RESUMEN
The lifetimes of the first excited 2^{+}, 4^{+}, and 6^{+} states in ^{98}Zr were measured with the recoil-distance Doppler shift method in an experiment performed at GANIL. Excited states in ^{98}Zr were populated using the fission reaction between a 6.2 MeV/u ^{238}U beam and a ^{9}Be target. The γ rays were detected with the EXOGAM array in correlation with the fission fragments identified by mass and atomic number in the VAMOS++ spectrometer. Our result shows a very small B(E2;2_{1}^{+}â0_{1}^{+}) value in ^{98}Zr, thereby confirming the very sudden onset of collectivity at N=60. The experimental results are compared to large-scale Monte Carlo shell model and beyond-mean-field calculations. The present results indicate the coexistence of two additional deformed shapes in this nucleus along with the spherical ground state.
RESUMEN
Both transcription factors albumin site d-binding protein (DBP) and thyrotroph embryonic factor (TEF) are elements of the "cell-clock". Their circadian accumulation in suprachiasmatic nucleus (SCN) and peripheral tissues such as liver, kidney and lung is thought to participate in controlling circadian regulation of downstream genes. TEF and DBP control elements have never been investigated in the insulin-secreting cells, but impairment of the circadian rhythm of the beta-cells might be involved in the development of diabetic state as type 2 diabetics have lost daily temporal variations of insulin secretion. We investigated the expression pattern of TEF and DBP in insulin-secreting cells. TEF and DBP transcripts are expressed at extremely high levels in human pancreatic islets compared to other tissues, suggesting a potentially important circadian regulation of these cells. Both TEF and DPB accumulate in a circadian way in insulin-secreting cells after a serum shock known to restore circadian rhythms in cultured cells. In addition, the expression of islet-specific genes involved in glucose sensing (glucose transporter 2 (Glut2), glucokinase), insulin production (insulin) and secretion (migration inhibitory factor (MIF), somatostatin and syntaxin 1A) were modulated in the same daily rhythm as well. The circadian deregulation of these genes could therefore participate in the diabetic state development.
Asunto(s)
Ritmo Circadiano , Proteínas de Unión al ADN/metabolismo , Insulina/biosíntesis , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Factores de Transcripción/metabolismo , Antígenos de Superficie/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Regulación de la Expresión Génica , Glucoquinasa/metabolismo , Transportador de Glucosa de Tipo 2 , Humanos , Secreción de Insulina , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas , Somatostatina/metabolismo , Núcleo Supraquiasmático/metabolismo , Sintaxina 1RESUMEN
X chromosome inactivation in mammals requires the Xist gene, which is exclusively expressed from the inactive X chromosome (Xi). The large heterogeneous Xist nuclear RNA colocalizes with Xi, most likely through nuclear protein interactions. The 5' region of the Xist RNA contains a series of well-conserved tandem repeats known to bind heteronuclear proteins in vitro and to enhance human XIST transcription. We show in an in vitro system that the conserved repeat element located in the 5' region of the mouse Xist gene (Xcr) represses three X-linked genes but has no effect on the autosomal genes Aprt, Ins, and the viral SV40 gene. The repression effect is not mediated by the conserved core sequence (Ccs) of Xcr, but requires the presence of the complete Xcr. This Xcr effect on X-linked genes suggests that Xcr transcript recognizes the genes to be silenced and is involved in the spreading of X inactivation.
Asunto(s)
Ligamiento Genético , ARN no Traducido/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Cromosoma X , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Cartilla de ADN , Glucosafosfato Deshidrogenasa/genética , Hipoxantina Fosforribosiltransferasa/genética , Ratones , Fosfoglicerato Quinasa/genética , ARN Largo no Codificante , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The mouse Xist gene is expressed exclusively from the inactive X chromosome and is involved in the initiation of X inactivation. We previously reported that the -1157/+917 region of the Xist promoter was ubiquitously functional in mammalian cells and that experiments in a transient expression system revealed no trans-acting element responsible for the inactive X specific expression of Xist. In somatic tissues, the 5' end of the silent Xist allele on the active X is known to be fully methylated whereas the expressed allele on the inactive X is unmethylated. In the present study we have used a bisulphite genomic sequencing method to evaluate DNA methylation at all cytosines including CpG dinucleotides within the Xist promoter. We report and confirm that methylation of specific sites plays a key role in Xist gene expression. In vitro DNA methylation of the 5'-region drastically reduced transcriptional activity in transiently transfected fibroblasts. Mobility shift assays showed that methylation does not inhibit Xist promoter activity by preventing the binding of transcription factors and that two distinct nuclear proteins bind in a sequence methyl-CpG-specific manner. Therefore, we suggest that Xist repression involves its promoter methylation and two distinct methylated DNA binding proteins.
Asunto(s)
Islas de CpG/genética , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas/genética , ARN no Traducido , Factores de Transcripción/genética , Animales , Azacitidina/farmacología , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa , Citosina , Metilación de ADN/efectos de los fármacos , Femenino , Regulación de la Expresión Génica , Masculino , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/metabolismo , ARN Largo no Codificante , Análisis de Secuencia de ADN , Timina , Cromosoma XRESUMEN
The mouse Xist gene is expressed exclusively from the inactive X chromosome and may be implicated in initiating X inactivation. To better understand the mechanisms underlying the control of Xist expression, we investigated the upstream regulatory region of the mouse Xist promoter. A 1.2-kb upstream region of the Xist gene was sequenced and promoter activity was studied by chloramphenicol acetyltransferase (CAT) assays after transfection in murine XX and XY cell lines. The region analyzed (-1157 to +917 showed no in vitro sex-specific promoter activity. However, a minimal constitutional promoter was assigned to a region from -81 to +1, and a cis element from -41 to -15 regulates promoter activity. We showed that a nuclear factor binds to an element located at -30 to -25 (TTAAAG). A second sequence at -41 to -15 does not act as an enhancer and is unable to confer transcriptional activity to the Xist gene on its own. A third region from -82 to -41 is needed for correct expression. Deletion of the segment -441 to -231 is associated with an increase in CAT activity and may represent a silencer element.
Asunto(s)
Aberraciones Cromosómicas , Trastornos de los Cromosomas , Ratones/genética , Regiones Promotoras Genéticas , ARN no Traducido , Factores de Transcripción/genética , Cromosoma X , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/biosíntesis , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos , Datos de Secuencia Molecular , ARN Largo no Codificante , Proteínas Recombinantes/biosíntesis , Caracteres Sexuales , TransfecciónRESUMEN
Acyclic nucleoside analogues with carboxamido- or nitro-substituted heterocyclic bases have been evaluated for their possible use as universal bases in oligodeoxynucleotides. The acyclic moiety endows the constructs with enough flexibility to allow good base stacking. The 5-nitroindazole analogue afforded the most stable duplexes among the acyclic derivatives with the least spread in Tm versus the four natural bases. In spite of the acyclic moiety, stabilities are comparable with those of duplexes incorporating the recently described 5-nitroindole nucleoside analogue, but considerably exceed those for the 3-nitropyrrole analogue.
Asunto(s)
Sondas de ADN/química , Indazoles/química , Conformación de Ácido Nucleico , Nucleósidos/química , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Datos de Secuencia Molecular , Desnaturalización de Ácido NucleicoRESUMEN
The direct approach in molecular diagnosis proposes evidences of the mutations underlying the investigated diseases. Due to its speed, specificity and low cost, the Polymerase Chain Reaction (PCR) has become the method of choice in most of these analyses. The direct approach can be either positive or negative. In the first case, the diagnosis is based on the presence of a pathognomonic PCR product, while in the latter, it is the absence of such a product that makes the diagnosis possible. It is evident that both methods have to be validated by several control reactions. Examples out of daily practice illustrate various diagnostic areas.
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Reacción en Cadena de la Polimerasa , Secuencia de Bases , Fibrosis Quística/genética , Ácido Graso Desaturasas/deficiencia , Femenino , Enfermedades Genéticas Congénitas/diagnóstico , Humanos , Enfermedad de Huntington/genética , Errores Innatos del Metabolismo Lipídico/genética , Masculino , Datos de Secuencia Molecular , Distrofias Musculares/genéticaRESUMEN
The goal of indirect molecular diagnostic techniques is the detection of a link between a specific allele of a polymorphic genomic marker and a given disease. This technique is used in two specific clinical situations: 1) when the gene is unknown and the search for a mutation or a gene product is impossible 2) when the gene is known but large and several mutations might be present. If none of the known mutations prevails in the local population the systematic and sequential search for every single mutation is not economical. A linkage study is required in those instances. The indirect analysis was up to the nineties based on markers detecting DNA-restriction fragments of various length. By the amplification of microsatellites by PCR the indirect approach has brought enormous progress. Together with the mapping by the Human Genome Project it will progress further. Although indirect molecular methodology can often avoid extended work it is only applicable to families with a member already affected by the disease. DNA from this individual is needed for the detection of a link of one allele to the disease. This means necessarily that the disease must be inherited and that the diagnosis is certain. Presymptomatic molecular diagnosis is illustrated by the analysis of a pedigree with familial adenomatous polyposis by microsatellite PCR techniques.