Trends in Genetics, 2003

Mitochondrion, 2003

The role of nuclear DNA (n DNA)-encoded proteins in the regulation of mitochondrial fission and fusion has been documented, yet the role of mitochondrial DNA (mt DNA) and encoded proteins in mitochondrial biogenesis remains unknown. Long-term treatment of a lymphoblastoid cell line Molt-4 with ethidium bromide generated mt DNA-deficient rho 0 mutants. Depletion of mt DNA in rho 0 cells produced functional and morphological changes in mitochondria without affecting the nuclear genome and encoded proteins. Indeed, the gene encoding subunit II of mitochondrial cytochrome c oxidase (COX II), a prototypical mitochondrial gene, was reduced in rho 0 mutants blunting the activity of mitochondrial cytochrome coxidase. Yet, the amount of the nuclear b-actin gene and the activity of citrate synthase, a mitochondrial matrix enzyme encoded by n DNA, remained unaffected in rho 0 cells. Loss of mt DNA in rho 0 cells was associated with significant distortion of mitochondrial structure, decreased electron density of the matrix and disorganized inner and outer membranes, resulting in the appearance of 'ghost-like' mitochondria. However, the number of mitochondria-like structures was not significantly different between mt DNA-deficient and parental cells. Thus, we conclude that cells lacking mt DNA still generate mitochondrial scaffolds, albeit with aberrant function.

American Journal of Physiology-cell Physiology, 2006

The past two decades have witnessed an evolving understanding of the mitochondrial genome's (mtDNA) role in basic biology and disease. From the recognition that mutations in mtDNA can be responsible for human disease to recent efforts showing that mtDNA mutations accumulate over time and may be responsible for some phenotypes of aging, the field of mitochondrial genetics has greatly benefited from the creation of cell and animal models of mtDNA mutation. In this review, we critically discuss the past two decades of efforts and insights gained from cell and animal models of mtDNA mutation. We attempt to reconcile the varied and at times contradictory findings by highlighting the various methodologies employed and using human mtDNA disease as a guide to better understand cell and animal mtDNA models. We end with a discussion of scientific and therapeutic challenges and prospects for the future of mtDNA transfection and gene therapy.

Human Molecular Genetics, 2006

Generation of various kinds of trans-mitochondrial mice, mito-mice, each carrying mtDNAs with a different pathogenic mutation, is required for precise investigation of the pathogenesis of mitochondrial diseases. This study used two respiration-deficient mouse cell lines as donors of mtDNAs with possible pathogenic mutations. One cell line expressed 45-50% respiratory activity due to mouse mtDNAs with a T6589C missense mutation in the COI gene (T6589C mtDNA) and the other expressed 40% respiratory activity due to rat (Rattus norvegicus) mtDNAs in mouse cells. By cytoplasmic transfer of these mtDNAs to mouse ES cells, we isolated respiration-deficient ES cells. We obtained chimeric mice and generated their F 6 progeny carrying mouse T6589C mtDNAs by its female germ line transmission. They were respiration-deficient and thus could be used as models of mitochondrial diseases caused by point mutations in mtDNA structural genes. However, chimeric mice and mito-mice carrying rat mtDNAs were not obtained, suggesting that significant respiration defects or some deficits induced by rat mtDNAs in mouse ES cells prevented their differentiation to generate mice carrying rat mtDNAs.

Journal of Biological Chemistry, 2013

Background: Nonviability of cells lacking mitochondrial DNA ligase suggests essential function of this enzyme. Results: We report the isolation of viable Lig3 Ϫ/Ϫ cells, which lack mtDNA. Conclusion: The lethality of the Lig3 knockout is mediated by the 0 phenotype. Significance: This is definitive proof that the essential function of LIG3 in mitochondria is limited to DNA transactions. Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knockout observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3deficient cells is conditional and secondary to the 0 phenotype. In mammalian cells, there are two DNA-containing organelles, the nucleus and the mitochondrion. Human mitochondrial DNA (mtDNA) is a 16,568-bp circular molecule (1) crucial for proper mitochondrial function and cellular ATP production. It encodes 13 proteins as follows: 11 polypeptide subunits of the mitochondrial electron transport chain complexes I, III, and IV and two subunits of the ATP synthase, complex V. These polypeptides are encoded using a genetic code distinct from that used in the nucleus and therefore require a separate translational apparatus, some components of which (22 tRNAs and 2 rRNAs) also are encoded in mtDNA (2). Therefore, it comes as no surprise that the loss of mtDNA integrity either through mutation or depletion may lead to serious, often fatal diseases (3, 4). The mitochondrial electron transport chain is a major cellular source for the production of reactive oxygen species. Although the exact rates of mitochondrial reactive oxygen

Proceedings of the National Academy of Sciences of the United States of America, 2022

Nucleic acids research, 2015

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic ...

American journal of human genetics, 1992

Current opinion in genetics & …, 2003