RESUMEN
Expansive growth of neural progenitor cells (NPCs) is a prerequisite to the temporal waves of neuronal differentiation that generate the six-layered neocortex, while also placing a heavy burden on proteins that regulate chromatin packaging and genome integrity. This problem is further reflected by the growing number of developmental disorders caused by mutations in chromatin regulators. ATRX gene mutations cause a severe intellectual disability disorder (α-thalassemia mental retardation X-linked (ATRX) syndrome; OMIM no. 301040), characterized by microcephaly, urogenital abnormalities and α-thalassemia. Although the ATRX protein is required for the maintenance of repetitive DNA within heterochromatin, how this translates to disease pathogenesis remain poorly understood and was a focus of this study. We demonstrate that Atrx(FoxG1Cre) forebrain-specific conditional knockout mice display poly(ADP-ribose) polymerase-1 (Parp-1) hyperactivation during neurogenesis and generate fewer late-born Cux1- and Brn2-positive neurons that accounts for the reduced cortical size. Moreover, DNA damage, induced Parp-1 and Atm activation is elevated in progenitor cells and contributes to their increased level of cell death. ATRX-null HeLa cells are similarly sensitive to hydroxyurea-induced replication stress, accumulate DNA damage and proliferate poorly. Impaired BRCA1-RAD51 colocalization and PARP-1 hyperactivation indicated that stalled replication forks are not efficiently protected. DNA fiber assays confirmed that MRE11 degradation of stalled replication forks was rampant in the absence of ATRX or DAXX. Indeed, fork degradation in ATRX-null cells could be attenuated by treatment with the MRE11 inhibitor mirin, or exacerbated by inhibiting PARP-1 activity. Taken together, these results suggest that ATRX is required to limit replication stress during cellular proliferation, whereas upregulation of PARP-1 activity functions as a compensatory mechanism to protect stalled forks, limiting genomic damage, and facilitating late-born neuron production.
Asunto(s)
ADN Helicasas/genética , Replicación del ADN , Heterocromatina/química , Neuronas/metabolismo , Proteínas Nucleares/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1 , Proteínas Portadoras/genética , Proliferación Celular/efectos de los fármacos , Proteínas Co-Represoras , ADN/genética , ADN/metabolismo , Daño del ADN , ADN Helicasas/deficiencia , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Células HeLa , Heterocromatina/efectos de los fármacos , Heterocromatina/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hidroxiurea/farmacología , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína Homóloga de MRE11 , Ratones , Ratones Noqueados , Chaperonas Moleculares , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Proteínas Nucleares/deficiencia , Proteínas Nucleares/metabolismo , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Prosencéfalo/citología , Prosencéfalo/efectos de los fármacos , Prosencéfalo/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Aberrant chromatin remodeling is involved in the pathogenesis of Huntington's disease (HD) but the mechanism is not known. Herein, we report that mutant huntingtin (mtHtt) induces the transcription of alpha thalassemia/mental retardation X linked (ATRX), an ATPase/helicase and SWI/SNF-like chromatin remodeling protein via Cdx-2 activation. ATRX expression was elevated in both a cell line model and transgenic model of HD, and Cdx-2 occupancy of the ATRX promoter was increased in HD. Induction of ATRX expanded the size of promyelocytic leukemia nuclear body (PML-NB) and increased trimethylation of H3K9 (H3K9me3) and condensation of pericentromeric heterochromatin, while knockdown of ATRX decreased PML-NB and H3K9me3 levels. Knockdown of ATRX/dXNP improved the hatch rate of fly embryos expressing mtHtt (Q127). ATRX/dXNP overexpression exacerbated eye degeneration of eye-specific mtHtt (Q127) expressing flies. Our findings suggest that transcriptional alteration of ATRX by mtHtt is involved in pericentromeric heterochromatin condensation and contributes to the pathogenesis of HD.
Asunto(s)
ADN Helicasas/metabolismo , Heterocromatina/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Animales , Secuencia de Bases , Factor de Transcripción CDX2 , Línea Celular , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , Drosophila , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Humanos , Proteína Huntingtina , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Masculino , Metilación , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
The hippocampus is a primary region of the brain controlling the formation of memories and learned behaviours. The ability to learn or form a memory requires a neuron to translate a transient signal into gene expression changes that have a long-lasting effect on synapse activity and connectivity. Numerous studies over the past decade have detailed changes in epigenetic modifications under various learning paradigms to support a role for chromatin remodelling in these processes. Moreover, the identification of mutations in epigenetic regulators as the cause of mental retardation or intellectual disability (MR/ID) disorders further strengthens their importance to learning and memory. Animal models for many of these disorders are emerging and advancing our understanding of the molecular mechanisms linking epigenetic regulation and cognitive function. Here, we review how chromatin remodelling proteins implicated in MR/ID contribute to the development of the hippocampus and memory formation.
Asunto(s)
Trastornos del Conocimiento/genética , Epigénesis Genética , Hipocampo/embriología , Hipocampo/fisiología , Discapacidad Intelectual/genética , Animales , Ensamble y Desensamble de Cromatina , Trastornos del Conocimiento/fisiopatología , Metilación de ADN , Hipocampo/metabolismo , Histonas/genética , Histonas/metabolismo , Aprendizaje/fisiología , Memoria/fisiología , Ratones , MutaciónAsunto(s)
Anomalías Múltiples/genética , Empalme Alternativo/genética , Proteínas Portadoras/genética , Exones/genética , Mutación/genética , Anomalías Múltiples/fisiopatología , Adulto , Secuencia de Bases , Canadá , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Nucléolo Celular/metabolismo , Análisis Mutacional de ADN , Femenino , Células HeLa , Humanos , Masculino , Fenotipo , Polirribosomas/genética , Polirribosomas/metabolismo , Transporte de Proteínas , Proteínas Represoras , SíndromeRESUMEN
Here we report the cloning of two cDNAs, Snf2h and Snf2l, encoding the murine members of the Imitation Switch (ISWI) family of chromatin remodeling proteins. To gain insight into their function we examined the spatial and temporal expression patterns of Snf2h and Snf2l during development. In the brain, Snf2h is prevalent in proliferating cell populations whereas, Snf2l is predominantly expressed in terminally differentiated neurons after birth and in adult animals, concomitant with the expression of a neural specific isoform. Moreover, a similar proliferation/differentiation relationship of expression for these two genes was observed in the ovaries and testes of adult mice. These results are consistent with a role of Snf2h complexes in replication-associated nucleosome assembly and suggest that Snf2l complexes have distinct functions associated with cell maturation or differentiation.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Envejecimiento , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Clonación Molecular , ADN Helicasas , Desarrollo Embrionario y Fetal , Etiquetas de Secuencia Expresada , Femenino , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neuronas/metabolismo , Ovario/metabolismo , Placenta/metabolismo , Embarazo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/metabolismo , Transcripción GenéticaRESUMEN
Mutations in the ATRX gene are associated with an X-linked mental retardation (XLMR) syndrome most often accompanied by alpha-thalassaemia (ATR-X syndrome). The ATRX gene encodes a predicted protein of 280 kDa featuring a PHD zinc finger motif and an ATPase/helicase domain of the SWI/SNF type; the vast majority of mutations in the ATRX gene fall within these two motifs. Although these domains are suggestive of a role for ATRX in transcriptional regulation by affecting chromatin structure and/or function, the precise cellular role of the ATRX protein remains undefined. Using indirect immunofluorescence and biochemical fractionation, we demonstrate that the ATRX protein has a punctate nuclear staining pattern and that it is tightly associated with the nuclear matrix at interphase. At the onset of M phase, the ATRX protein was associated mainly with condensed chromatin. The association of the ATRX protein with chromosomes at mitosis is concomitant with phosphorylation of the protein and its association with heterochromatin protein 1alpha (HP1alpha). The phosphorylation-dependent changes in localization between the nuclear matrix and condensed chromatin are consistent with a dual role for ATRX, possibly involving gene regulation at interphase and chromosomal segregation at mitosis.
Asunto(s)
Ciclo Celular/genética , Cromatina/metabolismo , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares , Factores de Transcripción/metabolismo , Animales , Células COS , Cromatina/genética , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Interfase/genética , Mitosis/genética , Matriz Nuclear/genética , Fosforilación , Pruebas de Precipitina , Factores de Transcripción/genética , Proteína Nuclear Ligada al Cromosoma XRESUMEN
ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with alpha-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.
Asunto(s)
Centrómero/química , ADN Helicasas , Proteínas de Unión al ADN/análisis , Heterocromatina/química , Proteínas Nucleares/análisis , Factores de Transcripción/análisis , Animales , Anticuerpos Monoclonales/inmunología , Células COS , Fraccionamiento Celular , Línea Celular Transformada , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Ovinos , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Proteína Nuclear Ligada al Cromosoma XAsunto(s)
Secuencia Conservada , Proteínas de Unión al ADN/genética , Proteínas Nucleares , Factores de Transcripción/genética , Adenosina Trifosfatasas/genética , Empalme Alternativo , Animales , ADN Helicasas/genética , Proteínas de Unión al ADN/química , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Estructura Terciaria de Proteína , Factores de Transcripción/química , Proteína Nuclear Ligada al Cromosoma XAsunto(s)
ADN Helicasas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Mutación , Proteínas Nucleares , Factores de Transcripción/química , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Globinas/genética , Humanos , Discapacidad Intelectual/genética , Ratones , Datos de Secuencia Molecular , Fenotipo , Homología de Secuencia de Aminoácido , Síndrome , Proteína Nuclear Ligada al Cromosoma X , Dedos de Zinc/genética , Talasemia alfa/genéticaRESUMEN
It was shown recently that mutations of the ATRX gene give rise to a severe, X-linked form of syndromal mental retardation associated with alpha thalassaemia (ATR-X syndrome). In this study, we have characterised the full-length cDNA and predicted structure of the ATRX protein. Comparative analysis shows that it is an entirely new member of the SNF2 subgroup of a superfamily of proteins with similar ATPase and helicase domains. ATRX probably acts as a regulator of gene expression. Definition of its genomic structure enabled us to identify four novel splicing defects by screening 52 affected individuals. Correlation between these and previously identified mutations with variations in the ATR-X phenotype provides insights into the pathophysiology of this disease and the normal role of the ATRX protein in vivo.
Asunto(s)
ADN Helicasas , Proteínas de Unión al ADN/genética , Discapacidad Intelectual/genética , Proteínas Nucleares , Factores de Transcripción/genética , Talasemia alfa/genética , Secuencia de Aminoácidos , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Síndrome , Proteína Nuclear Ligada al Cromosoma XRESUMEN
We have determined the cDNA and genomic structure of a gene (-14 gene) that lies adjacent to the human alpha-globin cluster. Although it is expressed in a wide range of cell lines and tissues, a previously described erythroid-specific regulatory element that controls expression of the alpha-globin genes lies within intron 5 of this gene. Analysis of the -14 gene promoter shows that it is GC rich and associated with a constitutively expressed DNase 1 hypersensitive site; unlike the alpha-globin promoter, it does not contain a TATA or CCAAT box. These and other differences in promoter structure may explain why the erythroid regulatory element interacts specifically with the alpha-globin promoters and not the -14 gene promoter, which lies between the alpha promoters and their regulatory element. Interspecies comparisons demonstrate that the sequence and location of the -14 gene adjacent to the alpha cluster have been maintained since the bird/mammal divergence, 270 million years ago.
Asunto(s)
Evolución Biológica , Regulación de la Expresión Génica , Globinas/genética , Familia de Multigenes , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Aminoácidos , Animales , Composición de Base , Secuencia de Bases , Aves , Línea Celular , Pollos , Secuencia Conservada , Cartilla de ADN , ADN Complementario , Exones , Globinas/biosíntesis , Humanos , Intrones , Mamíferos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Homología de Secuencia de Ácido Nucleico , TATA Box , Transfección , Células Tumorales CultivadasRESUMEN
We have examined the role of the major positive upstream regulatory element of the human alpha-globin gene locus (HS-40) in its natural chromosomal context. Using homologous recombination, HS-40 was replaced by a neo marker gene in a mouse erythroleukemia hybrid cell line containing a single copy of human chromosome 16. In clones from which HS-40 had been deleted, human alpha-globin gene expression was severely reduced, although basal levels of alpha 1 and alpha 2-globin mRNA expression representing less than 3% of the level in control cell lines were detected. Deletion of the neo marker gene, by using FLP recombinase/FLP recombinase target system, proved that the phenotype observed was not caused by the regulatory elements of this marker gene. In the targeted clones, deletion of HS-40 apparently does not affect long-range or local chromatin structure at the alpha promoters. Therefore, these results indicate that, in the experimental system used, HS-40 behaves as a strong inducible enhancer of human alpha-globin gene expression.
Asunto(s)
Globinas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Línea Celular , Desoxirribonucleasa I , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Mutagénesis Insercional , ARN Mensajero/genética , Mapeo RestrictivoRESUMEN
The ATR-X syndrome is an X-linked disorder comprising severe psychomotor retardation, characteristic facial features, genital abnormalities, and alpha-thalassemia. We have shown that ATR-X results from diverse mutations of XH2, a member of a subgroup of the helicase superfamily that includes proteins involved in a wide range of cellular functions, including DNA recombination and repair (RAD16, RAD54, and ERCC6) and regulation of transcription (SW12/SNF2, MOT1, and brahma). The complex ATR-X phenotype suggests that XH2, when mutated, down-regulates expression of several genes, including the alpha-globin genes, indicating that it could be a global transcriptional regulator. In addition to its role in the ATR-X syndrome, XH2 may be a good candidate for other forms of X-linked mental retardation mapping to Xq13.
Asunto(s)
Anomalías Múltiples/genética , ADN Helicasas/genética , Reparación del ADN , Regulación de la Expresión Génica , Discapacidad Intelectual/genética , Familia de Multigenes , Eliminación de Secuencia , Transcripción Genética , Cromosoma X , Talasemia alfa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Encéfalo/metabolismo , Mapeo Cromosómico , Secuencia Conservada , Cartilla de ADN , Feto , Biblioteca de Genes , Ligamiento Genético , Humanos , Escala de Lod , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , SíndromeRESUMEN
Mental handicap is a common clinical problem that has been a relatively neglected area of research. Though the causes are varied and complex, molecular biologists are making progress in understanding the mechanisms in some cases, particularly where there are distinguishing phenotypic or genetic markers. The fortuitous association of alpha thalassaemia with a form of mental retardation has allowed us to define a specific X-linked syndrome (ATR-X). Positional cloning was used to define a disease interval and examination of candidate genes demonstrated that mutations in a gene, XH2, showing homology to the SNF2 superfamily were responsible for this syndrome. The complex ATR-X phenotype suggests that this gene, when mutated, down-regulates the expression of several genes including the alpha-globin genes indicating that it could be a global transcriptional regulator. It is conceivable that this mechanism is involved in other forms of syndromal mental retardation.
Asunto(s)
Regulación de la Expresión Génica/genética , Discapacidad Intelectual/genética , Cromosoma X , Talasemia alfa/genética , Niño , Mapeo Cromosómico , Ligamiento Genético , Humanos , Masculino , Mutación , SíndromeRESUMEN
Hemophilia B Leyden is a rare form of inherited factor IX deficiency in which patients experience spontaneous postpubertal recovery of factor IX levels. The mutations resulting in this disorder are localized in a 40-nucleotide region encompassing the major transcriptional start site for factor IX. Here we report the further characterization of five cis-acting elements in the factor IX promoter and the effects on protein binding and transcriptional activation of five Leyden mutations (at nucleotides +13, -5, -6, -20, and -26) that occur within the proximal three elements (sites 1 through 3). Bandshift studies using nuclear extracts from four different rat tissues have shown that at least some of the proteins binding to each of the five sites are ubiquitous in nature. The pattern of DNA binding at site 1 suggests that this element plays an important role in mediating the liver-specific expression of factor IX. Additional studies with liver nuclear extracts obtained at several different points in development have shown an increase in DNA binding at sites 1, 4, and 5 between 1 day and 1 week. Using DNase I footprint analysis and competition bandshift studies, we have shown that the binding of nuclear proteins to each of the mutant sites is disrupted to a variable extent. There appears to be some, although reduced, protein binding to all of the mutant oligonucleotides apart from the -26 mutant. In vitro transcription assays have shown that each of the mutations reduces the global proximal promoter activity by approximately 40%. Two double mutant promoters did not show any additional downregulation in the in vitro transcription assay. In experiments designed to assess the relative transcriptional activity mediated from each of the five sites independently, we have tested artificial homopolymer promoters of each site in the in vitro transcription assay. These studies show that sites 4 and 5 are the strongest activators and that transactivation from site 5 is further enhanced by the albumin D site-binding protein. In summary, these investigations show deleterious effects of each of the Leyden mutations tested on the binding of trans-acting factors and also show disruption of transcriptional activation in a functional in vitro transcription assay. Our results also show that cis-acting elements 4 and 5 are the principal activators of this locus.
Asunto(s)
Factor IX/genética , Hemofilia B/genética , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Cartilla de ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Ratas , Factores de Transcripción/metabolismoRESUMEN
Haemophilia B Leyden is characterized by low childhood levels of factor IX which gradually increase after puberty, eventually resulting in a return to health. The disease is the result of single nucleotide substitutions within a 40 bp region encompassing the major transcriptional start site. We have characterized transcription factor binding sites within the factor IX promoter. Five sites were identified and a Leyden mutation at nucleotide -5 was shown to interfere with the binding of proteins to one of three newly identified sites. The correlation between the post-pubertal recovery of these mutants and the induction of the transcription factor DBP led us to the discovery of a synergistic interaction between DBP and C/EBP responsible for the recovery of normal transcriptional activity of the -5 mutant promoter and may play a role in the resolution of other Leyden mutants.
Asunto(s)
Proteínas de Unión al ADN/genética , Factor IX/genética , Hemofilia B/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Adolescente , Adulto , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Niño , Preescolar , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I , Humanos , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Unión Proteica , Pubertad , Factores de Transcripción/metabolismoRESUMEN
We have developed a novel technique that is useful for the rapid detection of previously characterized point mutations in the human genome. The method relies on the differential termination of primer extension being performed simultaneously on normal and mutant template molecules in the presence of a selectively limited complement of deoxynucleotide triphosphates. We have used this technique to determine the carrier status of two potential carriers of a hemophilia B mutation at codon 145 in the factor IX gene. In addition, the technique has been used to quantify the levels of mutant sequence in several tissues of a hemophilia B patient who is a somatic mosaic for a point mutation at codon 350.
Asunto(s)
Factor IX/genética , Tamización de Portadores Genéticos/métodos , Hemofilia B/genética , Mutación/genética , Secuencia de Bases , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
The molecular pathology that results in hemophilia B has been determined by many studies to be highly variable. Several point mutations have been identified in a 40-bp region within the Factor IX promoter. These include mutations at nucleotides -20, -6, +8, and +13. Here we report an A to T transversion at position -5 of the Factor IX gene. There is strong circumstantial evidence to support an important role for this region of the Factor IX promoter in the developmental regulation of the gene through the binding of specific transcription factors.
Asunto(s)
Factor IX/genética , Hemofilia B/genética , Mutación , Regiones Promotoras Genéticas , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
The deduced amino acid sequences for a major mitochondrial protein (designated P1, related to the 'chaperonin' family of proteins) from human and Chinese hamster cells show extensive similarity (greater than 60% identity observed over the entire length) with a related protein present in evolutionarily as divergent organisms as Escherichia coli, Coxiella burnetii, Mycobacterium species, cyanobacteria as well as in yeast mitochondria and higher plant chloroplasts. Of the different groups of bacteria for which sequence data is available, maximum similarity of the mammalian/yeast P1 protein is observed with the corresponding protein from purple bacteria (especially C. burnetii) while the protein from plant chloroplasts exhibited highest similarity with the corresponding protein from cyanobacteria. The sequence data for this protein thus support the contention that the endosymbiont that gave rise to mitochondrion was a member of purple bacteria, while plant chloroplast originated from a member of the cyanobacterial lineage.
Asunto(s)
Cloroplastos , Mitocondrias , Plantas , Proteínas , Secuencia de Aminoácidos , Animales , Evolución Biológica , Células Cultivadas , Chaperoninas , Coxiella , Cricetinae , Cricetulus , Cianobacterias , Escherichia coli , Humanos , Datos de Secuencia Molecular , Mycobacterium , Saccharomyces cerevisiaeRESUMEN
The complete cDNA sequence of a mitochondrial protein from Chinese hamster ovary cells, designated P1, which was originally identified as a microtubule-related protein (Gupta, R.S., Ho, T.K.W., Moffat, M.R.K., and Gupta, R. (1982) J. Biol. Chem. 257, 1071-1078), has been determined. The P1 cDNA encodes a protein of 60,983 Da including a 26-amino acid putative mitochondrial targeting sequence at its N-terminal end. The deduced amino acid sequence of Chinese hamster P1 shows 97% identity to the human P1 protein. Most interestingly, the amino acid sequences of mammalian P1 proteins show extensive sequence homology (42-60% identical residues and an additional 15-25% conservative replacements) to the "chaperonin" family of bacterial, yeast, and plant proteins (viz. groEL protein of Escherichia coli, hsp 60 protein of yeast, and ribulose-1,5-bisphosphate carboxylase subunit binding protein of plant chloroplasts) and to the 60-65-kDa major antigenic protein of mycobacteria and Coxiella burnetii. The homology between mammalian P1 and other proteins begins after the putative mitochondrial presequence and extends up to the C-terminal end. Furthermore, similar to the chaperonin family of proteins, P1 appears to exist in cells as a homooligomeric complex of seven subunits and shows ATPase activity. These observations strongly indicate that P1 protein is a member of the chaperonin family and that it may be involved in a similar function in mammalian cells.