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2.
J Biol Chem ; 276(23): 20719-26, 2001 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-11259421

RESUMO

In vivo studies in the mouse have revealed that the muscle promoter of the mouse dystrophin gene can target the right ventricle of the heart only, suggesting the need for other regulatory elements to target the skeletal muscle as well as other compartments of the heart. In this study we report the identification of the mouse dystrophin gene enhancer that is located approximately 8.5 kilobases downstream from the mouse dystrophin gene muscle promoter. The enhancer was tested in myogenic G8, H9-C2, and nonmyogenic 3T3 cell lines and is mostly active in G8 myotubes. Sequence analysis of the mouse dystrophin gene enhancer revealed the presence of four E-boxes numbered E1-E4, a putative mef-2 binding site, and a serum response element. Site-directed mutagenesis studies have shown that E-boxes 1, 2, and 3 as well as the serum response element are required for enhancer activity. Gel shift analysis revealed two binding activities at binding sites E1 and E3 which were specific to myotubes, and supershift assays confirmed that myoD binds at both these sites. Our study also shows that werum response factor binds the serum response element but in myoblasts and fibroblasts only, suggesting that serum response factor may repress enhancer function.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Distrofina/genética , Elementos Facilitadores Genéticos , Proteína MyoD/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sequência de Bases , Cromossomos Artificiais Bacterianos , DNA , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Homologia de Sequência do Ácido Nucleico , Fator de Resposta Sérica
3.
Muscle Nerve ; 22(1): 16-27, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9883853

RESUMO

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene, a complex gene that generates a family of distinct isoforms. In immature muscle cells, two dystrophin isoforms are expressed, Dp427 and Dp71. To characterize the function of Dp71 in myogenesis, we have examined the expression of Dp71 in myogenic cells. The localization of Dp71 in these cells is distinct from the localization of Dp427. Whereas Dp427 localizes to focal adhesions and surface membrane during myogenesis, Dp71 localizes to stress fiberlike structures in myogenic cells. Biochemical fractionation of myogenic cells demonstrates that Dp71 cosediments with the actin bundles thus confirming this interaction. Furthermore, transfection of C2C12 myoblasts with constructs encoding Dp71 fused to green fluorescent protein targeted the protein to the actin microfilament bundles. These results demonstrate involvement of Dp71 with the actin cytoskeleton during myogenesis and suggest a role for Dp71 that is distinct from Dp427.


Assuntos
Distrofina/análogos & derivados , Distrofina/fisiologia , Músculo Esquelético/metabolismo , Actinas/metabolismo , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Distrofina/análise , Distrofina/genética , Feto , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Microscopia de Fluorescência , Músculo Esquelético/química , Isoformas de Proteínas , Proteínas Recombinantes de Fusão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares , Transfecção
4.
Gene Ther ; 5(10): 1306-13, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9930335

RESUMO

The inhibitory effect of serum is one of the main obstacles to the in vivo use of cationic liposomes as a DNA delivery system. We have found that a novel liposome formulation, DODAC:DOPE (1:1) is totally resistant to the inhibitory effects of serum for transfection of cultured myoblasts and myotubes. Transfection with a lacZ reporter gene in the presence of 95% fetal bovine serum gave up to 25% beta-gal-positive cells in C2C12 myoblasts and about six-fold less in primary human myoblasts. The lower transgene expression in primary cells does not appear to be a result of less DNA uptake but might result from differences in intracellular trafficking of the complexes. DODAC-based liposomes are unique in their resistance to serum inhibition and may therefore be valuable for the systemic delivery of genetic information to muscle and other tissues.


Assuntos
Adjuvantes Imunológicos , Terapia Genética/métodos , Vetores Genéticos , Fosfatidiletanolaminas , Compostos de Amônio Quaternário , Transfecção/métodos , Animais , Linhagem Celular , DNA/metabolismo , Eletroforese em Gel de Ágar , Humanos , Lipossomos , Camundongos , Microscopia de Fluorescência , Músculo Esquelético/metabolismo , beta-Galactosidase/análise , beta-Galactosidase/genética
5.
Nucleic Acids Res ; 25(8): 1618-25, 1997 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-9092671

RESUMO

In previous studies we have described a 5.0 kb Hin dIII fragment downstream of muscle exon 1 that exhibits properties consistent with a muscle-specific transcriptional enhancer. The goal of this study has been to identify the sequence elements responsible for muscle-specific enhancer activity. Functional studies indicated that this enhancer is active in pre- and post-differentiated H9C2(2-1) myoblasts but functions poorly in L6 and C2C12 myotubes. The core enhancer region was delimited to a 195 bp Spe I- Acc I fragment and sequence analysis identified three MEF-1/E box and two MEF-2/AT-rich motifs as potential muscle-specific regulatory domains. EMSA competition and DNase footprinting indicated that sequences within a 30 bp region containing single adjoining MEF-1/E box and MEF-2/AT-rich motifs are target binding sites for trans -acting factors expressed in H9C2(2-1) myotubes but not in L6 or C2C12 myotubes. Site-specific mutations within these motifs resulted in a significant reduction in enhancer activity in H9C2(2-1) myotubes. These results suggest that the mechanisms governing DMD gene expression in muscle are similar to those identified in other muscle-specific genes. However, the myogenic profile of enhancer activity and trans -acting factor binding suggests a more specialized role for this enhancer that is consistent with its potential involvement in dystrophin gene regulation in cardiac muscle.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Distrofina/biossíntese , Distrofina/genética , Elementos Facilitadores Genéticos , Íntrons , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Adenina , Composição de Bases , Sequência de Bases , Sítios de Ligação , Encéfalo/metabolismo , Linhagem Celular , Cerebelo/metabolismo , Primers do DNA , Genes Reporter , Humanos , Fatores de Transcrição MEF2 , Dados de Sequência Molecular , Fatores de Regulação Miogênica , Reação em Cadeia da Polimerase , Células de Purkinje/metabolismo , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Timina , Transcrição Gênica
6.
Hum Mol Genet ; 5(10): 1559-66, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8894689

RESUMO

The dystrophin gene defective in Duchenne muscular dystrophy (DMD) is extreme in size and complexity with several promoters which direct expression of different isoforms in different tissues. In contrast with adult skeletal muscle which expresses 427 kDa dystrophin, fetal muscle tissue expresses the 71 kDa ubiquitous isoform Dp71 as well as 427 kDa muscle dystrophin. To examine Dp71 expression in fetal muscle further, we have monitored its expression pattern in differentiating myogenic cultures of human fetal muscle origin. The presence of transcripts initiated from the Dp71 promoter was demonstrated by quantitative RT-PCR. The level of transcript expressed from the Dp71 promoter did not change significantly during myogenic differentiation, consistent with the housekeeping nature of the promoter. Measurements to determine the stability of the Dp71 mRNA indicated that it has a half-life of -20 h and, therefore, is somewhat more stable than the larger 14 kb muscle dystrophin mRNA (t1/2 = 16 h). In contrast with the constant level of Dp71 transcript during myogenic differentiation, the level of Dp71 protein increased significantly, perhaps due to changes in translation efficiency or protein stability. These results demonstrate expression and posttranscriptional upregulation of Dp71 in human fetal myogenic cultures.


Assuntos
Distrofina/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Músculo Esquelético/metabolismo , Diferenciação Celular , Células Cultivadas , Distrofina/genética , Feminino , Humanos , Músculo Esquelético/embriologia , Distrofias Musculares/genética , Especificidade de Órgãos , Gravidez
7.
Hum Mol Genet ; 5(10): 1599-606, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8894694

RESUMO

The 14 kb muscle isoform of the Duchenne muscular dystrophy (DMD) gene is expressed primarily in skeletal and cardiac muscle. Transcription of the muscle isoform is induced as myoblasts differentiate into multinucleated myotubes and transcript levels are increased a further 10-fold in mature skeletal muscle. In previous studies we have demonstrated that the core muscle promoter of the human DMD gene contains sequences that regulate the induction of DMD gene expression with myoblast differentiation. However, direct injection studies have indicated that the activity of the core muscle promoter in mature skeletal muscle is 30-fold lower than in immature myotubes. This discrepancy between endogenous transcript levels and core promoter activity suggested that additional transcriptional elements are involved in the regulation of DMD gene expression in muscle. In this report we present evidence for the existence of a muscle-specific enhancer within intron 1 of the human DMD gene. Functional analysis of Hindill fragments from within a 36 kb region surrounding muscle exon 1 of the human DMD gene resulted in the identification of a 5.0 kb fragment within muscle intron 1 that consistently provided high levels of reporter gene expression in both immature and mature skeletal muscle. Sequences within this 5 kb fragment were shown to be functionally independent of position and orientation and to be inactive in fibroblasts, properties that are consistent with the definition of a muscle-specific enhancer. Although this enhancer provided a 30-fold increase in transcription from a SV40 viral promoter in mature skeletal muscle, only a 3-fold increase was observed from the DMD core muscle promoter. Intron 1 enhancer activity alone is therefore insufficient to account for the discrepancy between endogenous transcript levels and core muscle promoter activity in immature and mature skeletal muscle and points to the existence of additional enhancer elements in other regions of the DMD gene. This report provides the first evidence for the involvement of a transcriptional enhancer in DMD gene regulation in muscle and impacts on our understanding of the functional consequences of mutations at the 5'-end of gene. In this regard, deletions in this region in X-linked dilated cardiomyopathy patients provides indirect evidence for a role for this enhancer in regulating DMD gene expression in cardiac muscle.


Assuntos
Distrofina/genética , Elementos Facilitadores Genéticos/genética , Genoma Humano , Íntrons/genética , Distrofias Musculares/genética , Humanos , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Análise de Sequência , Transcrição Gênica
8.
Nucleic Acids Res ; 24(15): 3059-64, 1996 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-8760894

RESUMO

The human dystrophin gene has 79 exons spanning >2300 kb making it the largest known gene. In previous studies we showed that approximately 16 h are required to transcribe the gene in myogenic cultures [Tennyson, C.N., Klamut, H.J. and Worton, R.G. (1995) Nature Genet. 9, 184-190]. To estimate the half-life of the dystrophin mRNA, the decay of the transcript was monitored by quantitative RT-PCR in cultured human fetal myotubes following exposure to actinomycin D. Results from this analysis indicated that the half-life of the dystrophin mRNA is 15.6 +/- 2.8 h in these cultures. Transcript accumulation profiles were predicted using a mathematical model which incorporated the measured half-life. The modeled accumulation profiles were consistent with observed profiles supporting the half-life measured using actinomycin D. The kinetic model was then used to predict the relative amount of nascent and mature dystrophin transcript at steady state. Measurements by quantitative RT-PCR indicated that in adult skeletal muscle tissue the concentration of mature dystrophin mRNA is 5-10 molecules per nucleus, demonstrating, as expected, that it is a low abundance transcript. Furthermore the ratio of nascent to mature dystrophin transcript indicated that dystrophin synthesis may not be at steady state in the adult skeletal muscle we tested. Alternatively, the kinetics of transcript production in skeletal muscle tissue may be different from those observed in cultured fetal myogenic cells.


Assuntos
Distrofina/genética , Músculos/metabolismo , RNA Mensageiro/metabolismo , Adulto , Sequência de Bases , Células Clonais , Dactinomicina/farmacologia , Feto/citologia , Meia-Vida , Humanos , Cinética , Dados de Sequência Molecular , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculos/citologia , Reação em Cadeia da Polimerase , Processamento Pós-Transcricional do RNA , Transcrição Gênica/efeitos dos fármacos
9.
Gene Ther ; 3(5): 396-404, 1996 May.
Artigo em Inglês | MEDLINE | ID: mdl-9156800

RESUMO

Cationic liposomes provide a means to introduce genes into cells both ex vivo and in vivo. In the past few years their use has been described in several tissues, e.g. lungs, liver, endothelium, brain. In this study we evaluated a commercially available poly-cationic liposome formulation in delivering a reporter gene into cultured myogenic cell lines from mouse and rat, and primary fetal human myoblasts. We also examined the effect of serum on liposome-mediated transfection and designed a new procedure to enhance transfection efficiency, based on the pre-condensation of plasmid DNA with polylysine. Polylysine pre-condensation was particularly effective when transfecting the cells in the presence of serum, a finding that could be significant for in vivo transfections.


Assuntos
DNA/genética , Técnicas de Transferência de Genes , Músculo Esquelético , Plasmídeos/genética , Animais , Células Cultivadas , DNA/ultraestrutura , Sistemas de Liberação de Medicamentos , Humanos , Lipossomos , Camundongos , Polilisina , Ratos
10.
Nat Genet ; 9(2): 184-90, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7719347

RESUMO

The largest known gene is the human dystrophin gene, which has 79 exons spanning at least 2,300 kilobases (kb). Transcript accumulation was monitored from four regions of the gene following induction of expression in muscle cell cultures. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) results indicate that approximately 12 h are required for transcription of 1,770 kb (at an average elongation rate of 2.4 kb min-1), extrapolating to a transcription time of 16 h for the complete gene. Accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5' end before transcription is complete providing strong evidence for cotranscriptional splicing. The rate of transcript accumulation was reduced at the 3' end of the gene relative to the 5' end, perhaps due to premature termination of transcription complexes.


Assuntos
Distrofina/genética , Transcrição Gênica , Animais , Sequência de Bases , Diferenciação Celular , DNA Recombinante , Éxons , Expressão Gênica , Humanos , Dados de Sequência Molecular , Músculos/citologia , Reação em Cadeia da Polimerase/métodos , Fatores de Tempo
13.
CMAJ ; 148(9): 1455, 1993 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-8477364
15.
Am J Hum Genet ; 51(3): 562-70, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1496988

RESUMO

In a previous study we identified 14 cases with Duchenne muscular dystrophy (DMD) or its milder variant, Becker muscular dystrophy (BMD), with a deletion of exons 3-7, a deletion that would be expected to shift the translational reading frame of the mRNA and give a severe phenotype. We have examined dystrophin and its mRNA from muscle biopsies of seven cases with either mild or intermediate phenotypes. In all cases we detected slightly lower-molecular-weight dystrophin in 12%-15% abudance relative to the normal. By sequencing amplified mRNA we have found that exon 2 is spliced to exon 8, a splice that produces a frameshifted mRNA, and have found no evidence for alternative splicing that might be involved in restoration of dystrophin mRNA reading frame in the patients with a mild phenotype. Other transcriptional and posttranscriptional mechanisms such as cryptic promoter, ribosomal frameshifting, and reinitiation are suggested that might play some role in restoring the reading frame.


Assuntos
Deleção Cromossômica , Distrofina/genética , Mutação da Fase de Leitura/genética , Distrofias Musculares/genética , Adolescente , Adulto , Sequência de Bases , Southern Blotting , Western Blotting , Criança , Pré-Escolar , Distrofina/análise , Humanos , Masculino , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Reação em Cadeia da Polimerase
16.
Genomics ; 13(4): 1247-54, 1992 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1354642

RESUMO

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.


Assuntos
Aminoácidos/genética , Canais de Cálcio/genética , Hipertermia Maligna/genética , Proteínas Musculares/genética , Polimorfismo de Fragmento de Restrição , Receptores Colinérgicos/genética , Sequência de Bases , DNA , Eletroforese em Gel de Poliacrilamida , Feminino , Haplótipos , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Canal de Liberação de Cálcio do Receptor de Rianodina
17.
Hum Mutat ; 1(1): 3-12, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1301188

RESUMO

Tandem duplication of large regions of DNA, including duplication of whole genes, provides a substrate for genetic evolution. Tandem duplication of smaller regions involving parts of genes is now recognized as a contributor to the mutation spectrum that results in genetic disease. In this review, more than 30 unrelated partial gene duplications that have been implicated in the genesis of human genetic disease are presented and the pathogenic effects and frequency of such duplications are summarized. The mechanisms of duplication formation are analyzed with special emphasis on the molecular details of the nucleotide sequences at the duplication junctions. Evidence to date suggests that duplication may arise from either homologous (Alu-Alu) recombination or nonhomologous recombination, the latter possibly mediated by topoisomerases. For the dystrophin gene, in which most duplications have been identified, these recombination events are intrachromosomal, suggesting that unequal sister chromatid exchange is the major mechanism.


Assuntos
Doenças Genéticas Inatas/genética , Família Multigênica , Evolução Biológica , DNA/genética , Genética Médica , Humanos , Modelos Genéticos , Mutação
19.
J Inherit Metab Dis ; 15(4): 539-50, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1528015

RESUMO

The X-linked gene responsible for Duchenne muscular dystrophy encodes dystrophin, a high-molecular-weight cytoskeletal protein. Studies in several laboratories have revealed deletion of one or more exons in 60% of affected boys; quantitative analysis in our laboratory has detected duplication of exons in another 6%. The severe Duchenne phenotype is associated with deletions or duplications that shift the reading frame of the message, whereas the milder Becker muscular dystrophy is associated with deletions or duplications that maintain the reading frame. Patients who have neither deletion nor duplication may have nonsense mutations, one of which has been detected by predicting the site of the mutation from the size of the truncated protein. Rare females with the disease have a translocation that disrupts the dystrophin gene on one X chromosome and causes non-random inactivation of the normal X, resulting in the expression of the disease. The high frequency of new mutation provides an opportunity to study the mechanism of chromosomal rearrangement that is characteristic of the disease. Our laboratory has focused on the translocations in females and on duplications in affected males. The X-autosome translocations of affected females are all de novo events that originated in the paternal set of chromosomes. Molecular characterization of the translocation junctions revealed reciprocal translocation with both deletion and addition of nucleotides at the junction, suggestive of a breakage and reunion mechanism. Duplications studied to date are all tandem in nature and sequence analysis of duplication junctions has revealed both homologous and non-homologous recombination. Marker segregation analysis has revealed that five out of five duplications originated in a single X chromosome of one of the maternal grandparents, suggesting that the recombination event is unequal sister chromatid exchange.


Assuntos
Distrofias Musculares/genética , Mutação , Aberrações Cromossômicas , Distrofina/genética , Feminino , Humanos , Masculino , Cromossomo X
20.
Symp Soc Exp Biol ; 46: 189-201, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1341035

RESUMO

Malignant hyperthermia (MH) is an inherited, potentially lethal condition in which sustained muscle contracture with attendant hypermetabolism and hyperthermia is triggered in humans, heterozygous for the gene defect, by inhalational anaesthetics and skeletal muscle relaxants, and in pigs, homozygous for the defect, by stress. Because muscle contracture could result from a defective Ca2+ release channel, we have focussed our attention on the linkage of MH to defects in the gene (RYR1) encoding the skeletal muscle Ca2+ release channel. We have cloned and sequenced human RYR1 cDNA and found restriction fragment length polymorphisms (RFLPs) in the human gene. We also localized RYR1 to human chromosome 19q13.1. Studies of the cosegregation of MH with these RFLPs established RYR1/MH linkage on human chromosome 19q13.1 (lod score of 4.2; recombinant fraction 0.0). We then sequenced MH and normal porcine RYR1 cDNAs. Mutation of C1843 to T, leading to substitution of Cys for Arg615, was the sole amino acid change noted between MH and normal animals. Linkage of this mutation to MH was established in a study of 338 informative meioses (lod score of 102; recombinant fraction 0.0). We identified the corresponding mutation in 1 of 35 human MH families studied and found cosegregation of the mutation and MH. The combination of a high lod score with crossing of a species barrier supports the causal nature of this mutation. Future studies are aimed at finding the major human MH mutations and establishing assays for their accurate diagnosis.


Assuntos
Canais de Cálcio/genética , Hipertermia Maligna/genética , Proteínas Musculares/genética , Músculos/química , Sequência de Aminoácidos , Canais de Cálcio/química , Canais de Cálcio/fisiologia , Ligação Genética , Humanos , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina
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