Heteroplasmy of Short Tandem Repeats in Mitochondrial DNA of Atlantic Cod, Gadus Morhua

The mitochondrial DNA of the Atlantic cod (Gadus morhua) contains a tandem array of 40-bp repeats in the D-loop region of the molecule. Variation among molecules in the copy number of these repeats results in mtDNA length variation and heteroplasmy (the presence of more than one form of mtDNA in an individual). In a sample of fish collected from different localities around Iceland and off George's Bank, each individual was heteroplasmic for two or more mtDNAs ranging in repeat copy number from two (common) to six (rare). An earlier report on mtDNA heteroplasmy in sturgeon (Acipenser transmontanus) presented a competitive displacement model for length mutations in mtDNAs containing tandem arrays and the cod data deviate from this model. Depending on the nature of putative secondary structures and the location of D-loop strand termination, additional mechanisms of length mutation may be needed to explain the range of mtDNA length variants maintained in these populations. The balance between genetic drift and mutation in maintaining this length polymorphism is estimated through a hierarchical analysis of diversity of mtDNA length variation in the Iceland samples. Eighty percent of the diversity lies within individuals, 8% among individuals and 12% among localities. An estimate of theta = 2N(eo) mu greater than 1 indicates that this system is characterized by a high mutation rate and is governed primarily by deterministic dynamics. The sequences of repeat arrays from fish collected in Norway, Iceland and George's Bank show no nucleotide variation suggesting that there is very little substructuring to the North Atlantic cod population.     

Length and Sequence Variation in Evening Bat D-Loop Mtdna

Length variation in D-loop mitochondrial DNA was observed after amplification with the polymerase chain reaction (PCR) in 28% of 195 evening bats, Nycticeius humeralis, from seven colonies. Nucleotide sequences of PCR products show that this heteroplasmy is characterized by an 81-bp region which is tandemly repeated five to eight times. Southern blots using PCR products as probes on HaeIII genomic digests confirm the presence of heteroplasmy. Furthermore, densitometry of electrophoresed PCR products from 109 mother-offspring pairs indicate that heteroplasmy is stably transmitted from mother to offspring with one exception: a heteroplasmic offspring had a homoplastic mother and sib. Nucleotide sequences from this family reveal that a repeat duplication and deletion occurred. The observed mutation rate per generation, mu, for length polymorphism is comparable to an independent estimate, mu = 10(-2), based on hierarchical diversity statistics. With the exception of the repeat nearest the proline tRNA gene, sequence similarities between repeats within bats are consistent with a model of concerted evolution due to unidirectional replication slippage. Selection is inferred to act on the first repeat because in comparison to other repeats it has the least sequence divergence among bats, the fewest transversions, and the lowest minimum free energy associated with folding.     

Length Variation, Heteroplasmy and Sequence Divergence in the Mitochondrial DNA of Four Species of Sturgeon (Acipenser)

The extent of mtDNA length variation and heteroplasmy as well as DNA sequences of the control region and two tRNA genes were determined for four North American sturgeon species: Acipenser transmontanus, A. medirostris, A. fulvescens and A. oxyrhnychus. Across the Continental Divide, a division in the occurrence of length variation and heteroplasmy was observed that was concordant with species biogeography as well as with phylogenies inferred from restriction fragment length polymorphisms (RFLP) of whole mtDNA and pairwise comparisons of unique sequences of the control region. In all species, mtDNA length variation was due to repeated arrays of 78-82-bp sequences each containing a D-loop strand synthesis termination associated sequence (TAS). Individual repeats showed greater sequence conservation within individuals and species rather than between species, which is suggestive of concerted evolution. Differences in the frequencies of multiple copy genomes and heteroplasmy among the four species may be ascribed to differences in the rates of recurrent mutation. A mechanism that may offset the high rate of mutation for increased copy number is suggested on the basis that an increase in the number of functional TAS motifs might reduce the frequency of successfully initiated H-strand replications.     

Mitochondrial DNA Length Variation and Heteroplasmy in Populations of White Sturgeon (Acipenser Transmontanus)

Southern blot analysis was used to quantify the extent of mtDNA D-loop length variation in two populations of white sturgeon, Acipenser transmontanus. Over 42% of individuals were heteroplasmic for up to six different mtDNA length variants attributable to varying copy numbers of an 82-bp repeat sequence. Chi-square analyses revealed that the frequencies of length genotypes and the incidence of heteroplasmy were significantly different between Fraser and Columbia River sturgeon populations but not between restriction site haplotypes. Heteroplasmic fish have, on average, higher copy number than homoplasmic fish. Forty-five of 101 homoplasmic individuals carry only a single copy of the repeat, while none of the 73 heteroplasmic fish has the single repeat as the predominant variant. On the basis of differences in frequency distributions of copy number within and between fish, we suggest that (1) heteroplasmy is maintained by high recurrent mutation of multiple copy genomes, favoring increased copy number and (2) the mutation pressure toward higher copy number heteroplasmy is partially offset by selection to reduced genome size and segregation to the homoplasmic condition.     

Nuclear mitochondrial interplay in the modulation of the homopolymeric tract length heteroplasmy in the control (D-loop) region of the mitochondrial DNA

We have studied the genetic characteristics of a homopolymeric tract length heteroplasmy associated with the 16189C variant in the mtDNA D-loop control region to identify the factor(s) involved in the generation of the length heteroplasmy. The relative proportion of the various lengths of the polycytosines (i.e., the pattern of the length heteroplasmy) is maintained in an individual, and previous evidence shows that it is regenerated de novo following cell divisions. The pattern is maintained in maternally related individuals, suggestive of mtDNA determinants. Of the 38 individuals with the 16189C variant studied, 39% were found to exhibit the (16180)AAACCCCCCCCCCC(16193) variant associated with A16183C polymorphism [(11C)-group], while 53% showed the (16180)AACCCCCCCCCCCC(16193) variant associated with a further A16182C polymorphism [(12C)-group]. Haplotype analysis of the mtDNA revealed a specific association of the longer mean length of the poly[C] in the (12C)-group with haplogroup B. A similar association was also observed in the (11C)-group, but with a novel haplogroup. Cybrid constructions revealed that the involvement of nuclear factor(s) in the generation of the length heteroplasmy is prominent in homopolymeric tract of eight cytosines. The nuclearly coded factor(s) is/are presumably related to the fidelity of the nuclearly coded components of the mitochondrial DNA replication machinery.     

Mitochondrial genome size variation in New World and Old World populations of Drosophila melanogaster

Drosophila melanogaster originated in Africa, spread to Europe and Asia, and is believed to have colonized the New World in the past few hundred years. Levels of genetic variation are typically reduced in New World populations, consistent with a founder event following range expansion out of Africa and the Old World. We describe the patterns of mtDNA length variation within and among several populations of Drosophila melanogaster from the Old and New World. MtDNA length variation is due to insertion and deletion of tandem repeats in the control region (D-loop) of D. melanogaster mitochondrial genome. The distinct mutational dynamics of this system provide an opportunity to compare the patterns of variation in this marker to those of other markers with different mutational pressures and linkage relationships. The data show significantly more length variation in African and Asian samples than in New World samples. New World samples also show more pronounced skew of the length distribution. Our results are distinct from an earlier study that showed significantly higher levels of length variation and heteroplasmy. The level of heteroplasmy is highly correlated with the number of years that samples have been maintained in laboratory culture, suggesting that relaxed selection in small populations permits the accumulation of mtDNA length variation and heteroplasmy. Together, the data indicate that mtDNA length variants retain a signature of founder events and selection, and suggest that further investigation into the mutation-selection dynamics of the D-loop region of mtDNA would provide a distinct and informative marker for analysis of the recent history of populations.     

Lack of Structural Variation but Extensive Length Polymorphisms and Heteroplasmic Length Variations in the Mitochondrial DNA Control Region of Highly Inbred Crested Ibis,Nipponia nippon

The animal mitochondrial DNA (mtDNA) length polymorphism and heteroplasmy are accepted to be universal. Here we report the lack of structural variation but the presence of length polymorphism as well as heteroplasmy in mtDNA control region of an endangered avian species – the Crested Ibis (Nipponia nippon). The complete control region was directly sequenced while the distribution pattern and inheritance of the length variations were examined using both direct sequencing and genotyping of the PCR fragments from captive birds with pedigrees, wild birds and a historical specimen. Our results demonstrated that there was no structural variation in the control region, however, different numbers of short tandem repeats with an identical motif of CA₃CA₂CA₃ at the 3′-end of the control region determined the length polymorphisms among and heteroplasmy within individual birds. There were one to three predominant fragments in every bird; nevertheless multiple minor fragments coexist in all birds. These extremely high polymorphisms were suggested to have derived from the ‘replication slippage’ of a perfect microsatellite evolution following the step-wise mutational model. The patterns of heteroplasmy were found to be shifted between generations and among siblings but rather stable between blood and feather samples. This study provides the first evidence of a very extensive mtDNA length polymorphism and heteroplasmy in the highly inbred Crested Ibis which carries an mtDNA genome lack of structural genetic diversity. The analysis of pedigreed samples also sheds light on the transmission of mtDNA length heteroplasmy in birds following the genetic bottleneck theory. Further research focusing on the generation and transmission of particular mtDNA heteroplasmy patterns in single germ line of Crested Ibis is encouraged by this study.     

Widespread heteroplasmy in schistosomes makes an mtVNTR marker "nearsighted".

Mitochondrial markers are often hailed as the preferred DNA elements for analyses of population subdivision. To this end we have employed a mitochondrial repeat element to examine the population structure in Schistosoma mansoni (human blood flukes). Schistosome isolates were collected from each of 21 different patients representing seven different areas of a Brazilian village. These parasite isolates demonstrate substantial genetic polymorphism, with an average of 10 genotypes infecting each patient, which is more readily detected because of high levels of heteroplasmy (i.e., 72.5% of the individual worms exhibit multiple versions of this repeat region with different numbers of repeats). Due to the high number of common haplotypes in the population, this repeat element from S. mansoni has a large proportion (47%) of its genetic variation described by differences among mitochondrial genomes within individual worms. However, when only rare haplotypes are considered, population structure can be detected. It seems that heteroplasmy in the schistosome population of Melquiades is both the source of plentiful genetic variation and a confounding factor in the analysis of that variation. Thus the schistosome population in Melquiades may actually be more strongly subdivided than we are able to detect using this mitochondrial marker.     

Complete mtDNA of Meretrix lusoria (Bivalvia: Veneridae) reveals the presence of an atp8 gene, length variation and heteroplasmy in the control region

The complete nucleotide sequence of the mitochondrial genome of the clam Meretrix lusoria (Bivalvia: Veneridae) was determined. It comprises 20,268 base pairs (bp) and contains 13 protein-coding genes, including ATPase subunit 8 (atp8), two ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. The atp8 encodes a protein of 39 amino acids. All genes are encoded on the same strand. A putative control region (CR or D-loop) was identified in the major non-coding region (NCR) between the tRNA^Gly and tRNA^Gln. A 1087 bp tandem repeat fragment was identified that comprises nearly 11 copies of a 101 bp motif and accounts for approximately 41% of the NCR. The 101 bp tandem repeat motif of the NCR can be folded into a stem–loop secondary structure. Samples of eight individuals from Hainan and Fujian provinces were collected and their NCR regions were successfully amplified and sequenced. The data revealed a highly polymorphic VNTR (variable number of tandem repeats) associated with high levels of heteroplasmy in the D-loop region. The size of the CR ranged from 1942 to 3354 bp depending upon the copy number of the repeat sequence.     

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.     

Heteroplasmy in the mtDNA control region of sturgeon (Acipenser, Huso and Scaphirhynchus)

Data from 1238 fishes from 19 sturgeon species and 1 paddlefish were used to analyze heteroplasmy in sturgeon. Lengths of central repeat units ranged from 74 to 83 bp among sturgeon species. No repeat sequence was found in the paddlefish, Polyodon spathula. A general feature of the repeat units was the presence of termination associated sequence (TAS) motifs. About 50% of 138 interspecific mutations observed among the D-loop sequences are located 10 bp down- and upstream from these TAS motifs. Interestingly, most homoplasmic species showed deletions upstream to the TAS motifs, whereas deletions downstream to the TAS motifs observed in two species do not seem to preclude heteroplasmy. Calculations of secondary structures and thermal stabilities of repeat units showed DeltaG values for all heteroplasmic species to be <-8 and for most homoplasmic species DeltaG value to be >-8. Most heteroplasmic fishes had two and/or three repeat units. No homoplasmic sturgeon with >2 repeat units were observed. Molecular phylogeny based on the entire cytochrome b showed that heteroplasmy probably resulted from a single evolutionary event. Our data demonstrate that heteroplasmy is present in most sturgeon species and suggest that the thermal stability of the secondary structure of the repeat unit in combination with mutations downstream of the TAS sequences influences heteroplasmy.     

Analysis of heteroplasmy in the major noncoding region of mitochondrial DNA in the blood and atherosclerotic plaques of carotid arteries

For identification of somatic mitochondrial DNA (mtDNA) mutations, the mtDNA major noncoding region (D-loop) sequence in blood samples and carotid atherosclerosis plaques from patients with atherosclerosis was analyzed. Five point heteroplasmic positions were observed in 4 of 23 individuals (17%). Only in two cases could heteroplasmy have resulted from somatic mutation, whereas three heteroplasmic positions were found in both vascular tissue and blood. In addition, length heteroplasmy in a polycytosine stretches was registered at nucleotide positions 303–315 in 16 individuals, and also in the 16184–16193 region in four patients. The results suggest that somatic mtDNA mutations can occur during atherosclerosis, but some heteroplasmic mutations may appear in all tissues, possibly being inherited.     

Extensive mtDNA diversity in horses revealed by PCR–SSCP analysis

The hypervariable D-loop region of mitochondrial DNA (mtDNA) was amplified with the polymerase chain reaction using total horse DNA samples. Analysis of single strand conformation polymorphism (SSCP) of denatured amplification products was carried out by native polyacrylamide (8%) gel electrophoresis followed by silver staining. As many as 15 distinct SSCP variants were revealed when screening a total of 78 maternally unrelated horses representing five different breeds. All breeds showed a high degree of polymorphism and the estimated probability (PI_mt) that two maternally unrelated individuals have, by chance, identical SSCP variants varied between 0.14 and 0.30. We detected no heteroplasmy or deviations from strict and stable maternal inheritance when examining four maternal lineages, each represented by six to eight horses, separated by up to five generations from a common ancestral mare. The study establishes a simple screening method for detecting equine mtDNA types, which can be applied for tracing maternal genealogies and for association studies.     

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.     

Evolution of Repeated Sequence Arrays in the D-Loop Region of Bat Mitochondrial DNA

Analysis of mitochondrial DNA control region sequences from 41 species of bats representing 11 families revealed that repeated sequence arrays near the tRNA-Pro gene are present in all vespertilionine bats. Across 18 species tandem repeats varied in size from 78 to 85 bp and contained two to nine repeats. Heteroplasmy ranged from 15% to 63%. Fewer repeats among heteroplasmic than homoplasmic individuals in a species with up to nine repeats indicates selection may act against long arrays. A lower limit of two repeats and more repeats among heteroplasmic than homoplasmic individuals in two species with few repeats suggests length mutations are biased. Significant regressions of heteroplasmy, θ and π, on repeat number further suggest that repeat duplication rate increases with repeat number. Comparison of vespertilionine bat consensus repeats to mammal control region sequences revealed that tandem repeats of similar size, sequence and number also occur in shrews, cats and bighorn sheep. The presence of two conserved protein-binding sequences in all repeat units indicates that convergent evolution has occurred by duplication of functional units. We speculate that D-loop region tandem repeats may provide signal redundancy and a primitive repair mechanism in the event of somatic mutations to these binding sites.     

Frequency and Pattern of Heteroplasmy in the Control Region of Human Mitochondrial DNA

In this work, we present the results of the screening of human mitochondrial DNA (mtDNA) heteroplasmy in the control region of mtDNA from 210 unrelated Spanish individuals. Both hypervariable regions of mtDNA were amplified and sequenced in order to identify and quantify point and length heteroplasmy. Of the 210 individuals analyzed, 30% were fully homoplasmic and the remaining presented point and/or length heteroplasmy. The prevalent form of heteroplasmy was length heteroplasmy in the poly(C) tract of the hypervariable region II (HVRII), followed by length heteroplasmy in the poly(C) tract of hypervariable region I (HVRI) and, finally, point heteroplasmy, which was found in 3.81% of the individuals analyzed. Moreover, no significant differences were found in the proportions of the different kinds of heteroplasmy in the population when blood and buccal cell samples were compared. The pattern of heteroplasmy in HVRI and HVRII presents important differences. Moreover, the mutational profile in heteroplasmy seems to be different from the mutational pattern detected in population. The results suggest that a considerable number of mutations and, particularly, transitions that appear in heteroplasmy are probably eliminated by drift and/or by selection acting at different mtDNA levels of organization. Taking as a whole the results reported in this work, it is mandatory to perform a broad-scale screening of heteroplasmy to better establish the heteroplasmy profile which would be important for medical, evolutionary, and forensic proposes.     

Investigation of Heteroplasmy in the Human Mitochondrial DNA Control Region: A Synthesis of Observations from More Than 5000 Global Population Samples

Instances of point and length heteroplasmy in the mitochondrial DNA control region were compiled and analyzed from over 5,000 global human population samples. These data represent observations from a large and broad population sample, representing nearly 20 global populations. As expected, length heteroplasmy was frequently observed in the HVI, HVII and HVIII C-stretches. Length heteroplasmy was also observed in the AC dinucleotide repeat region, as well as other locations. Point heteroplasmy was detected in approximately 6% of all samples, and while the vast majority of heteroplasmic samples comprised two molecules differing at a single position, samples exhibiting two and three mixed positions were also observed in this data set. In general, the sites at which heteroplasmy was most commonly observed correlated with reported control region mutational hotspots. However, for some sites, observations of heteroplasmy did not mirror established mutation rate data, suggesting the action of other mechanisms, both selective and neutral. Interestingly, these data indicate that the frequency of heteroplasmy differs between particular populations, perhaps reflecting variable mutation rates among different mtDNA lineages and/or artifacts of particular population groups. The results presented here contribute to our general understanding of mitochondrial DNA control region heteroplasmy and provide additional empirical information on the mechanisms contributing to mtDNA control region mutation and evolution. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of an unusual DNA length polymorphism 5'' to the human antithrombin III gene

Nucleotide sequence analysis revealed that a DNA length polymorphism 5' to the human antithrombin III gene is due to the presence of 32bp or 108bp nonhomologous nucleotide sequences (variable segments) 345bp upstream from the translation initiation codon. Sequences at the 3' borders of both variable segments can form intrastrand inverted repeat structures with sequences further downstream. An inverted repeat is also found immediately 5' to the site where the variable segments are located. Thus, cruciform structures may form flanking the variable segments of both alleles of this DNA length polymorphism. DNA secondary structure may be detected with single strand specific nucleases. S1 nuclease sensitive sites were mapped in recombinant plasmids containing the cloned alleles of the ATIII length polymorphism. The site most sensitive to S1 is located upstream from the variable segments in an AT-rich segment flanked by 6bp direct repeats. A region of lesser nuclease sensitivity was also observed in the AT-rich loops formed between the inverted repeats 5' to the variable segments.     

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.