Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and
plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have
fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation
and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination
between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified
using three independent experimental approaches. First, recombinant molecules containing genetic markers
from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase
chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant
mtDNA were cloned and sequenced to identify the regions involved in the recombination events.
Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations
of polymorphic restriction sites and probes. This combined approach confirmed the existence of
heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important
implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and
forensic analyses based on mtDNA genotyping.