Mitochondrial DNA was purified from four species of higher primates (Guinea baboon, rhesus macaque, guenon, and human) and digested with 11 restriction endonucleases. A cleavage map was constructed for the mitochondrial DNA of each species. Comparison of the maps, aligned with respect to the origin and direction of DNA replication, revealed that the species differ from one another at most of the cleavage sites. The degree of divergence in nucleotide sequence at these sites was calculated from the fraction of cleavage sites shared by each pair of species. By plotting the degree of divergence in mitochondrial DNA against time of divergence, the rate of base substitution could be calculated from the initial slope of the curve. The value obtained, 0.02 substitutions per base pair per million years, was compared with the value for single-copy nuclear DNA. The rate of evolution of the mitochondrial MAnome appears to exceed that of the single-copy fraction of the nuclear genome by a factor of about 10. This high rate may be due, in part, to an elevated rate of mutation in mitochondrial DNA. Because of the high rate of evolution, mitochondrial DNA is likely to be an extremely useful molecule to employ for high-resolution analysis of the evolutionary process.

We cloned and sequenced a segment of mitochondrial DNA from human, chimpanzee, gorilla, orangutan, and gibbon. This segment is 896 bp in length, contains the genes for three transfer RNAs and parts of two proteins, and is homologous in all 5 primates. The 5 sequences differ from one another by base substitutions at 283 positions and by a deletion of one base pair. The sequence differences range from 9 to 19% among species, in agreement with estimates from cleavage map comparisons, thus confirming that the rate of mtDNA evolution in primates is 5 to 10 times higher than in nuclear DNA. The most striking new finding to emerge from these comparisons is that transitions greatly outnumber transversions. Ninety-two percent of the differences among the most closely related species (human, chimpanzee, and gorilla) are transitions. For pairs of species with longer divergence times, the observed percentage of transitions falls until, in the case of comparisons between primates and non-primates, it reaches a value of 45. The time dependence is probably due to obliteration of the record of transitions by multiple substitutions at the same nucleotide site. This finding illustrates the importance of choosing closely related species for analysis of evolutionary process. The remarkable bias toward transitions in mtDNA evolution necessitates the revision of equations that correct for multiple substitutions at the same site. With revised equations, we calculated the incidence of silent and replacement substitutions in the two protein-coding genes. The silent substitution rate is 4 to 6 times higher than the replacement rate, indicating strong functional constraints at replacement sites. Moreover, the silent rate for these two genes is about 10% per million years, a value 10 times higher than the silent rate for the nuclear genes studied so far. In addition, the mean substitution rate in the three mitochondrial tRNA genes is at least 100 times higher than in nuclear tRNA genes. Finally, genealogical analysis of the sequence differences supports the view that the human lineage branched off only slightly before the gorilla and chimpanzee lineages diverged and strengthens the hypothesis that humans are more related to gorillas and chimpanzees than is the orangutan.

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Mitochondrial DNA samples from each of 21 humans of diverse racial and geographic origin were digested with each of 18 restriction endonucleases. The sizes of the resulting DNA fragments were compared after gel electrophoresis. No differences among the samples were detected in digests with 7 of the enzymes. Analysis of digests with the remaining enzymes showed one or more differences. Each 76,[1967][1968][1969][1970][1971], Homo sapiens could have speciated or passed through a severe population constriction as recently as 180,000 years ago. The data suggest that group-specific patterns of cleavage exist. The high resolution and precision afforded by this method of analysis makes possible the investigation of many questions concerning human population genetics, evolution, and recent history.The mitochondrial genome of humans is a closed circular duplex DNA of ;16,500 nucleotide pairs (1, 2). The relative positions of the mitochondrial origin of replication and ribosomal genes have been shown to be the same among vertebrate species (3-5). It is likely that the order of the remaining mitochondrial genes will also prove to be highly conserved, at least among vertebrates (2, 6). Despite this conservative feature, the base sequence of mitochondrial DNA (mtDNA) has been shown to evolve rapidly (2, 7-9). The rate of base substitution appears to be 5-10 times faster than that of single-copy nuclear DNA (2). These properties and the relative ease of its preparation make mtDNA a useful molecule to employ in studies of population genetics and evolution.Human mtDNA samples were obtained from 21 individuals of diverse geographic and racial backgrounds (10). The fragments produced by digestion of mtDNA with 18 different restriction endonucleases have been analyzed by gel electrophoresis. The fragment patterns have been compared among the samples. An estimate for the average fraction of base pair substitutions in human mtDNA has been obtained. The amount of time elapsed since the human population was monomorphic for mtDNA has been calculated as the product of the substitution rate times this fraction. The result indicates that the human species may have passed through a severe population constriction ("bottleneck") relatively recently.Humans were chosen for study because they are genetically outbreeding and, originally, because a large amount of mtDNA could be obtained from each individual (from placental tissue), thus enabling many analyses to be performed per sample (10).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3605The same techniques have been successfully applied to the analysis of individual samples from much smaller animals (11,12). The use of radioactive labeling methods make multiple analyses on even the smallest animals possible.MATERIALS AND METHODS DNAs. Closed circular mtDNAs from HeLa cells (strain S3) and from human placentas were prep...

We determined the complete mtDNA sequence of the centipede Lithobius forficatus and found that only one of the 22 inferred tRNA genes encodes a fully paired aminoacyl acceptor stem. The other 21 genes encode tRNAs with up to five mismatches in these stems, and some of these overlap extensively with the downstream genes. Because a well-paired acceptor stem is required for proper tRNA functioning, RNA editing in the products of these genes was suspected. We investigated this hypothesis by studying cDNA sequences from eight tRNAs and found the editing of up to 5 nt at their 3 ends. This editing appears to occur by a novel mechanism with the 5 end of the acceptor stem being used as a template for the de novo synthesis of the 3 end, presumably by an RNA-dependent RNA polymerase. In addition, unusual secondary structures for several tRNAs were found, including those lacking a T⌿C (T) or a dihydrouridine (D) arm, and having an unusual number of base pairs in the acceptor or anticodon stems.