Accumulation of mitochondrial DNA deletions in myotubes cultured from muscles of patients with mitochondrial myopathies

 Myoblast cultures were established from muscle biopsies of two patients harboring heteroplasmic mitochondrial (mt) DNA deletions. The accumulation kinetics of the deleted mtDNA was followed during myoblast to myotube differentiation. The percent- age of deleted mtDNA was determined by quantitative PCR in myoblasts, myotubes, and muscle biopsies. The deleted form accounted for 65% of the mtDNA present in a muscle biopsy from a patient harboring a 5.6-kb deletion. The percentage of deleted mtDNA was 1.2% in myoblasts and increased progressively after differentiation, up to 12% at 21 days after the commitment time. In a second patient harboring a 2.8-kb deletion, the percentage of deleted mtDNA increased much more slowly: from 0.07% in myoblasts to 0.21% after 22 days of differentiation, as compared with 45% in the muscle biopsy. Thus, a three- and ten-fold increase, respectively, in the fraction of deleted mtDNA occurred during the differentiation of myoblasts to myotubes from the two patients. The faster accumulation of deleted mtDNA in the first patient’s cells was linked to an earlier myoblast to myotube differentiation, suggesting that the level of deleted mtDNA is inversely related to the rate of cell proliferation. Received: 16 April 1996/Accepted: 29 July 1996     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.