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.     

Analysis of Mitochondrial DNA Lineages in Yakuts

To study the mitochondrial gene pool structure in Yakuts, polymorphism of mtDNA hypervariable segment I (16,024–16,390) was analyzed in 191 people sampled from the indigenous population of the Sakha Republic. In total, 67 haplotypes of 14 haplogroups were detected. Most (91.6%) haplotypes belonged to haplogroups A, B, C, D, F, G, M*, and Y, which are specific for East Eurasian ethnic groups; 8.4% haplotypes represented Caucasian haplogroups H, HV1, J, T, U, and W. A high frequency of mtDNA types belonging to Asian supercluster M was peculiar for Yakuts: mtDNA types belonging to haplogroup C, D, or G and undifferentiated mtDNA types of haplogroup M (M*) accounted for 81% of all haplotypes. The highest diversity was observed for haplogroups C and D, which comprised respectively 22 (44%) and 18 (30%) haplotypes. Yakuts showed the lowest genetic diversity (H = 0.964) among all Turkic ethnic groups. Phylogenetic analysis testified to common genetic substrate of Yakuts, Mongols, and Central Asian (Kazakh, Kyrgyz, Uighur) populations. Yakuts proved to share 21 (55.5%) mtDNA haplotypes with the Central Asian ethnic groups and Mongols. Comparisons with modern Paleoasian populations (Chukcha, Itelmen, Koryaks) revealed three (8.9%) haplotypes common for Yakuts and Koryaks. The results of mtDNA analysis disagree with the hypothesis of an appreciable Paleoasian contribution to the modern Yakut gene pool.     

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.     

动物mtDNA控制区及保守与异质

本文通过文献综述,对动物线粒体DNA控制区进行了阐述.从线粒体控制区（control region）基因组的研究出发,重点介绍了动物线粒体控制区基因组结构特点.主要结论：由于碱基替换、插入和缺失以及重复序列数目的变异致使D-loop成为mtDNA中变异最多的区域,但突变和结构重排并不是发生在整个D-loop区域,而是在高变区;大多研究集中在mtDNA D-loop保守区和异质方面：对D-loop序列分析,能较好地阐明动物的起源,在动物亲缘关系鉴定、系统进化和物种形成方式的研究等领域具有广阔的研究和应用前景.     

Phylogeographic Analysis of Mitochondrial DNA in the Nogays: A Strong Mixture of Maternal Lineages from Eastern and Western Eurasia

Analysis of mtDNA markers in a population of the Nogays (n = 206), the people inhabiting the North Caucasus and speaking a Turkic language of the Altaic linguistic family, has revealed a high level of genetic diversity (H = 0.99). The identified haplotypes include all major West Eurasian haplogroups, with the prevalence of H and U clusters (22 and 21%, respectively), but the percentage of lineages specific for East Eurasian populations is the highest (40%). Some other mtDNA variants in the Nogay population belong to the M1 haplogroups typical of northeastern Africa and U2 characteristic of Indian populations. Thus, components of different origin have contributed to the gene pool of Nogays. An erratum to this article is available at .     

Mitochondrial DNA Genetics and the Heteroplasmy Conundrum in Evolution and Disease

The unorthodox genetics of the mtDNA is providing new perspectives on the etiology of the common “complex” diseases. The maternally inherited mtDNA codes for essential energy genes, is present in thousands of copies per cell, and has a very high mutation rate. New mtDNA mutations arise among thousands of other mtDNAs. The mechanisms by which these “heteroplasmic” mtDNA mutations come to predominate in the female germline and somatic tissues is poorly understood, but essential for understanding the clinical variability of a range of diseases. Maternal inheritance and heteroplasmy also pose major challengers for the diagnosis and prevention of mtDNA disease.     

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.     

西藏小型猪的线粒体DNA控制区分析

目的研究西藏小型猪的遗传标记以及与其他国内地方猪的亲缘关系。方法扩增102头西藏小型猪以及16头巴马小型猪、17头贵州香猪的线粒体DNA控制区,测序并与国内其他猪进行比较。结果西藏小型猪线粒体DNAD-loop区分三个区域。串联重复序列区处于中间位置,包含有15～29个10 bp的重复片段,分为A、B两种类型。D-loop 3′端340 bp,与国内其他猪的序列相同比较保守;5′端704 bp,共有22个变异位点。由22个变异位点中归纳出25个单倍型,其中有两种主要的单倍型,分别占34.4%和36.6%。根据三个转换位点：305、500、691,将西藏小型猪分成了两组,几乎与串联重复序列所分的A、B两组类型相对应。与西藏小型猪相比,巴马小型猪和贵州香猪D-loop 5′端变异位点较少,分别只有4种和2种单倍型,串联重复区也只有一个类型。结论西藏小型猪可能有两个母系祖先并且与我国西南地区的品种猪有较近的亲缘关系;不同的串联重复片段类型和5′端的变异位点可以联合组建西藏小型猪的遗传标记。     

Mitochondrial DNA in the Bark Weevils: Size, Structure and Heteroplasmy

Mitochondrial DNA of higher animals has been described as an example of extreme efficiency in genome structure and function. Where exceptionally large size molecules have been found (greater than 20 kb), most have occurred as rare variants within a species, suggesting that these variants arise infrequently and do not persist for long periods in evolutionary time. In contrast, all individuals of at least three species of bark weevil (Curculionidae: Pissodes) possess a mitochondrial genome of unusually large size (30-36 kb). The molecule owes its large size to a dramatically enlarged A + T-rich region (9-13 kb). Gene content and order outside of this region appear to be identical to that found in Drosophila. A series of 0.8-2.0-kb repeated sequences occur adjacent to the large A + T rich region and have perhaps played a role in the generation of the large size as well as an unprecedented frequency of size variant heteroplasmy. Every weevil sampled in all three species (n = 219) exhibits anywhere from two to five distinct size classes of mtDNA. The persistence of this large amount of size polymorphism through two speciation events combined with the abundant size variation within individuals suggests that these molecules may not be subject to strong selection for small overall size and efficiency of replication. This pattern of variation contrasts strongly with the conservation of gene content and arrangement in the coding region of the molecule.     

奶牛IL-6基因克隆与原核表达

目的:克隆新疆地区奶牛il-6基因.获得原核表达IL-6蛋白.方法:根据GenBank上牛IL-6的序列,用premier 5.0软件设计一对引物.以牛肺巨噬细胞提取的RNA反转录产物(cDNA)为模板扩增出567bp的条带,克隆到pBS-T载体,测序正确后,提取质粒经BamH Ⅰ和EcoR Ⅰ双酶切回收目的条带,亚克隆到pGEX-4T-1原核表达载体,转化DE3菌,经IPTG诱导表达.以表达产物免疫小白鼠血清为一抗榆测表达产物的反应原性.结果:成功克隆了新疆地区奶牛il-6基因,克隆部分编码188个氨基酸与GenBank公布的序列100%同源,表达出46 kDa的融合蛋白,免疫印迹检测显示原核表达牛IL-6有免疫原性.结论:新疆地区奶牛在IL-6的基因序列上与其他地方牛无差异,原核表达奶牛IL-6蛋白为在奶牛乳房炎疫苗中的佐剂效果研究打下了基础.     

Large Subunit Mitochondrial rRNA Secondary Structures and Site-Specific Rate Variation in Two Lizard Lineages

A phylogenetic-comparative approach was used to assess and refine existing secondary structure models for a frequently studied region of the mitochondrial encoded large subunit (16S) rRNA in two large lizard lineages within the Scincomorpha, namely the Scincidae and the Lacertidae. Potential pairings and mutual information were analyzed to identify site interactions present within each lineage and provide consensus secondary structures. Many of the interactions proposed by previous models were supported, but several refinements were possible. The consensus structures allowed a detailed analysis of rRNA sequence evolution. Phylogenetic trees were inferred from Bayesian analyses of all sites, and the topologies used for maximum likelihood estimation of sequence evolution parameters. Assigning gamma-distributed relative rate categories to all interacting sites that were homologous between lineages revealed substantial differences between helices. In both lineages, sites within helix G2 were mostly conserved, while those within helix E18 evolved rapidly. Clear evidence of substantial site-specific rate variation (covarion-like evolution) was also detected, although this was not strongly associated with specific helices. This study, in conjunction with comparable findings on different, higher-level taxa, supports the ubiquitous nature of site-specific rate variation in this gene and justifies the incorporation of covarion models in phylogenetic inference.Reviewing Editor: Dr. Yves Van de Peer     

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.     

African DNA Lineages in the Mitochondrial Gene Pool of Europeans

Mitochondrial DNA (mtDNA) nucleotide sequences of African origin are found in various European populations at a low frequency (on average, less than 1%). Data on mtDNA variation in Eurasian and African populations have been analyzed, and African mtDNA lineages have been found in Europeans. It has been demonstrated that, despite the high diversity of mtDNA haplotypes of African origin in Europeans, few monophyletic clusters of African lineages are characterized by long-term diversity formed in Europe. Only two such mtDNA clusters (from haplogroups L1b and L3b) have been found, their evolutionary age not exceeding 6500 years. European and African populations have been compared with respect to the frequency distributions of the alleles of autosomal microsatellite loci found in Russian carriers of African mtDNA haplotypes. It has been demonstrated that alleles typical of Europeans are characteristic of the autosomal genotypes of these Russian individuals.     

Mitochondrial DNA Heteroplasmy and Purifying Selection in the Mammalian Female Germ Line

Inherited mutations in the mitochondrial (mt)DNA are a major cause of human disease, with approximately 1 in 5000 people affected by one of the hundreds of identified pathogenic mtDNA point mutations or deletions. Due to the severe, and often untreatable, symptoms of many mitochondrial diseases, identifying how these mutations are inherited from one generation to the next has been an area of intense research in recent years. Despite large advances in our understanding of this complex process, many questions remain unanswered, with one of the most hotly debated being whether or not purifying selection acts against pathogenic mutations during germline development.     

中国荷斯坦牛和鲁西黄牛mtDNAD-loop序列多态性分析

为了从母系遗传角度深入阐明中国荷斯坦牛和鲁西黄牛的群体遗传多样性以及起源进化,本研究采用PCR测序法测定了9头中国荷斯坦牛和11头鲁西黄牛的线粒体DNA D-loop区的部分序列,并经剪切后进行生物信息学软件分析.结果表明,20个个体D-loop区共711 bp,共检测到19种单倍型和50个多态位点.核苷酸多样性(Pi)为0.021 33±0.004 54,单倍型多样性(Hd)为0.994±0.019,平均核苷酸差异数(k)为15.146 20.构建的NJ网络进化树共分为两大支系,其中部分鲁西黄牛与瘤牛聚为一支,而中国荷斯坦牛和部分鲁西黄牛与普通黄牛聚为一支.说明中国荷斯坦牛和鲁西黄牛群体遗传多样性均较高;鲁西黄牛同时含有瘤牛和普通黄牛的血统,而中国荷斯坦牛只含有普通黄牛的血统.     

人类线粒体DNA异质性研究进展及其法医学应用

线粒体DNA(mitochondrial DNA mtDNA)的异质性自从被发现以来,一直被遗传学、进化学、发育遗传学以及法医遗传学、分子生物学领域所重视。由于线粒体异质性的存在,使得很多涉及疾病、进化、系统发育线粒体基因组与核基因组的相互作用关系、线粒体DNA复制机制以及法医学运用线粒体DNA进行实际案件评估的问题变得复杂化。此外线粒体DNA异质性的发生原因以及对线粒体异质性的检测方法标准化问题还没有一个统一的答案。针对线粒体DNA异质性带来的种种问题,近年来国内外取得了不少研究进展。     

枯草杆菌启动子－信号肽序列的克隆及序列分析

利用含红霉素抗性基因和缺启动子－信号肽序列的氨苄青霉素抗性基因的双功能质粒ｐＧＰＢ１４为探针载体,克隆了枯草杆菌的启动子－信号肽序列并对克隆的片段进行序列分析。枯草杆菌染色体ＤＮＡ经Ｓａｕ３Ａ酶解后与ＢｏｍＨＩ酶切的质粒ｐＧＰＢ１４连接,转化大肠杆菌Ｃ６００,筛选抗氨苄青霉素及抗红霉素的转化子,从双抗性转化子中提取重组质粒并经酶切分析,显示克隆的ＤＮＡ片段在０．２７－１．５ｋｂ之间。用Ｓａｎｇｅｒ的双脱氧链终止法测定了１０个克隆片段的ＤＮＡ顺序,结果表明,克隆的片段都含有启动子、核糖体结合优点及信号肽序列。克隆片段可以在大肠杆菌和枯草杆菌中恢复氨苄青霉素抗性的表型。β－内酰胺酶活力测定结果证明：大肠杆菌的酶活力主要积累在周质空间内而枯草杆菌的酶活力主要分泌到胞外。     

中华鲟(Acipenser sinensis)间的长度变异与个体内的长度异质性

用PCR技术扩增中华鲟（Acipensersinensis）线粒体DNA（mtDNA）控制区（D－loop）时,发现中华鲟天然群体内存在个体间和个体内的mtDNA长度变异现象。DNA测序表明,长度变异发生在mtDNAryloop靠近tRANpro的位置,由长约82碱基对（bp）的重复序列串联形成的。由个体内mtDNA长度变异造成的异质性个体比例为57.4％,非异质性（同质性）个体的比例为426％。非异质性个体间的mtDNA的大小也不一样,存在长度变异。在非异质性个体中,有2、3、4、5个串联重复序列形成的4种分子类型的情况,其重复序列出现的频率从高到低的循序是3→2→4→5。在异质性个体中,同一个体由2种不同分子组合的异质体最普通,占77.78％3种不同分子组合的频率次之,占18.520。4种不同分子组合的异质体比例最少,占3．70％。没有发现由5种不同分子组合的异质体。对所有异质体混合分析表明,各种类型的重复序列出现的比例与非异质体的类似,即分子大小（含重复序列数）从高到低的顺序为3→2→4→5→1。对47尾中华鲟的个体内和个体间的遗传多样性指数分析发现,有65．3％遗传变异表现在群体内的个体间,有347％的遗传变异表现在个体内。由mtDNA长度异质性造成的个体内的多样性是中华鳍物种遗传多样性的另一途径。     

Sequence Similarity in Nuclear and Mitochondrial Gene Regions in Plants

A 114-basepair fragment at 5′-upstream of the gene oee2-A from Nicotiana tabacum encoding the 23kD polypeptide of oxygen-evolving complex of photosystem II was found, through database search, to have high sequence similarity with segments in the mitochondrial genome of Oenothera, watermelon and wheat. This observation is supported by the Southern hybridization data. Repeated sequences were also found in the nuclear-mitochondrial homologous region, However, the function of these repeats is yet to be determined.     

Length Variation and Heteroplasmy Are Frequent in Mitochondrial DNA from Parthenogenetic and Bisexual Lizards (Genus Cnemidophorus)

Samples of mtDNA isolated from each of 92 lizards representing all color pattern classes of Cnemidophorus tesselatus and two populations of C. tigris marmoratus were digested with the restriction endonucleases MboI, TaqI, RsaI and MspI. The mtDNA fragment sizes were compared after radioactive labeling and gel electrophoresis. Three features were notable in the comparisons: there was little variation due to gain or loss of cleavage sites, two fragments varied noticeably in length among the samples, one by a variable amount up to a maximum difference of approximately 370 base pairs (bp) and the other by a discrete amount of 35 bp, these two fragments occasionally varied within, as well as between, samples. Two regions that corresponded in size to these variants were identified by restriction endonuclease cleavage mapping. One of these is adjacent to the D-loop. Heteroplasmy, heretofore rarely observed, occurred frequently in these same two regions. Variability in the copy number of a tandemly repeated 64-bp sequence appears to be one component of the variation, but others (e.g., base substitutions or small additions/deletions) must also be involved. The frequent occurrence of these length variations suggests either that they can be generated rapidly or that they were inherited from a highly polymorphic ancestor. The former interpretation is favored.