Taenia hydatigena: isolation of mitochondrial DNA, molecular cloning, and physical mitochondrial genome mapping

Mitochondrial DNA was isolated from Taenia hydatigena, T. crassiceps, and Echinococcus granulosus using a cetyltrimethylammonium bromide precipitation technique. The technique is simple, rapid, reproducible, and does not require extensive high speed ultracentrifugation. The advantage of using mitochondrial DNA from taeniid cestodes for comparative restriction analysis was demonstrated. Mitochondrial DNA of T. hydatigena was isolated as covalently closed circular molecules. These were linearized by single digestion with BamHI and the molecular weight was estimated from the linear form of 17.6 kb. The mitochondrial DNA of T. hydatigena is therefore similar in size and structure to that of many other animal species. The entire mitochondrial genome was cloned into pBR322 in Escherichia coli and a restriction map of the recombinant molecule was constructed. The potential of using the cloned mitochondrial genome as a probe in speciation studies as well as for providing functional information on the role of the cestode mitochondrion is discussed.     

Analysis of sequence homology between human and mouse mitochondrial DNA

The base sequence homology between human and mouse mitochondrial DNA has been investigated by hybridization of highly labelled mitochondrial DNA probes with restriction fragments of mitochondrial DNA blotted according to the Southern technique. By this analysis, the homologous regions have been found to be widely distributed along the mitochondrial genome. Competition hybridization experiments with unlabelled HeLa mitochondrial RNAs have shown that most of the cross-hybridization involves the ribosomal and 4 S RNA genes.     

Mitochondrial genome in animal cells. Structure, organization, and evolution

In the past decade, the development of new DNA, RNA, and protein technologies has greatly incremented the knowledge about the organization and expression of mitochondrial DNA. The complete base sequence of mitochondrial DNA of several animals is known and many data are rapidly accumulating on the mitochondrial genomes of other systems. Here we discuss the results so far obtained that disclosed unexpected features of mitochondrial genetics. Furthermore, mitochondrial DNA has become established as a powerful tool for evolutionary studies in animals. Evidences are presented demonstrating that the evolution of mitochondrial DNA has proceeded in different ways in the various taxonomic groups. Data on heteroplasmic animals, which demonstrate the rapid evolution of mitochondrial DNA, are also presented.     

荧光-构象敏感凝胶电泳技术筛查上海市郊区野生小家鼠线粒体编码区SNP位点

利用通用引物荧光PCR方案, 应用荧光-构象敏感凝胶电泳(fluorescence-based conformation sensitive gel elec-trophoresis, F-CSGE)和DNA直接测序分型技术, 对上海地区64只野生小家鼠线粒体DNA(mtDNA)编码区进行序列分析, 在上海市郊区野生小家鼠群体中初步检测mtDNA编码区SNP, 以发现合适的遗传位标用于野生小家鼠遗传多态性分析。结果发现: F-CSGE所有存在SNP突变的峰图中同源双链和异源双链峰电泳泳动距离差异均较为明显, 在检测未知SNP中无假阳性出现, 检测效率高。F-CSGE检出野生小鼠mtDNA编码区SNP 24个, 其中新发现SNP为16个。结果表明, F-CSGE可用于mtDNA编码区SNP检测, 新发现的SNP可作为遗传位标用以研究整个上海地区野生小家鼠的遗传结构和遗传多态性。     

A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy

We report a rapid experimental procedure based on high-density in vivo psoralen inter-strand DNA cross-linking coupled to spreading of naked purified DNA, positive staining, low-angle rotary shadowing, and transmission electron microscopy (TEM) that allows quick visualization of the dynamic of heavy strand (HS) and light strand (LS) human mitochondrial DNA replication. Replication maps built on linearized mitochondrial genomes and optimized rotary shadowing conditions enable clear visualization of the progression of the mitochondrial DNA synthesis and visualization of replication intermediates carrying long single-strand DNA stretches. One variant of this technique, called denaturing spreading, allowed the inspection of the fine chromatin structure of the mitochondrial genome and was applied to visualize the in vivo three-strand DNA structure of the human mitochondrial D-loop intermediate with unprecedented clarity.     

A rapid method for detecting mitochondrial DNA variation in the brown trout, Salmo trutta

A method is described for the amplification and restriction digestion of two variable regions of mitochondrial DNA (mtDNA) in the brown trout ( Salmo trutta ) as a non-invasive sampling and rapid screening technique for measuring genetic variation in populations.     

Chromosomal DNA patterns and mitochondrial DNA polymorphism as tools for identification of enological strains of Saccharomyces cerevisiae

Summary Chromosomal DNA patterns using the transverse alternating field electrophoresis technique and mitochondrial DNA restriction profiles have been achieved for 22 enological strains of Saccharomyces cerevisiae. Both methods have evidenced a marked polymorphism of these strains. Twenty different karyotypes and 17 mitochondrial DNA banding patterns have been observed. Only three strains originating from the same vineyard could not be differentiated by either of the two methods. The polymorphism observed at the chromosomal and mitochondrial levels makes the techniques investigated powerful tools for identification and control of industrial strains.Offprint requests to: J.-N. Hallet     

The BAH domain, polybromo and the RSC chromatin remodelling complex

We isolated Rivulus marmoratus mitochondrial DNA by long-polymerase chain reaction with conserved primers, and sequenced it with 36 sets of internal conserved primers, which were designed from the extensive sequence similarities of mitochondrial DNA from several fish species. The R. marmoratus mitochondrial DNA has 17,329 bp with a conserved structural organization compared to those of other fish. Rivulus marmoratus mitochondrial DNA also has two nearly identical control regions. The basic characteristics of the R. marmoratus mitochondrial genome are discussed.     

Characterization of mitochondrial DNA in various Candida species: isolation, restriction endonuclease analysis, size, and base composition.

A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed. Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C. albicans, C. kefyr, C. lusitaniae, C. maltosa, C. parapsilosis, C. shehatae, and C. tropicalis) and Lodderomyces elongisporus revealed different cleavage patterns that appeared to be specific for the species. Few common restriction fragments were evident. The genome sizes of the mitochondrial DNAs ranged from 26.4 to 51.4 kilobase pairs, and the guanine-plus-cytosine contents ranged from 20.7 to 36.8 mol%. There was no correlation between the base compositions of nuclear and mitochondrial DNAs. Eight isolates of C. parapsilosis, including the type culture, and an ascosporogenous strain of L. elongisporus, which was once proposed as the teleomorph of C. parapsilosis, had similar mitochondrial DNA molecular sizes (30.2 and 28.8 kilobase pairs); however, restriction endonuclease patterns of these organisms were distinct. These data provide additional support for discrimination of these two species. The results of our experiments demonstrate that mitochondrial DNA analyses may provide useful criteria for the differentiation of yeast species.     

香菇野生菌株线粒体DNA多态性

利用PCR技术扩增了我国 7个省的 2 0个野生香菇菌株的线粒体DNA的 2个小区段 ,利用限制性酶切技术研究这 2个片段长度多型性 (RFLPs) ,结果显示 ,2 0个野生香菇菌株间的线粒体DNA在研究的区段上分为 2大类型 ,相似性为 70 % ,与栽培种相比存在较大的遗传差异。     

Isolation of mitochondrial DNA, purified of nuclear DNA, from animal tissues (degree of methylation and level of pyrimidine nucleotide clustering--criteria of purity)]

A method of preparation of mitochondria free of nuclear DNA and its fragments by treatment of mitochondria with DEAE-cellulose has been developed. This method is based on binding nuclear nucleic acids and nucleoproteins to DEAE-cellulose particles in the media used for isolation of mitochondria. Treatment with DEAE-cellulose under the conditions described does not induce any visible degradation of mitochondria and mitochondrial DNA. The mitochondrial DNA preparations obtained from beef and rat liver are represented with closed circular molecules of contour length about 5.5 mu. The 5-methylcytosine content in beef and rat mitochondrial DNA (3.03 and 2.0 mole %, respectively) is twice as much as in corresponding nuclear DNA. Besides, mitochondrial DNA strongly differs from nuclear ones by a lower degree of pyrimidine clustering: the amount of mono- and dipyrimidine fragments (about 32 mole %) in mitochondrial DNA is 1.5 times as large and the content of long pyrimidine clusters (hexa- and others) is 2--4 times as low as those in nuclear DNA. The methylation level and the pyrimidine clustering degree may be used as criteria for the purity of mitochondrial DNA from nuclear DNA.     

Molecular analysis of variability within the toxigenic Aspergillus ochraceus species

Genetic variability of Aspergillus ochraceus was examined at the DNA level. Based on the HaeIII-Bg/II generated mitochondrial DNA restriction profiles, most isolates could be classified into two distinct groups. These two groups could also be distinguished by the random amplified polymorphic DNA technique, and with telomeric PCR amplifications. Phylogenetic analysis of sequences of the intergenic transcribed spacer region of some of the strains resulted in a dendrogram with the same topology as that based on mitochondrial DNA and amplified DNA data. None of the isolates with type 2 mtDNA profiles produce ochratoxins. Some strains (e.g., A. ochraceus ICMP 939) displayed strain-specific mitochondrial DNA patterns, and their amplified DNA profiles were also different from all other A. ochraceus strains examined.     

Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia.

The in vivo cellular impact of age-associated mitochondrial DNA mutations is unknown. We hypothesized that mitochondrial DNA deletion mutations contribute to the fiber atrophy and loss that cause sarcopenia, the age-related decline of muscle mass and function. We examined 82,713 rectus femoris muscle fibers from Fischer 344 x Brown Norway F1 hybrid rats of ages 5, 18, and 38 months through 1000 microns by serial cryosectioning and histochemical staining for cytochrome c oxidase and succinate dehydrogenase. Between 5 and 38 months of age, the rectus femoris muscle in the hybrid rat demonstrated a 33% decrease in mass concomitant with a 30% decrease in total fibers at the muscle mid-belly. We observed significant increases in the number of mitochondrial abnormalities with age from 289 +/- 8 ETS abnormal fibers in the entire 5-month-old rectus femoris to 1094 +/- 126 in the 38-month-old as calculated from the volume density of these abnormalities. Segmental mitochondrial abnormalities contained mitochondrial DNA deletion mutations as revealed by laser capture microdissection and whole mitochondrial genome amplification. Muscle fibers harboring mitochondrial deletions often displayed atrophy, splitting and increased steady-state levels of oxidative nucleic damage. These data suggest a causal role for age-associated mitochondrial DNA deletion mutations in sarcopenia.     

不同生理状态下膜状急纤虫大核DNA和线粒体DNA的多态性比较

为探索纤毛虫在营养及休眠条件下两套遗传系统的作用关系,对膜状急纤虫(Tachysomapellionella)营养细胞和休眠包囊大核DNA、线粒体DNA进行了RAPD比较。结果显示,在所选用的34条随机引物中,大核DNA共扩增出203条片段,其中以休眠包囊大核DNA为模板扩增出45条特有片段,以营养细胞大核DNA为模板扩增出36条特有片段,两者存在40%的差异。在所选用的32条随机引物中,线粒体DNA共扩增出216条片段,其中以休眠包囊线粒体DNA为模板扩增出35条特有片段,以营养细胞线粒体DNA为模板扩增出47条特有片段,两者有38%的差异。结果表明,膜状急纤虫休眠包囊与营养期的大核DNA结构存在显著的差异;两者的线粒体DNA结构也存在较大差异。这表明,膜状急纤虫在包囊形成过程中,大核及线粒体DNA结构可能都发生了一定的变化,并且这些变化可能与包囊形成过程中的形态结构和代谢活动等剧烈变化以及休眠状态下的生理生化变化密切相关。     

Mitochondrial DNA polymerase from Drosophila melanogaster embryos: kinetics, processivity, and fidelity of DNA polymerization

The mitochondrial DNA polymerase from embryos of Drosophila melanogaster has been examined with regard to template-primer utilization, processivity, and fidelity of nucleotide polymerization. The enzyme replicates predominantly single-stranded and double-stranded DNAs: the rate of DNA synthesis is greatest on the gapped homopolymeric template poly(dA).oligo(dT), while the highest substrate specificity is observed on single-stranded DNA templates of natural DNA sequence. Kinetic experiments and direct physical analysis of DNA synthetic products indicate that the Drosophila DNA polymerase gamma polymerizes nucleotides by a quasi-processive mechanism. The mitochondrial enzyme demonstrates a high degree of accuracy in nucleotide incorporation which is nearly identical with that of the replicative DNA polymerase alpha from Drosophila embryos. Thus, the catalytic properties of the near-homogeneous Drosophila DNA polymerase gamma are consistent with the in vivo requirements for mitochondrial DNA synthesis as described in a variety of animal systems.     

Isolation of organellar DNA from Codium fragile (Codiaceae, Codiales, Ulvophyceae)

Organellar DNA was isolated from Codium fragile (Suringar) Hariot (Codiaceae, Codiales, Ulvophyceae) by CsCI-buoyant density centrifugation in the presence of Hoechst dye 33258. Three bands were formed by ultracentrifugation and each fraction of DNA was identified by Southern hybridization. The uppermost fraction was identified as chloroplast DNA, the middle fraction was nuclear DNA and the bottom fraction was mitochondrial DNA. Nuclear rDNA was isolated in the same fraction as mitochondrial DNA. The estimated genome size of mitochondrial DNA by analysis with restriction endonucleases was more than 141.6 kb, which was larger than that of microalgae but smaller than land plants. Restriction endonuclease analysis of the chloroplast DNA showed no difference with that known of C. fragile in New York.     

Small, repetitive DNAs contribute significantly to the expanded mitochondrial genome of cucumber

Closely related cucurbit species possess eightfold differences in the sizes of their mitochondrial genomes. We cloned mitochondrial DNA (mtDNA) fragments showing strong hybridization signals to cucumber mtDNA and little or no signal to watermelon mtDNA. The cucumber mtDNA clones carried short (30-53 bp), repetitive DNA motifs that were often degenerate, overlapping, and showed no homology to any sequences currently in the databases. On the basis of dot-blot hybridizations, seven repetitive DNA motifs accounted for >13% (194 kb) of the cucumber mitochondrial genome, equaling >50% of the size of the Arabidopsis mitochondrial genome. Sequence analysis of 136 kb of cucumber mtDNA revealed only 11.2% with significant homology to previously characterized mitochondrial sequences, 2.4% to chloroplast DNA, and 15% to the seven repetitive DNA motifs. The remaining 71.4% of the sequence was unique to the cucumber mitochondrial genome. There was <4% sequence colinearity surrounding the watermelon and cucumber atp9 coding regions, and the much smaller watermelon mitochondrial genome possessed no significant amounts of cucumber repetitive DNAs. Our results demonstrate that the expanded cucumber mitochondrial genome is in part due to extensive duplication of short repetitive sequences, possibly by recombination and/or replication slippage.     

Mouse models of mitochondrial disease, oxidative stress, and senescence.

During the course of normal respiration, reactive oxygen species are produced which are particularly detrimental to mitochondrial function. This is shown by recent studies with a mouse that lacks the mitochondrial form of superoxide dismutase (Sod2). Tissues that are heavily dependent on mitochondrial function such as the brain and heart are most severely affected in the Sod2 mutant mouse. Recent work with a mouse mutant for the heart/muscle specific isoform of the mitochondrial adenine nuclear translocator (Ant1) demonstrates a potential link between mitochondrial oxidative stress and mitochondrial DNA mutations. These mutations can be detected by Long-extension PCR, a method for detecting a wide variety of mutations of the mitochondrial genome. Such mutations have also been observed in the mitochondrial genome with senescence regardless of the mean or maximal lifespan of the organism being studied. Mutations have been detected with age in Caenorhabditis elegans, mice, chimpanzees, and humans. This implies that a causal relationship may exist between mitochondrial reactive oxygen species production, and the senescence specific occurrence of mitochondrial DNA mutations.     

Age-associated change in mitochondrial DNA damage

There is an age-associated decline in the mitochondrial function of the Wistar rat heart. Previous reports from this lab have shown a decrease in mitochondrial cytochrome c oxidase (COX) activity associated with a reduction in COX gene and protein expression and a similar decrease in the rate of mitochondrial protein synthesis. Damage to mitochondrial DNA may contribute to this decline.

Using the HPLC-Coularray system (ESA, USA), we measured levels of nuclear and mitochondrial 8-oxo-2'-deoxyguanosine (8-oxodG) from 6-month (young) and 23-month-old (senescent) rat liver DNA. We measured the sensitivity of the technique by damaging calf thymus DNA with photoactivated methylene blue for 30s up to 2h. The levels of damage were linear over the entire time course including the shorter times which showed levels comparable to those expected in liver. For the liver data, 8-oxodG was reported as a fraction of 2-deoxyguanosine (2-dG). There was no change in the levels of 8-oxodG levels in the nuclear DNA from 6 to 23-months of age. However, the levels of 8-oxodG increased 2.5-fold in the mitochondrial DNA with age. At 6 months, the level of 8-oxodG in mtDNA was 5-fold higher than nuclear and increased to approximately 12-fold higher by 23 months of age. These findings agree with other reports showing an age-associated increase in levels of mtDNA damage; however, the degree to which it increases is smaller. Such damage to the mitochondrial DNA may contribute to the age-associated decline in mitochondrial function.     

Molecular genetic diagnostics of some Daphnia species (Crustacea,Cladocera) from the Volga river

Population genetic structure of the widespread Daphnia species from Kuibyshev and Saratov reservoirs was examined by use of RAPD-PCR technique with four mitochondrial DNA (16S rRNA) specific primers. One of the examined Daphnia clones from the Volga region appeared to be conspecific to the North American Daphnia galeata, while another clone was most likely a hybrid between D. galeata and D. cucullata.