After the introduction of mitochondria with a mixture of mutant and wild-type mitochondrial DNA (mtDNA) into a human p0 cell line (143B.206), Yoneda et al. [Yoneda, M., Chomyn, A., Martinuzzi, A., Hurko, 0. & Attardi, G. (1992) Proc. Natl. Acad. Sci. USA 89, [11164][11165][11166][11167][11168] observed a shift in the proportion of the two mitochondrial genotypes in a number of cybrid clones. In every case where a shift was observed, there was an increase in the proportion of mutant mtDNA. By using the same cell line (143B.206 p°), we also generated cybrids that were either stable in their mitochondrial genotype or showed an increase in the proportion of mutant mtDNA. However, temporal analysis of the same mutant mtDNA type in another p0 cell line revealed a quite distinct outcome. Those clones that showed a change shifted toward higher levels of wild-type rather than mutant mtDNA. These results indicate that the nuclear genetic background of the recipient (p0) cell can influence the segregation of mutant and wild-type mitochondrial genomes in cell cybrids.Disease-associated mitochondrial DNA (mtDNA) mutations were first recognized in 1988 (1). These mutations were large deletions, accounting for 15-50% of the 16.6-kb genome. All the patients showed the proliferation of skeletal muscle mitochondria characteristic of mitochondrial myopathy (2), and in each case, the partially deleted genomes were found to coexist with apparently wild-type genomes. Subsequently, a number of disease-associated mtDNA point mutations were identified (3-6). One of these, an A -> G point mutation in the mitochondrial tRNALeU(UUR) gene at bp 3243, accounts for the majority of cases of MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) (6). As with partial deletions of mtDNA, wild-type mtDNA molecules coexisted with the mutant molecules.A human cell line devoid of endogenous mtDNA (p°) was generated in 1989 (7) and was used to provide evidence that mitochondrial genomes with the bp-3243 mutation caused respiratory deficiency (8, 9). Mitochondria from patients with MELAS were introduced into p0 cells and cybrid cell lines were established. Clonal cell lines with high levels of mutant mtDNA showed decreased in vitro mitochondrial translation products and whole-cell oxygen consumption.Yoneda et al. (10) have described clonal cybrid lines that were derived from the fusion of cytoplasts containing MELAS patient mitochondria with a recipient p0 cell line. Some of the resulting cybrids contained a mixture of mutant and wild-type mitochondrial genomes. Temporal analysis revealed that in 5 of 13 clones investigated, the proportion of mutant mtDNA increased with time. In no case was a shift toward wild-type mtDNA observed. In addition, the level of mutant mtDNA reached 95% in only one cybrid line, and in this case, respi-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734...

143B.206 rho degrees cells were repopulated with mitochondria from a MELAS patient harbouring a mixture of 3243G:C and 3243A:T mitochondrial DNA. A number of biochemical assays were performed on selected cybrids with various proportions of the two types of mitochondrial DNA. These assays revealed a marked decrease in oxygen consumption with pyruvate, a complex I substrate, in cybrids containing 60% to 90% 3243G:C mitochondrial DNA. Moreover, these cybrids showed decreased synthesis of a number of polypeptides in a mitochondrial in vitro translation assay. A cybrid line with a very high level of 3243G:C mitochondrial DNA (95%) had additional deficiencies in complexes III and IV and there was a marked generalised decrease in mitochondrial translation in this cybrid. The observation of complex I deficiency is consistent with previously reported enzymatic measurements of muscle homogenates from MELAS patients with the 3243G:C mutation.

Mitochondrial DNA from a 38 year old male with diabetes mellitus and features of mitochondrial dysfunction was analysed and shown to include a population with a partial duplication. The partially duplicated mitochondrial DNA molecules were evident in both muscle and blood. The region of mitochondrial DNA duplicated includes the origin of heavy strand replication, but not the light strand origin. This patient has features in common with other cases of partial direct tandem duplications and with a family which was reported to harbour a 10.4 kb mtDNA deletion. Initial restriction enzyme analysis of our case produced results consistent with a partial deletion of mitochondrial DNA. This leads us to propose that the rarity of reports of partial mitochondrial DNA duplications may stem in part from the classification of such mutants as partial deletions.

No abstract

No abstract