Synthesis of mitochondrial DNA in permeabilised human cultured cells.
The mechanisms that underlie the maintenance of and increase in mutant mitochondrial DNA (mtDNA) are central to our understanding of mitochondrial disease. We have therefore developed a technique based on saponin permeabilisation that allows the study of mtDNA synthesis in intact cells. Permeabilisation of cells has been extensively used in an established method both for studying transcription and DNA replication in the nucleus and for measuring respiratory chain activities in mitochondria. We have quantitatively studied incorporation of radiolabelled DNA precursors into mtDNA in human cell lines derived from controls and from patients with mitochondrial DNA disease. Total cell DNA is extracted, restriction digested and Southern blotted, newly synthesised mtDNA being proportional to the label incorporated in each restriction band. A rate of synthesis can then be derived by estimating the relative steady-state mtDNA after probing with full-length mtDNA. Where co-existing mutant and wild-type mtDNA (heteroplasmy) can be distinguished using restriction digestion, their rates of synthesis can be compared within a single cell line. This will be particularly useful in elucidating the pathophysiology of mtDNA diseases in which the distribution of mutant and wild-type mtDNA in cell lines in patient tissues may evolve with time.
Emmerson
C F
CF
Department of Paediatrics and Department of Biochemistry, University of Oxford, UK.
Brown
G K
GK
Poulton
J
J
D018929:Cell Culture Techniques / Q000379:methods; D002461:Cell Line, Transformed; D002463:Cell Membrane Permeability / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004272:DNA, Mitochondrial / Q000096:biosynthesis* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006822:Hybrid Cells; D017241:MELAS Syndrome / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D017354:Point Mutation; D011544:Pseudogenes; D015203:Reproducibility of Results; D012503:Saponins; D014407:Tumor Cells, Cultured
D004272:DNA, Mitochondrial; D009711:Nucleotides; D010761:Phosphorus Radioisotopes; D012503:Saponins