Nucleotide sequence variation is frequent in the mitochondrial DNA displacement loop region of individual human tumor cells

The mitochondrial DNA (mtDNA) displacement loop (D-loop) regions of 76 various tumor cell lines were examined to investigate the existence of a specific relationship between a somatic mtDNA sequence and initiation and/or progression of a tumor. Based on molecular cloning-sequencing analysis, a nucleotide sequence in the D-loop region in each cell line was found to be homoplasmic. Several site-specific nucleotide variations were found in stomach and liver tumor cell lines more frequently than those in other tumor cell lines. Subsequently, 20 pairs of noncancerous and cancerous parts from stomach and liver tumor tissues were examined. In the liver tumor tissue, 80% of the noncancerous parts exhibited slightly higher heterogeneity than the corresponding cancerous parts. Several site-specific nucleotide variations found in 76 tumor cell lines were also detected in noncancerous or cancerous parts of stomach and liver tumor tissues. However, it remains unclear why the mtDNA D-loop sequence is homoplasmic in each tumor cell line. The data indicate that mtDNA exhibits heterogeneity even in the noncancerous part and a slight decrease in heterogeneity during tumorigenesis and/or tumor progression. Homoplasmy of the mtDNA population in the tumor cell line would be acquired in the cloning process of establishing a cell line. Site-specific nucleotide substitutions might not be directly involved in the tumorigenesis process.     

Rapid concerted evolution in animal mitochondrial DNA

Recombinational genetic processes are thought to be rare in the uniparentally inherited mitochondrial (mt) DNA molecules of vertebrates and other animals. Here, however, we document extremely rapid concerted microevolution, probably mediated by frequent gene conversion events, of duplicated sequences in the mtDNA control region of mangrove killifishes (Kryptolebias marmoratus). In local populations, genetic distances between paralogous loci within an individual were typically smaller (and often zero) than those between orthologous loci in different specimens. These findings call for the recognition of concerted evolution as a microevolutionary process and gene conversion as a likely recombinational force in animal mtDNA. The previously unsuspected power of these molecular phenomena could greatly impact mtDNA dynamics within germ cell lineages and in local animal populations.     

Rapid evolution of a heteroplasmic repetitive sequence in the mitochondrial DNA control region of carnivores

We describe a repetitive DNA region at the 3 end of the mitochondrial DNA (mtDNA) control region and compare it in 21 carnivore species representing eight carnivore families. The sequence and organization of the repetitive motifs can differ extensively between arrays; however, all motifs appear to be derived from the core motif ACGT. Sequence data and Southern blot analysis demonstrate extensive heteroplasmy. The general form of the array is similar between heteroplasmic variants within an individual and between individuals within a species (varying primarily in the length of the array, though two clones from the northern elephant seal are exceptional). Within certain families, notably ursids, the array structure is also similar between species. Similarity between species was not apparent in other carnivore families, such as the mustelids, suggesting rapid changes in the organization and sequence of some arrays. The pattern of change seen within and between species suggests that a dominant mechanism involved in the evolution of these arrays is DNA slippage. A comparative analysis shows that the motifs that are being reiterated or deleted vary within and between arrays, suggesting a varying rate of DNA turnover. We discuss the evolutionary implications of the observed patterns of variation and extreme levels of heteroplasmy.By acceptance of this article, the publisher acknowledges the right of the US Government to retain non-exclusive, royalty-free license in and to any copyright covering the article. Correspondence to: A.R. Hoetzel     

The bovine mitochondrial endonuclease prefers a conserved sequence in the displacement loop region of mitochondrial DNA

The purified endonuclease of bovine heart mitochondria shows a remarkable preference for a specific site in bovine mtDNA. This site was identified using a recombinant DNA template which includes a 4.9-kilobase portion of the 16-kilobase bovine mitochondrial sequence encompassing all of the displacement loop region. The endonucleolytic target corresponds to an evolutionarily conserved sequence tract of 12 consecutive guanine residues that is found in an otherwise highly divergent domain of the mitochondrial displacement loop region. The preference for this site is several hundred-fold greater than other less favored sites. Unlike other less prominent cleavage loci, the conserved sequence tract is readily nicked in either or both DNA strands, even at 0 degrees C. These findings suggest that there is a specific interaction of the endonuclease with this site in vivo that may be important for mtDNA replication.     

Nonneutral evolution of tandem repeats in the mitochondrial DNA control region of lagomorphs

The mitochondrial DNA of the European rabbit (Oryctolagus cuniculus) contains a tandem array of 153-bp repeats in the vicinity of the replication origin of the H-stand. Variation among molecules in the number of these repeats results in inter- and intraindividual length polymorphism (heteroplasmy). Generally, in an individual, one predominant molecular type is observed, the others representing a low percentage of the mtDNA content. At the tissue level, we observe a particular distribution of this polymorphism in the gonads compared with liver, kidneys, or brain, implying a relationship between the differentiation status of the cells and the types of new mtDNA molecules which appear and accumulate during lifetime. Similar tandem repeats were also found in the mtDNA noncoding region of European hares (Lepus europaeus), a cottontail (Sylvilagus floridanus), and a pika (Ochotona rufescens). The lengths and the sequences of these units evolve rapidly and in a concerted way, but the number of repeats is maintained in a narrow range, and an internal 20-bp segment is highly conserved. Constraints restrict the evolution of the primary sequence of these repeated units, the number of which is probably controlled by a stabilizing selection.      

Tempo and mode of mitochondrial DNA evolution in vertebrates at the amino acid sequence level: Rapid evolution in warm-blooded vertebrates

Summary By using complete sequence data of mitochondrial DNAs, three Markov models (Day-hoff, Proportional, and Poisson models) for amino acid substitutions during evolution were applied in maximum likelihood analyses of mitochondrially encoded proteins to estimate a phylogenetic tree depicting human, cow, whale, and murids (mouse and rat), with chicken, frog, and carp as outgroups. A cow/whale clade was confirmed with a more than 99.8% confidence level by any of the three models, but the branching order among human, murids, and the cow/whale clade remained uncertain. It turned out that the Dayhoff model is by far the most appropriate model among the alternatives in approximating the amino acid substitutions of mitochondrially encoded proteins, which is consistent with a previous analysis of a more limited data set. It was shown that the substitution rate of mitochondrially encoded proteins has increased in the order of fishes, amphibians, birds, and mammals and that the rate in mammals is at least six times, probably an order of magnitude, higher than that in fishes. The higher evolutionary rate in birds and mammals than in amphibians and fishes was attributed to relaxation of selective constraints operating on proteins in warm-blooded vertebrates and to high mutation rate of bird and mammalian mitochondrial DNAs.Offprint requests to: M. Hasegawa     

Mapping of genomic DNA loop organization in a 500-kilobase region of the Drosophila X chromosome by the topoisomerase II-mediated DNA loop excision protocol.

The recently developed procedure of chromosomal DNA loop excision by topoisomerase II-mediated DNA cleavage at matrix attachment sites (S. V. Razin, R. Hancock, O. Iarovaia, O. Westergaard, I. Gromova, and G. P. Georgiev, Cold Spring Harbor Symp. Quant. Biol. 58:25-35, 1993; I. I. Gromova, B. Thompsen, and S. V. Razin, Proc. Natl. Acad. Sci. USA 92:102-106, 1995) has been employed for mapping the DNA loop anchorage sites in a 500-kb region of the Drosophila melanogaster X chromosome. Eleven anchorage sites delimiting 10 DNA loops ranging in size from 20 to 90 kb were found within this region. Ten of these 11 anchorage sites colocalize with previously mapped scaffold attachment regions. However, a number of other scaffold attachment regions are found to be located in loop DNA.     

Origin and evolution of homologous repeated sequences in the mitochondrial DNA control region of shrews

The complete mitochondrial DNA (mtDNA) control region was amplified and directly sequenced in two species of shrew, Crocidura russula and Sorex araneus (Insectivora, Mammalia). The general organization is similar to that found in other mammals: a central conserved region surrounded by two more variable domains. However, we have found in shrews the simultaneous presence of arrays of tandem repeats in potential locations where repeats tend to occur separately in other mammalian species. These locations correspond to regions which are associated with a possible interruption of the replication processes, either at the end of the three-stranded D-loop structure or toward the end of the heavy-strand replication. In the left domain the repeated sequences (R1 repeats) are 78 bp long, whereas in the right domain the repeats are 12 bp long in C. russula and 14 bp long in S. araneus (R2 repeats). Variation in the copy number of these repeated sequences results in mtDNA control region length differences. Southern blot analysis indicates that level of heteroplasmy (more than one mtDNA form within an individual) differs between species. A comparative study of the R2 repeats in 12 additional species representing three shrew subfamilies provides useful indications for the understanding of the origin and the evolution of these homologous tandemly repeated sequences. An asymmetry in the distribution of variants within the arrays, as well as the constant occurrence of shorter repeated sequences flanking only one side of the R2 arrays, could be related to asymmetry in the replication of each strand of the mtDNA molecule. The pattern of sequence and length variation within and between species, together with the capability of the arrays to form stable secondary structures, suggests that the dominant mechanism involved in the evolution of these arrays in unidirectional replication slippage.      

Fragment comparison of hamster mitochondrial DNA: general conclusions about the evolution of mitochondrial DNA

Mitochondrial DNA from heart, liver and kidney of two hamster species, Mesocricetus auratus and Cricetulus griseus has been digested with the restriction endonucleases Bam HI, Bgl I, Eco RI, Hae III, Hind III, Hpa II and Xba I. Cleavage patterns for Hpa II are identical for both species, while only two of seven bands are common with Hae III. All other endonucleases give a species-specific cleavage pattern. The results suggest a fairly high phylogenetic differentiation of the mitochondrial genome between the two hamster species. The large differences in mitochondrial DNA variability between different species is discussed as a function of mutation rate and species-specific generation time.     

Sequence variation and evolution of the mitochondrial DNA control region in the musk shrew, Suncus murinus

The complete mitochondrial DNA (mtDNA) control region was cloned and sequenced in the musk shrew, Suncus murinus, Insectivora. The general aspect was similar to that found in other mammals. We have found in two locations of this region the presence of arrays of tandem repeats like those in other shrew species. One array was located in the left domain containing the termination-associated sequences (TAS) and the length of a copy was 77 bp. The other repeats were situated upstream from the recognition site for the end of H-strand replication in the right domain and were 20 bp long. The left halves of the control region containing the former repeats were sequenced and compared in several laboratory lines and wild animals from different localities, variations in copy number of repeated sequences were found both among individuals and within an individual. A comparative study of repeated sequences provides useful indication for the origin and evolution of tandem repeated sequences. Strand slippage and mispairing during replication of mtDNA with concerted manner is currently regarded as a dominant theory to account molecular mechanism for tandemly repeated sequences, and the pattern of sequence and length variation in our study supports this theory. Our results, however, suggest that the evolution of the repeated sequences containing the TAS in the musk shrew might go through the process of two steps; at the first step one complete repeated and several incomplete repeated sequences had reproduced in common ancestor of the shrew, and the second stage step-up of complete repeated sequences occurred with concerted evolution after differentiation into continental and insular groups.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

The evolution of mitochondrial DNA

Although the massive sequencing of mitochondrial DNA from various organisms, together with studies of a different nature, has contributed enormously to the knowledge of the organization and function of this cytoplasmic genome, many issues, mainly the relationships with the nuclear genome, remain unsolved. This review critically evaluates the most recent advances in research on the evolution of the mitochondrial DNA from a qualitative and quantitative point of view, underlining the multiplicity of structures and genetic organization of this genome, which contrasts with its reduced, but rather constant, information content in various organisms. It also highlights the role that mitochondrial DNA is now playing, particularly in metazoans, in different disciplines and application fields. Among these, particular attention is focused on the discovery of the mitochondrial origin of several diseases affecting primarily the neuromuscular system.     

Structure and evolution of the avian mitochondrial control region

The structural and evolutionary characteristics of the mitochondrial control region were studied by using control region sequences of 68 avian species. The distribution of the variable nucleotide positions within the control region was found to be genus specific and not dependant on the level of divergence, as suggested before. Saturation was shown to occur at the level of divergence of 10% in pairwise comparisons of the control region sequences, as has also been reported for the third codon positions in ND2 and cytochrome b genes of mtDNA. The ratio of control region vs cytochrome b divergence in pairwise comparisons of the sequences was shown to vary from 0.13 to 21.65, indicating that the control region is not always the most variable region of the mtDNA, but also that there are differences in the rate of divergence among the lineages. Only two of the conserved sequence blocks localized earlier for other species, D box and CSB-1, were found to show a considerable amount of sequence conservation across the avian and mammalian sequences. Additionally, a novel avian-specific sequence block was found.     

Calibration of mitochondrial DNA evolution in geese

Summary Mitochondrial DNA was purified from five American species of geese representing the generaAnser andBranta, which have fossil records. The results of electrophoretic comparisons of about 75 fragments per individual produced by 14 restriction enzymes imply that the mean extent of sequence divergence between species ofAnser andBranta is about 9%. Fossil evidence suggests that these two groups of geese had a common ancestor 4–5 million years ago. Thus, the mean rate of sequence divergence in goose mitochondiral DNA is not far from 2% per million years, the value in mammals.     

Rapid step-gradient purification of mitochondrial DNA

A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5-end labeling, gel retention assays, and various types of hybridization.