Restriction enzyme analysis of mitochondrial DNA in aging human cells

Human diploid fibroblasts show a limited lifespan in vitro. To investigate the integrity of mitochondrial DNA (mtDNA) in aging fibroblasts, whole cell DNA samples from the human cell line IMR-90 have been prepared at 36, 22, and 3 population doublings (PD) from the end of the lifespan (63 PD). These DNA samples were then digested separately with 19 different restriction endonucleases, and the resulting fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose filters. Fragment sizes were revealed by hybridization to 32P-labelled mouse mtDNA and autoradiography, and were compared with computer maps of fragments generated from the known sequence of human mtDNA. These 19 enzymes recognize a total of 297 recognition sites comprising 1315 nucleotide base pairs (bp), approximately 8% of the human mtDNA (16 569 bp). Control experiments reveal that a minor component representing as little as 5% of the total mtDNA can be detected. No changes were seen in the restriction fragment pattern with fibroblast cell age. It is concluded that there are no large deletions, insertions, or rearrangements in human mtDNA, and no single base changes in the detectable regions. This suggests efficient maintenance of mtDNA molecules and/or elimination of damaged mtDNA during fibroblast cell lifespan.