Variation in repeat length and heteroplasmy of the mitochondrial DNA control region along a core-edge gradient in the eastern spadefoot toad (Pelobates syriacus)

Peripheral populations are those situated at the distribution margins of a species and are often subjected to more extreme abiotic and biotic conditions than those at the core. Here, we hypothesized that shorter repeat length and fewer heteroplasmic mitochondrial DNA (mtDNA) copies, which are associated with more efficient mitochondrial function, may be related to improved survival under extreme environmental conditions. We sampled eastern spadefoot toads (mostly as tadpoles) from 43 rain pools distributed along a 300-km gradient from core to edge of the species' distribution. We show that mean pool tandem repeat length and heteroplasmy increase from edge to core, even after controlling for body size. We evaluate several alternative hypotheses and propose the Fisher hypothesis as the most likely explanation. However, additional sequential sampling and experimental studies are required to determine whether selection under extreme conditions, or alternative mechanisms, could account for the gradient in heteroplasmy and repeat length in the mtDNA control region.     

Individual human hair mitochondrial DNA control region heteroplasmy proportions in mothers and children

Due to maternal inheritance, lack of recombination and a high polymorphic density, the mtDNA control region hypervariable (HV) regions are well suited for forensic identification using a maternal relative as the known sample. This analysis can be performed in hair, however, heteroplasmy in this tissue is not rare and can result in an apparent sequence mismatch that complicates this application. There is little data comparing mother and child mtDNA-CR heteroplasmic proportions in hair. In this study, we assayed four hairs per individual in 26 mother-child pairs by TTGE for heteroplasmy across HV1. Single nucleotide heteroplasmy was detected in seven families, and in four families at least two hairs were heteroplasmic. In each of the latter families, sequencing and PCR-RFLP confirmed single nucleotide heteroplasmy in proportions of the variant ranging from < or =10 to > or =90% in the mothers, with far less variability in their children. Sequencing alone would have revealed apparent homoplasmic differences at one nucleotide in these families, possibly resulting in an 'inconclusive' verdict for relatedness of child and mother. However, mother-child heteroplasmic variability did not exceed intra-individual variability in the mothers alone.     

Polymorphic sequence in the D-loop region of equine mitochondrial DNA

The D-loop regions in equine mitochondrial DNA were cloned from three thoroughbred horses by polymerase chain reaction (PCR). The total number of bases in the D-loop region were 1114bp, 1115bp and 1146bp. The equine D-loop region is A/T rich like many other mammalian D-loops. The large central conserved sequence block and small conserved sequence blocks 1, 2 and 3, that are common to other mammals, were observed. Between conserved sequence blocks 1 and 2 there were tandem repeats of an 8bp equine-specific sequence TGTGCACC, and the number of tandem repeats differed among individual horses. The base composition in the unit of these repeats is G/C rich as are the short repeats in the D-loops of rabbit and pig. Comparing DNA sequences between horse and other mammals, the difference in the D-loop region length is mostly due to the difference in the number of DNA sequences at both extremities. The similarities of the DNA sequences are in the middle part of the D-loop. In comparison of the sequences among three thoroughbred horses, it was determined that the region between tRNA^Pro and the large central conserved sequence block was the richest in variation. PCR primers in the D-loop region were designed and the expected maternal inheritance was confirmed by PCR-RFLP (restriction fragment length polymorphism).     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

Mitochondrial topoisomerase I sites in the regulatory D-loop region of mitochondrial DNA

Mitochondrial DNA (mtDNA) is required for mitochondrial activities because it encodes key proteins for oxidative phosphorylation and the production of cellular ATP. We previously reported the existence of a specific mitochondrial topoisomerase gene, Top1mt, in all vertebrates. The corresponding polypeptide contains an N-terminal mitochondrial targeting sequence and is otherwise highly homologous to the nuclear topoisomerase I (Top1). In this study, we provide biochemical evidence of the presence of an endogenous Top1mt polypeptide in human mitochondria. Using novel antibodies against Top1mt, we detected the corresponding 70 kDa polypeptide in mitochondria but not in nuclear fractions. This polypeptide could be trapped to form covalent complexes with mtDNA when mitochondria from human cells were treated with camptothecin. Mapping of Top1mt sites in the regulatory D-loop region of mtDNA in mitochondria revealed the presence of an asymmetric cluster of Top1mt sites confined to a 150 bp segment downstream from, and adjacent to, the site at which replication is prematurely terminated, generating an approximately 650-base (7S DNA) product that forms the mitochondrial D-loop. Moreover, we show that inhibition of Top1mt by camptothecin reduces the level of formation of the 7S DNA. These results suggest novel roles for Top1mt in regulating mtDNA replication.     

Analysis of mitochondrial DNA mutations in D-loop region in thyroid lesions

Background

Mitochondrial defects have been associated with various human conditions including cancers.

Methods

We analyzed the mutations at the mitochondrial DNA (mtDNA) in patients with different thyroid lesions. In particular, in order to investigate if the accumulation of mtDNA mutations play a role in tumor progression, we studied the highly variable main control region of mtDNA, the displacement-loop (D-loop) in patients with non-tumor nodular goiters, with benign thyroid adenomas, and with malignant thyroid carcinomas. Total thyroid tumor or goiter samples were obtained from 101 patients, matched with nearby normal tissue and blood from the same subject.

Results

Noticeably, mitochondrial microsatellite instability (mtMSI) was detected in 2 of 19 nodular goiters (10.53%), and 8 of 77 (10.39%) malignant thyroid carcinomas. In addition, 6 patients, including 5 (6.49%) with malignant thyroid carcinomas and 1 (5.26%) with nodular goiter, were found to harbor point mutations. The majority of the mutations detected were heteroplasmic.

General significance

Our results indicate that mtDNA alterations in the D-loop region could happen before tumorigenesis in thyroid, and they might also accumulate during tumorigenesis.     

Mutations in the mitochondrial DNA D-loop region are frequent in cervical cancer

Background  

Mitochondrial DNA (mtDNA) is known for high mutation rates caused by lack of protective histones, inefficient DNA repair systems, and continuous exposure to mutagenic effects of oxygen radicals. Alterations in the non-coding displacement (D) loop of mitochondrial DNA are present in many cancers. It has been suggested that the extent of mitochondrial DNA mutations might be useful in the prognosis of cancer outcome and/or the response to certain therapies. In order to investigate whether a high incidence of mutations exist in mitochondrial DNA of cervical cancer patients, we examined the frequency of mutations in the D-loop region in 19 patients of cervical cancer.     

Haplotype diversity of the mitochondrial DNA D-loop region in Calomys musculinus (Rodentia,Muridae) detected by PCR-RFLP

In order to contribute to knowledge of colonization patterns in the rodent Calomys musculinus, a natural reservoir of the virus producing Argentine hemorrhagic fever (AHF), we studied the haplotype diversity of the mitochondrial DNA D-loop region in five natural populations from central Argentina. Digestion with eight restriction enzymes (RsaI, MseI, Tsp509I, AluI, AciI, HaeIII, NlaIII, and AseI) revealed polymorphism in the 1300 bp fragment amplified by PCR. Twenty different composite haplotypes were detected. Hierarchical analyses indicated that almost all variation (94%) is contained within local populations. Haplotypes 1 and 2, shared by all populations, were the most frequent. Nonsignificant genetic differentiation was found among populations of the endemic and nonendemic areas of AHF: All locations sampled presented exclusive haplotypes in spite of their geographic proximity, which would support previous observations indicating restricted gene flow among C. musculinus populations.     

Sea urchin egg mitochondrial DNA contains a short displacement loop (D-loop) in the replication origin region.

Based on solution hybridization using single-stranded probes, native mitochondrial DNA extracted from sea urchin eggs contains a displacement-loop (D-loop) of approximately 70-80 nt. This maps to the single extended unassigned sequence of the genome, between the genes for tRNA(thr) and tRNA(pro), which also appears to contain the origin of first-strand replication. The D-loop commences at or close to a site of supercoil-dependent S1 nuclease hypersensitivity, adjacent to a run of 20 consecutive C residues, terminates near to the boundary of tRNA(thr), and appears to be composed at least partly of RNA, based on the sensitivity of the assays to RNase H. These experiments imply that the mechanisms of replication initiation in sea urchin and vertebrate mtDNAs are very similar, and suggest that the developmental restriction on mtDNA synthesis in eggs and embryos is maintained at the level of D-loop extension.     

Phylogenetic relationships of intraspecific forms of the house mouse Mus musculus: Analysis of variability of the control region (D-loop) of mitochondrial DNA

Analysis of the control region of mitochondrial DNA (mtDNA) or D-loop of 96 house mice (Mus musculus) from Russia, Moldova, Armenia, Azerbaijan, Kazakhstan, and Turkmenistan has been used to reconstruct the phylogenetic relationships and phylogeographic patterns of intraspecific forms. New data on the phylogenetic structure of the house mouse are presented. Three phylogroups can be reliably distinguished in the eastern part of the M. musculus species range, the first one mainly comprising the haplotypes of mice from Transcaucasia (Armenia); the second one, the haplotypes of mice from Kazakhstan; and the third one, the haplotypes of mice from Siberia and some other regions. The morphological subspecies M. m. wagneri and M. m. gansuensis have proved to be genetically heterogeneous and did not form discrete phylogroups in the phylogenetic tree.     

DNA sequence variation in the mitochondrial control region of red-backed voles (Clethrionomys)

The complete mitochondrial DNA (mtDNA) control region was sequenced for 71 individuals from five species of the rodent genus Clethrionomys both to understand patterns of variation and to explore the existence of previously described domains and other elements. Among species, the control region ranged from 942 to 971 bp in length. Our data were compatible with the proposal of three domains (extended terminal associated sequences [ETAS], central, conserved sequence blocks [CSB]) within the control region. The most conserved region in the control region was the central domain (12% of nucleotide positions variable), whereas in the ETAS and CSB domains, 22% and 40% of nucleotide positions were variable, respectively. Tandem repeats were encountered only in the ETAS domain of Clethrionomys rufocanus. This tandem repeat found in C. rufocanus was 24 bp in length and was located at the 5' end of the control region. Only two of the proposed CSB and ETAS elements appeared to be supported by our data; however, a "CSB1-like" element was also documented in the ETAS domain.     

A family exhibiting heteroplasmy in the human mitochondrial DNA control region reveals both somatic mosaicism and pronounced segregation of mitotypes

A family exhibiting heteroplasmy at position 16 355 in hypervariable region I of the human mtDNA control region has been identified. This family consists of a mother, daughter, and son. DNA samples extracted from blood stains, buccal swabs, and hairs from these individuals were amplified by PCR and sequenced utilizing fluoresence-labeled dye terminator chemistry in an automated DNA sequencer. In both the daughter and mother, heteroplasmy was observed in DNA extracted from blood stains, buccal swabs, and hairs. In the blood stains, the proportion of cytosine was greater than thymine in both individuals. Buccal swab extracts showed a more balanced contribution from the two nucleotides. Telogenic hair root and hair shaft samples exhibited a wide range of nucleotide contributions at this position, from predominately cytosine in some samples to predominately thymine in others. The apparent stochastic segregation of mitotypes in hair samples is discussed from a forensic viewpoint, and the mechanism of mtDNA heteroplasmy is considered. Received: 6 November 1996 / Accepted: 13 February 1997     

Transmission of mitochondrial DNA heteroplasmy in normal pedigrees

The presence of multiple mitochondrial genotypes (heteroplasmy) has been studied in normal individuals. Six multigenerational normal families were screened for heteroplasmy by PCR of the mitochondrial control region and the cytochrome c oxidase intergenic regions. Two individuals from different families exhibited multiple length polymorphisms in a homopolymeric tract at positions 16 184–16 193 and a grandmother in a third family was heteroplasmic for both cytosine and thymidine at position 15 945. Although the 15 945 T variant comprised 28% of the grandmother’s mitochondrial DNA, this sequence was not present in any of her descendants. Heteroplasmy was detected in 2.5% of the 96 mother-offspring pairs, consistent with the possibility that it may not be rare. Received: 18 August 1997 / Accepted: 10 November 1997     

Naturally occurring mitochondrial DNA heteroplasmy in the MRL mouse

The MRL/MpJ mouse is an inbred laboratory strain of Mus musculus, known to exhibit enhanced autoimmunity, increased wound healing, and increased regeneration properties. We report the full-length mitochondrial DNA (mtDNA) sequence of the MRL mouse (Accession # EU450583), and characterize the discovery of two naturally occurring heteroplasmic sites. The first is a T3900C substitution in the TPsiC loop of the tRNA methionine gene (tRNA-Met; mt-Tm). The second is a heteroplasmic insertion of 1-6 adenine nucleotides in the A-tract of the tRNA arginine gene (tRNA-Arg; mt-Tr) at positions 9821-9826. The level of heteroplasmy varied independently at these two sites in MRL individuals. The length of the tRNA-Arg A-tract increased with age, but heteroplasmy at the tRNA-Met site did not change with age. The finding of naturally occurring mtDNA heteroplasmy in an inbred strain of mouse makes the MRL mouse a powerful new experimental model for studies designed to explore therapeutic measures to alter the cellular burden of heteroplasmy.     

Evolution of the cetacean mitochondrial D-loop region.

We sequenced the mitochondrial DNA D-loop regions from two cetacean species and compared these with the published D-loop sequences of several other mammalian species, including one other cetacean. Nucleotide substitution rates, DNA sequence simplicity, possible open reading frames (ORFs), and potential RNA secondary structure were investigated. The substitution rate is an order of magnitude lower than would be expected on the basis of reports on human sequence variation in this region but are consistent with interspecific primate and rodent D-loop sequence variation and with estimates of substitution rates from whole mitochondrial genomes. Deletions/insertions are less common in the cetacean D-loop than in other vertebrate species. Areas of high sequence simplicity (clusters of short repetitive motifs) across the region correspond to areas of high sequence divergence. Three regions predicted to form secondary structures are homologous to such putative structures in other species; however, the presumptive structures most conserved in cetaceans are different from those reported for other taxa. While all three species have possible long ORFs, only a short sequence of seven amino acids is shared with other mammalian species, and those changes that had occurred within it are all nonsynonymous. We conclude that DNA slippage, in addition to point mutation, contributes to the evolution of the D-loop and that regions of conserved secondary structure in cetaceans and an ORF are unlikely to contribute significantly to the conservation of the central region.     

Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

A methylation protection assay was used in a novel manner to demonstrate a specific bovine protein-mitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a approximately 48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end.     

Analysis of canine transmissible veneral tumor genotypes using the D-loop region of mitochondrial DNA

Canine transmissible venereal tumor (CTVT) is the only neoplasm that can be spread among dogs through cell transplantation. Therefore, this tumor does not originate from host cell transformation. Although CTVT has a monophyletic origin, several studies have shown the presence of genetic diversity which was probably acquired after the development of its original clone. To investigate the genetic diversity of CTVT in Mexico and its relation with CTVTs disseminated worldwide, we sequenced a fragment of mitochondrial DNA in 50 tumor samples and matched blood samples from dog hosts from Mexico. We found ten new haplotypes in tumor samples, which were all distinct from their matched host. The TVT1 haplotype was the most frequent in our samples, suggesting that it could be the origin of the others. We found that haplotypes in Mexico and other countries are distributed in two well-defined clusters. Our data also suggest a close relationship among American haplotypes (Mexico, USA, Chile and Brazil). Interestingly, these American haplotypes were also closely related to Asian haplotypes. Taking into account the estimated timing of the origin of CTVT, we propose that CTVT might have originated in Asia; consequently, haplotypes currently present in America could descend from Asiatic lineages.     

Length and restriction site heteroplasmy in the mitochondrial DNA of american shad (alosa sapidissima)

Restriction endonuclease analysis was used to assess mitochondrial DNA (mtDNA) variation in American shad (Alosa sapidissima) collected from 14 rivers ranging from Florida to Quebec. Two types of heteroplasmy were observed, one involving a major length polymorphism and the other a single restriction site. Shad mtDNA occurred in two principal size classes, 18.3 and 19.8 kb. Of 244 shad examined, 30 were heteroplasmic and carried both size classes of mtDNA in varying proportions; the remainder were homoplasmic for the smaller size class of mtDNA. The large mtDNA variant occurred most frequently at the southern end of the range, and except for two individuals from Nova Scotia, was not detected among shad from rivers north of the Delaware. In contrast, ten shad heteroplasmic for a SalI restriction site originated from rivers ranging from South Carolina to Nova Scotia. DNA mapping and hybridization experiments indicated that the length polymorphism is in the D-loop-containing region and consists of a tandemly repeated 1.5-kb DNA sequence occurring in two and three copies, respectively, in the two major size classes of shad mtDNA. Continuous length variation up to approximately 40 bp occurs among copies of the repeat both within and among individuals. Restriction site data support the conclusion that both forms of heteroplasmy in shad mtDNA have originated more than once.