Similarity of mutation spectra of the mitochondrial DNA hypervariable segment 1 in Homo and Pan species

The mutation spectrum of mtDNA hypervariable segment 1 (HVS1) was compared for east chimpanzee Pan troglodytes schweigfurthi and human. The two HVS1 had much the same nucleotide composition, and their mutation spectra were similar in major characteristics (substantial prevalence of transitions over transversions, pyrimidine transitions over purine ones, and C --> T over T --> C). DNA strand displacement (dislocation) during replication was identified as a major mechanism of context-dependent mutagenesis in human and chimpanzee mtDNAs. Nucleotide positions with mutations fitting the model of dislocation mutagenesis accounted for 21% of all variable positions in the chimpanzee HVS1. Variable motifs proved to be similar in the chimpanzee and human HVS1. Comparison of the Neanderthal and modern human HVS1 nucleotide sequences showed that most variable nucleotides are in DNA sites allowing context-dependent mutagenesis.     

Comparison of the Mutation Spectrum of Hypervariable Segment 1 for Phylogeographical Groups of Human Mitochondrial DNA

The nucleotide sequence variation of hypervariable segment 1 (HVS1) was analyzed for mtDNAs of 88 phylogeographical clusters characteristic of African, West Eurasian, or East Eurasian populations. A significant difference in the distribution of mutations was revealed for the mitochondrial gene pools of the regional human populations. The HVS1 positions were identified whose instability is explained by strand displacement during mtDNA replication. Strand displacement was assumed to be a major mechanism of context-dependent mutagenesis associated with the regional differentiation of human populations.     

Similarity of Mutation Spectra of the Mitochondrial DNA Hypervariable Segment 1 in Homo and Pan Species

The mutation spectrum of mtDNA hypervariable segment 1 (HVS1) was compared for east chimpanzee Pan troglodytes schweigfurthi and human. The two HVS1 had much the same nucleotide composition, and their mutation spectra were similar in major characteristics (substantial prevalence of transitions over transversions, pyrimidine transitions over purine ones, and C T over T C). DNA strand displacement (dislocation) during replication was identified as a major mechanism of context-dependent mutagenesis in human and chimpanzee mtDNAs. Nucleotide positions with mutations fitting the model of dislocation mutagenesis accounted for 21% of all variable positions in the chimpanzee HVS1. Variable motifs proved to be similar in the chimpanzee and human HVS1. Comparison of the Neanderthal and modern human HVS1 nucleotide sequences showed that most variable nucleotides are in DNA sites allowing context-dependent mutagenesis.     

Analysis of phylogenetically reconstructed mutational spectra in human mitochondrial DNA control region

Analysis of mutations in mitochondrial DNA is an important issue in population and evolutionary genetics. To study spontaneous base substitutions in human mitochondrial DNA we reconstructed the mutational spectra of the hypervariable segments I and II (HVS I and II) using published data on polymorphisms from various human populations. An excess of pyrimidine transitions was found both in HVS I and II regions. By means of classification analysis numerous mutational hotspots were revealed in these spectra. Context analysis of hotspots revealed a complex influence of neighboring bases on mutagenesis in the HVS I region. Further statistical analysis suggested that a transient misalignment dislocation mutagenesis operating in monotonous runs of nucleotides play an important role for generating base substitutions in mitochondrial DNA and define context properties of mtDNA. Our results suggest that dislocation mutagenesis in HVS I and II is a fingerprint of errors produced by DNA polymerase gamma in the course of human mitochondrial DNA replication     

Genetic Characterization of Balkars and Karachays Using mtDNA Data

Russian Journal of Genetics - To determine mtDNA haplogroups in the populations of Balkars (N = 235) and Karachays (N = 123), the nucleotide sequence of the hypervariable segment 1 (HVS1) and...     

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.     

Release of replication termination controls mitochondrial DNA copy number after depletion with 2',3'-dideoxycytidine

Although cellular mitochondrial DNA (mtDNA) copy number varies widely among cell lines and tissues, little is known about the mechanism of mtDNA copy number control. Most nascent replication strands from the leading, heavy-strand origin (O_H) are prematurely terminated, defining the 3′ boundary of the displacement loop (D-loop). We have depleted mouse LA9 cell mtDNA to ~20% of normal levels by treating with 2′,3′-dideoxycytidine (ddC) and subsequently allowed recovery to normal levels of mtDNA. A quantitative ligation-mediated PCR assay was used to determine the levels of both terminated and extended nascent O_H strands during mtDNA depletion and repopulation. Depleting mtDNA leads to a release of replication termination until mtDNA copy number approaches a normal level. Detectable total nascent strands per mtDNA genome remain below normal. Therefore, it is likely that the level of replication termination plays a significant role in copy number regulation in this system. However, termination of D-loop strand synthesis is persistent, indicating formation of the D-loop structure has a purpose that is required under conditions of rapid recovery of depleted mtDNA.     

Mitochondrial DNA in the sea urchin Arbacia lixula: nucleotide sequence differences between two polymorphic molecules indicate asymmetry of mutations.

Two polymorphic forms of mitochondrial DNA (mtDNA) extracted from Arbacia lixula eggs were cloned and the nucleotide sequences of specific regions determined. A comparison of the sequences of the sense strand of the two molecules demonstrates that all the differences are transitions and only of the A----G type. A change such as G----A (or A----G) on the sense mtDNA strand results from either a direct G----A (or A----G) mutation on that strand or a C----T (or T----C) on the complementary strand. None of the C----T (or T----C) changes were detected on the sense strand, which implies that the A----G mutation bias on the sense strand is not reversed for the other strand. Our observation indicates the existence of mechanisms acting asymmetrically on the two mtDNA strands, possibly during mtDNA replication.     

The Effect of the Nucleotide Context on Induction of Mutations in Hypervariable Segment 1 of Human Mitochondrial DNA

The distribution of unstable nucleotide positions with a higher frequency of homoplastic mutations was analyzed in hypervariable segment 1 (HVS1) of the major noncoding region of human mtDNA. Three motifs (GTAC, ACCC, CCTC) proved to be associated with a higher rate of point substitutions at unstable positions. The motifs were often arranged in direct, including tandem, repeats. Motifs CCTC and ACCC were found in extended poly(C) tracts, which form direct repeats associated with deletions and tandem duplications. The results suggested that the inconstancy of the human mitochondrial genome is to a great extent determined by context-dependent mutations.     

Distribution of nucleotide substitutions in human mitochondrial DNA genes

To analyze the distribution pattern of nucleotide substitutions in human mitochondrial DNA (mtDNA), mutational spectra of the mitochondrial genes were reconstructed. The reconstruction procedure is based on the mutation distribution data for 47 monophyletic mtDNA clusters, to which 794 examined mtDNA sequences encoding for tRNAs, rRNAs, and mitochondrial proteins are attributed. One of specific features of mitochondrial mutational spectra revealed was homoplasy of the mutations (the mean mutation number per variable nucleotide site in the coding region varied from 1.09 to 1.43). It was established that in the mtDNA genes maximum mutational constraint fell onto the guanine bases, albeit the content of these bases in the mtDNA L-chains was minimal. Maximal bias towards parallel G to A transitions was observed for rRNA genes, with the protein-and tRNA-encoding genes ranking next. Despite the fact that the differences in the average G-nucleotides content and variability between the genes of two mtDNA segments located between the OriH and OriL were statistically significant, the results did not provide the conclusion that the G-nucleotide instability observed in the mtDNA L-spectra was determined by the mechanism of asynchronous mtDNA replication, along with the deamination of cytosines in the H-chain regions, which remained single-stranded during replication.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 93–99.Original Russian Text Copyright © 2005 by Malyarchuk.     

The effect of the nucleotide context on induction of mutations in hypervariable segment 1 of the human mitochondrial DNA

The distribution of unstable nucleotide positions with a higher frequency of homoplastic mutations was analyzed in hypervariable segment 1 (HVS1) of the major noncoding region of human mtDNA. Three motifs (GTAC, ACCC, CCTC) proved to be associated with a higher rate of point substitutions at unstable positions. The motifs were often arranged in direct, including tandem, repeats. Motifs CCTC and ACCC were found in extended poly(C) tracts, which form direct repeats associated with deletions and tandem duplications. The results suggested that the inconstancy of the human mitochondrial genome is to a great extent determined by context-dependent mutations.     

Mitochondrial DNA replication in human T lymphocytes is regulated primarily at the H-strand termination site.

The most unique feature in the replication of mitochondrial DNA (mtDNA) is that most of the newly synthesized heavy strands (H-strands) terminate prematurely, resulting in the formation of displacement loop (D-loop) strands. Only the H-strand which proceeds past the termination site is a true nascent H-strand leading to the overall replication on a circular mtDNA molecule. The physiological significance of the D-loop formation has long been unclear. To examine the role of premature termination in mtDNA replication, we therefore developed a method for selectively measuring both the total amount of nascent H-strands and the amount of true nascent H-strands using ligation-mediated polymerase chain reaction, which, for the first time, enabled us to estimate the frequency of premature termination. The stimulation of cell proliferation with interleukin 2 and phytohemagglutinin in human peripheral T lymphocytes caused an increase in the net replication rate of mtDNA. In stimulated cells, in comparison to resting ones, the amount of true nascent H-strands increased approx. 2.6-fold while the total amount of nascent H-strands remained unchanged, indicating that premature termination decreased while the initiation of replication remained the same. Our findings thus demonstrate the first clear example that premature termination plays a primary role in the up-regulation of the net rate of mtDNA replication in human cells.     

Distribution of nucleotide substitutions in human mitochondrial DNA genes

To analyze the distribution pattern of nucleotide substitutions in human mitochondrial DNA (mtDNA), mutational spectra of the mitochondrial genes were reconstructed. The reconstruction procedure is based on the mutation distribution data for 47 monophyletic mtDNA clusters, to which 794 examined mtDNA sequences encoding for tRNAs, rRNAs, and mitochondrial proteins are attributed. One of specific features of mitochondrial mutational spectra revealed was homoplasy of the mutations (the mean mutation number per variable nucleotide site in the coding region varied from 1.09 to 1.43). It was established that in the mtDNA genes maximum mutational constraint fell onto the guanine bases, albeit the content of these bases in the mtDNA L-chains was minimal. Maximal bias towards parallel G to A transitions was observed for rRNA genes, with the protein- and tRNA-encoding genes ranking next. Despite the fact that the differences in the average G-nucleotides content and variability between the genes of two mtDNA segments located between the OriH and OriL were statistically significant, the results did not provide the conclusion that the G-nucleotide instability observed in the mtDNA L-spectra was determined by the mechanism of asynchronous mtDNA replication, along with the deamination of cytosines in the H-chain regions, which remained single-stranded during replication.     

The role of nucleotide context in the induction of mutations in human mitochondrial DNA genes

Based on the mutations distribution patterns in the mitochondrial DNA (mtDNA) genes, context analysis of the regions, including mutable positions characterized by the appearance of more than two parallel mutations, was performed. It was demonstrated that the mechanism of dislocation mutagenesis, leading to the appearance of mismatches within the frameshift regions of either primer or template mtDNA chains during replication, accounts for the induction of 21% of unstable positions in the mtDNA genes. Context analysis showed that pyrimidine bases in the positions +1 and +2 (gYRNS, gYY, and gR consensuses, where g is mutable position) had the highest influence on the induction of mutations in G positions of the mtDNA genes. The highest effect on the mutagenesis in T positions was excreted by the bases in the positions -1 and +1 (RyT and tA consensuses, where t is mutable position). In general, these data point to the prevalence of the context-dependant mechanisms of the mutations induction in human mitochondrial genome.     

Three novel mtDNA restriction site polymorphisms allow exploration of population affinities of African Americans

To develop informative tools for the study of population affinities in African Americans, we sequenced the hypervariable segments I and II (HVS I and HVS II) of mitochondrial DNA (mtDNA) from 96 Sierra Leoneans; European Americans; rural, Gullah-speaking African Americans; urban African Americans living in Charleston, South Carolina; and Jamaicans. We identified single nucleotide polymorphisms (SNPs) exhibiting ethnic affinities, and developed restriction endonuclease tools to screen these SNPs. Here we show that three HVS restriction site polymorphisms (RSPs), EcoRV, FokI, and MfeI, exhibit appreciable differences in frequency (average delta = 0.4165) between putative African American parental populations (i.e., extant Africans living in Sierra Leone and European Americans). Estimates of European American mtDNA admixture, calculated from haplotypes composed of these three novel RSPs, show a cline of increasing admixture from Gullah-speaking African American (m = 0.0300) to urban Charleston African American (m = 0.0689) to West Coast African American (m = 0.1769) populations. This haplotype admixture in the Gullahs is the lowest recorded to date among African Americans, consistent with previous studies using autosomal markers. These RSPs may become valuable new tools in the study of ancestral affinities and admixture dynamics of African Americans.     

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.     

塔克拉玛干沙漠腹地隔离人群线粒体DNA序列多态性分析

对新疆塔克拉玛干沙漠腹地的75名克里雅人线粒体DNA的高可变I区的15996—16401的片段进行了序列分析,在所测定的75个个体中,共检测到68个位点存在变异,界定了71种不同的单倍型。克里雅人群的核昔酸变异度和平均核苷酸差异都介于所报道的东方人群和西方人群之间。根据Neighbor-joining法构建系统发育树,发现中亚的各人群均处于东方人群的亚洲谱系和西方人群的欧洲谱系之间,并且克里雅与新疆维吾尔和境外维吾尔之间的遗传距离最近,表明他们之间有很密切的亲缘关系。     

The role of nucleotide context in the induction of mutations in human mitochondrial DNA genes

Based on the mutations distribution patterns in the mitochondrial DNA (mtDNA) genes, context analysis of the regions, including mutable positions characterized by the appearance of more than two parallel mutations, was performed. It was demonstrated that the mechanism of dislocation mutagenesis, leading to the appearance of mismatches within the frameshift regions of either primer or template mtDNA chains during replication, accounts for the induction of 21% of unstable positions in the mtDNA genes. Context analysis showed that pyrimidine bases in the positions +1 and +2 (gYRNS, gYY, and gR consensuses, where g is mutable position) had the highest influence on the induction of mutations in G positions of the mtDNA genes. The highest effect on the mutagenesis in T positions was excreted by the bases in the positions –1 and +1 (RtY and tA consensuses, where t is mutable position). In general, these data point to the prevalence of the context-dependant mechanisms of the mutations induction in human mitochondrial genome.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 385–390.Original Russian Text Copyright © 2005 by Malyarchuk.