Cloning of neuronal mtDNA variants in cultured cells by synaptosome fusion with mtDNA-less cells

Synaptosome cybrids were used to confirm the presence of heteroplasmic mtDNA sequence variants in the human brain. Synaptosomes contain one to several mitochondria, and when fused to mtDNA-deficient (ρ°) mouse or human cell lines result in viable cybrid cell lines. The brain origin of mouse synaptosome cybrid mtDNAs was confirmed using sequence polymorphisms in the mtDNA COIII, ND3 and tRNA^Arg genes. The brain origin of the human synaptosome cybrids was confirmed using a rare mtDNA MboI polymorphism. Fusion of synaptosomes from the brain of a 35-year-old woman resulted in 71 synaptosome cybrids. Sequencing the mtDNA control region of these cybrid clones revealed differences in the number of Cs in a poly C track between nucleotide pairs (nps) 301 and 309. Three percent of the cybrid clones had mtDNAs with 10 Cs, 76% had nine, 18% had eight and 3% had seven Cs. Comparable results were obtained by PCR amplification, cloning and sequencing of mtDNA control regions directly from the patient’s brain tissue, but not when the control region was amplified and cloned from a synaptosome cybrid homoplasmic for a mtDNA with nine Cs. Thus, we have clonally recovered mtDNA control region length variants from an adult human brain without recourse to PCR, and established the variant mtDNAs within living cultured cells. This confirms that some mtDNA heteroplasmy can exist in human neurons, and provides the opportunity to study its functional significance.     

不同野化训练条件下朱鹮的行为差异

2010年7月至201 1年1月,在陕西省洋县朱鹮生态园和华阳镇朱鹮野化训练基地,采用瞬时扫描取样法和行为取样法,对2处野化训练大网笼中朱鹮(Nipponia nippon)(n洋县=30只;n华阳=22只)的行为进行研究,同时调查2处大网笼野化训练条件的不同.结果表明,在觅食行为的时间分配中,秋季洋县群的划动寻觅、探...     

Structure and Polymorphism of the Major Histocompatibility Complex Class II Region in the Japanese Crested Ibis,Nipponia nippon

The major histocompatibility complex (MHC) is a highly polymorphic genomic region that plays a central role in the immune system. Despite its functional consistency, the genomic structure of the MHC differs substantially among organisms. In birds, the MHC-B structures of Galliformes, including chickens, have been well characterized, but information about other avian MHCs remains sparse. The Japanese Crested Ibis (Nipponia nippon, Pelecaniformes) is an internationally conserved, critically threatened species. The current Japanese population of N. nippon originates from only five founders; thus, understanding the genetic diversity among these founders is critical for effective population management. Because of its high polymorphism and importance for disease resistance and other functions, the MHC has been an important focus in the conservation of endangered species. Here, we report the structure and polymorphism of the Japanese Crested Ibis MHC class II region. Screening of genomic libraries allowed the construction of three contigs representing different haplotypes of MHC class II regions. Characterization of genomic clones revealed that the MHC class II genomic structure of N. nippon was largely different from that of chicken. A pair of MHC-IIA and -IIB genes was arranged head-to-head between the COL11A2 and BRD2 genes. Gene order in N. nippon was more similar to that in humans than to that in chicken. The three haplotypes contained one to three copies of MHC-IIA/IIB gene pairs. Genotyping of the MHC class II region detected only three haplotypes among the five founders, suggesting that the genetic diversity of the current Japanese Crested Ibis population is extremely low. The structure of the MHC class II region presented here provides valuable insight for future studies on the evolution of the avian MHC and for conservation of the Japanese Crested Ibis.     

Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography

In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1–2 kb, digested in fragments of 90–600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA^Leu(UUR) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA^Leu(UUR) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.     

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.     

朱鹮游荡期对人类干扰的耐受性

随着野生动物旅游业的发展,野生动物对人类干扰的耐受性研究在科学管理野生动物旅游业,提高游客体验,促进野生动物保护等方面具有重要的实践意义.2007年10～11月应用可操纵试验法在陕西汉中朱鹮(Nipponia nippon)国家级自然保护区研究了朱鹮游荡期对人类干扰的耐受性.研究结果表明,朱鹮游荡期对人类干扰具有较强的耐受性并表现出一定的适应性.朱鹮的警戒距离为(38.57±14.01)m,惊飞距离为(23.84±9.45)m,警戒距离是惊飞距离的1.6倍左右,两者存在着显著相关性.干扰者的衣着颜色是影响朱鹮对人类干扰的耐受性的主要因子,朱鹮对鲜艳衣物敏感.随着距居民点距离的减小,朱鹮对人类干扰的耐受性逐渐增强,但朱鹮对道路有一定回避效应.建议最小接近区域的面积应该是以警戒距离(AD)为半径的圆形区域,朱鹮最小接近区域的面积=∏×38.572≈4700m2,该区域范围内限制游客进入,并禁止游客穿着鲜艳色衣物进入自然保护区.     

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.     

2016至2019年朱鹮野生种群收容救护现状分析

朱鹮(Nipponia nippon)野生种群目前仅分布在陕西省洋县及邻近的城固、西乡、南郑、汉台、勉县、宁强、留坝、汉阴等9个县区,数量达到2 500余只,但对其野外伤病和收容救治状况报道较少.陕西汉中朱鹮国家级自然保护区在2016至2019年的巡护和监测过程中,共发现并收容救治野生朱鹮伤病个体362只.本文利用双因...     

基于人工巢试验分析黄腹角雉巢卵捕食者

2007年至今,我国在陕西宁陕、铜川和千阳,以及河南董寨和浙江德清初步建立了朱鹮(Nipponia nippon)野化放归种群。鸟类的繁殖行为是其生活史策略的重要组成部分,可以反映动物个体的营养状态、所处栖息地的质量,以及对栖息地的适应等。本文报道浙江德清朱鹮野化放归种群的繁殖行为,旨在了解其在我国南方的适应情况,为最终建立中国朱鹮南方种群提供科学支撑。本研究于2018年4和5月及2020年3至5月对浙江德清野化放归朱鹮的3个繁殖巢进行实时监测,记录孵卵期和育雏期繁殖行为,总记录时长为134 d,有效数据时长为2 958 h。利用线性混合模型探究朱鹮衔巢材频率、孵卵期以及育雏期行为的影响因子,采用Spearman相关分析检验朱鹮翻卵频率与孵卵进程的关系,建立Logistic回归模型探究朱鹮亲鸟暖雏时长随雏鸟日龄的变化情况,并利用one-way ANOVA分析德清朱鹮与陕西洋县野生种群和其他野化放归种群的换巢频率差异。结果表明,德清朱鹮平均窝卵数3.7 ± 0.3,孵化成功率90.9%,雏鸟出飞率100.0%;繁殖的不同时期、营巢条件和亲鸟性别均显著影响朱鹮的衔巢材频率;朱鹮的翻卵频率随孵卵进程显著下降;亲鸟的暖雏时长在雏鸟11日龄时下降率最大;育雏的不同时期对朱鹮的暖雏时长、换巢频率以及喂雏频率有极显著的影响;环境温度较高可能是导致德清朱鹮比洋县野生种群提前产卵的原因之一;较高的平均窝卵数与出飞成功率表明,朱鹮对我国南方的栖息地较为适应。鉴于本研究发现利用人工巢筐营巢的亲鸟花费更多时间补充巢材,表明人工巢筐的结构和大小有必要优化,建议在今后设计时加大筐壁仰角并减小深度、扩大外径,使人工巢筐更接近于自然巢的盘状结构,同时,应将巢筐底部设计成多孔透气、利于排水的结构,以适应我国长江中下游地区湿热多雨的气候。     

Reduced Dependence of Crested Ibis on Winter-Flooded Rice Fields: Implications for Their Conservation

The Crested Ibis Nipponia nippon was once thought to be extinct in the wild until seven birds were discovered in a remote mountain village in China in 1981. Studies suggested that winter-flooded rice fields play an essential role in nest site selection by the Crested Ibis and hence in their survival. Considerable efforts were therefore made to conserve the winter-flooded rice fields, but these have caused conflicts between the agricultural and conservation communities. The population and geographical range of the wild Crested Ibis has expanded greatly since 1981, but there is no spatial information on the winter-flooded rice fields, nor on the current association of nest sites and winter-flooded rice fields. We mapped winter-flooded rice fields across the entire current range of Crested Ibis using innovative remote sensing and geographical information systems (GIS) techniques. The spatial relationships between the nest site clusters and winter-flooded rice fields were quantified using Ward''s hierarchical clustering method and Ripley''s K-function. We show that both have significantly clumped distribution patterns and that they are positively associated. However, the dependence of Crested Ibis on the winter-flooded rice fields varied significantly among the nest site clusters and has decreased over the years, indicating the absence of winter-flooded rice fields is not constraining their recovery and population expansion. We therefore recommend that efforts should be made to protect the existing winter-flooded rice fields and to restore the functionality of natural and semi-natural wetlands, to encourage both in-situ conservation and the re-introduction of the Crested Ibis. In addition, we recommend that caution should be exercised when interpreting the habitat requirements of species with a narrow distribution, particularly when that interpretation is based only on their current habitat.     

北戴河朱鹮野化种群非繁殖期日间活动时间分配和行为节律

于2019年7月-2020年1月对北戴河朱鹮野化种群非繁殖期的日间活动时间分配和行为节律进行了分析。结果表明,休息（41.8%）、觅食（39.7%）和理羽（13.3%）是北戴河朱鹮非繁殖期的主要行为。在行为节律上,有3个觅食高峰、2个休息高峰和2个理羽高峰。成鸟和幼鸟行为分配相似,但成鸟的警戒和社群等其他行为比例显著较高。与野生种群相比,北戴河野化种群的觅食时间显著较少,主要原因是野生个体需要花费较多时间搜寻食物,且飞行等活动的能耗较高。朱鹮在冬季的休息和理羽行为比例高于夏秋季,觅食行为则相反,这样可以在低温和大风天气降低能耗,是对北戴河气候环境的有效适应。为了提高朱鹮对多种气候环境的适应能力,为今后北戴河朱鹮的再引入奠定基础,我们建议在冬季要确保朱鹮有充足的适宜觅食地,同时在野化网笼中进行必要的环境丰容,如在网笼内增加常绿树种和阔叶树种,部分侧网上安装防风板,或在网笼中设置防风避寒的伞棚等设施,以供朱鹮进行选择。     

Mitochondrial DNA evolution in lagomorphs: Origin of systematic heteroplasmy and organization of diversity in European rabbits

Summary A characterization was conducted on mitochondrial DNA (mtDNA) molecules extracted separately from 107 European rabbits (Oryctolagus cuniculus) both wild and domestic, 13 European hares (Lepus capensis), and 1 eastern cottontail (Sylvilagus floridanus). Experimentally this study took into account restriction site polymorphism, overall length variation of the noncoding region, and numbers of repeated sequences. Nucleotide divergences indicate that the mtDNAs from the three species derived from a common ancestor some 6–8 million years (Myr) ago. Every animal appeared heteroplasmic for a set of molecules with various lengths of the noncoding region and variable numbers of repeated sequences that contribute to them. This systematic heteroplasmy, most probably generated by a rate of localized mtDNA rearrangements high enough to counterbalance the cellular segregation of rearranged molecules, is a shared derived character of leporids.The geographic distribution of mtDNA polymorphism among wild rabbit populations over the western European basin shows that two molecular lineages are represented, one in southern Spain, the second over northern Spain, France, and Tunisia. These two lineages derived from a common ancestor some 2 Myr ago. Their present geographical distribution may be correlated to the separation of rabbits into two stocks at the time of Mindel glaciation.Finally the distribution of mtDNA diversity exhibits a mosaic pattern both at inter- and intrapopulation levels.     

Structure of the mitochondrial control region of the Eurasian otter (Lutra lutra; Carnivora, Mustelidae): patterns of genetic heterogeneity and implications for conservation of the species in Italy

In this study we determined the complete sequence of the mitochondrial DNA (mtDNA) control region of the Eurasian otter (Lutra lutra). We then compared these new sequences with orthologues of nine carnivores belonging to six families (Mustelidae, Mephitidae, Canidae, Hyaenidae, Ursidae, and Felidae). The comparative analyses identified all the conserved regions previously found in mammals. The Eurasian otter and seven other species have a single location with tandem repeats in the right domain, while the spotted hyena (Hyaenidae) and the tiger (Felidae) have repeated sequences in both the right and left domains. To assess the degree of genetic heterogeneity of the Eurasian otter in Italy we sequenced two fragments of the gene and analyzed length polymorphisms of repeated sequences and heteroplasmy in 32 specimens. The study includes 23 museum specimens collected in northern, central, and southern Italy; most of these specimens are from extinct populations, while the southern Italian samples belong to the sole extant Italian population of the Eurasian otter. The study also includes all the captive-reared animals living in the colony "Centro Lontra, Caramanico Terme" (Pescara, central Italy). The colony is maintained for reintroduction of the species. We found a low level of genetic polymorphism; a single haplotype is dominant, but our data indicate the presence in central and southern Italy of two slightly divergent haplotypes. One haplotype belongs to an extinct population, the other is present in the single extant Italian population. Analyses of length polymorphisms and heteroplasmy indicate that the autochthonous Italian samples are characterized by a distinct array of repeated sequences from captive-reared animals.     

朱鹮迁地保护研究

朱鹮（Nipponia mppon）是当今世界最濒危的鸟类之一,现存总数不过 40余只。由于生存环境的改变和种群生命力的脆弱,朱鹮数量急剧下降。中国自1986年开始人工繁殖朱鹮,1992年人工孵化并育活3只幼鸟,这是人工繁殖朱鹮成功的首次记录。这篇文章总结了朱鹮的野外生态,实验室研究及人工饲养、繁殖。     

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.     

Stock Structure and Homing Fidelity in Gulf of Mexico Sturgeon (Acipenser Oxyrinchus Desotoi) Based on Restriction Fragment Length Polymorphism and Sequence Analyses of Mitochondrial DNA

Efforts have been proposed worldwide to restore sturgeon populations through the use of hatcheries to supplement natural reproduction and to reintroduce sturgeon where they have become extinct. We examined the population structure and inferred the extent of homing in the anadromous Gulf of Mexico (Gulf) sturgeon (Acipenser oxyrinchus desotoi). Restriction fragment length polymorphism and control region sequence analyses of mitochondrial DNA (mtDNA) were used to identify haplotypes of Gulf sturgeon specimens obtained from eight drainages spanning the subspecies' entire distribution from Louisiana to Florida. Significant differences in haplotype frequencies indicated substantial geographic structuring of populations. A minimum of four regional or river-specific populations were identified (from west to east): (1) Pearl River, LA and Pascagoula River, MS, (2) Escambia and Yellow rivers, FL, (3) Choctawhatchee River, FL, and (4) Apalachicola, Ochlockonee, and Suwannee rivers, FL. Estimates of maternally mediated gene flow between any pair of the four regional or river-specific stocks ranged between 0.15 to 1.2. Tandem repeats in the mtDNA control region of Gulf sturgeon were not perfectly conserved. This result, together with an absence of heteroplasmy and length variation in Gulf sturgeon mtDNA, indicates that the molecular mechanisms of mtDNA control region sequence evolution differ among acipenserids.     

Detecting Heteroplasmy from High-Throughput Sequencing of Complete Human Mitochondrial DNA Genomes

Heteroplasmy, the existence of multiple mtDNA types within an individual, has been previously detected by using mostly indirect methods and focusing largely on just the hypervariable segments of the control region. Next-generation sequencing technologies should enable studies of heteroplasmy across the entire mtDNA genome at much higher resolution, because many independent reads are generated for each position. However, the higher error rate associated with these technologies must be taken into consideration to avoid false detection of heteroplasmy. We used simulations and phiX174 sequence data to design criteria for accurate detection of heteroplasmy with the Illumina Genome Analyzer platform, and we used artificial mixtures and replicate data to test and refine the criteria. We then applied these criteria to mtDNA sequence reads for 131 individuals from five Eurasian populations that had been generated via a parallel tagged approach. We identified 37 heteroplasmies at 10% frequency or higher at 34 sites in 32 individuals. The mutational spectrum does not differ between heteroplasmic mutations and polymorphisms in the same individuals, but the relative mutation rate at heteroplasmic mutations is significantly higher than that estimated for all mutable sites in the human mtDNA genome. Moreover, there is also a significant excess of nonsynonymous mutations observed among heteroplasmies, compared to polymorphism data from the same individuals. Both mutation-drift and negative selection influence the fate of heteroplasmies to determine the polymorphism spectrum in humans. With appropriate criteria for avoiding false positives due to sequencing errors, next-generation technologies can provide novel insights into genome-wide aspects of mtDNA heteroplasmy.     

朱鹮野化种群对冬季低温的行为响应

低温对鸟类的生长发育、新陈代谢和生活状态有显著的影响,鸟类往往通过改变行为模式来适应温度的变化。朱鹮(Nipponia nippon)是东亚特有的濒危鸟类,2018年从秦岭南麓的陕西洋县引入到河北北戴河,建立了全球纬度最高的野化种群。本文以北戴河和洋县两个野化种群为研究对象,采用瞬时扫描法观察分析了季节和温度等因素对朱鹮休息行为的影响。结果表明,双腿站立露喙是朱鹮在游荡期最常见的休息行为(70.9%),但在越冬期显著降低(55.6%),而单腿站立和隐喙行为比例相应升高。温度对双腿站立隐喙、单腿站立露喙和单腿站立隐喙3种行为的比例有显著影响。可见,朱鹮在冬季通过增加保护裸露部位的行为比例来适应低温。在冬季,随着温度的下降,朱鹮趴卧在树干上的夜宿行为比例逐渐增加,而这一行为在白天没有记录到。温度对成鸟和幼鸟冬季的休息行为有显著影响。由此表明,趴卧行为很可能是朱鹮对极端耐受低温的一种应对,在保护监测过程中需警惕。朱鹮在北戴河越冬过程中没有使用网笼中的遮挡伞棚和庇护场所,表明其能够自主适应-20℃的低温。为了确保野化朱鹮顺利越冬,建议在野化网笼中增加一些松科等常绿树种,为朱鹮提供更加优良的夜宿环境,并用稻草搭建风挡,减少夜间大风的负面影响。     

陕西汉中野生朱鹮雏鸟的生长发育特征

2014年和2015年监测朱鹮保护区野生朱鹮(Nipponia nippon)15只雏鸟的生长发育,拟合其生长发育数据的生长曲线和相对生长公式,与1989年研究结果进行对比。结果显示,野生朱鹮雏鸟的生长发育不受孵化顺序的影响,雄性体长的生长速度快于雌性。目前朱鹮雏鸟体重、体长、翅长、嘴峰长、跗跖长和中趾长的生长均符合逻辑斯谛生长曲线,雏鸟体重生长率在13日龄左右达到最大。与1989年的研究结果比较发现,目前野生朱鹮幼鸟总体生长发育有所放缓。这可能与近年野生朱鹮种群的增长和扩散导致的环境压力加大以及繁殖期食物资源不足有关。     

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.