RESUMEN
Cofilin is a widely-distributed, intracellular, actin binding protein which is involved in the translocation of actin-cofilin complex from cytoplasm to nucleus. We have cloned a non-muscle-type cofilin (CFL1) from a human promyelocytic cDNA library and mapped this to human chromosome 11 by PCR amplification of 3' untranslated sequence in a panel of rodent-human somatic cell hybrids, and to the interval 11q12-q13.2 in a chromosome 11 somatic cell hybrid mapping panel. Confirmation of regional localisation to 11q13 has been obtained by fluorescent in situ hybridisation of genomic cosmid clones, by demonstration of the presence of both SEA (the human homologue of avian retrovirus proviral tyrosine kinase, 11q13) and CFL1 in some of these clones and by close linkage of CFL1 to SEA in a panel of high-dose irradiation hybrids. We have identified human muscle-type cofilin sequences by comparison of human expressed sequence tags with M-type cofilins of other species and we have mapped the human M-type cofilin, CFL2, to chromosome 14.
Asunto(s)
Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 14/genética , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso/genética , Factores Despolimerizantes de la Actina , Secuencia de Aminoácidos , Mapeo Cromosómico , Clonación Molecular , Cofilina 2 , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Granulocitos , Humanos , Células Híbridas/metabolismo , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Músculos/química , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
Defects in gp91-phox, the large subunit of cytochrome b558 (b-245) give rise to X-linked chronic granulomatous disease (CGD), a rare inherited condition characterized by an extreme susceptibility to bacterial and fungal infection. In the majority of cases, the phagocytes are unable to generate any superoxide owing to complete absence of the flavocytochrome. However, a small minority of these patients do have some phagocytic oxidase activity. We describe here an analysis of the molecular basis of the disease in three such variant patients with lesions in the gene coding for gp91-phox on the X chromosome. Three different genetic lesions were found, resulting in the substitution of tyrosine for cysteine 244, a deletion of one of three lysines 313 through 315, and the deletion of the six C-terminal amino acids, respectively. The functional consequences of these defects on oxidase activity was a reduction to 12%, 3.6%, and 2.1% of the normal levels, respectively. Corresponding levels of gp91-phox were 20%, 8%, and 16% of normal classifying these patients as X91-. Microbicidal assays showed that killing of Staphylococcus aureus was grossly impaired in cells in which there was 12% normal activity. This implies that if gene therapy is to be applied, it must restore oxidase activity to a much higher level than that present in the cells of this patient. The sites of two of the mutations were analyzed on a model of the C-terminal half of the gp91-phox, based on the crystal structure of the homologous protein ferrodoxin NADP reductase. Possible structural consequences of the mutations were examined.
Asunto(s)
Grupo Citocromo b/genética , Enfermedad Granulomatosa Crónica/genética , Glicoproteínas de Membrana/genética , NADH NADPH Oxidorreductasas/química , Mutación Puntual , Conformación Proteica , Eliminación de Secuencia , Cromosoma X , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Grupo Citocromo b/química , Grupo Citocromo b/deficiencia , Análisis Mutacional de ADN , ADN Complementario/genética , Variación Genética , Humanos , Lisina , Sustancias Macromoleculares , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/deficiencia , Modelos Moleculares , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/deficiencia , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 2 , NADPH Oxidasas , Reacción en Cadena de la Polimerasa , Superóxidos/metabolismoRESUMEN
The human parvovirus, adeno-associated virus-2 (AAV-2), has many attributes that recommend its use as a gene transfer vehicle, including a broad tissue tropism, the ability to integrate stably into the host genome, and efficient transduction of cells which proliferate slowly. However, application to human gene therapy is currently limited by existing methods for generation of recombinant AAV (rAAV), resulting in relatively low transducing titres. In an attempt to overcome some of these problems, we have developed a defective adenoviral vector which improves the efficiency of rAAV vector delivery to cells in which rAAV is propagated, and from which the rAAV genome can be efficiently rescued. A functional copy of the p47phox gene was successfully transferred to cell lines derived from patients with autosomal recessive chronic granulomatous disease (CGD) by rAAV recovered in this way, and function of the NADPH-oxidase was restored to levels which were stable for at least 8 weeks. This method for generation of rAAV, although still limited by the need for cotransfection of AAV Rep and Cap functions, may permit recovery of higher titre transducing stocks from cell lines in which these genes are stably incorporated, and significantly reduces the risk of contamination with wild-type adenovirus (wtAd).
Asunto(s)
Adenoviridae/genética , ADN Viral/farmacología , Vectores Genéticos , NADH NADPH Oxidorreductasas/genética , Secuencia de Bases , Línea Celular , ADN Recombinante/farmacología , ADN Viral/genética , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/biosíntesis , NADPH OxidasasRESUMEN
For somatic gene therapy to become a realistic therapeutic strategy for chronic granulomatous disease (CGD), we have to be able to assign the molecular lesion to a specific component of the NADPH oxidase and to confirm that transfer of a functional copy of the corresponding defective gene will result in correction of the cellular defect. We used an adenovirus vector expressing p47phox to transduce monocytes from patients with CGD. We showed by nitroblue-tetrazolium staining that NADPH-oxidase activity was restored to these cells. This technique offers a rapid means for molecular diagnosis. In the short term, this approach may have therapeutic potential.
Asunto(s)
Técnicas de Transferencia de Gen , Enfermedad Granulomatosa Crónica/terapia , Monocitos/metabolismo , Adenoviridae/genética , Mapeo Cromosómico , Femenino , Terapia Genética , Vectores Genéticos , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasas , Neutrófilos/metabolismo , Nitroazul de Tetrazolio , Coloración y EtiquetadoRESUMEN
Chronic granulomatous disease (CGD) comprises a heterogeneous group of inherited conditions characterized biochemically by disordered function of a unique multicomponent enzyme system present in phagocytic cells, the NADPH-oxidase. Clinically, it is characterized by recurrent bacterial and fungal infections that are relatively resistant to treatment by conventional means. Curative bone marrow transplantation has been successfully achieved in a small number of cases, but the wider application of this procedure is limited by availability of suitable donor material. Somatic gene therapy would overcome this problem, and several groups have now shown correction of the biochemical defect in hematopoietic cells by retrovirus-mediated gene transfer. However, the failure of the current generation of retroviral vectors to efficiently transduce quiescent cells greatly restricts their potential for gene transfer to pluripotent hematopoietic stem cells. Given these limitations, we have constructed vectors based on adeno-associated virus and used these to transfer a functional copy of the p47phox gene to immortalized B cells derived from patients with p47phox-deficient autosomal recessive CGD. We show stable expression of protein and restoration of NADPH-oxidase function in these cells in the absence of selection. Adeno-associated virus vectors may overcome some of the limitations of retroviral gene delivery systems and may therefore be a useful vehicle for curative gene therapy of CGD and other primary immunodeficiencies.
Asunto(s)
Dependovirus/genética , Terapia Genética , Vectores Genéticos , Enfermedad Granulomatosa Crónica/terapia , NADH NADPH Oxidorreductasas/genética , Linfocitos B/enzimología , Linfocitos B/virología , Southern Blotting , Western Blotting , Línea Celular Transformada , Células Cultivadas , Dependovirus/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Enfermedad Granulomatosa Crónica/enzimología , Enfermedad Granulomatosa Crónica/genética , Humanos , NADH NADPH Oxidorreductasas/biosíntesis , NADH NADPH Oxidorreductasas/deficiencia , NADPH Oxidasas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Estallido Respiratorio , Superóxidos/metabolismo , TransfecciónRESUMEN
The NADPH oxidase of phagocytes is known to be expressed in Epstein-Barr-virus-transformed B-lymphocytes, albeit at levels only approx. 5% of those found in neutrophils. We have investigated the basis of this low level of expression and find that all four specific components of the NADPH oxidase are expressed in B-lymphocytes, but only p47-phox protein attains levels equivalent with those found in neutrophils. This component was shown to phosphorylate and translocate to the membrane normally on activation. The other cytosolic component, p67-phox, did show a deficit, and by supplementing a B-cell cytosol extract with recombinant p67-phox, this was shown to account for the somewhat reduced activity of B-cell cytosol in a cell-free oxidase system. The cell-free analysis also clearly located the major deficiency in superoxide-generating capacity of B-lymphocytes to the membrane. Western blotting of membrane proteins revealed major reductions in the amount of cytochrome b558. Analysis of the levels of mRNA for both subunits of cytochrome b558, however, showed levels greater than expected. Significantly more mRNA for gp91-phox was present in B-cells than in undifferentiated HL60 cells, although it was not quite as abundant as in differentiated HL60 cells, which are capable of producing large amounts of superoxide. We conclude that the failure of B-lymphocytes to generate amounts of superoxide equivalent to those generated by neutrophils is primarily due to a post-transcriptionally determined block to the accumulation of cytochrome b558.
Asunto(s)
Linfocitos B/enzimología , Grupo Citocromo b/genética , Herpesvirus Humano 4 , NADH NADPH Oxidorreductasas/metabolismo , Transcripción Genética , Linfocitos B/virología , Western Blotting , Línea Celular Transformada , Humanos , NADPH Deshidrogenasa/genética , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasas , Neutrófilos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Superóxidos/metabolismo , Células Tumorales CultivadasRESUMEN
Human red blood cells (RBCs) that are deficient in an integral membrane-associated protein ("stomatin") of apparent molecular mass 31 Kd show a catastrophic increase in passive membrane permeability to the univalent cations Na+ and K+ and are stomatocytic in shape. We have purified this protein from normal RBC membranes and isolated a cDNA clone coding for it. The deduced protein sequence is unrelated to that of any known ion-transport-related protein. Selective solubilization studies using detergents show that while the protein is strongly associated with the phospholipid bilayer, it also binds to the cytoskeleton. The predicted polypeptide has a single trans-membranous hydrophobic segment near the N-terminus, which would locate it in the membrane; the large C-terminal domain is hydrophilic and cytoplasmic in orientation and is presumed to be responsible for the attachment to the cytoskeleton. By inference, the protein has the function of closing a latent ion channel. The messenger RNA encoding this protein is ubiquitously distributed in different human cell types and tissues and is thus presumably a widely distributed regulator of transmembrane cation fluxes. As a membrane-bound inhibitor protein of Na+ and K+ transport, it is unique among the known components of membrane-transport proteins.
Asunto(s)
Proteínas Sanguíneas/genética , ADN/aislamiento & purificación , Membrana Eritrocítica/química , Eritrocitos Anormales/metabolismo , Proteínas de la Membrana/genética , Potasio/sangre , Sodio/sangre , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Membrana Eritrocítica/metabolismo , Humanos , Proteínas de la Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Mutación , ARN Mensajero/análisisRESUMEN
A novel EF-hand Ca(2+)-binding protein we have called grancalcin has been identified and characterized. This protein is particularly abundant in neutrophils and monocytes, with relatively small amounts in lymphocytes. The cDNA for this protein has been cloned and sequenced. The sequence predicts that the protein is composed of 217 amino acids, with a molecular mass of 24,010 daltons. It contains four EF-hand calcium-binding motifs and exhibits strong homology to sorcin, one of two proteins overexpressed in multidrug-resistant cells whose function is unknown. There are potentially one phosphorylation and two glycosylation sites. The 1.65-kilobase mRNA is detected in bone marrow and is present in neutrophils, monocytes, macrophages, B and T lymphocytes, and the promyelocytic cell line HL60s. The protein displays a Ca(2+)-dependent translocation to the granules and plasma membrane of neutrophils, suggesting that it might play an effector role in the specialized functions of these cells.
Asunto(s)
Proteínas de Unión al Calcio/genética , Monocitos/metabolismo , Neutrófilos/metabolismo , Secuencia de Aminoácidos , Anticuerpos , Northern Blotting , Calcio/farmacología , Proteínas de Unión al Calcio/sangre , Proteínas de Unión al Calcio/aislamiento & purificación , Línea Celular , Membrana Celular/metabolismo , Clonación Molecular/métodos , Gránulos Citoplasmáticos/metabolismo , Citosol/metabolismo , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Peso Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
Chronic granulomatous disease (CGD) is a rare inherited condition rendering neutrophils incapable of killing invading pathogens. This condition is due to the failure of a multicomponent microbicidal oxidase that normally yields a low-midpoint-potential b cytochrome (cytochrome b245). Although defects in the X chromosome-linked cytochrome account for the majority of CGD patients, as many as 30% of CGD cases are due to an autosomal recessive disease. Of these, greater than 90% have been shown to be defective in the synthesis of a 47-kDa cytosolic component of the oxidase. We demonstrate here in three unrelated cases of autosomal recessive CGD that the identical underlying molecular lesion is a dinucleotide deletion at a GTGT tandem repeat, corresponding to the acceptor site of the first intron-exon junction. Slippage of the DNA duplex at this site may contribute to the high frequency of defects in this gene.
Asunto(s)
Deleción Cromosómica , Grupo Citocromo b/genética , Fosfatos de Dinucleósidos/análisis , Genes Recesivos , Enfermedad Granulomatosa Crónica/genética , Secuencias Repetitivas de Ácidos Nucleicos , Cromosoma X , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , ADN/genética , ADN/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Valores de Referencia , Mapeo RestrictivoRESUMEN
The chromosomal location of the alpha subunit (23-kDa protein) of human cytochrome b-245 was analyzed by Southern blot hybridization using DNA isolated from a panel of 12 independent human-rodent somatic cell hybrids. The results indicate that this protein is encoded at a single locus on chromosome 16.
Asunto(s)
Cromosomas Humanos Par 16 , Grupo Citocromo b/genética , Animales , Southern Blotting , Mapeo Cromosómico , Genes , Humanos , Células Híbridas , RoedoresRESUMEN
A full-length cDNA clone was isolated for the 47-kilodalton (kDa) subunit of the NADPH oxidase system, whose absence is responsible for the most common form of autosomally inherited chronic granulomatous disease (CGD). It encodes a 44.7-kDa polypeptide, which contains two src homology (SH3) domains and several possible sites for phosphorylation by protein kinase C. We speculate that the SH3 domains may interact with the Rap1 protein associated with cytochrome b-245 (M.T. Quinn, C.A. Parkes, L. Walker, S. Orkin, M. Dinauer, and A. Jesaitis, Nature [London] 342:198-200, 1989). An antiserum raised to the predicted C terminus of the protein detects a polypeptide with an apparent molecular mass of 47 kDa in normal neutrophil granulocytes but not in those from patients with autosomal CGD. The antibody has been used to show that the protein associates with the vacuolar membrane and is phosphorylated in response to phorbol ester treatment. Analysis of a number of tissue types and cell lines shows that expression of the gene is confined to phagocytic cells and B lymphocytes. This observation suggests that patients with CGD may also have a defect in lymphocyte function. p47 protein and mRNA levels increase during retinoic acid-induced neutrophil differentiation of HL60 cells. Nuclear run-on transcription assays show that the gene for p47 is induced at the transcriptional level in a cycloheximide-insensitive manner. These data indicate that this gene is a primary target for regulation by retinoic acid.
Asunto(s)
Enfermedad Granulomatosa Crónica/fisiopatología , NADH NADPH Oxidorreductasas/genética , Fosfoproteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Peso Molecular , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas , Neutrófilos/fisiología , Fosfoproteínas/metabolismo , ARN Mensajero/genética , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
A 47 kDa protein in the cytosol of phagocytic cells becomes heavily phosphorylated and translocates to the cell membrane upon stimulation. This protein was isolated from the cytosol of human neutrophils by chromatography on ion-exchange and hydroxyapatite resins. Polyclonal antibodies to this protein demonstrated that it was present in the neutrophils of two patients with X-linked chronic granulomatous disease (CGD) but not in those of three patients with the autosomal recessive pattern of inheritance. A sequence of amino acids was determined from a tryptic peptide of this protein: Glu-Met-Phe-Pro-Ile-Glu-Ala-Gly-Ala-Ile-Asn-Xaa-Glu. This served to establish that the phosphoprotein isolated here is the same as a protein of a similar molecular mass identified by other workers. These studies confirm the involvement of this 47 kDa phosphoprotein in the molecular pathology of autosomal recessive CGD and describe a method for the purification of the native protein.
Asunto(s)
NADH NADPH Oxidorreductasas/sangre , Neutrófilos/enzimología , Fosfoproteínas/sangre , Secuencia de Aminoácidos , Western Blotting , Cromatografía , Cromatografía por Intercambio Iónico , Durapatita , Electroforesis en Gel de Poliacrilamida , Humanos , Hidroxiapatitas , Datos de Secuencia Molecular , Peso Molecular , NADH NADPH Oxidorreductasas/aislamiento & purificación , NADPH Oxidasas , Fosfoproteínas/aislamiento & purificaciónRESUMEN
Amphibian limb regeneration is a process in which it has been suggested that cells of one differentiated type may dedifferentiate and give rise to cells of another type in the regenerate. We have used two tissue-specific hypomethylations in the newt cardioskeletal myosin heavy chain gene as lineage markers to follow the fate of cells during limb regeneration. Analysis of genomic DNA from different muscle cell populations allowed the assignment of one marker to the muscle (Hypo A) lineage and the other, more tentatively, to the 'connective tissue' (Hypo B) component of muscle. The contribution to regenerated limb cartilage and limb blastemal tissue by cells carrying these markers was estimated by quantitative analysis of Southern blot hybridizations using DNA from regenerate tissues. The results are consistent with a contribution of cells from both muscle and connective tissue lineages to cartilage in regenerated limbs. In addition, removal of the humerus at the time of amputation (eliminating any contribution from pre-existing cartilage), has provided evidence for an increased representation of cells carrying the connective tissue marker in regenerate cartilage but did not affect the representation of cells carrying the muscle cell marker.
Asunto(s)
ADN/genética , Extremidades/fisiología , Regeneración , Animales , Southern Blotting , Cartílago/citología , Diferenciación Celular , Células Cultivadas , Células del Tejido Conectivo , ADN/metabolismo , Marcadores Genéticos , Metilación , Músculos/citología , Notophthalmus viridescensRESUMEN
As part of our studies on the fate of the muscle lineage during amphibian limb regeneration, we have isolated genomic and cDNA sequences from a myosin heavy chain in the newt (Notophthalmus viridescens). Notwithstanding the technical problems inherent in analysing the large newt genome, genomic and cDNA sequences have been isolated and subjected to analysis by restriction mapping. Northern hybridization, Southern hybridization and DNA sequencing. We believe these to be the first single copy newt gene sequences to have been subjected to this type of analysis. The newt gene sequences showed a striking difference from mammalian myosins in both the estimated sizes of the gene and its intervening sequences; these being much larger than in the mammalian models, it is speculated that this could contribute to the exceptional size of the newt genome. By contrast, the coding sequences displayed very high levels of sequence homology to mammalian myosins. In particular, the amino acid sequence of the newt myosin was found to have greatest homology with rat and human myosin isotypes having a similar cardio-skeletal muscle expression pattern. Despite a long evolutionary separation, newt and mammalian cardio-skeletal myosins have remained more similar to each other than have the human or rat cardiac forms to skeletal myosins within their own respective species.
Asunto(s)
Genes , Miosinas/genética , Salamandridae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARNRESUMEN
A method is described here that can be used to provide qualitative data on the expression of a wide number of genes in a variety of different tissues or cell types. This approach can further be utilized to enable tissue-specific or temporally regulated genes to be identified and isolated. The possibility of obtaining molecular probes for these types of genes should enable us to further our understanding of the molecular events underlying the process of differentiation.