RESUMEN
Huntington's disease (HD) is a progressive neurological disorder caused by a CAG/polyglutamine repeat expansion. We have previously generated the R6/2 mouse model that expresses exon 1 of the human HD gene containing CAG repeats in excess of 150. These mice develop a progressive neurological phenotype with a rapid onset and progression. We show here that it is impossible to establish fibroblast lines from these mice at 12 weeks of age, whilst this can be achieved without difficulty at 6 and 9 weeks. Cultures derived from mice at 12 weeks contained a high frequency of dysmorphic cells, including cells with an aberrant nuclear morphology and a high frequency of micronuclei and large vacuoles. All of these features were also present in a line derived from a juvenile HD patient. Fibroblast lines derived from R6/2 mice and from HD patients were found to have a high frequency of multiple centrosomes which could account for all of the observed phenotypes including a reduced mitotic index, high frequency of aneuploidy and persistence of the midbody. We were unable to detect large insoluble polyglutamine aggregates in either the mouse or human lines. We have identified a novel progressive HD pathology that occurs in cells of non-central nervous system origin. An investigation of the pathological consequences of the HD mutation in these cells will provide insight into cellular basis of the disease.
Asunto(s)
Centrosoma/metabolismo , Fibroblastos/metabolismo , Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Aneuploidia , Animales , Western Blotting , Encéfalo/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Senescencia Celular/genética , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Replicación del ADN/genética , Endocitosis , Endosomas/metabolismo , Femenino , Fibroblastos/citología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/patología , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Microscopía Fluorescente , Índice Mitótico , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Expansión de Repetición de Trinucleótido/genética , Repeticiones de Trinucleótidos/genéticaRESUMEN
Huntington's disease (HD) is one of a class of inherited progressive neurodegenerative disorders that are caused by a CAG/polyglutamine repeat expansion. We have previously generated mice that are transgenic for exon 1 of the HD gene carrying highly expanded CAG repeats which develop a progressive movement disorder and weight loss with similarities to HD. Neuronal inclusions composed of the exon 1 protein and ubiquitin are present in specific brain regions prior to onset of the phenotype, which in turn occurs long before specific neurodegeneration can be detected. In this report we have extended the search for polyglutamine inclusions to non-neuronal tissues. Outside the central nervous system (CNS), inclusions were identified in a variety of post-mitotic cells. This is consistent with a concentration-dependent nucleation and aggregation model of inclusion formation and indicates that brain-specific factors are not necessary for this process. To possibly gain insights into the wasting that is observed in the human disease, we have conducted a detailed analysis of the timing and progression of inclusion formation in skeletal muscle and an investigation into the cause of the severe muscle atrophy that occurs in the mouse model. The formation of inclusions in non-CNS tissues will be particularly useful with respect to in vivo monitoring of pharmaceutical agents selected for their ability to prevent polyglutamine aggregation in vitro, without the requirement that the agent can cross the blood-brain barrier in the first instance.
Asunto(s)
Enfermedad de Huntington/genética , Músculo Esquelético/ultraestructura , Atrofia Muscular/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Péptidos/genética , Animales , Núcleo Celular/genética , Núcleo Celular/ultraestructura , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiopatología , Modelos Animales de Enfermedad , Humanos , Proteína Huntingtina , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Atrofia Muscular/fisiopatología , Neuronas/patologíaRESUMEN
HAP1 (huntingtin associated protein) has previously been found to interact with huntingtin (htt) in a glutamine length dependent manner and has been proposed to play a role in the cell specific neurodegeneration observed in Huntington's disease (HD). We have isolated mouse HAP1 (hap1) and have shown that expression is not enriched in areas specifically affected in HD. We have used the yeast two hybrid system to demonstrate that htt amino acids 171-230 are necessary for the hap1-htt binding and that hapl does not interact with the transgene exon 1 protein in a transgenic model of HD.
Asunto(s)
Enfermedad de Huntington/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Expresión Génica , Humanos , Proteína Huntingtina , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Unión Proteica , Ratas , Relación Estructura-ActividadRESUMEN
The puffer fish ( Fugu rubripes ) has a compact genome of 400 Mbp which is approximately 7.5-fold smaller than the human genome. It contains a similar number of genes but is deficient in intergenic, intronic and dispersed repetitive sequences. Fugu is becoming established as the model vertebrate genome for the identification and characterisation of novel human genes and conserved regulatory sequences. It has also been proposed that Fugu genes may provide natural mini-genes for the production of transgenic mice. We have used the Fugu homologue of the Huntington's disease (HD) gene to test this possibility. The human and Fugu HD genes cover 170 kb and 23 kb respectively and have previously been sequenced in their entirety. In Fugu tissue, the Fugu HD gene was found to be expressed as predicted from the gene sequence but three differentially spliced forms were also detected. Despite the absence of conserved promoter sequences, the Fugu promoter was found to be functional in mouse cells. We have generated mice transgenic for the Fugu HD gene and conducted a detailed expression analysis across the entire 10 kb transcript. This revealed the presence of many aberrant splice forms which would be incompatible with the production of the Fugu huntingtin protein. The Fugu HD gene is incorrectly processed in mouse cells both in vitro and in vivo which sheds doubt on the usefulness of Fugu genes for transgenesis.
Asunto(s)
Peces Venenosos/genética , Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Línea Celular , Proteína Huntingtina , Ratones , Ratones Transgénicos , Regiones Promotoras GenéticasRESUMEN
Renin is an aspartyl protease that is involved in the conversion of angiotensinogen to angitensin II and hence participates in the regulation of blood pressure. Mice are polymorphic for the number of renin genes with some strains harbouring two renin genes, Ren-1d and Ren-2. To study the role of renin Ren-1d in regulating cardiovascular homeostasis, mice with a disrupted Ren-1d gene were created. Analyses of kidney renin mRNA expression in Ren-1d-/-/Ren-2+/+ mice demonstrated that only Ren-2 transcripts were present. Mean arterial blood pressures of Ren-1d+/+/Ren-2+/+, Ren-1d+/-/Ren-2+/+ and Ren-1d-/-/Ren-2+/+ mice showed no significant differences. These observations demonstrate that the Ren-1d gene product is not essential for normal blood pressure maintenance under normal physiological conditions.
Asunto(s)
Presión Sanguínea/genética , Ratones Transgénicos/fisiología , Renina/genética , Animales , Presión Sanguínea/fisiología , Southern Blotting , Femenino , Heterocigoto , Homocigoto , Riñón/metabolismo , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Recombinación Genética , Células Madre/fisiología , Transcripción GenéticaRESUMEN
The human thyroglobulin (hTg)-encoding gene is a single-copy gene of more than 300 kb and composed of at least 42 exons. After studying the 5' portion of the Tg cDNA (from nucleotides (nt) 1 to 842), we have previously suggested the existence of alternative splicing of the hTg pre-mRNA in human thyroid tissue. Here, we describe four alternative splicings in the central region of the hTg pre-mRNA between nt 3597 and 4653. We have deduced from these results the exonic structure of this region (exons 16 to 22). Finally, the identification of alternative splicings has also allowed us to demonstrate the existence of a strong 5' splice site inside intron 16 of Tg.
Asunto(s)
Empalme Alternativo , Exones/genética , ARN Mensajero/genética , Tiroglobulina/genética , Secuencia de Bases , Amplificación de Genes , Humanos , Intrones/genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Distribución TisularRESUMEN
We have studied the binding of purified TTF-1 on the bovine thyroglobulin gene promoter. DNase I footprinting experiments revealed three binding sites which corresponded in location to the A, B and C sites found in the rat thyroglobulin promoter. Mutants in the A and C regions showing reduced binding of TTF-1, also exhibited largely decreased promoter activity in transient expression experiments in primary-cultured dog thyrocytes. Two mutants in the B site that exhibited a reduced capacity to bind TTF-1 also displayed a drastically affected transcriptional activity in transient assays. As in the rat, sites A and C only are critical for promoter activity, these results suggest that full occupancy of the B site is required for thyroglobulin promoter activity in the cow only.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Tiroglobulina/genética , Factores de Transcripción/genética , Nucleótidos de Adenina/genética , Animales , Secuencia de Bases , Sitios de Unión/fisiología , Bovinos , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/genética , Nucleótidos de Citosina/genética , Perros , Datos de Secuencia Molecular , Mutagénesis , Proteínas Nucleares/química , Unión Proteica , Ratas , Relación Estructura-Actividad , Glándula Tiroides/citología , Factor Nuclear Tiroideo 1 , Factores de Transcripción/químicaRESUMEN
Previous reports on human thyroglobulin (hTg) modifications in thyroid carcinomas prompted us to study hTg mRNA in thyroid adenomas and carcinomas. The quantification of hTg mRNA showed a decrease in its levels of expression in both pathological conditions which differed by a factor of 2 between adenomas and carcinomas. Furthermore, PCR was used to analyse the characteristics of hTg mRNA by amplifying 4 regions of the hTg mRNA. When applied to 2 normal, 17 benign and 13 malignant pathological tissue specimens, PCR showed no modification in the size of Tg mRNA. However, abnormal sized cDNAs appeared in all tissues with no distinction between the pathologies; the Restriction Fragment Length Polymorphism study of these cDNAs suggests the existence of alternate splicing patterns in thyroglobulin mRNAs.