Synthesis and turnover of Euglena gracilis mitochondrial DNA

Abstract

Replication of mitochondrial DNA was investigated by a density transfer experiment in a strain of Euglena gracilis lacking chloroplast DNA. DNA was uniformly labeled in a medium containing 32P-labeled inorganic phosphate and [ 3H]adenine in the presence of the heavy-density label and transferred to a medium containing 32P-labeled inorganic phosphate but no [ 3H]adenine following removal of the heavy-density label. Replication of nuclear DNA within these cells was used as an internal control. The densities and ratios of the peaks of nuclear DNA were those expected for a strict semiconservative replication. In contrast, replication of mitochondrial DNA was dispersive, as illustrated by the following results: (1) both native and denatured mitochondrial DNA exhibited a single density peak at 1.1 and 2.2 cell doublings after the density transfer. (2) The specific activity of 3H-labeled DNA varied across the peak of native or denatured DNA, indicating a heterogeneous population of molecules exhibiting different degrees of density and radioisotope labeling. This dispersive replication could involve either multiple recombination events or extensive turnover of the DNA or a mixture of both. Extensive dispersion of the sample obtained at 1.1 cell doublings after the density transfer is shown by the persistence of the same peak density for duplex DNA reduced to a molecular weight of 6 × 10 5 by shearing. Two measures of the rate of replication of mitochondrial DNA were obtained from the densities of native duplex DNA and the rate of decrease in 3H-specific activities of duplex DNA during the experiment. The average of these rates indicates that mitochondrial DNA replicates at least 1.5 times as fast as nuclear DNA. Since there is a constant ratio of mitochondrial DNA:nuclear DNA in a logarithmic culture, mitochondrial DNA was calculated to have a half-life of 1.8 cell doublings.