Aging-Dependent Large Accumulation of Point Mutations in the Human mtDNA Control Region for Replication
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; M. Corral-Debrinski et al. ibid. p. 324; M. Hayakawa
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; D. C. Wallace C. Stugard D. Murdock T. Schurr M. D. Brown ibid. p. 14900.
Y. Michikawa unpublished observations. The methods used are available at (38).
Marchington D. R., Hartshorne G. M., Barlow D., Poulton J., Am. J. Hum. Genet. 60, 408 (1997).
See (38) for methods used for total DNA and mtDNA isolation.
See (38) for primers used and details about the PCR amplification.
As shown in Fig. 1C before the enzyme treatment the nuclear DNA present in the total cell DNA sample migrated in an agarose gel as a band moving more slowly than the 23.1-kb λ DNA Hind III fragment and the heterogeneous RNA migrated faster than the 2-kb λ DNA fragment. After the three digestion steps no ethidium bromide–stained material was recognizable in the gel.
Skin fibroblasts from normal Caucasian individuals were either obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden NJ) (20 wf 1 y 10 y 13 y 19 y 24 y 25 y 26 y 42 y 48 y 78-1 y 98 y) or from the National Institute of Aging Cell Respository (Camden NJ) {nine twice-sampled longitudinal study (LS) individuals [LS1 (52 y–71 y) LS2 (59 y–75 y) LS3 (63 y–78-2 y) LS4 (64 y–80 y) LS5 (65 y–74-1 y) LS6 (66 y–82 y) LS7 (70 y–86-2 y) LS8 (73 y–89 y) LS9 (77 y–90 y)] and 74-2 y} or from the Institute of Clinical Neurology of the University of Milan Italy (86-1 y 100-1 y 100-2 y 100-3 y 101 y). The latter set of individuals all from retirement homes were free of any neurological or muscular pathology. All fibroblast cultures had undergone less than 10 passages when received and were in general used within two or three additional passages.
See (38) for methods for fibroblast culture and first DGGE analysis.
Methods for mtDNA cloning and second-DGGE analysis can be found in (38) .
The method for sequencing is described in (38).
A table summarizing the sequence data obtained for the cloned DLP6 and DLP4 fragments analyzed in this work is available at (38).
A cloned wild-type DLP6 mtDNA fragment from the 25-year-old individual and a cloned wild-type DLP4 mtDNA fragment from the 78-year-old individual were PCR reamplified and the recloned PCR products and 48 cloned fragments thus isolated were then subjected to a second DGGE screening.
A repeat of the cloning and sequencing of the DLP6 fragments of the two LS6 samples gave results that when combined with the previous ones yielded a coefficient of variation for the frequency of the specific point mutations of ∼20% supporting the reproducibility of the estimates. Therefore it is possible that the observed decrease in the T414G mutation frequency in the LS6-82 y sample results from a difference in the biopsy site or from handling of the original specimen.
The method for allele-specific termination of primer extension is described in (38).
Y. Michikawa and G. Attardi data not shown.
Ghivizzani S. C., Madsen C. S., Nelen M. R., Ammini C. V., Hauswirth W. W., Mol. Cell. Biol. 14, 7717 (1994).
Supplemental material is available at the Science Web site (www.sciencemag.org/feature/data/1040395.shl).
Supported by National Institute on Aging (NIH) grant AG12117-03 (to G.A.). We thank A. Chomyn and G. Villani for valuable discussions M. Lai for support A. Aman R. Steinberger and M. Scott for their help with experiments and A. Drew B. Keeley R. Zedan and C. Lin for expert technical assistance.