RESUMEN
A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation groups that have been suspected to represent different genes. ATM, which has a transcript of 12 kilobases, was found to be mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3' kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification of AT heterozygotes, who are at increased risk of cancer.
Asunto(s)
Ataxia Telangiectasia/genética , Cromosomas Humanos Par 11 , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Serina-Treonina Quinasas , Proteínas/genética , Secuencia de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular , Proteínas de Ciclo Celular , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Clonación Molecular , ADN Complementario/genética , Proteínas de Unión al ADN , Femenino , Prueba de Complementación Genética , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Masculino , Meiosis , Datos de Secuencia Molecular , Neoplasias/genética , Hibridación de Ácido Nucleico , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Proteínas/química , Proteínas/fisiología , Tolerancia a Radiación , Eliminación de Secuencia , Transducción de Señal , Proteínas Supresoras de TumorRESUMEN
Ataxia-telangiectasia (A-T) is a genetic disorder caused by mutational inactivation of the ATM gene. A-T patients display a pleiotropic phenotype and suffer primarily from progressive ataxia caused by degeneration of cerebellar Purkinje and granule neurons. Disruption of the mouse Atm locus creates a murine model of A-T that exhibits most of the clinical features of the human disease. We previously hypothesized that some aspects of A-T, such as the preferential loss of certain neurons, could result from a continuous state of increased oxidative stress (G. Rotman and Y. Shiloh, Cancer Surv., 29: 285-304, 1997; G. Rotman and Y. Shiloh, BioEssays, 19: 911-917, 1997). The present work tests this hypothesis by analyzing markers of redox state in brains of Atm-deficient mice. We found alterations in the levels of thiol-containing compounds in Atm (-/-) brains, as well as significant changes in the activities of thioredoxin, catalase, and manganese superoxide dismutase in Atm (-/-) cerebella. These changes are indicative of increased levels of reactive oxygen species, which are seen primarily in the cerebellum of Atm-deficient mice. Our findings support the hypothesis that the absence of functional ATM results in oxidative stress, which may be an important cause of the degeneration of cerebellar neurons in A-T.
Asunto(s)
Ataxia Telangiectasia/metabolismo , Cerebelo/metabolismo , Estrés Oxidativo/fisiología , Proteínas Serina-Treonina Quinasas/deficiencia , Telencéfalo/metabolismo , Animales , Antioxidantes/metabolismo , Ataxia Telangiectasia/enzimología , Proteínas de la Ataxia Telangiectasia Mutada , Catalasa/metabolismo , Proteínas de Ciclo Celular , Cerebelo/enzimología , Cisteína/metabolismo , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Glutatión/metabolismo , Peroxidación de Lípido , Ratones , Proteínas del Tejido Nervioso/metabolismo , Oxidación-Reducción , Proteínas Serina-Treonina Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Telencéfalo/enzimología , Tiorredoxinas/metabolismo , Proteínas Supresoras de TumorRESUMEN
The ATM protein kinase is the product of the gene responsible for the pleiotropic recessive disorder ataxia-telangiectasia. ATM-deficient cells show enhanced sensitivity and greatly reduced responses to genotoxic agents that generate DNA double strand breaks (DSBs), such as ionizing radiation and radiomimetic chemicals, but exhibit normal responses to DNA adducts and base modifications induced by other agents. Therefore, DSBs are most likely the predominant signal for the activation of ATM-mediated pathways. Identification of the ATM gene triggered extensive research aimed at elucidating the numerous functions of its large multifaceted protein product. While ATM has both nuclear and cytoplasmic functions, this review will focus on its roles in the nucleus where it plays a central role in the very early stages of damage detection and serves as a master controller of cellular responses to DSBs. By activating key regulators of multiple signal transduction pathways, ATM mediates the efficient induction of a signaling network responsible for repair of the damage, and for cellular recovery and survival.
Asunto(s)
Daño del ADN/genética , Daño del ADN/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular , Supervivencia Celular , Cromatina/genética , Cromatina/metabolismo , Daño del ADN/efectos de la radiación , Reparación del ADN/genética , Reparación del ADN/fisiología , Proteínas de Unión al ADN , Humanos , Proteínas Supresoras de TumorRESUMEN
Cells from patients with the genetic disorder ataxia-telangiectasia (A-T) are hypersensitive to ionizing radiation and radiomimetic agents, both of which generate reactive oxygen species capable of causing oxidative damage to DNA and other macromolecules. We describe in A-T cells constitutive activation of pathways that normally respond to genotoxic stress. Basal levels of p53 and p21(WAF1/CIP1), phosphorylation on serine 15 of p53, and the Tyr15-phosphorylated form of cdc2 are chronically elevated in these cells. Treatment of A-T cells with the antioxidant alpha-lipoic acid significantly reduced the levels of these proteins, pointing to the involvement of reactive oxygen species in their chronic activation. These findings suggest that the absence of functional ATM results in a mild but continuous state of oxidative stress, which could account for several features of the pleiotropic phenotype of A-T.
Asunto(s)
Antioxidantes/farmacología , Ataxia Telangiectasia/tratamiento farmacológico , Ataxia Telangiectasia/metabolismo , Ácido Tióctico/farmacología , Ataxia Telangiectasia/patología , Proteína Quinasa CDC2/efectos de los fármacos , Proteína Quinasa CDC2/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/efectos de los fármacos , Ciclinas/metabolismo , Cicloheximida/farmacología , Daño del ADN/efectos de los fármacos , Humanos , Estrés Oxidativo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Tirosina/metabolismoRESUMEN
We recently identified a genomic domain at chromosome 10q26 that is highly amplified in the gastric carcinoma cell lines KATO III and SNU-16 and contains the BEK/K-sam gene, which encodes several growth factor receptors. A contiguous segment of 200 kb spanning this gene was amplified in five of 139 (3.6%) primary gastric carcinomas, all of them classified as poorly differentiated tumors. There was no amplification of this genomic region in a variety of other solid tumors. The overall frequency of gene amplification among the gastric carcinomas rose to 19.4% when MYC, ERBB2, and INT2 were included in the analysis, with significant association with advanced tumor stage. Amplification of various genomic regions in solid tumors may be more frequent than previously estimated.
Asunto(s)
Carcinoma/genética , Cromosomas Humanos Par 10 , Exones/genética , Amplificación de Genes/genética , Neoplasias Gástricas/genética , Genes myc/genética , Humanos , Proto-Oncogenes/genéticaRESUMEN
Positional cloning of rare disease genes depends on the availability of highly polymorphic markers near the disease loci. The most abundant class of polymorphic markers in the human genome is CA-repeats. We have developed a strategy for the rapid isolation of highly polymorphic CA-repeats from YAC clones. Total DNA of yeast clones containing partly overlapping YACs is digested with frequent cutter restriction enzymes, blotted and hybridized with a poly(CA/GT) probe under high stringency conditions that enable preferential detection of long CA-repeats. The repeats detected in this way are isolated by PCR using vectorette linkers, sequenced, and appropriate flanking markers are constructed for genotyping. All of the CA-repeats identified using this approach were highly polymorphic. This simple and rapid approach should allow the development of highly polymorphic markers at any genomic region cloned in YACs.
Asunto(s)
Southern Blotting/métodos , Cromosomas Artificiales de Levadura/genética , ADN Satélite/aislamiento & purificación , Marcadores Genéticos , Secuencias Repetitivas de Ácidos Nucleicos , Enzimas de Restricción del ADN/química , Genoma Fúngico , Genoma Humano , Humanos , Polidesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Análisis de SecuenciaRESUMEN
In a study of ataxia-telangiectasia (A-T) in the UK, patients in 10 out of 60 families were shown to have a much lower level of chromosomal radiosensitivity compared with the majority of patients. In some patients the level of radiosensitivity was hardly distinguishable from normal. Patients in this group, however, could be distinguished clinically from the majority either by the later onset of severe cerebellar features or the slower rate of progress of the disorder. By using highly polymorphic microsatellite repeat markers a chromosome 11q22-23 haplotype common to the majority of these patients, and not occurring in any non-A-T chromosome in 60 families, was identified on one chromosome. The haplotype probably defines the region of the A-T gene in these families and the mutation associated with this haplotype may be much less severe than the second mutation thereby producing the slightly milder phenotype.
Asunto(s)
Ataxia Telangiectasia/genética , Haplotipos , Tolerancia a Radiación , Adolescente , Adulto , Niño , Cromosomas Humanos Par 11 , Femenino , Variación Genética , Humanos , Masculino , Reino UnidoRESUMEN
Genetic heterogeneity in ataxia-telangiectasia (A-T) points to four different genes responsible for this disease. The two major A-T genes, ATA and ATC, were localized by genetic analysis close to each other on chromosome 11q22-23, prompting efforts of positional cloning. Essential steps in positional cloning are long-range cloning of the genomic region of interest, and derivation of highly polymorphic markers that would allow further reduction of the interval carrying the A-T gene. We constructed genomic contigs across the D11S611-D1S424 region harbouring the ATA and ATC genes in yeast artificial chromosome (YAC) vectors. These contigs were used as a fine mapping tool and enabled us to localize along the A-T region, eight microsatellite markers generated randomly by genome mapping centres. In addition, we used specific YAC clones to generate five new microsatellite markers based on polymorphic CA repeats. Recombination mapping based on Israeli A-T families indicates that the ATC gene is distal to the locus D11S1817. Further linkage analysis using these markers is expected to reduce the major A-T locus considerably to a size appropriate for cosmid cloning and identification of transcribed sequences.
Asunto(s)
Ataxia Telangiectasia/genética , Mapeo Cromosómico , Cromosomas Humanos Par 11 , Secuencia de Bases , Clonación Molecular , ADN Satélite/análisis , ADN Satélite/genética , Femenino , Ligamiento Genético , Marcadores Genéticos , Haplotipos , Humanos , Masculino , Datos de Secuencia Molecular , LinajeRESUMEN
The genotyping data given localize the major A-T gene to an approximately 850 kb region. They also localize the group A A-T gene (ATA) to a region that contains the approximately 850 kb region. They are compatible with linking A-TFresno to 11q22-23. NBS-V2 does not link to this region. Four non-linking families contain only single affecteds, suggesting that these may be spontaneous mutations rather than evidence for an A-T gene outside the 11q22-23 region. Finally, two other non-linking families contain recombinant haplotypes that are compatible with a second A-T gene at 11q22-23, slightly distal to the approximately 850 kb region. However, convincing evidence for a second gene is still lacking.
Asunto(s)
Ataxia Telangiectasia/genética , Cromosomas Humanos Par 11 , Haplotipos , Adulto , Secuencia de Bases , Niño , Mapeo Cromosómico , Consanguinidad , Salud de la Familia , Femenino , Ligamiento Genético , Genotipo , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Linaje , FenotipoRESUMEN
There is growing evidence that amplification of specific genes is associated with tumor progression. While several proto-oncogenes are known to be activated by amplification, it is clear that not all the genes involved in DNA amplification in human tumors have been discovered. Our approach to the identification of such genes is based on the 'reverse genetics' methodology. Anonymous amplified DNA fragments are cloned by virtue of their amplification in a given tumor. These sequences are mapped in the normal genome and hence define a new genetic locus. The amplified domain is isolated by long-range cloning and analyzed along three lines of investigation: new genes are sought that can explain the biological significance of the amplification; the structure of the domain is studied in normal cells and in the amplification unit in the cancer cell; attempts are made to identify molecular probes of diagnostic value within the amplified domain. This application of genome technology to cancer biology is demonstrated in our study of a new genomic domain at chromosome 10q26 which is amplified specifically in human gastric carcinomas.
Asunto(s)
Cromosomas Humanos Par 10 , Amplificación de Genes/genética , Proteínas de Neoplasias/genética , Proteínas Tirosina Quinasas Receptoras , Receptores de Factores de Crecimiento de Fibroblastos , Neoplasias Gástricas/genética , Southern Blotting , Clonación Molecular , Genoma Humano , Humanos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
BACKGROUND AND PURPOSE: Pathological angiogenesis is associated with various human diseases, such as cancer, autoimmune diseases and retinopathy. The angiopoietin (Ang)-Tie2 system plays critical roles in several steps of angiogenic remodelling. Here, we have investigated the anti-angiogenic effect of a novel angiopoietin-derived peptide. EXPERIMENTAL APPROACH: Using computational methods, we identified peptides from helical segments within angiopoietins, which were predicted to inhibit their activity. These peptides were tested using biochemical methods and models of angiogenesis. The peptide with best efficacy, A11, was selected for further characterization as an anti-angiogenic compound. KEY RESULTS: The potent anti-angiogenic activity of A11 was demonstrated in a multicellular assay of angiogenesis and in the chorioallantoic membrane model. A11 bound to angiopoietins and reduced the binding of Ang-2 to Tie2. A11 was also significantly reduced vascular density in a model of tumour-induced angiogenesis. Its ability to inhibit Ang-2 but not Ang-1-induced endothelial cell migration, and to down-regulate Tie2 levels in tumour microvessels, suggests that A11 targets the Ang-Tie2 pathway. In a rat model of oxygen-induced retinopathy, A11 strongly inhibited retinal angiogenesis. Moreover, combination of A11 with an anti-VEGF antibody showed a trend for further inhibition of angiogenesis, suggesting an additive effect. CONCLUSIONS AND IMPLICATIONS: Our results indicate that A11 is a potent anti-angiogenic compound, through modulation of the Ang-Tie2 system, underlining its potential as a therapeutic agent for the treatment of ocular and tumour neovascularization, as well as other pathological conditions that are dependent on angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Péptidos/uso terapéutico , Neovascularización Retiniana/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Angiopoyetinas/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Pollos , Membrana Corioalantoides/irrigación sanguínea , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Femenino , Células HCT116 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Ratones , Ratones Desnudos , Neovascularización Patológica/patología , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Neovascularización Retiniana/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The identification of ATM , the gene responsible for the pleiotropic recessive disease ataxia telangiectasia, has initiated extensive research to determine the functions of its multifaceted protein product. The ATM protein belongs to a family of protein kinases that share similarities at their C-terminal region with the catalytic domain of phosphatidylinositol 3-kinases. Studies with ataxia telangiectasia (A-T) cells and Atm-deficient mice have shown that ATM is a key regulator of multiple signaling cascades which respond to DNA strand breaks induced by damaging agents or by normal processes, such as meiotic or V(D)J recombination. These responses involve the activation of cell cycle checkpoints, DNA repair and apoptosis. Other roles outside the cell nucleus might be carried out by the cytoplasmic fraction of ATM. In addition, ATM appears to function as a 'caretaker', suppressing tumorigenesis in specific T cell lineages.
Asunto(s)
Ataxia Telangiectasia/genética , Proteínas Serina-Treonina Quinasas , Proteínas/genética , Proteínas/fisiología , Animales , Ataxia Telangiectasia/fisiopatología , Proteínas de la Ataxia Telangiectasia Mutada , Dominio Catalítico/genética , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Femenino , Genes Supresores de Tumor , Genotipo , Trastornos del Crecimiento/genética , Humanos , Masculino , Meiosis/genética , Ratones , Ratones Noqueados , Mutación , Neoplasias Experimentales/etiología , Degeneración Nerviosa/genética , Fenotipo , Tolerancia a Radiación/genética , Proteínas Supresoras de TumorRESUMEN
The autosomal recessive disorder ataxia-telangiectasia (AT) is highly pleiotropic. It is characterized by gradual loss of Purkinje cells in the cerebellum, leading to progressive neuromotor deterioration, immunodeficiency, developmental defects in specific tissues, profound predisposition to malignancy and acute sensitivity to ionizing radiation. AT cells show chromosomal instability, premature senesence, radiosensitivity and defects in cell cycle checkpoints activated by ionizing radiation. Several radiation induced pathways that regulate the cell cycle seem to be defective in AT cells, at least one of which is mediated by TP53. Extensive characterization of the cellular defects of AT cells, together with the recent isolation of the ATM gene, has provided some insight into the possible physiological roles of the ATM protein. Several lines of evidence, including the nature of the agents that elicit the hypersensitivity of AT cells, point to the possibility of a defect in the response to damage induced by oxidative stress, which affects various cellular macromolecules. The ATM protein might have a role in activating defence mechanisms against oxidative stress. This hypothesis broadens the previous concept of the AT defect and explains several aspects of the AT phenotype that cannot be accounted for by defective processing of DNA damage.
Asunto(s)
Ataxia Telangiectasia/genética , Ciclo Celular/genética , Daño del ADN , Estrés Oxidativo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular , Rotura Cromosómica , Proteínas de Unión al ADN , Genes p53 , Humanos , Proteínas Quinasas/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de TumorRESUMEN
Ataxia-telangiectasia (A-T) is a pleiotropic recessive disorder characterized by cerebellar ataxia, immunodeficiency, specific developmental defects, profound predisposition to cancer and acute radiosensitivity. Functional inactivation of a single gene product, ATM, accounts for this compound phenotype. We suggest that ATM acts as a sensor of reactive oxygen species and/or oxidative damage of cellular macromolecules, including DNA. In turn, ATM induces signalling through multiple pathways, thereby coordinating acute phase stress responses with cell cycle checkpoint control and repair of oxidative damage. Absence of ATM is proposed to limit the repair of insidious oxidative damage that can occur under normal physiological conditions, ultimately leading to apoptosis of particularly sensitive cells, such as neurons and thymocytes.
Asunto(s)
Ataxia Telangiectasia/genética , Ataxia Telangiectasia/fisiopatología , Daño del ADN , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas , Proteínas/genética , Proteínas/metabolismo , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Humanos , Leucina Zippers , Modelos Biológicos , Neuronas/fisiología , Transducción de Señal , Linfocitos T/fisiología , Proteínas Supresoras de TumorRESUMEN
Defects in regulation of the cellular life cycle may lead to premature cellular death or malignant transformation. Most of the proteins known to be involved in these processes are mediators of mitogenic signals or components of the cell cycle machinery. It has recently become evident, however, that systems responsible for ensuring genome stability and integrity are no less important in maintaining the normal life cycle of the cell. These systems include DNA repair enzymes and a recently emerging group of proteins that alert growth regulating mechanisms to the presence of DNA damage. These signals slow down the cell cycle while DNA repair ensues. Ataxia telangiectasia (A-T) is a genetic disorder whose clinical and cellular phenotype points to a defect in such a signaling system. A-T is characterized by neurodegeneration, immunodeficiency, radiosensitivity, cancer predisposition, and defective cell cycle checkpoints. The responsible gene, ATM, was recently cloned and sequenced. ATM encodes a large protein with a region highly similar to the catalytic domain of PI 3-kinases. The ATM protein is similar to a group of proteins in various organisms which are directly involved in the cell cycle response to DNA damage. It is expected to be part of a protein complex that responds to a specific type of DNA strand break by conveying a regulatory signal to other proteins. Interestingly, the immune and nervous systems, which differ markedly in their proliferation rates, are particularly sensitive to the absence of ATM function. The identification of the ATM gene highlights the growing importance of signal transduction initiated in the nucleus rather than in the external environment, for normal cellular growth.
Asunto(s)
Ataxia Telangiectasia/genética , Ataxia Telangiectasia/inmunología , Ciclo Celular/genética , Genes Relacionados con las Neoplasias/inmunología , Síndromes de Inmunodeficiencia/genética , Animales , Ataxia Telangiectasia/fisiopatología , Humanos , Síndromes de Inmunodeficiencia/fisiopatologíaRESUMEN
VL30 sequences are a murine dispersed multigene family with several "retrovirus-like" characteristics. Notably, they share basic structural features with retrovirus proviruses and 30S RNA transcripts of these genes are capable of efficient packaging in C-type virions and may be subsequently transmitted to other cells. It is not known whether VL30 information is genetically related to endogenous proviruses or to cellular elements. We extended our studies concerning evolutionary conservation and genetic relationships of VL30 sequences within and outside the genus Mus. The following observations were made: (i) Although VL30 DNA sequences were detected in all mice examined, analysis of VL30 reiteration disclosed up to a 100-fold difference among different Mus species. For example, only 1-3 VL30 copies were detected in M. pahari compared to approximately 200 VL30 copies in certain strains of M. musculus. (ii) Using low-stringency hybridization conditions, nucleotide sequences homologous to mouse VL30 DNA were detected in the DNAs of other animal cells such as rat and human. (iii) The cross-hybridization between mouse VL30 DNA and rat genomic DNA was fully accounted for by the cross-homology between the respective VL30 elements. The homologous regions were mapped and were found confined within a small fragment (less than 1kb) in both mouse VL30 and rat 30S DNA (as well as in Ha-MSV). The data suggested differential conservation of subsets of VL30 information. (iv) A subset of VL30 information was found in the mouse genome in molecular linkages other than "standard" VL30 units (that is a segment of VL30 DNA flanked by non-VL30 sequences). Results are discussed in terms of the possible evolution of VL30 sequences.
Asunto(s)
ADN/análisis , Genes , Muridae/genética , Retroviridae/genética , Animales , Secuencia de Bases , Evolución Biológica , Enzimas de Restricción del ADN , Genes Virales , Humanos , Ratones , Ratones Endogámicos/genética , Hibridación de Ácido Nucleico , Ratas/genética , Especificidad de la EspecieRESUMEN
LTR units associated with cellular retrovirus-like elements are abundantly present in chromosomal DNA of animal cells. We have analyzed the promoter and enhancer activities of diverse LTR units associated with different members of the murine retrovirus-like family known as VL30. We report here that the structurally heterogenous VL30 LTRs displayed highly variable promoter/enhancer activities. The most active VL30 LTR (designated VL3) promoted CAT activity to levels six-fold higher than the LTR of the strongly transforming retrovirus, MSV. This VL30 transcription unit, containing a unique U3 region, was further characterized by S1 nuclease mapping. VL3 LTR functioned as an enhancer in CAT constructs containing a SV40 promoter. In addition, a defined U3 segment was shown to augment expression of CAT in an orientation independent manner. VL3 LTR also served as an efficient promoter and enhancer in heterologous monkey cells. These results suggest that certain resident LTRs possess promoter and enhancer capacities greater than those possessed by LTRs of infectious retroviruses.
Asunto(s)
Elementos de Facilitación Genéticos , Genes Reguladores , Regiones Promotoras Genéticas , Retroviridae/genética , Acetiltransferasas/genética , Animales , Línea Celular , Cloranfenicol O-Acetiltransferasa , Chlorocebus aethiops , Clonación Molecular , Genes , Genes Virales , Riñón , Células L/enzimología , Ratones , Ratones Endogámicos BALB C , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , TransfecciónRESUMEN
VL30 genetic elements constitute a murine multicopy gene family that is retrovirus-like, despite the lack of sequence homology with any known retrovirus. Over one hundred copies of VL30 units are dispersed throughout the mouse genome. We report here that the mouse genome also contains 'solo' VL30 long terminal repeats (LTRs). These are structures which contain the LTR detached from the rest of the VL30 sequences. The isolation of solo LTRs from a mouse embryonic gene library with the aid of sub-genomic VL30 probes is described. Direct DNA sequencing established that the solo LTR unit is grossly similar to a standard VL30 LTR and that the LTR is flanked by a 4-base pair duplication. The analogy to the occurrence of solitary LTR units of transposable elements is discussed.
Asunto(s)
Transformación Celular Neoplásica , Clonación Molecular , Genes Virales , Genes , Retroviridae/genética , Animales , Enzimas de Restricción del ADN , ADN Recombinante/análisis , Ratones , Ratones Endogámicos BALB C , Hibridación de Ácido Nucleico , Plásmidos , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
This paper reports a detailed investigation of the pif region of the F factor responsible for inhibition of development of T7 and related "female-specific" phages. We have mapped a series of pif::Tn5 insertions to a region between 39.6 and 42.8 kilobases on the physical map of F. All pif::Tn5 insertions plated T7 at full efficiency; most were clustered in a 1.8-kilobase interval on both sides of the EcoRI site located at F coordinate 40.3 kilobases. A 5.2-kilobase Pst-I fragment with F coordinates 38.9 to 44.1 has been cloned into a pSC101 vector to create the Pif+ plasmid pGS103. A series of Pif- deletion mutants and nonsense mutants were isolated from pGS103. Using minicells carrying pGS103 or its derivatives, we have identified a 70,000-dalton pif protein.
Asunto(s)
Proteínas Bacterianas/genética , Clonación Molecular , Escherichia coli/fisiología , Factor F , Enzimas de Restricción del ADN , Escherichia coli/genética , Genotipo , Mutación , PlásmidosRESUMEN
Ataxia-telangiectasia (AT) is a human disease caused by mutations in the ATM gene. The neural phenotype of AT includes progressive cerebellar neurodegeneration, which results in ataxia and eventual motor dysfunction. Surprisingly, mice in which the Atm gene has been inactivated lack distinct behavioral ataxia or pronounced cerebellar degeneration, the hallmarks of the human disease. To determine whether lack of the Atm protein can nonetheless lead to structural abnormalities in the brain, we compared brains from male Atm-deficient mice with male, age-matched controls. Atm-deficient mice exhibited severe degeneration of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, and their terminals in the striatum. This cell loss was accompanied by a large reduction in immunoreactivity for the dopamine transporter in the striatum. A reduction in dopaminergic neurons also was evident in the ventral tegmental area. This effect was selective in that the noradrenergic nucleus locus coeruleus was normal in these mice. Behaviorally, Atm-deficient mice expressed locomotor abnormalities manifested as stride-length asymmetry, which could be corrected by peripheral application of the dopaminergic precursor L-dopa. In addition, these mice were hypersensitive to the dopamine releasing drug D-amphetamine. These results indicate that ATM deficiency can severely affect dopaminergic neurons in the central nervous system and suggest possible strategies for treating this aspect of the disease.