A novel system for assigning the mode of inheritance in mitochondrial disorders using cybrids and rhodamine 6G.

When normal human cultured skin fibroblasts were treated with the fluorescent dye rhodamine 6G (R6G), there was a drastic reduction in numbers of intact mitochondria and electron transport chain enzyme activities, despite the fact that mitochondrial DNA (mtDNA) was still present in treated cells. We used this observation to develop a novel system for generating cybrids. When cultured skin fibroblast cells from a patient with the mitochondrial encephalopathy and ragged-red fibers (MERRF) syndrome harboring the A8344G mtDNA mutation and which showed a severe reduction in cytochrome c oxidase activity were treated with R6G and fused to enucleated HeLaCOT cells, the resulting cybrid clones showed recovery of cytochrome c oxidase activity, and were shown to have mtDNA derived solely from the HeLaCOT cell line. R6G has significant advantages over ethidium bromide in removing the mitochondrial elements from cultured cells, and the results reported here demonstrate that this strategy can be used to determine the origin of the genetic defect in patients with electron transport chain abnormalities.

[1]  J. Hayashi,et al.  Identification of Inheritance Modes of Mitochondrial Diseases by Introduction of Pure Nuclei from mtDNA-less HeLa Cells to Patient-derived Fibroblasts* , 1997, The Journal of Biological Chemistry.

[2]  G. Attardi,et al.  Relationship of genotype to phenotype in fibroblast-derived transmitochondrial cell lines carrying the 3243 mutation associated with the MELAS encephalomyopathy: shift towards mutant genotype and role of mtDNA copy number. , 1996, Human molecular genetics.

[3]  V. Tiranti,et al.  Nuclear DNA origin of cytochrome c oxidase deficiency in Leigh's syndrome: genetic evidence based on patient's-derived rho degrees transformants. , 1995, Human molecular genetics.

[4]  D. Wallace,et al.  Cytoplasmic transfer of the mtDNA nt 8993 T-->G (ATP6) point mutation associated with Leigh syndrome into mtDNA-less cells demonstrates cosegregation with a decrease in state III respiration and ADP/O ratio. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Anthony H.V. Schapira,et al.  Mitochondrial Disorders in Neurology , 1994 .

[6]  D. Rideout,et al.  Mechanism of inhibition of fadu hypopharyngeal carcinoma cell growth by tetraphenylphosphonium chloride , 1994, International journal of cancer.

[7]  V. Tiranti,et al.  Defective respiratory capacity and mitochondrial protein synthesis in transformant cybrids harboring the tRNA(Leu(UUR)) mutation associated with maternally inherited myopathy and cardiomyopathy. , 1994, The Journal of clinical investigation.

[8]  R. W. Logan,et al.  Malonyl coenzyme A decarboxylase deficiency. , 1993, Archives of disease in childhood.

[9]  J. Cooper,et al.  Nuclear complementation restores mtDNA levels in cultured cells from a patient with mtDNA depletion. , 1993, American journal of human genetics.

[10]  D. Birch,et al.  Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. , 1992, Nucleic acids research.

[11]  S. Dimauro.  Mitochondrial Encephalomyopathies , 1992, Archives of neurology.

[12]  R. Reddel,et al.  Finite life span of hybrids formed by fusion of different simian virus 40-immortalized human cell lines , 1992, Journal of Virology.

[13]  J. Hayashi,et al.  Introduction of disease-related mitochondrial DNA deletions into HeLa cells lacking mitochondrial DNA results in mitochondrial dysfunction. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. King,et al.  Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. , 1989, Science.

[15]  Shirley A. Miller,et al.  A simple salting out procedure for extracting DNA from human nucleated cells. , 1988, Nucleic acids research.

[16]  R. Hull,et al.  Mitochondrial modulation of maternally transmitted antigen: analysis of cell hybrids. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[17]  R. Morais,et al.  Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts , 1985, Molecular and cellular biology.

[18]  M. Melamed,et al.  Rhodamine 123 alters the mitochondrial ultrastructure of cultured L1210 cells. , 1985, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[19]  F. Sanger,et al.  Sequence and organization of the human mitochondrial genome , 1981, Nature.

[20]  A. Gear.  Rhodamine 6G. A potent inhibitor of mitochondrial oxidative phosphorylation. , 1974, The Journal of biological chemistry.

[21]  P. Zuurendonk,et al.  Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver cells. , 1974, Biochimica et biophysica acta.

[22]  B. Kadenbach,et al.  Synthesis of mitochondrial proteins. The synthesis of cytochrome c in vitro. , 1967, Biochimica et biophysica acta.

[23]  D. Wallace,et al.  Production of transmitochondrial mouse cell lines by cybrid rescue of rhodamine-6G pre-treated L-cells , 1996, Somatic cell and molecular genetics.

[24]  D. Wallace,et al.  Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. , 1996, Methods in enzymology.

[25]  A. Chomyn.  In vivo labeling and analysis of human mitochondrial translation products. , 1996, Methods in enzymology.

[26]  J. Cooper,et al.  The structural organization of the mitochondrial respiratory chain , 1994 .

[27]  D. Wallace,et al.  Oxidative Phosphorylation Diseases , 1990 .

[28]  E. Wills,et al.  Electron microscopy in the diagnosis of percutaneous fine needle aspiration specimens. , 1987, Ultrastructural pathology.

[29]  M. Wilson,et al.  Mitochondria: a practical approach. , 1987 .

[30]  R. Ian Freshney,et al.  Culture of Animal Cells , 1983 .

[31]  R. Davidson,et al.  Elimination of mitochondrial elements and improved viability in hybrid cells , 1981, Somatic cell genetics.

[32]  P. Garland,et al.  [2] Citrate synthase from rat liver: [EC 4.1.3.7 Citrate oxaloacetage-lyase (CoA-acetylating)] , 1969 .

[33]  A. Tzagoloff,et al.  [45] Cytochrome oxidase from beef heart mitochondria , 1967 .