Sequence Evolution of Drosophila Mitochondrial DNA

We have compared nucleotide sequences of corresponding segments of the mitochondrial DNA (mtDNA) molecules of Drosophila yakuba and Drosophila melanogaster, which contain the genes for six proteins and seven tRNAs. The overall frequency of substitution between the nucleotide sequences of these protein genes is 7.2%. As was found for mtDNAs from closely related mammals, most substitutions (86%) in Drosophila mitochondrial protein genes do not result in an amino acid replacement. However, the frequencies of transitions and transversions are approximately equal in Drosophila mtDNAs, which is in contrast to the vast excess of transitions over transversions in mammalian mtDNAs. In Drosophila mtDNAs the frequency of C----T substitutions per codon in the third position is 2.5 times greater among codons of two-codon families than among codons of four-codon families; this is contrary to the hypothesis that third position silent substitutions are neutral in regard to selection. In the third position of codons of four-codon families transversions are 4.6 times more frequent than transitions and A----T substitutions account for 86% of all transversions. Ninety-four percent of all codons in the Drosophila mtDNA segments analyzed end in A or T. However, as this alone cannot account for the observed high frequency of A----T substitutions there must be either a disproportionately high rate of A----T mutation in Drosophila mtDNA or selection bias for the products of A----T mutation. --Consideration of the frequencies of interchange of AGA and AGT codons in the corresponding D. yakuba and D. melanogaster mitochondrial protein genes provides strong support for the view that AGA specifies serine in the Drosophila mitochondrial genetic code.     

Sequence Characterization of a Unique Intergenic Spacer in Gadiformes Mitochondrial DNA

The nucleotide sequences of intergenic spacers located between the tRNA^Thr and tRNA^Pro genes in mitochondrial DNA of cod fishes (order Godiformes) were determined. Spacers from eight species representing two families of cod fishes were analyzed and found to vary in size from 25 to 99 bp. Each spacer sequence contains one or two copies of a conserved 17-bp motif. Four to five central nucleotides of this motif constitute a substitutional hot spot as observed from interspecific and intraspecific comparisons. The substitution rate of the spacer is approximately twice that of the variable part I of the mitochondrial DNA control region, making this sequence region interesting as a molecular marker in population studies or stock assessments of cod fishes. We propose that the spacer originated in a duplication event and evolved into a functional domain, perhaps by binding regulatory proteins. Accepted February 26, 1999     

贵州水族人群线粒体DNA序列多态分析

研究了我国贵州水族64个个体线粒体DNA控制区第一高变区序列的变异情况,在所测定的495bp序列中,共检测到73个点存在变异,界定了48种不同的单倍型,单倍型的系统发育分析表明水族中存在一些古老的单倍型类型,并且其中的几个单倍型在欧亚人群中也存在分布,来自群体历史动态分析估算的水族群体扩张时间距今约6万年,这些结果提示水族是一个古老的民族群体,结合前期测定的广西壮族以及其他报道的民族人群的比较分析发现,水族在总体上表现出与壮族相近,但又不同于壮族这一典型的南方民族群体。     

Uniparental Inheritance and Replacement of Mitochondrial DNA in Neurospora Tetrasperma

This study tested mechanisms proposed for maternal uniparental mitochondrial inheritance in Neurospora: (1) exclusion of conidial mitochondria by the specialized female reproductive structure, trichogyne, due to mating locus heterokaryon incompatibility and (2) mitochondrial input bias favoring the larger trichogyne over the smaller conidium. These mechanisms were tested by determining the modes of mitochondrial DNA (mtDNA) inheritance and transmission in the absence of mating locus heterokaryon incompatibility following crosses of uninucleate strains of Neurospora tetrasperma with trichogyne (trichogyne inoculated by conidia) and without trichogyne (hyphal fusion). Maternal uniparental mitochondrial inheritance was observed in 136 single ascospore progeny following both mating with and without trichogyne using mtDNA restriction fragment length polymorphisms to distinguish parental types. This suggests that maternal mitochondrial inheritance following hyphal fusions is due to some mechanism other than those that implicate the trichogyne. Following hyphal fusion, mututally exclusive nuclear migration permitted investigation of reciprocal interactions. Regardless of which strain accepted nuclei following seven replicate hyphal fusion matings, acceptor mtDNA was the only type detected in 34 hyphal plug and tip samples taken from the contact and acceptor zones. No intracellular mtDNA mixtures were detected. Surprisingly, 3 days following hyphal fusion, acceptor mtDNA replaced donor mtDNA throughout the entire colony. To our knowledge, this is the first report of complete mitochondrial replacement during mating in a filamentous fungus.     

Sequence Variation in the tRNA Genes of Human Mitochondrial DNA

Recent analyses have shown that nonsynonymous variation in human mitochondrial DNA (mtDNA) contains nonneutral variants, suggesting the presence of mildly deleterious mutations. Many of the disease-causing mutations in mtDNA occur in the genes encoding the tRNAs. Nucleotide sequence variation in these genes has not been studied in human populations, nor have the structural consequences of nucleotide substitutions in tRNA molecules been examined. We therefore determined the nucleotide sequences of the 22 tRNA genes in the mtDNA of 477 Finns and, also, obtained 435 European sequences from the MitoKor database. No differences in population polymorphism indices were found between the two data sets. We assessed selective constraints against various tRNA domains by comparing allele frequencies between these domains and the synonymous and nonsynonymous sites, respectively. All tRNA domains except the variable loop were more conserved than synonymous sites, and T stem and D stem were more conserved than the respective loops. We also analyzed the energetic consequences of the 96 polymorphisms recovered in the two data sets or in the Mitomap database. The minimum free energy (ΔG) was calculated using the free energy rules as implemented in mfold version 3.1. The ΔG’s were normally distributed among the 22 wild-type tRNA genes, whereas the 96 polymorphic tRNAs departed significantly from a normal distribution. The largest differences in ΔG between the wild-type and the polymorphic tRNAs in the Finnish population tended to be in the polymorphisms that were present at low frequencies. Allele frequency distributions and minimum free energy calculations both suggested that some polymorphisms in tRNA genes are nonneutral.Reviewing Editor: Dr. Rüdiger Cerff     

广西壮族人群线粒体ＤＮＡ序列遗传多态性分析

测定了８３例广西壮族人的线粒体ＤＮＡ（mtDNA）第一高变区５１５bp的序列,并从这一母系遗传标记的角度对壮族人群的起源和群体的遗传结构进行了探讨,在所测定的８３个个体中,共检测到６６个单倍型,包括７１个多态位点,显示了高度的遗传多样性,对单倍型的系统发育分析表明：壮族人群内部存在一定的分化,不同地区的壮族人群在mtＤＮＡ遗传变异上有一定的差异,其中柳州和河池地区的个体有一半以上属于聚类簇Ⅱ,而南宁和百色地区的个体属于聚类族Ⅰ的较多,结合已相关报道的人群比较分析显示,壮族和我国北方民族群体亲缘关系较远,而与南方的少数民族及南方汉族亲缘关系较近,壮族人群中单倍型辐射型体分布,系统树中壮族人群与其他人群聚类先后次序都表明壮族是一个较为典型的南方群体。     

金丝猴属的DNA序列变异及进化与保护遗传学研究

金丝猴的分类及系统发育存在许多争议。本文测定了２只川金丝猴、８只滇金丝猴、１只越南金丝猴和１只灰叶猴的２５３ｂｐ的线粒体细胞色素ｂ基因的序列。其中４７个位点（１９％）检出变异。我们采用简约法、最大似然法和距离法构建了一系列的分子系统树,得到相同的拓扑结构,从而可能在分子水平澄清了金丝猴属的系统发育。结果表明,云南金丝猴与越南金丝猴间的关系较与川金丝猴的为近。金丝猴属的分化大约发生在２～６百万年以前。这３种金丝猴均是独立的种,且都应归入金丝猴属。对８只来自野外的滇金丝猴（其中包括了昆明动物研究所圈养群体的所有６只创立者）的非损伤性遗传分析提示,编号为ＹＫ２的母猴是维持该圈养群体遗传多样性的关键猴。我们建立的这种非损伤性遗传分析方法广泛适用于珍稀濒危动物的遗传多样性及遗传管理研究。     

塔克拉玛干沙漠腹地隔离人群线粒体DNA序列多态性分析

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

Mitochondrial DNA Variant Discovery and Evaluation in Human Cardiomyopathies through Next-Generation Sequencing

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We “shotgun” sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche''s 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300× average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20× while heteroplasmic variants required >200× coverage. Several Sanger “misses” were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.     

扬子鳄饲养种群线粒体DNA控制区的序列多态性

扬子鳄(Alligator sinensis)是中国特有的珍稀爬行动物,至2000年,野生扬子鳄的个体数已不足150条,作为保护这一物种的措施之一,先后于80年代初建起了2个养殖场,现人工繁殖的扬子鳄总数已达9000余条。为揭示扬子鳄种群遗传多样性,从两个饲养种群中采集了42个个体的样品,其中宣州样品33个(xZSP),长兴样品9个(CxSP),用PCR方法扩增mtDNA控制区,扩增产物纯化后直接用ABI310全自动遗传分析仪荧光标记测序,得到其中39个个体的血DNA控制区5’端462bP的序列。经比对发现,39个个体间的5’端mtDNA控制区没有任何变异位点,共享一种单元型,提示扬子鳄饲养种群的遗传多样性非常贫乏,造成这一结果的主要原因是近50年来,扬子鳄种群衰退和数量迅速减少导致的遗传多样性丢失,其次是人工繁殖的群体同时受到始创者数量较少产生的瓶颈效应影响。针对扬子鳄遗传多样性的现状,作者最后就这一濒危动物遗传多样性的保护对策提出3点建议。     

Mitochondrial DNA sequences reveal extensive cryptic diversity within a western American springsnail

We analysed cytochrome c oxidase subunit I and NADH dehydrogenase subunit I sequence variation among 29 populations of a widely ranging southwestern springsnail (Pyrgulopsis micrococcus) and 18 regional congeners. Cladistic analyses of these sequences depict P. micrococcus as a polyphyletic composite of five well-supported clades. Sequence divergences among these clades and subclades imply the possible occurrence of as many as seven or eight cryptic species in addition to P. micrococcus. Our finding that P. micrococcus contains multiple, genetically distinct and geographically restricted lineages suggests that diversification within this highly speciose aquatic genus has been structured in large part by the operation of terrestrial barriers to gene flow. However, these sequence data also indicate that recent dispersal among hydrographically separated areas has occurred within one of these lineages, which we attribute to passive transport on migratory waterbirds.     

Mitochondrial and nuclear DNA diversity within six species ofPhytophthora

The extent of intraspecific mitochondrial DNA (mtDNA) diversity was investigated in isolates ofPhytophthora capsici,P. citricola, P. citrophthora, P. megakarya, P. palmivora, andP. parasitica that represented a wide range of host plants and geographic origins. Phenograms were constructed following the analysis of restriction fragment patterns that were generated by several endonucleases. The six species showed different degrees of mtDNA diversity. Restriction fragment patterns inP. palmivora andP. parasitica were very uniform. Distinct subgroups could be distinguished inP. megakarya andP. citrophthora that correlated with the geographic origin or the host plant, respectively. These subgroups did not seem to be closely related to each other. High degrees of diversity were also evident inP. citricola andP. capsici. Although some isolates ofP. capsici had identical mtDNA patterns, no distinct subgroups were found that could be correlated with either a specific host plant or geographic origin. InP. capsici andP. parasitica variation in nuclear DNA was much more pronounced as compared to mtDNA. InP. capsici both types of analysis correlated well. Because of very limited variation of mtDNA inP. parasitica a comparison between the two phenograms was difficult.     

Mitochondrial DNA data reveal cryptic species within Taenia krabbei

Cysticerci of Taenia sp. from two elks (Alces alces) in Finland were characterized using morphological criteria and sequences of two mitochondrial DNA regions. The host species, size, structure and location of the cysticerci indicated that they might belong to Taenia krabbei, a circumpolar species occurring in a sylvatic life cycle in wild canids and cervids. Based on the number, length and shape of the rostellar hooks, the specimens could not be unambiguously defined as belonging to T. krabbei, T. cervi, T. ovis or T. solium. In the phylogenetic analysis, based on mitochondrial nucleotide sequence data, Taenia sp. was placed as a sister species of T. solium, distant from T. krabbei isolates previously characterized from Svalbard. This indicates that the Finnish and the Svalbard isolates, resembling T. krabbei, cannot represent a single species. The results suggest that careful morphological and genetic analyses of further isolates from intermediate and definitive hosts are required to define the taxonomic status of these two cryptic species.     

Mitochondrial DNA Sequence Divergence among Lycopersicon and Related Solanum Species

Sequence divergence among the mitochondrial (mt) DNA of nine Lycopersicon and two closely related Solanum species was estimated using the shared fragment method. A portion of each mt genome was highlighted by probing total DNA with a series of plasmid clones containing mt-specific DNA fragments from Lycopersicon pennellii. A total of 660 fragments were compared. As calculated by the shared fragment method, sequence divergence among the mtDNAs ranged from 0.4% for the L. esculentum-L. esculentum var. cerasiforme pair to 2.7% for the Solanum rickii-L. pimpinellifolium and L. cheesmanii-L. chilense pairs. The mtDNA divergence is higher than that reported for Lycopersicon chloroplast (cp) DNA, which indicates that the DNAs of the two plant organelles are evolving at different rates. The percentages of shared fragments were used to construct a phenogram that illustrates the present-day relationships of the mtDNAs. The mtDNA-derived phenogram places L. hirsutum closer to L. esculentum than taxonomic and cpDNA comparisons. Further, the recent assignment of L. pennellii to the genus Lycopersicon is supported by the mtDNA analysis.     

One Step Isolation of Bovine Asialoglycoprotein Receptor and Its Characterization by Sequence Analysis and MALDI Mass Spectrometry

The asialoglycoprotein receptor (ASGP-R), which is responsible for the uptake of partially deglycosylated serum glycoproteins was isolated from bovine liver. The receptor was purified in one step from solubilized plasma membranes by affinity chromatography on 6-(-D-lactosyl)-n-hexylamine coupled to N-hydroxysuccinimide activated Sepharose with a coupling degree of 7.6 mol/ml gel. The preparation yielded two distinct polypeptides with apparent molecular weights of 48 and 43 kDa as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. A polyclonal antibody raised against the human ASGP-R recognized the bovine 43 kDa protein in Western blot analysis. The 48 and 43 kDa polypeptides were digested by trypsin and the digests were subsequently analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Sequence analysis of four tryptic fragments, two each of the 48 kDa and of the 43 kDa polypeptides revealed that these were highly homologous to ASGP-R subunits from man, mouse and rat.     

云南保山猪线粒体DNA D-loop区序列初步分析

采用低小牛血清、低叶酸、pH 值较高的培养基,对50例白血病患儿进行rJL色休脆性浑位分析。发 现染色体畸变率及脆性部位表达率明显高于正常对照组, 且表达之脆性部位与痛断裂点及;zEt基因座位 密切相关。本文就脆性部位与白血病类型关系进行了讨沱。     

云南保山猪线粒体DNA D-loop区序列初步分析 Primary Analysis of Mitochondrial DNA D-loop Region Sequence in Baoshan Pig

摘要：为了解云南保山猪(Baoshan pig)的遗传多样性及其遗传背景,我们测定了19个个体线粒体DNA D-loop高变区I 15 363～15 801片段序列438 bp。检测到10种单倍型,包括8个多态位点,其中5次T/C转换、1次G/A转换、1次G/C颠换和1次A/T颠换,其A、T、G、C碱基的平均含量分别为35.4%、26.9%、13.2%和24.5%,A+T含量(62.3%)明显高于G+C含量(37.7%)。对于保山猪的保种及其持续利用有着重要的理论指导意义。 Abstract:To investigate the genetic diversity and genetic data of Baoshan pig in Yunnan province,the mitochondrial DNA D-loop hypervariable segment I sequences 15 363～15 801 (438 bp) in 19 individuals of Baoshan pig were sequenced.Ten mitochondrial haplotypes were identified in the samples,with 8 sites showing polymorphism,which were 5 T/C and 1 G/A transitions,1 G/C and 1 A/T transversions.The contents of A,T,G and C were 35.4%,269%,13.2% and 24.5%,respectively.The content of A+T (62.3%) was significantly higher than that of G+C (37.3%).It will be of importance to conservation and sustainable utilization in Baoshan pig.     

Postmortem Miscoding Lesions in Sequence Analysis of Human Ancient Mitochondrial DNA

Genetic miscoding lesions can cause inaccuracies during the interpretation of ancient DNA sequence data. In this study, genetic miscoding lesions were identified and assessed by cloning and direct sequencing of degraded, amplified mitochondrial DNA (mtDNA) extracted from human remains. Forty-two individuals, comprising nine collections from five geographic locations, were analyzed for the presence of DNA damage that can affect the generation of a correct mtDNA profile. In agreement with previous studies, high levels (56.5% of all damage sites) of proposed hydrolytic damage products were observed. Among these, type 2 transitions (cytosine → thymine or guanine → adenine), which are highly indicative of hydrolytic deamination, were observed in 50% of all misincorporations that occurred. In addition to hydrolytic damage products, oxidative damage products were also observed in this study and were responsible for approximately 43.5% of all misincorporations. This level of misincorporation is in contrast to previous studies characterizing miscoding lesions from the analysis of bone and teeth, where few to no oxidative damage products were observed. Of all the oxidative damage products found in this study, type 2 transversions (cytosine → adenine/guanine → thymine or cytosine → guanine/guanine → cytosine), which are commonly formed through the generation of 8-hydroxyguanine, accounted for 30.3% of all genetic miscoding lesions observed. This study identifies the previously unreported presence of oxidative DNA damage and proposes that damage to degraded DNA templates is highly specific in type, correlating with the geographic location and the taphonomic conditions of the depositional environment from which the remains are recovered. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.