Nucleotide sequence preservation of human mitochondrial DNA ( mutation / evolution / recombinant DNA / DNA

Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mphl; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both betweenand within-individual differences were identified. Betweenindividual differences were identified in -1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation ofmammalian mitochondrial DNA sequences in single organisms. A change in the DNA sequence of somatic cells has frequently been invoked as a mechanism controlling cellular differentiation. It has also been suggested that an accumulation of somatic mutations during or after the completion of development may play a role in aging and tumorigenesis. A major difficulty in testing these hypotheses is the lack of sensitivity of several now classical techniques for measuring the relatedness ofDNA sequences from an individual: DNA hybridization, electron microscopy, and restriction endonuclease mapping. None of these techniques is sufficiently sensitive or informative to allow the identification, characterization, and quantitation of DNA sequence differences occurring with a frequency of -1% in a population of DNA molecules. Restriction endonuclease mapping is particularly insensitive when nucleotide sequence differences are randomly distributed. For example, restriction endonuclease mapping may fail to detect randomly distributed nucleotide sequence changes that involve as many as 10% of all nucleotide positions in a population of DNA molecules. The development of recombinant DNA and nucleotide sequencing techniques now allows detection of randomly distributed DNA sequence differences occurring with a frequency of <0.01%. As a first application of these techniques to the question of nucleotide sequence divergence within individuals, we have quantitated and characterized the amount of nucleotide sequence divergence within the mitochondrial DNA (mtDNA) population of normal humans. We chose to study human mtDNA for three reasons: it is well-characterized (1), it can be easily isolated from cells, and many nucleotide sequence differences are known to exist between mtDNAs isolated from different humans (2). We determined the nucleotide sequence of a large number of independently isolated recombinant DNA clones containing the same mtDNA segment from each of five normal humans. The mtDNA region studied contained the gene for cytochrome oxidase subunit III (COIII). The COIII protein, of 261 amino acids, is one of seven different polypeptides that form the cytochrome oxidase complex (3). It is an essential component of the respiratory electron transport chain. The surprising finding of this study was the virtual absence of nucleotide sequence divergence within the mtDNA population of normal individuals: only one within-individual difference was identified in 49 kilobases (kb) of sequence information from 248 independently isolated clones. This finding suggests the existence of a mechanism for limiting the development of nucleotide sequence divergence in the mammalian mtDNA population derived from a single zygote, thus maintaining the identity of mitochondrial nucleotide sequences among the differentiated cells of an individual.