RESUMO
The ß decays of neutron-rich nuclei near the doubly magic (78)Ni were studied at the Holifield Radioactive Ion Beam Facility using an electromagnetic isobar separator. The half-lives of (82)Zn (228±10 ms), (83)Zn (117±20 ms), and (85)Ga (93±7 ms) were determined for the first time. These half-lives were found to be very different from the predictions of the global model used in astrophysical simulations. A new calculation was developed using the density functional model, which properly reproduced the new experimental values. The robustness of the new model in the (78)Ni region allowed us to extrapolate data for more neutron-rich isotopes. The revised analysis of the rapid neutron capture process in low entropy environments with our new set of measured and calculated half-lives shows a significant redistribution of predicted isobaric abundances strengthening the yield of A>140 nuclei.
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Establishing how and when large N/Z values require modified or new theoretical tools is a major quest in nuclear physics. Here we report the first measurement of the lifetime of the 2(1)+ state in the near-dripline nucleus 20C. The deduced value of τ(2(1)+)=9.8±2.8(stat)(-1.1)(+0.5)(syst) ps gives a reduced transition probability of B(E2; 2(1)+â0(g.s.)+)=7.5(-1.7)(+3.0)(stat)(-0.4)(+1.0)(syst) e2 fm4 in good agreement with a shell model calculation using isospin-dependent effective charges.
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By studying the (109)Xeâ(105)Teâ(101)Sn superallowed α-decay chain, we observe low-lying states in (101)Sn, the one-neutron system outside doubly magic (100)Sn. We find that the spins of the ground state (J=7/2) and first excited state (J=5/2) in (101)Sn are reversed with respect to the traditional level ordering postulated for (103)Sn and the heavier tin isotopes. Through simple arguments and state-of-the-art shell-model calculations we explain this unexpected switch in terms of a transition from the single-particle regime to the collective mode in which orbital-dependent pairing correlations dominate.
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Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.
Assuntos
Códon sem Sentido/genética , Éxons/genética , Genes BRCA1/genética , Mutação de Sentido Incorreto/genética , Splicing de RNA/genética , Motivos de Aminoácidos , Substituição de Aminoácidos/genética , Proteína BRCA1/química , Proteína BRCA1/genética , Sequência de Bases , Elementos Facilitadores Genéticos/genética , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Fases de Leitura Aberta/genética , Fenótipo , Fosfoproteínas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Fatores de Processamento de Serina-ArgininaRESUMO
Exonic splicing enhancers (ESEs) are important cis elements required for exon inclusion. Using an in vitro functional selection and amplification procedure, we have identified a novel ESE motif recognized by the human SR protein SC35 under splicing conditions. The selected sequences are functional and specific: they promote splicing in nuclear extract or in S100 extract complemented by SC35 but not by SF2/ASF. They can also function in a different exonic context from the one used for the selection procedure. The selected sequences share one or two close matches to a short and highly degenerate octamer consensus, GRYYcSYR. A score matrix was generated from the selected sequences according to the nucleotide frequency at each position of their best match to the consensus motif. The SC35 score matrix, along with our previously reported SF2/ASF score matrix, was used to search the sequences of two well-characterized splicing substrates derived from the mouse immunoglobulin M (IgM) and human immunodeficiency virus tat genes. Multiple SC35 high-score motifs, but only two widely separated SF2/ASF motifs, were found in the IgM C4 exon, which can be spliced in S100 extract complemented by SC35. In contrast, multiple high-score motifs for both SF2/ASF and SC35 were found in a variant of the Tat T3 exon (lacking an SC35-specific silencer) whose splicing can be complemented by either SF2/ASF or SC35. The motif score matrix can help locate SC35-specific enhancers in natural exon sequences.
Assuntos
Elementos Facilitadores Genéticos , Éxons , Proteínas Nucleares/metabolismo , Splicing de RNA , Ribonucleoproteínas , Animais , Sequência de Bases , Sítios de Ligação , Núcleo Celular/metabolismo , Sistema Livre de Células , Sequência Consenso , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA , Fatores de Processamento de Serina-ArgininaRESUMO
A two-hybrid screening in yeast for proteins interacting with the human hnRNP A1, yielded a nuclear protein of 917 amino acids that we termed hnRNP A1 associated protein (HAP). HAP contains an RNA binding domain (RBD) flanked by a negatively charged domain and by an S/K-R/E-rich region. In in vitro pull-down assays, HAP interacts with hnRNP A1, through its S/K-R/E-rich region, and with several other hnRNPs. HAP was found to be identical to the previously described Scaffold Attachment Factor B (SAF-B) and to HET, a transcriptional regulator of the Heat Shock Protein 27 gene. We show that HAP is a bona fide hnRNP protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm the complete set of hnRNP proteins. Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly modified in heat-shocked HeLa cells. Heat-shock treatment at 42 degrees C causes a transcription-dependent recruitment of HAP to a few large nuclear granules that exactly coincide with sites of accumulation of Heat Shock Factor 1 (HSF1). The recruitment of HAP to the granules is temporally delayed with respect to HSF1 and persists for a longer time during recovery at 37 degrees C. The hnRNP complexes immunoprecipitated from nucleoplasm of heat-shocked cells with anti-HAP antibodies have an altered protein composition with respect to canonical complexes. Altogether our results suggest an involvement of HAP in the cellular response to heat shock, possibly at the RNA metabolism level.
Assuntos
Núcleo Celular/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Ciclo Celular , Imunofluorescência , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Cinética , Modelos Genéticos , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência de Aminoácidos , Temperatura , Fatores de Tempo , Transcrição GênicaRESUMO
Emerin, the protein whose production is altered in the X-linked form of Emery-Dreifuss muscular distrophy, has been hypothesized to be associated with the nuclear matrix on the basis of biochemical studies. In addition, immunocytochemical data reported its localization at the nuclear periphery, on the nuclear lamina, in sections of several normal tissues. We investigated the association of emerin with the nuclear matrix, by using cultured cells (SaOS-2, MG63 and HeLa-S3) and their in situ extracted matrix as a model, and immunocytochemical methods, both at the light and electron microscope level. Our results show a normal presence of emerin in the cultured cells and the specific persistence of emerin on the lamina of the in situ extracted nuclear matrix. This suggests a tight binding between emerin and the nuclear lamina independently from the interactions between the C-terminal hydrophobic domain of the protein and the inner nuclear membrane.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Distrofias Musculares/metabolismo , Timopoietinas/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Técnica Direta de Fluorescência para Anticorpo , Células HeLa , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Microscopia Eletrônica , Distrofias Musculares/genética , Distrofias Musculares/patologia , Proteínas Nucleares , Timopoietinas/genéticaRESUMO
Emery-Dreifuss muscular dystrophy (EMD) is an inherited myopathy characterised by muscle contractures, progressive muscle wasting and weakness, with humeroperoneal distribution. Cardiac arrhythmia and heart conduction block are also important characteristics of this disease. The X-linked form of EMD is caused by the absence of emerin, encoded by the STA gene (Xq28). Emerin is normally localized in muscle and other tissues at the nuclear rim. Currently, muscle and skin biopsies are used for the immunohistochemical diagnosis. We demonstrate that emerin is present in the cheek oral mucosa, in the exfoliating epithelial cells, and we propose the collection of these cells as a new method for the diagnosis of X-linked EMD patients and the detection of carriers by immunofluorescence techniques: smears from healthy subjects contained about 98% emerin-positive cells, those from X-linked EMD patients contained none and those from carriers contained about 45%. The technique is completely non-invasive, simple, repeatable and inexpensive.
Assuntos
Triagem de Portadores Genéticos , Ligação Genética , Mucosa Bucal/patologia , Distrofias Musculares/genética , Cromossomo X , Adolescente , Adulto , Estudos de Casos e Controles , Bochecha , Criança , Citodiagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Distrofias Musculares/patologia , Distrofia Muscular de Emery-DreifussRESUMO
We have raised an anti-emerin polyclonal antibody against a fusion protein encompassing most of the hydrophilic portion of emerin. Using this antibody, we have analyzed emerin expression in Emery-Dreifuss muscular dystrophy (EDMD) patients and controls, by immunocytochemistry, in skeletal muscle and skin, and by immunoblot, in peripheral blood mononuclear cells and lymphoblasts. Emerin was localized on the surfaces of nuclei in control skeletal muscle and skin but was absent or reduced in patient skeletal muscle, was absent from the skin of patients, and was expressed only in a few nuclei in a patient's mother. Immunoblot of peripheral blood cells from EDMD patients showed absence of the emerin band, altered-size emerin, or a protein of normal molecular mass but slightly reduced quantity. The diagnosis of X-linked EDMD is normally confirmed by genetic analysis of the STA gene coding for emerin. We propose immunocytochemical evaluation of emerin expression in skin biopsies as a sensitive and more convenient tool for diagnosing X-linked EDMD and, in particular, for distinguishing it from the autosomal dominant form. This technique may be applied to suspected EDMD patients, especially sporadic cases or those with incomplete clinical phenotype, and also suspected carriers. Immunoblot of peripheral blood cells is also useful, but it may not unequivocally identify carriers and some patients.
Assuntos
Leucócitos Mononucleares/metabolismo , Linfócitos/metabolismo , Proteínas de Membrana/análise , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Pele/patologia , Timopoietinas/análise , Cromossomo X , Adolescente , Adulto , Biomarcadores , Biópsia , Pré-Escolar , Feminino , Humanos , Imuno-Histoquímica , Laminas , Leucócitos Mononucleares/patologia , Linfócitos/patologia , Masculino , Proteínas de Membrana/biossíntese , Músculo Esquelético/metabolismo , Distrofias Musculares/sangue , Distrofias Musculares/patologia , Distrofia Muscular de Emery-Dreifuss , Proteínas Nucleares/análise , Valores de Referência , Pele/citologia , Pele/metabolismo , Timopoietinas/biossínteseRESUMO
Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked inherited disease characterized by early contracture of the elbows, Achilles tendons and post-cervical muscles, slow progressive muscle wasting and weakness and cardiomyopathy presenting with arrhythmia and atrial paralysis: heart block can eventually lead to sudden death. The EDMD geneencodes a novel ubiquitous protein, emerin, which decorates the nuclear rim of many cell types. Amino acid sequence homology and cellular localization suggested that emerin is a member of the nuclear lamina-associated protein family. These findings did not explain the role of emerin nor account for the skeletal muscle- and heart-specific clinical manifestations associated with the disorder. Now we report that emerin localizes to the inner nuclear membrane, via its hydrophobic C-terminal domain, but that in heart and cultured cardiomyocytes it is also associated with the intercalated discs. We propose a general role for emerin in membrane anchorage to the cytoskeleton. In the nuclear envelope emerin plays a ubiquitous and dispensable role in association of the nuclear membrane with the lamina. In heart its specific localization to desmosomes and fasciae adherentes could account for the characteristic conduction defects described in patients.
Assuntos
Desmossomos/química , Proteínas de Membrana/análise , Proteínas Musculares/análise , Distrofias Musculares/genética , Miocárdio/química , Membrana Nuclear/química , Timopoietinas/análise , Arritmias Cardíacas/etiologia , Adesão Celular , Proteínas do Citoesqueleto/análise , Citoesqueleto/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Microscopia Imunoeletrônica , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Distrofias Musculares/complicações , Distrofias Musculares/metabolismo , Distrofias Musculares/fisiopatologia , Distrofia Muscular de Emery-Dreifuss , Miocárdio/ultraestrutura , Proteínas Nucleares , Fosforilação , Processamento de Proteína Pós-Traducional , Timopoietinas/genética , Timopoietinas/fisiologia , Cromossomo XRESUMO
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant nuclear polypeptides, most likely involved in different steps of pre-mRNA processing. Protein A1 (34 kDa), a prominent member of the hnRNP family, seems to act by modulating the RNA secondary structure and by antagonizing some splicing factors (SR proteins) in splice-site selection and exon skipping/inclusion. A role of A1 in the nucleo-cytoplasmic transport of RNA has also been proposed. These activities might depend not only on the RNA-binding properties of the protein but also on specific protein-protein interactions. Here we report that A1 can indeed selectively interact, in vitro, both with itself and with other hnRNP basic "core" proteins. Such selective binding is mediated exclusively by the Gly-rich C-terminal domain, where a novel protein-binding motif constituted by hydrophobic repeats can be envisaged. The same domain is necessary and sufficient to promote specific interaction in vivo, as assayed by the yeast two-hybrid assay. Moreover, an in vitro interaction with some SR proteins was also observed. These observations suggest that diverse and specific protein-protein interactions might contribute to the different functions of the hnRNP A1 protein in mRNA maturation.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Sequência de Bases , Sítios de Ligação , Glicina/química , Glicina/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas , Modelos Moleculares , Dados de Sequência Molecular , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Fenóis/química , Conformação Proteica , Splicing de RNA , Proteínas de Ligação a RNA/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/química , Serina/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Leveduras/metabolismoRESUMO
hnRNP protein A1 (34 kDa, pl 9.5) is a prominent member of the family of proteins (hnRNP proteins) that associate with the nascent transcripts of RNA polymerase II and that accompany the hnRNA through the maturation process and the export to the cytoplasm. New evidence suggests an active and specific role for some of these proteins, including protein A1, in splicing and transport. Contrary to the other hnRNP proteins, the intracellular level of protein A1 was reported to change as a function of proliferation state and cell type. In this work we analyse the A1 gene expression in different cells under different growth and differentiation conditions. Proliferation dependent expression was observed in lymphocytes and fibroblasts while purified neurons express high A1 mRNA levels both in the proliferative (before birth) and in the quiescent (after birth) state. Transformed cell lines exhibit very high (proliferation independent) A1 mRNA levels compared to differentiated tissues. A structural and functional characterization of the A1 gene promoter was carried out by means of DNase I footprinting and CAT assays. The observed promoter features can account for both elevated and regulated mRNA transcription. At least 12 control elements are contained in the 734 nucleotides upstream of the transcription start site. Assays with the deleted and/or mutated promoter indicate a co-operation of multiple transcriptional elements, distributed over the entire promoter, in determining the overall activity and the response to proliferative stimuli (serum).