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1.
Artigo em Inglês | MEDLINE | ID: mdl-16869783

RESUMO

We have developed methods for the automation of transfection-grade DNA preparation, high-throughput retroviral preparation, and highly parallel phenotypic screens to establish approaches that will allow investigators to examine in an unbiased manner the roles of proteins in mammalian cells. These methods have been used to raise or lower the levels of individual kinases in individual micro-well cultures either by cDNA or short hairpin RNA expression and will allow investigators to treat mammalian cells in culture in manners that are analogous to genetic screens in yeast. Our proof-of-principle experiments have been performed in human cells using repositories that represent over 75% of the protein, nucleotide, carbohydrate, lipid, and amino acid kinases in the human genome. These initial experiments have demonstrated the feasibility of two general types of screens. We have performed phenotypic screens to identify proteins with specific roles in a chosen function and genetic interaction screens to establish epistatic relations between different proteins. The results suggest that any phenotype that can be scored by a robust assay in tissue culture is amenable to these types of screens and that interactions between mammalian proteins can be established. These results point to the near-term goal of establishing comprehensive, unbiased screens that will allow queries on the roles of all human proteins.


Assuntos
DNA Complementar/genética , Interferência de RNA , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Células Cultivadas , Expressão Gênica , Testes Genéticos , Genômica , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Fenótipo , Fosfotransferases/genética , Retroviridae/genética , Supressão Genética , Transfecção
2.
J Neurosci ; 21(23): 9112-23, 2001 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11717344

RESUMO

Neurons in Huntington's disease exhibit selective morphological and subcellular alterations in the striatum and cortex. The link between these neuronal changes and behavioral abnormalities is unclear. We investigated relationships between essential neuronal changes that predict motor impairment and possible involvement of the corticostriatal pathway in developing behavioral phenotypes. We therefore generated heterozygote mice expressing the N-terminal one-third of huntingtin with normal (CT18) or expanded (HD46, HD100) glutamine repeats. The HD mice exhibited motor deficits between 3 and 10 months. The age of onset depended on an expanded polyglutamine length; phenotype severity correlated with increasing age. Neuronal changes in the striatum (nuclear inclusions) preceded the onset of phenotype, whereas cortical changes, especially the accumulation of huntingtin in the nucleus and cytoplasm and the appearance of dysmorphic dendrites, predicted the onset and severity of behavioral deficits. Striatal neurons in the HD mice displayed altered responses to cortical stimulation and to activation by the excitotoxic agent NMDA. Application of NMDA increased intracellular Ca(2+) levels in HD100 neurons compared with wild-type neurons. Results suggest that motor deficits in Huntington's disease arise from cumulative morphological and physiological changes in neurons that impair corticostriatal circuitry.


Assuntos
Comportamento Animal , Córtex Cerebral/fisiopatologia , Corpo Estriado/fisiopatologia , Doença de Huntington/fisiopatologia , Neurônios/metabolismo , Idade de Início , Animais , Cálcio/metabolismo , Núcleo Celular/patologia , Córtex Cerebral/patologia , Corpo Caloso/fisiopatologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Dendritos/patologia , Modelos Animais de Doenças , Progressão da Doença , Eletrofisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Heterozigoto , Proteína Huntingtina , Doença de Huntington/patologia , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Receptores de N-Metil-D-Aspartato/metabolismo , Expansão das Repetições de Trinucleotídeos
3.
J Virol ; 75(20): 9857-71, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11559819

RESUMO

Baculovirus infection has extended the capabilities for transfection of exogenous genes into a variety of mammalian cell types. Because rat hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented chemically defined medium are an excellent model system for studying liver function in vitro, we investigated the ability of baculoviruses to infect and deliver exogenous genes to cells in this culture system. Efficient delivery to hepatocytes in short-term culture becomes restricted to peripheral cells, or "edge" cells, as the hepatocytes acquire intercellular junctions and form islands with time in culture. This barrier to baculovirus entry can be overcome, and the percentage of internal cells within the hepatocyte islands that are infected with the baculovirus can be increased more than 100-fold, when cells are subjected to transient calcium depletion before and during infection. These findings suggest that at least in some cell types, such as hepatocytes, baculovirus entry may require contact with the basolateral surface. We conclude from this study that recombinant baculovirus infection following transient depletion of extracellular calcium results in delivery of exogenous genes to at least 75% of hepatocytes in long-term DMSO culture, thereby making it possible for the first time to carry out gain-of-function and loss-of-function studies in this cell system.


Assuntos
Baculoviridae/fisiologia , Junções Intercelulares/virologia , Fígado/virologia , Animais , Baculoviridae/genética , Cálcio/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Dimetil Sulfóxido , Junções Intercelulares/metabolismo , Ratos , Transfecção
4.
Exp Mol Med ; 33(1): 46-53, 2001 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-11322486

RESUMO

The insect baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been evaluated as a vector for gene delivery to human tumor cells. A human osteogenic sarcoma cell line, Saos-2, was found to be highly susceptible to infection with a baculoviral vector, with nearly 100% of Saos-2 cells being able to express a lacZ reporter gene after a brief exposure to the virus at a m.o.i. of 30 pfu/cell. The production of beta-galactosidase protein was 18-times greater than that in HepG2 cells which were previously thought to be the mammalian cells most susceptible to the baculovirus. The possibility of developing a baculovirus as a cytotoxic vector for p53-defective cancer was tested by destruction of Saos-2 cells (p53-/-) with a recombinant baculovirus containing the wild type p53 gene (BV-p53) in vitro. The p53 baculovirus induced apoptotic cell death in tumor cells in a dose-dependent manner with approximately 60% killing at an m.o.i. of 160 pfu/cell. Combined treatments of gene therapy (p53) and chemotherapy (adriamycin) resulted in synergistic and potent killing of the osteogenic sarcoma cells. For example, greater than 95% of Saos-2 cells were killed by the combination of BV-p53 (m.o.i. of 100) and adriamycin (35 ng/ml), whereas approximately 50% and approximately 55% cells were killed by BV-p53 and adriamycin alone, respectively. These results indicate that a baculoviral gene delivery vector can be used to efficiently target certain types of mammalian cells and the combination treatment of gene-therapy mediated by a baculovirus and chemotherapy may enhance induction of apoptosis in cancer cells.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/terapia , Doxorrubicina/farmacologia , Terapia Genética/métodos , Osteossarcoma/terapia , Animais , Apoptose/genética , Baculoviridae/genética , Neoplasias Ósseas/virologia , Vetores Genéticos , Humanos , Osteossarcoma/virologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
J Neurosci ; 19(3): 964-73, 1999 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9920660

RESUMO

Neuronal intranuclear inclusions are found in the brains of patients with Huntington's disease and form from the polyglutamine-expanded N-terminal region of mutant huntingtin. To explore the properties of inclusions and their involvement in cell death, mouse clonal striatal cells were transiently transfected with truncated and full-length human wild-type and mutant huntingtin cDNAs. Both normal and mutant proteins localized in the cytoplasm, and infrequently, in dispersed and perinuclear vacuoles. Only mutant huntingtin formed nuclear and cytoplasmic inclusions, which increased with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features (membrane blebbing, shrinkage, cellular fragmentation), but those with mutant huntingtin generated the most cell fragments (apoptotic bodies). The caspase inhibitor Z-VAD-FMK significantly increased cell survival but did not diminish nuclear and cytoplasmic inclusions. In contrast, Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but did not increase survival. A series of N-terminal products was formed from truncated normal and mutant proteins and from full-length mutant huntingtin but not from full-length wild-type huntingtin. One prominent N-terminal product was blocked by Z-VAD-FMK. In summary, the formation of inclusions in clonal striatal cells corresponds to that seen in the HD brain and is separable from events that regulate cell death. N-terminal cleavage may be linked to mutant huntingtin's role in cell death.


Assuntos
Corpo Estriado/metabolismo , Mutação/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/fisiologia , Western Blotting , Inibidores de Caspase , Sobrevivência Celular/fisiologia , Células Clonais , Corpo Estriado/citologia , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Proteína Huntingtina , Corpos de Inclusão/fisiologia , Camundongos/embriologia , Neurônios/fisiologia , Oligopeptídeos/farmacologia , Distribuição Tecidual , Transfecção
6.
Hum Gene Ther ; 8(17): 2011-8, 1997 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-9414250

RESUMO

Baculovirus vectors recently have been shown to be capable of efficient transduction of human hepatoma cells and primary hepatocytes in culture. This paper describes the generation of a novel recombinant baculovirus (VGZ3) in which the vesicular stomatitis virus glycoprotein G (VSV G) is present in the viral envelope. The gene encoding VSV G was inserted into the baculovirus genome under the control of the polyhedrin promoter such that it was expressed at very high levels in infected insect cells but not in mammalian cells. Expression of the lacZ reporter gene was driven by a promoter that is functional in mammalian cells (the Rous sarcoma virus long terminal repeat). We show by Western analysis that VSV G protein was present in purified baculovirus preparations. A VSV G monoclonal antibody blocked transduction of mammalian cells by VGZ3. This virus was morphologically distinct from baculovirus lacking VSV G, with virions adopting an oval rather than rod-shaped morphology. VGZ3 transduced human hepatoma cells in vitro at an efficiency roughly 10-fold greater than baculovirus lacking VSV G (the virus Z4). VGZ3 was also capable of transducing cell lines that could not be transduced efficiently by Z4. We provide evidence that VSV G protein may enhance transduction by increasing the efficiency of escape of baculovirus from intracellular vesicles rather than by increasing cell binding or uptake of the virus. The possible use of this and related baculoviruses in gene therapy is discussed.


Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Glicoproteínas de Membrana , Transformação Genética , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Animais , Baculoviridae/ultraestrutura , Linhagem Celular , Núcleo Celular/virologia , Chlorocebus aethiops , Técnicas de Transferência de Genes , Humanos , Óperon Lac , Camundongos , Spodoptera/citologia , Células Tumorais Cultivadas , Vírus da Estomatite Vesicular Indiana/ultraestrutura
7.
Neurobiol Aging ; 17(2): 191-203, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8744400

RESUMO

The classic hallmarks of Alzheimer's disease are the deposition of amyloid in plaques and in the cerebrovasculature, and the emergence of neurofibrillary tangles in neurons. The interplay between these two pathologic processes, on the one hand, and the degeneration of neurons and loss of cognitive functions on the other, remains incompletely understood. We have proposed that one crucial component of this interplay is a fragment of the Alzheimer amyloid protein precursor (APP) comprising the carboxyterminal 100 amino acids of this molecule, which we term APP-C100 (or, more simply, C100). This fragment, which comprises the 42-amino acid amyloid protein (A beta) and an additional 58 amino acids carboxyterminal to it, was found to be toxic specifically to nerve cells in vitro. We developed transgenic mouse models to test the hypothesis that APP-C100 causes Alzheimer's disease neuropathology. APP-C100 was delivered to the mouse brain via a transgene expressing C100 under the control of the dystrophin brain promoter. These transgenic animal models for the action of APP-C100 in the brain exhibited some of the neuropathological features characteristic of Alzheimer disease brain. The animal models that we have created can be used to test hypotheses concerning the mechanism by which C100 interacts with a neuronal receptor to kill neurons.


Assuntos
Precursor de Proteína beta-Amiloide/biossíntese , Química Encefálica/genética , Degeneração Neural/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/patologia , Distrofina/biossíntese , Distrofina/genética , Hipocampo/patologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transgenes/genética
8.
Proc Natl Acad Sci U S A ; 93(6): 2348-52, 1996 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-8637876

RESUMO

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.


Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Nucleopoliedrovírus/genética , Animais , Células Cultivadas , Expressão Gênica , Terapia Genética/métodos , Humanos , Fígado , Lisossomos/fisiologia , Ratos , Sequências Reguladoras de Ácido Nucleico
9.
J Virol ; 70(1): 62-7, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8523582

RESUMO

Retroviral vectors that contain the tetracycline-inducible (Tet) system were developed. The two components of the Tet system were organized within the vectors in a manner that stringently maintains tetracycline-dependent regulation. Regulated expression of an indicator gene inserted into the retroviral vectors was examined in several different cell types. In infected NIH 3T3 cells, levels of induction in the absence of tetracycline were observed to be as much as 336-fold higher than levels in the presence of tetracycline, which were extremely low. Tetracycline-dependent regulation was observed in all other transduced cell types and ranged from 24- to 127-fold. The generation of retroviral vectors containing regulatory elements that allow for the regulated expression of heterologous genes and that have the ability to infect virtually all dividing target cells should greatly facilitate the biochemical and genetic examination of a broad range of genes. Moreover, these inducible retroviral vectors should prove useful in gene therapy applications.


Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Retroviridae/genética , Tetraciclina/farmacologia , Células 3T3 , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Transformada , Vetores Genéticos/efeitos dos fármacos , Luciferases/metabolismo , Mamíferos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Retroviridae/efeitos dos fármacos , Retroviridae/crescimento & desenvolvimento , Transativadores , Células Tumorais Cultivadas
11.
Proc Natl Acad Sci U S A ; 89(22): 10857-61, 1992 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-1438289

RESUMO

The deposition of amyloid in senile plaques and along the walls of the cerebral vasculature is a characteristic feature of Alzheimer disease. The peptide comprising the carboxyl-terminal 100 amino acids of the beta-amyloid precursor protein (beta APP) has been shown to aggregate into amyloid-like fibrils in vitro and to be neurotoxic, suggesting that this fragment may play a role in the etiology of Alzheimer disease. To address this question, we expressed this carboxyl-terminal 100-amino acid peptide of beta APP in transgenic mice under the control of the brain dystrophin promoter. We used an antibody to the principal component of amyloid, beta/A4, to demonstrate cell-body and neuropil accumulation of beta/A4 immunoreactivity in the brains of 4- and 6-month-old transgenic mice. Only light cytoplasmic staining with this antibody was visible in control mice. In addition, immunocytochemical analysis of the brains with an antibody to the carboxyl terminus of beta APP revealed abnormal aggregation of this epitope of beta APP within vesicular structures in the cytoplasm and in abnormal-appearing neurites in the CA2/3 region of the hippocampus in transgenic mice, similar to its aggregation in the cells of Alzheimer disease brains. Thioflavin S histochemistry suggested accumulations of amyloid in the cerebrovasculature of transgenic mice with the highest expression of the beta APP-C100 transgene. These observations suggest that expression of abnormal carboxyl-terminal subfragments of beta APP in vivo may cause amyloidogenesis and specific neuropathology.


Assuntos
Precursor de Proteína beta-Amiloide/biossíntese , Hipocampo/patologia , Neurônios/patologia , Proteínas Recombinantes de Fusão/biossíntese , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/genética , Animais , Clonagem Molecular , Cruzamentos Genéticos , Distrofina/genética , Feminino , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Neurônios/citologia , Regiões Promotoras Genéticas , Tratos Piramidais/citologia , Tratos Piramidais/metabolismo , Tratos Piramidais/patologia , Proteínas Recombinantes de Fusão/análise , Valores de Referência , Mapeamento por Restrição , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura
12.
Genomics ; 13(4): 991-8, 1992 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1505990

RESUMO

The microtubule-associated protein 1B (MAP1B) locus has been mapped in close proximity to spinal muscular atrophy (SMA) on chromosome 5q13. We have identified a second microsatellite within a MAP1B intron, which increases the heterozygosity of this locus to 94%. Two unambiguous recombination events establish MAP1B as a closely linked, distal flanking marker for the disease locus, while a third recombinant establishes D5S6 as the proximal flanking marker. The combination of key recombinants and linkage analysis place the SMA gene in an approximately 2-cM interval between loci D5S6 and MAP1B. Physical mapping and cloning locate MAP1B within 250 kb of locus D5S112. The identification and characterization of a highly polymorphic gene locus tightly linked to SMA will facilitate isolation of the disease gene, evaluation of heterogeneity, and development of a prenatal test for SMA.


Assuntos
Mapeamento Cromossômico , Proteínas Associadas aos Microtúbulos/genética , Atrofia Muscular Espinal/genética , Sequência de Bases , Cromossomos Fúngicos , Cromossomos Humanos Par 5 , DNA , Eletroforese em Gel de Campo Pulsado , Feminino , Biblioteca Gênica , Ligação Genética , Marcadores Genéticos , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico
13.
J Biol Chem ; 267(13): 9281-8, 1992 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-1339456

RESUMO

Conserved sequences of dystrophin, beta-spectrin, and alpha-actinin were used to plan a set of degenerate oligonucleotide primers with which we amplified a portion of a human alpha-actinin gene transcript. Using this short clone as a probe, we isolated and characterized full-length cDNA clones for two human alpha-actinin genes (ACTN2 and ACTN3). These genes encode proteins that are structurally similar to known alpha-actinins with approximately 80% amino acid identity to each other and to the previously characterized human nonmuscle gene. ACTN2 is the human homolog of a previously characterized chicken gene while ACTN3 represents a novel gene product. Northern blot analysis demonstrated that ACTN2 is expressed in both skeletal and cardiac muscle, but ACTN3 expression is limited to skeletal muscle. As with other muscle-specific isoforms, the EF-hand domains in ACTN2 and ACTN3 are predicted to be incapable of binding calcium, suggesting that actin binding is not calcium sensitive. ACTN2 was mapped to human chromosome 1q42-q43 and ACTN3 to 11q13-q14 by somatic cell hybrid panels and fluorescent in situ hybridization. These results demonstrate that some of the isoform diversity of alpha-actinins is the result of transcription from different genetic loci.


Assuntos
Actinina/genética , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 1 , Músculos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , DNA/genética , Distrofina/genética , Fluorescência , Humanos , Células Híbridas , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Espectrina/genética , Transcrição Gênica
14.
Proc Natl Acad Sci U S A ; 88(17): 7873-6, 1991 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-1881920

RESUMO

A polyclonal antiserum directed against the C-terminal domain of dystrophin was used to isolate a cDNA clone encoding an antigenically cross-reactive protein, microtubule-associated protein 1B (MAP-1B). Physical mapping of the human MAP-1B locus places its chromosomal location at 5q13, in proximity to the spinal muscular atrophy (SMA) locus. SMA is a degenerative disorder primarily affecting motor neurons. Genetic linkage analysis of SMA families using a human dinucleotide repeat polymorphism just 3' of the MAP-1B gene has shown tight linkage to SMA mutations. These mapping data together with the postulated role of MAP-1B in neuronal morphogenesis and its localization in anterior horn motor neurons suggest a possible association with SMA.


Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 5 , Proteínas Associadas aos Microtúbulos/genética , Atrofia Muscular Espinal/genética , Distrofia Muscular Animal/genética , Animais , Sequência de Bases , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Distrofina/genética , Feminino , Ligação Genética , Humanos , Masculino , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Músculos/fisiologia , Músculos/fisiopatologia , Sondas de Oligonucleotídeos , Linhagem , Valores de Referência
15.
Somat Cell Mol Genet ; 17(4): 327-39, 1991 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1653460

RESUMO

We have examined the ability of SV40 to induce changes in drug or temperature resistance in mouse, hamster, and mouse-human hybrid cells. SV40 induced a substantial increase of cells resistant to 5-bromodeoxyuridine + trifluorothymidine in Balb/c 3T3 cells and induced an increase of hybrid cells resistant to 6-thioguanine. SV40 was found to be nonmutagenic or weakly mutagenic in other test systems. The 3T3 cells were T-antigen positive, exhibited a marked reduction in TK activity, were heterogeneous for [3H]BrdU incorporation by autoradiography, and exhibited instability of the drug-resistance phenotype, suggesting that SV40 may be inducing resistance by an epigenetic process. SV40-induced 6-thioguanine resistance in the hybrids appears to occur predominantly by chromosome loss.


Assuntos
Células Híbridas/metabolismo , Células L/patologia , Mutagênese , Ovário/patologia , Vírus 40 dos Símios/genética , Animais , Antígenos Virais de Tumores/análise , Bromodesoxiuridina/metabolismo , Linhagem Celular Transformada/patologia , Cricetinae , Cricetulus , Resistência a Medicamentos/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Temperatura , Tioguanina/farmacologia , Timidina/análogos & derivados , Timidina/farmacologia , Timidina Quinase/metabolismo , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/patologia
16.
Am J Hum Genet ; 48(3): 621-7, 1991 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1998344

RESUMO

To identify CA repeats in genomic sequences which had been previously subcloned into plasmids, we performed PCR using a (CA)n primer and a flanking vector primer on the genomic inserts. By incorporation of a restriction enzyme site into the (CA)n primer, we have been able to subclone the genomic DNA so that the sequence flanking the CA repeat is readily determined. Primers can then be designed to amplify across the CA repeat in patient DNA samples. Application of this technique to genomic DNAs surrounding the upstream "brain" promoter of the dystrophin gene has led to the discovery of four new CA repeats. Three of these repeats are highly polymorphic, with PICs ranging from .586 to .768. The location of these markers at the extreme 5' terminus of the dystrophin gene, together with their high degree of polymorphism and ease of assay, makes them ideal for linkage analysis in families with Duchenne muscular dystrophy.


Assuntos
DNA/química , Distrofina/genética , Ligação Genética , Distrofias Musculares/genética , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Feminino , Marcadores Genéticos , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Plasmídeos , Reação em Cadeia da Polimerase
17.
Proc Natl Acad Sci U S A ; 88(4): 1276-80, 1991 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-1996328

RESUMO

Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is expressed in brain as well as muscle. The role of dystrophin in the brain is not clear, though one-third of Duchenne muscular dystrophy patients exhibit some degree of mental retardation. We have isolated the genomic region encoding the alternative 5' terminus of dystrophin used in the brain. Primer extension and polymerase chain reaction assays on RNA demonstrate that this region contains an alternative promoter for dystrophin used in the brain. Physical mapping of this region indicates that this brain promoter is located greater than 90 kilobases 5' to the promoter used in muscle and 400 kilobases from exon 2 to which it is spliced. The large physical distance between the promoters, taken together with their known tissue selectivities, suggests that in certain patients a deletion of either dystrophin promoter might give rise to reduced dystrophin expression selective to brain or muscle. We have identified one such individual with specific deletion of the dystrophin muscle promoter, giving rise to Becker muscular dystrophy, and we predict that specific loss of the brain promoter may be one cause of X chromosome-linked mental retardation.


Assuntos
Encéfalo/metabolismo , Distrofina/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Elementos Antissenso (Genética) , Sequência de Bases , Linhagem Celular , DNA/genética , DNA/isolamento & purificação , Feminino , Feto , Biblioteca Genômica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Transfecção
19.
Hum Genet ; 86(1): 45-8, 1990 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2253937

RESUMO

We describe oligonucleotide primer sequences that can be used to amplify eight exons plus the muscle promoter of the dystrophin gene in a single multiplex polymerase chain reaction (PCR). When used in conjunction with an existing primer set, these two multiplex reactions detect about 98% of deletions in patients with Duchenne or Becker muscular dystrophy (DMD, BMD). Furthermore, these primers amplify most of the exons in the deletion prone "hot spot" region around exons 44 to 53, allowing determination of deletion endpoints and prediction of mutational effects on the translational reading frame. Thus, use of these PCR-based assays will allow deletion detection and prenatal diagnosis for most DMD/BMD patients in a fraction of the time required for Southern blot analysis.


Assuntos
Deleção Cromossômica , Distrofina/genética , Distrofias Musculares/genética , Sequência de Bases , Éxons , Doenças Fetais/diagnóstico , Humanos , Dados de Sequência Molecular , Distrofias Musculares/diagnóstico , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Diagnóstico Pré-Natal
20.
Science ; 244(4910): 1342-4, 1989 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-2499928

RESUMO

A technique for the transfer of endothelial cells and expression of recombinant genes in vivo could allow the introduction of proteins of therapeutic value in the management of cardiovascular diseases. Porcine endothelial cells expressing recombinant beta-galactosidase from a murine amphotropic retroviral vector were introduced with a catheter into denuded iliofemoral arteries of syngeneic animals. Arterial segments explanted 2 to 4 weeks later contained endothelial cells expressing beta-galactosidase, an indication that they were successfully implanted on the vessel wall.


Assuntos
Endotélio Vascular/citologia , Galactosidases/biossíntese , beta-Galactosidase/biossíntese , Animais , Cateterismo Periférico , DNA Recombinante , Endotélio Vascular/enzimologia , Endotélio Vascular/transplante , Feminino , Vetores Genéticos , Artéria Ilíaca/citologia , Retroviridae , Suínos , Porco Miniatura
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