Intraspecific DNA sequence variation of the mitochondrial control region of white sturgeon (Acipenser transmontanus)

Intraspecific sequence variation in the D-loop region of mtDNA in white sturgeon (Acipenser transmontanus), a relict North American fish species, was examined in 27 individuals from populations of the Columbia and Fraser rivers. Thirty-three varied nucleotide positions were present in a 462-nucleotide D-loop sequence, amplified using the polymerase chain reaction. Bootstrapped neighbor-joining and maximum- parsimony trees of sequences from 19 haplotypes suggest that the two populations have recently diverged. This is consistent with the hypothesis that the Columbia River, a Pleistocene refugium habitat, was the source of founders for the Fraser River after the last glacial recession. On the basis of a divergence time of 10-12 thousand years ago, the estimated substitution rate of the white sturgeon D-loop region is 1.1-1.3 x 10(-7) nucleotides/site/year, which is comparable to rates for hypervariable sequences in the human D-loop region. Furthermore, the ratio of mean percent nucleotide differences in the D- loop (2.27%) to that in whole mtDNA (0.54%, as estimated from restriction-enzyme data) is 4.3, which is similar to the fourfold-to- fivefold-higher substitution rate estimated for the human D-loop. The high nucleotide substitution rate of the hypervariable region indicates that the vertebrate D-loop has potential as a genetic marker in molecular population studies.      

Pattern of nucleotide substitution and rate heterogeneity in the hypervariable regions I and II of human mtDNA.

This study provides a comprehensive survey of the complex pattern of nucleotide substitution in the control region of human mtDNA, which is of central importance to the studies of human evolution. A total of 1229 different hypervariable region I (HVRI) and 385 different hypervariable region II (HVRII) sequences were analyzed using a complex substitution model. Moreover, we suggest a new method to assign relative rates to each site in the sequence. Estimates are based on maximum-likelihood methods applied to randomly selected subsets of sequences. Our results indicate that the rate of substitution in HVRI is approximately twice as high as in HVRII and that this difference is mainly due to a higher frequency of pyrimidine transitions in HVRI. However, rate heterogeneity is more pronounced in HVRII.     

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.     

Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA.

The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA (mtDNA) from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA. Both D-loop regions are bounded by the genes for tRNAPhe and tRNAPro. This region contains the most highly divergent sequences in mtDNA with the exceptions of three small conserved sequence blocks near the 5' ends of D-loop strands, a 225 nucleotide conserved sequence block in the center of the D-loop strand template region, and a short sequence associated with the 3' ends of D-loop strands. A sequence similar to that associated with the 3' termini of D-loop strands overlaps one of the conserved sequence blocks near the 5' ends of D-loop strands. The large, central conserved sequence probably does not code for a protein since no open reading frames are discretely conserved. Numerous symmetric sequences and potential secondary structures exist in these sequences, but none appear to be clearly conserved between species.     

Nucleotide sequence identity of mitochondrial DNA from different human tissues

Recombinant DNA techniques have been used to search for mitochondrial (mt) nucleotide (nt) sequence differences between human tissues within an individual. mtDNA isolated from brain, heart, liver, kidney, and skeletal muscle of two different individuals was cleaved with SacI and XbaI, and then cloned in bacteriophage M13. Partial nt sequence determination of 121 independently isolated recombinant M13 clones containing either the cytochrome oxidase subunit III gene or the D-loop region of human mtDNA revealed base substitution differences between individuals, and between each individual and the published human mtDNA sequence. A majority of these base substitutions were transitions. No systematic nt sequence differences were identified between tissues within an individual, however. These results suggest that mtDNA sequence alterations do not accompany organogenesis, and that somatic mutations do not accumulate in the mtDNA of different human tissues to a level of greater than one nt substitution per molecule.     

Nucleotide Substitution Rate of Mammalian Mitochondrial Genomes

We present here for the first time a comprehensive study based on the analysis of closely related organisms to provide an accurate determination of the nucleotide substitution rate in mammalian mitochondrial genomes. This study examines the evolutionary pattern of the different functional mtDNA regions as accurately as possible on the grounds of available data, revealing some important ``genomic laws.' The main conclusions can be summarized as follows. (1) High intragenomic variability in the evolutionary dynamic of mtDNA was found. The substitution rate is strongly dependent on the region considered, and slow- and fast-evolving regions can be identified. Nonsynonymous sites, the D-loop central domain, and tRNA and rRNA genes evolve much more slowly than synonymous sites and the two peripheral D-loop region domains. The synonymous rate is fairly uniform over the genome, whereas the rate of nonsynonymous sites depends on functional constraints and therefore differs considerably between genes. (2) The commonly accepted statement that mtDNA evolves more rapidly than nuclear DNA is valid only for some regions, thus it should be referred to specific mitochondrial components. In particular, nonsynonymous sites show comparable rates in mitochondrial and nuclear genes; synonymous sites and small rRNA evolve about 20 times more rapidly and tRNAs about 100 times more rapidly in mitochondria than in their nuclear counterpart. (3) A species-specific evolution is particularly evident in the D-loop region. As the divergence times of the organism pairs under consideration are known with sufficient accuracy, absolute nucleotide substitution rates are also provided. Received: 11 May 1998 / Accepted: 2 September 1998     

Relative rates of evolution in the coding and control regions of African mtDNAs

Reduced median networks of African haplogroup L mitochondrial DNA (mtDNA) sequences were analyzed to determine the pattern of substitutions in both the noncoding control and coding regions. In particular, we attempted to determine the causes of the previously reported (Howell et al. 2004) violation of the molecular clock during the evolution of these sequences. In the coding region, there was a significantly higher rate of substitution at synonymous sites than at nonsynonymous sites as well as in the tRNA and rRNA genes. This is further evidence for the operation of purifying selection during human mtDNA evolution. For most sites in the control region, the relative rate of substitution was similar to the rate of neutral evolution (assumed to be most closely approximated by the substitution rate at 4-fold degenerate sites). However, there are a number of mutational hot spots in the control region, approximately 3% of the total sites, that have a rate of substitution greater than the neutral rate, at some sites by more than an order of magnitude. It is possible either that these sites are evolving under conditions of positive selection or that the substitution rate at some sites in the control region is strongly dependent upon sequence context. Finally, we obtained preliminary evidence for "nonideal" evolution in the control region, including haplogroup-specific substitution patterns and a decoupling between relative rates of substitution in the control and coding regions.     

MtDNA substitution rate and segregation of heteroplasmy in coding and noncoding regions

The mitochondrial DNA (mtDNA) substitution rate and segregation of heteroplasmy were studied for the non-coding control region (D-loop) and 500 bp of the coding region between nucleotide positions 5550 and 6050, by sequence analysis of blood samples from 194 individuals, representing 33 maternal lineages. No homoplasmic nucleotide substitutions were detected in a total of 292 transmissions. The estimated substitution rate per nucleotide per million years for the control region (micro>0.21, 95% CI 0-0.6) was not significantly different from that for the coding region (micro>0.54, 95% CI 0-1.0). Variation in the length of homopolymeric C streches was observed at three sites in the control region (positions 65, 309 and 16,189), all of which were in the heteroplasmic state. Segregation of heteroplasmic genotypes between generations was observed in several maternal pedigrees. At position 309, a longer poly C tract length was strongly associated with a higher probability for heteroplasmy and rapid segregation between generations. The length heteroplasmy at positions 65 and 16,189 was found at low frequency and was confined to a few families.     

Sequence variation and structural conservation in the D-loop region and flanking genes of mitochondrial DNA from Japanese pond frogs

The nucleotide sequences of the D-loop region and its flanking genes of the mitochondrial DNA (mtDNA) from Japanese pond frogs were determined by the methods of PCR, cloning, and sequencing. The frogs belonged to two species, one subspecies, and one local race. The gene arrangements adjacent to the D-loop region were analyzed. The frogs shared a unique mitochondrial gene order that was found in Rana catesbeiana; i.e., cyt b--D-loop region--tRNA(Leu(CUN))--tRNA(Thr)--tRNA(Pro)--tRNA(Phe)--12S rRNA. The arrangements of the three tRNA genes of these frogs were different from those of X. laevis, a species which has the same overall structure as in mammals. Highly repetitive sequences with repeat units (16-bp or 17-bp sequence specific for each taxon) were found in the D-loop region. The length of repetitive sequences varied from 0.6 kbp to 1.2 kbp, and caused the extensive size variation in mtDNA. Several short sequence elements such as putative TAS, OH, CSB-1, and CSB-2 were found in the D-loop region of these frogs. The sequences of these short regulatory elements were conserved in R. catesbeiana, X. laevis, and also in human. The comparison of sequence divergences of the D-loop region and its adjacent genes among various taxa revealed that the rates of nucleotide substitutions depend on genes. The nucleotide sequences of the 3'-side segment of the D-loop region were the most variable among taxa, whereas those of the tRNA and 12S rRNA genes were the most conservative.     

Human Mitochondrial DNA Variation and Evolution: Analysis of Nucleotide Sequences from Seven Individuals

We have analyzed nucleotide sequence variation in an approximately 900-base pair region of the human mitochondrial DNA molecule encompassing the heavy strand origin of replication and the D-loop. Our analysis has focused on nucleotide sequences available from seven humans. Average nucleotide diversity among the sequences is 1.7%, several-fold higher than estimates from restriction endonuclease site variation in mtDNA from these individuals and previously reported for other humans. This disparity is consistent with the rapidly evolving nature of this noncoding region. However, several instances of convergent or parallel gain and loss of restriction sites due to multiple substitutions were observed. In addition, other results suggest that restriction site (as well as pairwise sequence) comparisons may underestimate the total number of substitutions that have occurred since the divergence of two mtDNA sequences from a common ancestral sequence, even at low levels of divergence. This emphasizes the importance of recognizing the large standard errors associated with estimates of sequence variability, particularly when constructing phylogenies among closely related sequences. Analysis of the observed number and direction of substitutions revealed several significant biases, most notably a strand dependence of substitution type and a 32-fold bias favoring transitions over transversions. The results also revealed a significantly nonrandom distribution of nucleotide substitutions and sequence length variation. Significantly more multiple substitutions were observed than expected for these closely related sequences under the assumption of uniform rates of substitution. The bias for transitions has resulted in predominantly convergent or parallel changes among the observed multiple substitutions. There is no convincing evidence that recombination has contributed to the mtDNA sequence diversity we have observed.     

A reanalysis of the ancient mitochondrial DNA sequences recovered from Neandertal bones

Recent reports analyzing mitochondrial DNA sequences from Neandertal bones have claimed that Neandertals and modern humans are different species. The phylogenetic analyses carried out in these articles did not take into account the high substitution rate variation among sites observed in the human mitochondrial D-loop region and also lack an estimation of the parameters of the nucleotide substitution model. The separate phylogenetic position of Neandertals is not supported when these factors are considered. Our analysis shows that Neandertal-Human and Human-Human pairwise distance distributions overlap more than what previous studies suggested. We also show that the most ancient Neandertal HVI region is the most divergent when compared with modern human sequences. However, the opposite would be expected if the sequence had not been modified since the death of the specimen. Such incongruence is discussed in the light of diagenetic modifications in ancient Neandertal DNA sequences.     

Hypervariability of the D-loop region in mitochondrial DNA of Russian sturgeon <Emphasis Type="Italic">Acipenser gueldenstaedtii</Emphasis> (Acipenseriformes,Acipenseridae)

The structure of the D-loop region in mitochondrial DNA (mtDNA) of Russian sturgeon Acipenser gueldenstaedtii from the Azov Sea population was studied with the method of direct sequencing. Interindividual heteroplasmy of the length of mtDNA in the region of D-loop realized by the presence of a different number of tandem repeats (82 pairs of bases) was found. Analysis of tandem repeats in the D-loop region in mtDNA in the studied sample (28 individuals) revealed eight mitotypes differed in the pattern of nucleotide substitution and in the number of tandem repeats (2, 3, and 4 repeats). Revealed mitotypes can be considered as potential genetic markers for different biological groups, schools, or seasonal races of A. gueldenstaedtii.     

How rapidly does the human mitochondrial genome evolve?

The results of an empirical nucleotide-sequencing approach indicate that the evolution of the human mitochondrial noncoding D-loop is both more rapid and more complex than is revealed by standard phylogenetic approaches. The nucleotide sequence of the D-loop region of the mitochondrial genome was determined for 45 members of a large matrilineal Leber hereditary optic neuropathy pedigree. Two germ-line mutations have arisen in members of one branch of the family, thereby leading to triplasmic descendants with three mitochondrial genotypes. Segregation toward the homoplasmic state can occur within a single generation in some of these descendants, a result that suggests rapid fixation of mitochondrial mutations as a result of developmental bottlenecking. However, slow segregation was observed in other offspring, and therefore no single or simple pattern of segregation can be generalized from the available data. Evidence for rare mtDNA recombination within the D-loop was obtained for one family member. In addition to these germ-line mutations, a somatic mutation was found in the D-loop of one family member. When this genealogical approach was applied to the nucleotide sequences of mitochondrial coding regions, the results again indicated a very rapid rate of evolution.     

Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees

Examining the pattern of nucleotide substitution for the control region of mitochondrial DNA (mtDNA) in humans and chimpanzees, we developed a new mathematical method for estimating the number of transitional and transversional substitutions per site, as well as the total number of nucleotide substitutions. In this method, excess transitions, unequal nucleotide frequencies, and variation of substitution rate among different sites are all taken into account. Application of this method to human and chimpanzee data suggested that the transition/transversion ratio for the entire control region was approximately 15 and nearly the same for the two species. The 95% confidence interval of the age of the common ancestral mtDNA was estimated to be 80,000-480,000 years in humans and 0.57-2.72 Myr in common chimpanzees.      

Application of mtDNA d-loop region for the study of Russian sturgeon population structure from Iranian coastline of the Caspian Sea

The population structure of the Russian sturgeon ( Acipenser gueldenstaedtii ) was investigated using PCR amplification of the mtDNA D-loop region. Seven composite haplotypes were detected and average nucleotide and haplotype diversity over all samples were found to be 0.05 ± 0.00 and 0.75 ± 0.00 (mean ± SE) respectively. Restriction digest of the mtDNA D-loop region detected two genotypes A and B with a relative high frequencies of 0.5 and 0.36 respectively. These two genotypes (A and B) can be considered as potential genetic markers for different biological groups, stocks or seasonal races of Russian sturgeon. A maximum of 10.8% nucleotide diversity was found between haplotypes AAAAB and BBBBF. 100% heteroplasmy was found in Russian sturgeon and restriction digest of the mtDNA D-loop region also exhibited site heteroplasmy.     

Substitution rate variation among sites in mitochondrial hypervariable region I of humans and chimpanzees

Mitochondrial D-loop hypervariable region I (HVI) sequences are widely used in human molecular evolutionary studies, and therefore accurate assessment of rate heterogeneity among sites is essential. We used the maximum-likelihood method to estimate the gamma shape parameter alpha for variable substitution rates among sites for HVI from humans and chimpanzees to provide estimates for future studies. The complete data of 839 humans and 224 chimpanzees, as well as many subsets of these data, were analyzed to examine the effect of sequence sampling. The effects of the genealogical tree and the nucleotide substitution model were also examined. The transition/transversion rate ratio (kappa) is estimated to be about 25, although much larger and biased estimates were also obtained from small data sets at low divergences. Estimates of alpha were 0.28-0.39 for human data sets of different sizes and 0.20-0.39 for data sets including different chimpanzee subspecies. The combined data set of both species gave estimates of 0.42-0.45. While all those estimates suggest highly variable substitution rates among sites, smaller samples tend to give smaller estimates of alpha. Possible causes for this pattern were examined, such as biases in the estimation procedure and shifts in the rate distribution along certain lineages. Computer simulations suggest that the estimation procedure is quite reliable for large trees but can be biased for small samples at low divergences. Thus, an alpha of 0.4 appears suitable for both humans and chimpanzees. Estimates of alpha can be affected by the nucleotide sites included in the data, the overall tree length (the amount of sequence divergence), the number of rate classes used for the estimation, and to a lesser extent, the included sequences. The genealogical tree, the substitution model, and demographic processes such as population expansion do not have much effect.     

The rate and pattern of nucleotide substitution in Drosophila mitochondrial DNA.

The nucleotide sequences of a segment of mitochondrial DNA (mtDNA) have been determined for nine species or subspecies of the subgenus Drosophila of the genus Drosophila. This segment contains two complete protein-coding genes (i.e., NADH dehydrogenase subunit 1 and cytochrome b) and a transfer RNA gene (tRNA(ser)). The G+C content at third-codon positions for the two protein-coding genes was 1.5 times higher than that in the D. melanogaster species group, which belongs to the subgenus Sophophora. However, there was a substantial difference between the nucleotide frequencies of G and C. The number of nucleotide substitutions per silent site was more than three times higher than that for nuclear DNA, although it was only 60% of that for mammalian mtDNA. Both parametric and nonparametric analyses revealed a strong transition-transversion bias in nucleotide substitution, as was observed in mammalian mtDNA. Moreover, the rate of substitution of A and T for G and C is higher than that for the opposite direction. This bias seems to be responsible for the extremely A+T-rich base composition of Drosophila mtDNA. It is also noted that the rate of transitional change between A and G is higher than that between T and C.     

High frequency of mitochondrial DNA D-loop mutations in Ewing’s sarcoma

Somatic mutations and polymorphisms in the noncoding displacement (D)-loop of mitochondrial DNA (mtDNA) are present in a variety of human cancers. To investigate whether Ewing’s sarcoma (EWS) harbors genetic alterations within the D-loop region and their potential association with EWS carcinogenesis, we analyzed and compared the complete mtDNA D-loop sequences from 17 pairs of tumor tissues and corresponding peripheral blood samples using the direct DNA sequencing method. Our results revealed that 12 of the 17 EWS tumor specimens (70.6%) carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletions. Among the tested 17 patients, we screened a total of 40 germline polymorphisms including one novel sequence variant in the D-loop fragment. Most of these identified mutations and germline variations were clustered within two hypervariable segments (HVS1 and HVS2) as well as the homopolymeric C stretch between nucleotide position 303 and 309. In addition, there was no significant correlation between mtDNA D-loop mutations and various clinicopathological factors of EWS. In conclusion, our study reports for the first time that mtDNA D-loop mutations occur at a high frequency in EWS. These data provide evidence of mtDNA alterations’ possible involvement in the initiation and/or progression of this rare malignancy.     

Intra- and interbreed genetic variations of mitochondrial DNA major non-coding regions in Japanese native dog breeds [Canis familiaris)

Mitochondrial DNA (mtDNA) major non-coding regions were amplified from 73 dogs of eight Japanese native dog breeds and from 21 dogs of 16 non-Japanese dog breeds by the polymerase chain reaction and their DNA sequences were determined. A total of 51 nucleotide positions within the non-coding region (969–972 base pairs) showed nucleotide variations of which 48 were caused by transition. These nucleotide substitutions were abundant in the region proximate to tRNA^Pro. In addition to the nucleotide substitutions, the dog mtDNA D-loop sequences had a heteroplasmic repetitive sequence (TACACGTÀCG) involving size variation. The DNA sequences of the non-coding region were classified into four different groups by phylogenetic analysis and the deepest branchpoints of this dog phylogeny was calculated to about 100 000 years before the present. Phylogenetic analysis showed that Japanese native dog breeds could not be clearly delimited as distinct breeds. Many haplotypes found in members of some clustering groups were seen in each dog breed, and interbreed nucleotide differences between Japanese dog breeds were almost the same as the intrabreed nucleotide diversities.