RESUMEN
In this article we develop a model for the organization and maintenance of mitochondrial DNA (mtDNA) in mammalian somatic cells, based on the idea that the unit of genetic function comprises a group of mtDNA molecules that are semi-permanently associated as a mitochondrial nucleoid. Different mtDNA molecules within a nucleoid need not be genetically identical. We propose that nucleoids replicate faithfully via a kind of mitochondrial mitosis, generating daughter nucleoids that are identical copies of each other, but which can themselves segregate freely. This model can account for the very slow rates of mitotic segregation observed in cultured, heteroplasmic cell-lines, and also for the apparently poor complementation observed between different mutant mtDNAs co-introduced into rho(0) cells (cells that lack endogenous mtDNA). It also provides a potential system for maintaining the mitochondrial genetic fitness of stem cells in the face of a presumed high somatic mutation rate of mtDNA and many rounds of cell division in the absence of phenotypic selection. BioEssays 22:564-572, 2000.
Asunto(s)
ADN Mitocondrial/genética , Modelos Genéticos , Animales , Evolución Biológica , Mamíferos/genéticaRESUMEN
The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3'-5' (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.
Asunto(s)
ADN Mitocondrial/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Mitocondrias/enzimología , Sustitución de Aminoácidos , Secuencia de Bases , Línea Celular , ADN Polimerasa gamma , ADN Mitocondrial/química , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Partículas Submitocóndricas/enzimología , Transfección , Repeticiones de TrinucleótidosRESUMEN
The mitochondrial genotype of heteroplasmic human cell lines containing the pathological np 3243 mtDNA mutation, plus or minus its suppressor at np 12300, has been followed over long periods in culture. Cell lines containing various different proportions of mutant mtDNA remained generally at a consistent, average heteroplasmy value over at least 30 wk of culture in nonselective media and exhibited minimal mitotic segregation, with a segregation number comparable with mtDNA copy number (>/=1000). Growth in selective medium of cells at 99% np 3243 mutant mtDNA did, however, allow the isolation of clones with lower levels of the mutation, against a background of massive cell death. As a rare event, cell lines exhibited a sudden and dramatic diversification of heteroplasmy levels, accompanied by a shift in the average heteroplasmy level over a short period (<8 wk), indicating selection. One such episode was associated with a gain of chromosome 9. Analysis of respiratory phenotype and mitochondrial genotype of cell clones from such cultures revealed that stable heteroplasmy values were generally reestablished within a few weeks, in a reproducible but clone-specific fashion. This occurred independently of any straightforward phenotypic selection at the individual cell-clone level. Our findings are consistent with several alternate views of mtDNA organization in mammalian cells. One model that is supported by our data is that mtDNA is found in nucleoids containing many copies of the genome, which can themselves be heteroplasmic, and which are faithfully replicated. We interpret diversification and shifts of heteroplasmy level as resulting from a reorganization of such nucleoids, under nuclear genetic control. Abrupt remodeling of nucleoids in vivo would have major implications for understanding the developmental consequences of heteroplasmy, including mitochondrial disease phenotype and progression.
Asunto(s)
ADN Mitocondrial/genética , Mutación , Selección Genética , Secuencia de Bases , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Cartilla de ADN , Dimetilsulfóxido/farmacología , Genotipo , Humanos , Fenotipo , Células Tumorales CultivadasRESUMEN
Long-extension PCR (LX-PCR), followed by Southern hybridization to probes for two different regions of the mitochondrial genome, was used to evaluate the presence of deleted mtDNA molecules in heart muscle samples from alcoholic cardiomyopathy patients compared with age-matched controls. Two different primer pairs capable of amplifying the entire genome, as well as a variety of other primer pairs predicted to amplify the genome in large, overlapping fragments, were tested. Products indicating the presence of a variety of subgenomic, deleted molecules were detected in variable amounts from patient and control myocardial samples alike. Most of these hybridized with a probe for the 16S/ND1 region, but not with a probe for the ND4/ND5 region that is commonly deleted. Dilution of a given template DNA in which deleted products were prominent resulted in the disappearance of the subgenomic bands in favour of the full-length, undeleted product. Therefore, the appearance and amount of such products is subject to template concentration or quality. The results indicate that the application of LX-PCR to the detection and quantitation of deleted mtDNAs is inherently unreliable, and findings using this technique should be treated with caution unless supported by an independent method.
Asunto(s)
Artefactos , Cardiomiopatía Alcohólica/genética , ADN Mitocondrial/química , ADN Mitocondrial/genética , Reacción en Cadena de la Polimerasa/métodos , Eliminación de Secuencia , Adulto , Anciano , Secuencia de Bases , Southern Blotting/métodos , Mapeo Cromosómico , Cartilla de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias Musculares/química , Mitocondrias Musculares/metabolismo , Valores de Referencia , Reproducibilidad de los ResultadosRESUMEN
In cybrid cells carrying the mitochondrial A3243G MELAS mutation, which were also heteroplasmic for the G12300A suppressor mutation, we observed a transient episode of heteroplasmic instability, resulting in a wide diversification in G12300A heteroplasmy levels and a shift in the average heteroplasmy level from 11 to 29%. These cells were found to be trisomic for chromosome 9, whereas a minority of cells that retained disomy-9 showed no instability. Coculture experiments implied that trisomy-9 cells exhibited a significant growth advantage, but neither heteroplasmy levels, respiratory phenotype nor trisomy-9 itself had direct selective value under standard culture conditions. Mitochondrial nucleoid number was the same (50-100) in cells that had or had not experienced transient heteroplasmic instability, but 1-2 orders of magnitude less than the segregation number in such cells. These findings support the idea that mtDNA partition is under nuclear genetic control, and implicate a locus on chromosome 9 in this regulation.
Asunto(s)
Cromosomas Humanos Par 9 , Trisomía , Células Cultivadas , Técnicas de Cocultivo , ADN Mitocondrial/genética , Humanos , Hibridación Fluorescente in Situ , Síndrome MELAS/genética , Mutación , Reacción en Cadena de la PolimerasaRESUMEN
We have investigated the representation of structural isoforms of the two mitochondrial leucyl tRNAs in lung carcinoma cybrid cell lines containing the np 3243 (MELAS) mtDNA mutation, alone or in combination with the np 12300 suppressor mutation. The mutant tRNALeu(UUR) is aminoacylated very poorly or not at all, whereas the suppressor tRNALeu(CUN) is efficiently aminoacylated. Deacylated mitochondrial tRNALeu(CUN) is present, in all human cells tested, in two structural isoforms that are separable on denaturing gels, indicating a difference in primary structure. The ratio of the two isoforms differs between cell types and is strongly biased towards one isoform in lung carcinoma cybrids containing high levels of the np 3243 mutation, compared with control cybrids. We propose that structural modification of tRNALeu(CUN) could be a natural suppression mechanism for the np 3243 and other mitochondrial tRNALeu(UUR) mutations and could underlie some of the phenotypic variability of np 3243 disease.
Asunto(s)
ADN Mitocondrial/genética , Mitocondrias/genética , ARN de Transferencia de Leucina/genética , Acilación , Anticodón/genética , Anticodón/metabolismo , Secuencia de Bases , Humanos , Neoplasias Pulmonares/patología , Mutación Puntual , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia de Leucina/análisis , Supresión Genética , Aminoacilación de ARN de Transferencia/genética , Células Tumorales CultivadasRESUMEN
Mitochondrial mutations are associated with a wide spectrum of human diseases. A common class of point mutations affects tRNA genes, and mutations in the tRNA-leu(UUR) gene (MTTL1) are the most frequently detected. In earlier studies, we showed that lung carcinoma cybrid cells containing high levels (greater than 95%) of mutated mtDNA from a patient with the pathological nucleotide pair (np) 3243 tRNA-leu(UUR) mutation can remain genotypically stable over time, and exhibit severe defects in mitochondrial respiratory metabolism. From such a cybrid containing 99% mutated mtDNA, we have isolated a spontaneous derivative that retains mutant mtDNA at this level but which has nevertheless reverted to the wild-type phenotype, based on studies of respiration, growth in selective media, mitochondrial protein synthesis and biogenesis of mitochondrial membrane complexes. The cells are heteroplasmic for a novel anticodon mutation in tRNA-leu(CUN) at np 12300, predicted to generate a suppressor tRNA capable of decoding UUR leucine codons. The suppressor mutation represents approximately 10% of the total mtDNA, but was undetectable in a muscle biopsy sample taken from the original patient or in the parental cybrid. These results indicate that the primary biochemical defect in cells with high levels of np 3243 mutated mtDNA is the inability to translate UUR leucine codons.
Asunto(s)
Mitocondrias/genética , ARN de Transferencia de Leucina/genética , Anticodón/genética , Anticodón/fisiología , Northern Blotting , Análisis Mutacional de ADN , ADN Mitocondrial/análisis , ADN Mitocondrial/genética , ADN Mitocondrial/aislamiento & purificación , Humanos , Fenotipo , Mutación Puntual/genética , Mutación Puntual/fisiología , Reacción en Cadena de la Polimerasa , ARN de Transferencia de Leucina/análisis , ARN de Transferencia de Leucina/fisiología , Supresión Genética/fisiología , Células Tumorales CultivadasRESUMEN
Terminal differentiation of myocytes involves withdrawal from the cell cycle, induction of myogenin expression, and finally formation of myotubes. To study the factors that regulate the initial phase of muscle differentiation, we analyzed the binding activities of transcription factors AP-1, Sp-1, and NF-kappa B in L6, C2C12, and rhabdomyosarcoma BA-Han-1C cells. Temporal changes in transcription factor binding activities were compared to the activation of myogenin promoter-driven CAT reporter gene and the expression level of myogenin, a master gene of myogenic differentiation. We observed a prominent decrease in the nuclear binding activities of AP-1, Sp-1, and NF-kappa B already 12 to 24 h after the transfer of cells to differentiation medium. The response was very similar in L6 and C2C12 myocytes and in BA-Han-1C rhabdomyosarcoma cells. The down-regulation clearly preceded the activation of myogenin promoter and the induction of myogenin and retinoblastoma expression, as well as the initiation of myocyte fusion. Cholera toxin and okadaic acid, established inhibitors of myogenin expression and muscle differentiation, strongly up-regulated the binding activities of AP-1, Sp-1, and NF-kappa B in differentiation medium. Myogenin expression and myocyte fusion were also inhibited. Levels of nuclear c-Fos and c-Jun proteins, components of the AP-1 complex, showed a prominent decrease already after 12 h in differentiation medium. These results show that the down-regulation of the proliferation-promoting transcription factors is a prerequisite to the initiation of myocyte differentiation.
Asunto(s)
Regulación hacia Abajo , Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , FN-kappa B/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Diferenciación Celular , Fusión Celular , Toxina del Cólera/farmacología , Ratones , Músculo Esquelético/citología , Miogenina/biosíntesis , Ácido Ocadaico/farmacología , Unión Proteica , Ratas , Proteína de Retinoblastoma/biosíntesis , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Functional capacity of mitochondria declines during aging and this impairment may have a major role in aging process. Several observations indicate that transcriptional efficiency is reduced during aging. Our purpose was to find out whether aging and cellular senescence affect the nuclear binding activities of transcription factors which bind to OXBOX-REBOX sequence present in promoter regions of numerous nuclear genes encoding mitochondrial proteins. These factors regulate and coordinate the expression of mitochondrial proteins. We observed a strong down-regulation in the nuclear binding activities of OXBOX-REBOX factors in replicatively senesced human WI-38 and IMR-90 fibroblasts. On the contrary, SV-40 immortalization highly increased the nuclear binding activities. A considerable down-regulation of OXBOX-REBOX factors was also observed in UVB-irradiated WI-38 fibroblasts. Irradiation induced photoaging in fibroblasts which involved cell cycle arrest and senescent morphology. Interestingly, the nuclear binding activities of OXBOX-REBOX factors were also prominently decreased in the liver of Wistar rats at the age of 30 months but not yet at the age of 18 months. Our results suggest that the down-regulation of OXBOX-REBOX factors could affect the expression level of mitochondrial proteins encoded in nucleus and hence induce disturbances in mitochondrial function and promote the cellular aging process.
Asunto(s)
Senescencia Celular , Regulación hacia Abajo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Línea Celular Transformada , ADN , Humanos , Datos de Secuencia Molecular , Unión Proteica , Ratas , Ratas WistarRESUMEN
Both the aging of animals and the senescence of cultured cells involve an altered pattern of gene expression, suggesting changes in transcription factor regulation. We studied age-related changes in transcription factors nuclear factor (NF)-kappa B, activator protein factor-1 (AP-1) and Sp-1 by using electrophoretic mobility shift binding assays; we also analysed changes in the protein components of NF-kappa B complex with Western blot assays. Nuclear and cytoplasmic extracts were prepared from heart, liver, kidney and brain of young adult and old NMRI mice and Wistar rats as well as from presenescent, senescent and simian virus 40-immortalized human WI-38 fibroblasts. Aging of both mice and rats induced a strong and consistent increase in the nuclear binding activity of NF-kappa B factor in all tissues studied, whereas those of AP-1 and Sp-1 decreased, e.g. in liver. Protein levels of p50, p52 and p65 components of the NF-kappa B complex did not show any age-associated changes in the cytoplasmic fraction but in the nuclear fraction the level of p52 strongly increased in heart and liver during aging. The protein levels of inhibitory I kappa B-alpha and Bcl-3 components were not affected by aging in any of the tissues studied. Replicative cellular senescence of human WI-38 fibroblasts induced a strong decrease in nuclear NF-kappa B, AP-1 and Sp-1 binding activities. Protein levels of p50 and p52 components of NF-kappa B complex were decreased in the nuclear fraction of senescent WI-38 fibroblasts but in the cytoplasm of senescent fibroblasts the level of p65 protein was increased. Cellular senescence also slightly decreased the protein levels of I kappa B-alpha and Bcl-3. Transfection assays with NF-kappa B-enhancer-driven chloramphenicol acetyltransferase reporter gene showed a significant down-regulation of NF-kappa B promoter activity in senescent WI-38 fibroblasts. Results suggest that the aging process might be regulated differently in tissues and cultured fibroblasts, perhaps reflecting differences between mitotic and post-mitotic cells. In tissues, aging seems to involve specific changes in the regulation of NF-kappa B components and perhaps also changes in the DNA-binding affinities of the NF-kappa B complex.
Asunto(s)
Envejecimiento/metabolismo , Senescencia Celular , Regulación del Desarrollo de la Expresión Génica , Hígado/metabolismo , Miocardio/metabolismo , FN-kappa B/biosíntesis , Animales , Western Blotting , División Celular , Línea Celular , Línea Celular Transformada , Femenino , Corazón/crecimiento & desarrollo , Humanos , Hígado/crecimiento & desarrollo , Pulmón , Masculino , Ratones , Ratas , Ratas Wistar , Proteínas Recombinantes/biosíntesis , Virus 40 de los Simios , TransfecciónRESUMEN
The accumulation of lipofuscin to cardiomyocytes is a classical parameter of aging and is believed to reflect oxidative stress. NF-kB transcription factor complex is one of the cellular sensors which responds to oxidative stress and regulates gene expression. Our purpose was to study whether aging affects the level and distribution of DNA binding activities of NF-kB transcription factors both in cardiac sarcoplasm and nuclear extracts. We used electrophoretic mobility shift assays (EMSA) to characterize the DNA binding activities of NF-kB and two other transcription factors. AP-1 and Sp-1, in the myocardium of 4 months and 24 months old male and female NMRI-mice. The protein levels of p50, p52, and p65 components of NF-kB-complex and an inhibitory IkB-alpha/MAD-3 were assayed with Western blots. Surprisingly, aging upregulated by 123% the nuclear NF-kB binding activity in the male and female mice. The sarcoplasmic NF-kB activity, activated by deoxycholate, did not show any change during aging. Aging-induced increase in nuclear NF-kB protein-DNA binding activity was observed both by gel retardation and UV-crosslinking assays. In immunoblotting, the level of p52 component but not those of p50 and p65 components of NF-kB-complex was slightly increased in nuclear fractions. Aging did not affect the sarcoplasmic levels of p50, p52, and p65 proteins. Supershift EMSA assays showed that the nuclear NF-kB complex contained p50, p52, and p65 components. The level of inhibitory IkB-alpha/MAD-3 protein was unaffected by aging both in nuclear and sarcoplasmic fractions. Aging down-regulated the nuclear Sp-1 binding activities but did not affect AP-1 binding activities. Statistically significant sex-related differences did not appear in the aging responses of transcription factors. These results indicate that NF-kB transcription factor pathway is activated during aging in cardiac muscle and could be the signaling route regulating gene expression. However, the activation mechanism of NF-kB during aging whether oxidative stress responsive or not in vivo needs further studies.