , Origen de la Vida , Purinas/síntesis química , Pirimidinonas/síntesis química , CatálisisRESUMEN
The basis for the choice of translational position of a histone octamer on DNA is poorly understood. To gain further insights into this question we have studied the translational and rotational settings of core particles assembled on a simple repeating 20 bp positioning sequence. We show that the translational positions of the core particles assembled on this sequence are invariant with respect to the DNA sequence and occur at 20 bp intervals. Certain modifications of the original sequence reduce the spacing of possible dyads to 10 bp. At least one of these alters both the translational and rotational settings. We conclude that the translational position of a core particle is specified by sequence determinants additional to those specifying rotational positioning. The rotational settings on either side of the dyads of core particles assembled on the wild-type and a mutant sequence differ by +2 bp, corresponding to an overall helical periodicity of approximately 10.15 bp. The average helical periodicity of the central two to four turns is 10.5-11 bp whilst that of the flanking DNA is closer to 10 bp. The DNA immediately flanking the dyad is also characterised by a more extensive susceptibility to cleavage by hydroxyl radical.
Asunto(s)
Mutación/genética , Conformación de Ácido Nucleico , Nucleosomas/química , Nucleosomas/genética , Biosíntesis de Proteínas , Animales , Secuencia de Bases , Sitios de Unión , Pollos , ADN/química , ADN/genética , ADN/metabolismo , Huella de ADN , Eritrocitos , Exodesoxirribonucleasas/metabolismo , Histonas/metabolismo , Radical Hidroxilo/metabolismo , Ensayos de Protección de Nucleasas , Nucleosomas/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos/genética , Rotación , Termodinámica , Proteínas ViralesRESUMEN
Although the crystal structure of nucleosome core particle is essentially symmetrical in the vicinity of the dyad, the linker histone binds asymmetrically in this region to select a single high-affinity site from potentially two equivalent sites. To try to resolve this apparent paradox we mapped to base-pair resolution the dyads and rotational settings of nucleosome core particles reassembled on synthetic tandemly repeating 20 bp DNA sequences. In agreement with previous observations, we observed (1) that the helical repeat on each side of the dyad cluster is 10 bp maintaining register with the sequence repeat and (2) that this register changes by 2 bp in the vicinity of the dyad. The additional 2 bp required to effect the change in the rotational settings is accommodated by an adjustment immediately adjacent to the dyad. At the dyad the hydroxyl radical cleavage is asymmetric and we suggest that the inferred structural asymmetry could direct the binding of the linker histone to a single preferred site.
Asunto(s)
ADN/química , Nucleosomas/química , Nucleosomas/genética , Animales , Sitios de Unión , Histonas/química , Histonas/metabolismo , Unión Proteica , XenopusRESUMEN
In Saccharomyces cerevisiae, imbalance of the genes coding for the heterochromatin components Sir3p and histone H4 (namely, overdosage of SIR3 and lack of one of the two genes coding for H4) causes modifications in telomere length and telomere sequence organization, favoring the insertion of Y' elements into a stably shortened (C1-3A)n repeat tract. We report here that the newly inserted Y' elements are unstable and are lost with high frequency, generating clonal subpopulations with short telomeres, as revealed by the analysis of a specific telomere (LIII) and of the overall population of telomeres. Moreover, the growth rates of the subpopulations with and without Y' elements on LIII are different, the Y'-less individuals reproducing 20% more slowly than individuals bearing Y' elements. When grown together with Y'-bearing individuals, the subpopulations with the normal LIII telomere (which are viable and genetically stable if grown alone) are rapidly competed out. Hence, genetic imbalance for the structural components of heterochromatin results in a complex and rapidly changing mixture of subpopulations in such cultures. Thus, in situations where subpopulations are allowed to compete, heterochromatin-based differential growth rates result in neo-Darwinian clonal selection.
Asunto(s)
Saccharomyces cerevisiae/genética , Selección Genética , Telómero/genética , Genes Fúngicos , Heterocromatina/genética , Modelos Genéticos , Mutación , Fenotipo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Glucose depletion derepresses the Saccharomyces cerevisiae ADH2 gene; this metabolic change is accompanied by chromatin structural modifications in the promoter region. We show that the ADR6/SWI1 gene is not necessary for derepression of the wild type chromosomal ADH2, whereas the transcription factor Adr1p, which regulates several S. cerevisiae functions, plays a major role in driving nucleosome reconfiguration and ADH2 expression. When we tested the effect of individual domains of the regulatory protein Adr1p on the chromatin structure of ADH2, a remodeling consisting of at least two steps was observed. Adr1p derivatives were analyzed in derepressing conditions, showing that the Adr1p DNA binding domain alone causes an alteration in chromatin organization in the absence of transcription. This alteration differs from the remodeling observed in the presence of the Adr1p activation domain when the promoter is transcriptionally active.
Asunto(s)
Alcohol Deshidrogenasa/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Represión Enzimática , Regulación Fúngica de la Expresión Génica , Modelos Genéticos , Unión Proteica , Saccharomyces cerevisiae/enzimología , TATA Box , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Telomeric heterochromatin plays an essential role in telomere function, including the regulation of telomere length. We observe that in Saccharomyces cerevisiae an imbalance in the dosage of genes for two protein components of heterochromatin (namely Sir3p and histone H4) causes modifications in telomere length and telomere sequence organization. The effects of Sir3p/H4 imbalance were analyzed in yeast strains in which the wild-type SIR3 gene (normally a single-copy gene) was either absent or present in 20-30 copies, and both histone H4 genes (HHF1 and HHF2) were present or HHF1 was deleted, thus covering a wide range of viable gene-dosage combinations. Modifications of telomeres and of subtelomeric regions were identified by analyzing both the overall telomere population and by focusing on two single telomeric regions: the left telomere of chromosome III (LIII) and the right telomere of chromosome XI (RXI). The modifications induced by alteration of the Sir3p/H4 ratio consist of a reduction in the length and an increase in the instability of the terminal block of (C(1-3)A)n repeats and in susceptibility to insertion of Y' elements into this repeat element. Restoration of the wild-type gene ratio (by removal of the extra copies of SIR3 or by complementation with the missing second copy of HHF) restored the original telomere organization, both with respect to the length of the (C(1-3)A)n repeat stretch and the absence of Y' elements. This behavior shows that the stability of the wild-type sequence organization requires maintenance of the normal structure of telomeric heterochromatin.
Asunto(s)
Cromosomas Fúngicos , Proteínas Fúngicas/genética , Dosificación de Gen , Heterocromatina , Histonas/genética , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Telómero , Transactivadores/genética , Regulación Fúngica de la Expresión Génica , FenotipoRESUMEN
The chromatin organization of eukaryotic telomeres is essential for telomeric function and is currently receiving great attention. In yeast, the structural organization of telomeres involves a complex interplay of telomeric proteins that results in the formation of heterochromatin. This telomeric heterochromatin involves homotypic and heterotypic protein interactions that have been summarized in a general model. Recent analyses have focused on the study of the structural complexity at yeast telomeres to the level of specific nucleosomes and of the distribution of protein complexes in a natural telomeric region (LIII). In this report, we further analyze the structural complexity of LIII and the implication of this structure on telomeric silencing. It is shown that the establishment of repressive heterochromatin structures at LIII requires the recruitment of Sir3p through interaction with the N terminus of histone H4. The establishment of such structures does not require acetylation of any of four lysines located in the H4 N terminus (lysines 5, 8, 12, and 16).
Asunto(s)
Proteínas Fúngicas/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Telómero/metabolismo , Transactivadores/metabolismo , Acetilación , Heterocromatina/química , Heterocromatina/genética , Mutación , Conformación Proteica , Saccharomyces cerevisiae/metabolismoRESUMEN
We have determined the sites that are preferentially cleaved by Mn(T4MPyP) (where T4MPyP is the dianion of 5, 10, 15, 20, tetrakis (4-N-methylpyridine)porphyrin) on synthetic DNAs and on both intrinsically curved and average-shaped natural DNA sequences. On the basis of cleavage selectivity and of DNase I footprinting we show that the recognition specificity by this compound is based on steric properties: the preferred conformation is a DNA minor groove narrower than average and dimensionally defined. This conclusion is reached on the basis of: (i) the localization of the preferential cleavage sites at the 3' extremity of short A-tracts, known to undergo minor groove directional narrowing; (ii) the effects of temperature on cleavage specificity on curved sequences; (iii) the localization of cleavage sites in synthetic constructs whose crystal and solution structure was previously defined, and in programmed sequence variants thereoff; (iv) the effects of base substitutions on cleavage efficiency; (v) DNase I footprinting analysis. Several of these evidences argue against the possibility that Mn(T4MPyP)/DNA site selection occurs on the basis of electrostatic potential effects.Mn(T4MPyP) provides a tool for the analysis of DNA conformation whose selectivity is complementary to that of DNase I and hydroxyl radicals.
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ADN/química , Manganeso/química , Conformación de Ácido Nucleico , Compuestos Organometálicos/química , Porfirinas/química , Animales , Técnicas Biosensibles , Crithidia fasciculata , Huella de ADN , ADN de Cinetoplasto/química , Desoxirribonucleasa I/metabolismo , Oligodesoxirribonucleótidos/químicaRESUMEN
The effects of the rotational information of DNA in determining the in vivo localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5 S rRNA repeat gene. The distribution of the phased series of flexibility signals present in this DNA has been altered by inserting in its centre a 25 bp tract. The effects of such alteration on the in vivo distribution of the helically phased, alternatively located ncps have been determined relative to a reference 21 bp insertion mutant. The results show that the answers provided in vitro and in vivo by the yeast 5 S rRNA gene sequence to specific modifications of the DNA rotational frame are similar, thus pointing to the relevance of DNA rotational information in vivo.
Asunto(s)
Cromosomas Fúngicos , ADN de Hongos/ultraestructura , Conformación de Ácido Nucleico , Nucleosomas/ultraestructura , Saccharomyces cerevisiae/genética , Enzimas de Restricción del ADN/genética , Elementos Transponibles de ADN , ADN de Hongos/química , ADN de Hongos/genética , Nucleosomas/genética , Plásmidos , ARN Ribosómico 5S/genéticaRESUMEN
We have defined the in vivo heterochromatin structure of the left telomere of Saccharomyces cerevisiae chromosome III (LIII). Analysis of heterochromatin of a single telomere was so far lacking, due to the difficulties intrinsic to the highly repetitive nature of telomeric sequences. In LIII, the terminal (C1-3A)n repetitive sequences are followed by a complete X element and by the single copy Ty5-1 retrotransposon. Both the telosome and the X element exhibit overall resistance to micrococcal nuclease digestion reflecting their tight chromatin structure organization. The X element contains protein complexes and irregularly distributed but well localized nucleosomes. In contrast, a regular array of phased nucleosomes is associated with the promoter region of Ty5-1 and with the more centromere-proximal sequences. The lack of a structural component of yeast telomeres, the SIR3 protein, does not alter the overall tight organization of the X element but causes a nucleosome rearrangement within the promoter region of Ty5-1 and releases Ty5-1 silencing. Thus, Sir3p links the modification of the heterochromatin structure with loss of transcriptional silencing.
Asunto(s)
Proteínas Fúngicas/genética , Heterocromatina/genética , Regiones Promotoras Genéticas , Retroelementos , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Telómero/genética , Transactivadores/genética , Cromosomas Fúngicos/química , ADN de Hongos/química , Heterocromatina/química , Nucleasa Microcócica , Nucleosomas/genética , Secuencias Repetitivas de Ácidos Nucleicos , Telómero/químicaRESUMEN
Induction of transcription in eukaryotic promoters is accompanied by removal or remodeling of nucleosomes. Given that this process causes release of torsional stress, the question is asked relative to its fate and to its effects on local DNA conformation. Is it dispersed by free rotation through surrounding nucleosomes or does it stay locally to be used in the modulation or activation of the transcription machinery? The results of the calculations relative to the onset of writhing suggest that the free energy made available by removal of nucleosomes is in the range of values that corresponds to the transition linking difference, thus pointing to a possible regulatory mechanism for the local use of free energy in promoters.
Asunto(s)
ADN/química , Nucleosomas/química , Animales , Fenómenos Biomecánicos , Electroquímica , Conformación de Ácido Nucleico , Termodinámica , XenopusRESUMEN
We describe the reaction of formamide with 2'-deoxycytidine to give pyrimidine ring opening by nucleophilic addition on the electrophilic C(6) and C(4) positions. This information is confirmed by the analysis of the products of formamide attack on 2'-deoxycytidine, 5-methyl-2'-deoxycytidine, and 5-bromo-2'-deoxycytidine, residues when the latter are incorporated into oligonucleotides by DNA polymerase-driven polymerization and solid-phase phosphoramidite procedure. The increased sensitivity of 5-bromo-2'-deoxycytidine relative to that of 2'-deoxycytidine is pivotal for the improvement of the one-lane chemical DNA sequencing procedure based on the base-selective reaction of formamide with DNA. In many DNA sequencing cases it will in fact be possible to incorporate this base analogue into the DNA to be sequenced, thus providing a complete discrimination between its UV absorption signal and that of the thymidine residues. The wide spectrum of different sensitivities to formamide displayed by the 2'-deoxycytidine analogues solves, in the DNA single-lane chemical sequencing procedure, the possible source of errors due to low discrimination between C and T residues.
Asunto(s)
Desoxicitidina/química , Desoxicitidina/metabolismo , Formamidas , Análisis de Secuencia de ADN/métodos , Bromodesoxicitidina/química , Bromodesoxicitidina/metabolismo , Desoxicitidina/análogos & derivados , Sensibilidad y Especificidad , Timidina/química , Timidina/metabolismoRESUMEN
We have determined the chromatin organization of the Saccharomyces cerevisiae DNA topoisomerase I promoter. Three nucleosomal core particles have been mapped at nucleotide level over the promoter region, encompassing the presumptive TATA sequence and the two RNA initiation sites; the most upstream nucleosome particle forms on to a 29 bp-long poly(dA-dT) element. This simple organization remains constant throughout both the logarithmic and the linear phase of growth, with the exception of an increased accessibility to micrococcal nuclease of the nucleosome covering the TATA box and the RNA initiation sites during the diauxic shift (the switching from the fermentative to the respiratory metabolism) in parallel with an increase of the DNA topoisomerase I mRNA. In addition, a strong disorganization of the bulk chromatin structure in the late stationary phase is also reported.
Asunto(s)
Cromatina/genética , ADN-Topoisomerasas de Tipo I/genética , Regulación Enzimológica de la Expresión Génica , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Nucleosomas , ARN de Hongos/análisis , ARN Mensajero/análisis , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The chromatin structure of the Saccharomyces cerevisiae ADH2 gene is modified during the switch from repressing (high glucose) to derepressing (low glucose) conditions of growth. Loss of protection toward micrococcal nuclease cleavage for the nucleosomes covering the TATA box and the RNA initiation sites (-1 and +1, respectively) is the major modification taking place and is strictly dependent on the presence of the transcriptional activator ADR1. To identify separate functions involved in the transition from a repressed to a transcribing promoter, we have analyzed the ADH2 chromatin organization in various genetic backgrounds. Deletion of the CCR4 gene coding for a general transcription factor impaired ADH2 expression without affecting chromatin remodeling. Growing yeast at 37 degrees C also resulted in chromatin remodeling at the ADH2 locus even under glucose repressing conditions. However, although this temperature-induced remodeling was dependent on the ADR1 protein, no ADH2 mRNA was observed. In addition, inactivating RNA polymerase II (and therefore, elongation) was found to have no effect on the ability to reconfigure nucleosomes. Taken together, these data indicate that chromatin remodeling by itself is insufficient to induce transcription at the ADH2 promoter.