Human Molecular Genetics, Vol 5, 197-205, Copyright © 1996 by Oxford University Press
HA Bentlage and G Attardi
Transmitochondrial cell lines were isolated by fusing mtDNA-less rho
degrees 206 cells with enucleated fibroblasts derived from four members of
a pedigree carrying in their muscle varying proportions of the mutation at
position 3243 in the tRNA(Leu(UUR)) gene associated with the MELAS
encephalomyopathy. The mitochondrial transformants derived from an
asymptomatic individual were all homoplasmic for wild-type mtDNA. The
proportion of wild-type transformants derived from clinically affected
members of the pedigree appeared to decrease in correspondence with an
increase in severity of the clinical symptoms of the cell donor.
Furthermore, the average proportion of wild-type mtDNA in the transformants
derived from each member of the pedigree was very similar to that found in
mtDNA from the fibroblasts of that individual, suggesting that the
distribution of genotypes in the transformants reflected fairly closely
that in the fibroblasts. The genotype and phenotype of ten transformants
derived from one severely affected individual were investigated during
continuous culture up to 17-24 weeks after the transformation step. Six
heteroplasmic clones showed a progressive increase in the proportion of
mutant mtDNA, whereas the mitochondrial genotype remained constant in four
clones apparently homoplasmic for wild-type mtDNA or nearly homoplasmic for
mutant mtDNA. An analysis of the rate of repopulation of rho degrees 206
cells with fibroblast-derived mtDNA revealed a large variability among
different transformants, with the full re-establishment of the control
ratio of mtDNA to nuclear DNA being observed between approximately 6 weeks
and more than 22 weeks after the transformation step. An increase in rate
of O2 consumption generally accompanied the increase in mtDNA copy number
of the transformants, pointing to the important role of the mtDNA copy
number in determining the phenotype of a cell. The observation that a very
small amount of wild-type mtDNA (2 to 5% of the control level), coexisting
with strongly predominant mutant mtDNA, conferred upon the transformants a
substantial respiratory capacity (50% or more) and the evidence of
proportionality between O2 consumption rate and mtDNA copy number, which
occurred at widely different mutant to wild-type mtDNA ratios, strongly
suggest a contribution of the mutant mtDNA to the cell respiratory
competence.
ARTICLES
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
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
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