Figure 2.

Competition between human point-mutated mtDNA and primate wild-type mtDNA. (A) Southern analysis of fusion products between a human cell line harboring a pathogenic G5703A mutation and gorilla’s cytoplasts. DNA from nine clones able to grow in galactose medium were digested with XbaI and probed with a whole human mtDNA (positions 10–16,496) probe. (B) Analysis of the G5703A mutation by PCR and RFLP. A DNA fragment encompassing the tRNA^Asn gene was amplified by PCR and digested with DdeI as described (Hao and Moraes, 1997). There are five DdeI sites in the human DNA fragment, but the one at position 5699 allows the differentiation of human mutated and wild-type molecules, whereas two additional absent sites allow the identification of gorilla mtDNA. The small levels of gorilla mtDNA observed by Southern analysis of clone gW72.1 cannot be observed in the PCR and RFLP analysis, probably because the human-based PCR primers may be slightly biased toward human templates due of to a few mismatches in the 5′ region of the oligonucleotides. Note the absence of heteroplasmic or recombinant clones.