Preferential binding of cisplatin to mitochondrial DNA of Chinese hamster ovary cells

Some chemical carcinogens localize preferentially in mitochondrial DNA (mtDNA) when compared with genomic DNA (gDNA). Here we compare the ability of cisplatin (cis-diamminedichloroplatimum[II]) to induce DNA adducts in both genomic and mtDNA of Chinese hamster ovary (CHO) cells in culture. Cytotoxicity was examined by cell survival 4, 8 and 24 h afer exposure to 50 μM cisplatin. Cisplatin-DNA adducts were measured in DNA from nuclear and mitochondrial fractions by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), a sensitive competitive microtiter-based immunoassay utilizing antiserum elicited against cisplatin-modified DNA. An additional comparison of cisplatin-DNA binding in both compartments was performed by immunoelectron microscopy using the cisplatin-DNA antiserum and colloidal gold. DELFIA analysis of cisplatin-DNA adducts in gDNA and mtDNA showed a six-fold higher incorporation of drug into mtDNA as compared to gDNA. Morphometric studies of colloidal gold distribution in photomicrographs of CHO cells showed mtDNA to contain a four-fold higher concentration of cisplatin as compared to nuclear DNA. Therefore, both methods demonstrated a preferential binding of cisplatin to mtDNA versus gDNA.     

DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.     

一种改良的大豆线粒体DNA提取方法

以大豆(Glycine max (L.) Merrill)黄化苗为材料,通过优化提取线粒体DNA(mtDNA)时差速离心过程中的离心力和离心时间,以及纯化过程中设置不同的蔗糖密度梯度和裂解液浓度,结合高盐法去除蛋白质,改良大豆mtDNA的提取方法。结果表明,该方法提取的mtDNA浓度和纯度较高,无叶绿体和核基因组DNA的污染,可用于后续大豆线粒体基因组的相关研究。     

Simultaneous extraction of nuclear and mitochondrial DNA from human blood

A method of simultaneous isolation of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) from human blood has been proposed by improvising Lahiri's method of isolation of nuclear DNA. The approach presented here provides selectively enriched fractions and eliminates the need for two different methods or separate reagent sets for the extraction of nDNA and mtDNA. It employs an initial nuclear/ cytoplasm partitioning, followed by the similar procedural steps for the two fractions separately. It gives good quality and quantity of the nDNA as well as the mtDNA, suitable for processes like PCR amplification and sequencing and may prove to be useful for people studying population genetics and evolution using molecular markers maximizing the available resources, especially in cases where a large database needs to be generated from limited amount of blood sample. From 3 ml of blood, the yields of mtDNA salvaged from the supernatant were sufficient to set approximately 4x10(5) reactions (starting with 250 fg DNA per reactions) of mtDNA loci which otherwise would have been discarded as per original Lahiri's procedure. The quality of mtDNA from the mitochondrial fraction was suitable for all major downstream processes as confirmed by locus specific PCR amplifications and sequencing. Through this procedure, the wastage of nDNA can be avoided when mtDNA loci is studied.     

A decreased mitochondrial DNA content is related to insulin resistance in adolescents

The aim of this study was to investigate whether mitochondrial DNA (mtDNA) content is associated with insulin resistance (IR) in a sample of adolescents with features of metabolic syndrome. We further studied the link between polymorphisms in three genes involved in mitochondrial biogenesis and the presence of deleted mtDNA and mtDNA content. Data and blood samples were collected from 175 adolescents out of a cross-sectional, population-based study of 934 high school students. On the basis of the median value of homeostasis model assessment of IR (HOMA-IR) of the whole sample (2.2), the population was divided into two groups: noninsulin resistance (NIR) and IR. mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out using a real-time quantitative PCR method. Genotyping for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) (pro12Ala), PPAR- gamma coactivator-1alpha (PGC-1alpha) (Gly482Ser), and Tfam (rs1937 and rs12247015) polymorphisms was performed by PCR-based restriction fragment length polymorphism. Long-extension PCR was performed to amplify the whole mitochondrial genome. The mtDNA/nDNA ratio was significantly lower in the IR group (median: 9.08, range: 68.94) in comparison with the NIR group (12.24, 71.92) (P<0.03). Besides, the mtDNA/nDNA ratio was inversely correlated with HOMA (R: -0.18, P<0.02), glucose (R: -0.21, P<0.008), and uric acid (R: -0.18, P<0.03). Genotypes for the PPAR- gamma, PGC-1alpha, and Tfam variants were not associated with the mtDNA/nDNA ratio. Long-extension PCR did not show significant levels of mtDNA deletions. In conclusion, our findings indicate that reduced mtDNA content in peripheral leukocytes is associated with IR. This result seems not to be related with the previously mentioned variants in genes involved in the regulation of mitochondrial biogenesis.     

Isolation and characterization of DNA from the entomopathogenBeauveria bassiana

Beauveria bassiana chromosomal (chr) and mitochondrial (mt) DNA were isolated, purified, and characterized. Modification of a recent rapid method of total DNA extraction was followed by DNA purification on CsCl-bisbenzimide gradients. Cytosine methylation at 5′-CpG-3′ doublets was undetectable in the total DNA as demonstrated byMspI andHpaII restriction enzyme digests. The chrDNA had a bouyant density of 1.7130 g/ml (G + C ratio of 53.4%) and a T_m of 92.6°C (G + C ratio of 56.9%). Mitochondrial DNA was isolated from total DNA or purified mitochondria and had a buoyant density of 1.7120 g/ml (G + C ratio of 52.3%) and a biphasic DNA melting curve. A minor melt at 76.2°C (16.8% G + C ratio) yielded a 10% increase in absorbance while the major melt at 93.1°C (58% G + C ratio) yielded a 30% increase in absorbance. The mtDNA was estimated by gel electrophoresis to be 17.6 ± 0.6 MDa and determined to be 28.5 kb by restriction enzyme analysis. Electron microscopy of the mtDNA revealed circular molecules having an average contour length of 8.84 ± 0.51 μm. A physical map of the mtDNA was generated by double restriction enzyme digestion using the restriction enzymesClaI,EcoRI,SalI,BstEII, andXhoI.     

小麦线粒体DNA的高效提取方法

以小麦黄化苗为材料, 通过简单差速离心、DNaseⅠ处理得到无核DNA杂质的线粒体, 用SDS和蛋白酶K裂解线粒体, 经酚/氯仿抽提除去蛋白, 并用RNase A消化而得到单纯线粒体DNA(mtDNA)。对所提取的mtDNA进行紫外吸收光度分析, A₂₆₀/A₂₈₀ 平均为1.92, A260/A230 平均为2.09, 平均每克黄化苗可提取mtDNA 26.85 mg; 并对mtDNA进行琼脂糖凝胶电泳和RAPD扩增, 均得到清晰的电泳图谱。结果表明: 此提取方法得到的mtDNA, 不但产率高、结构完整, 而且能有效去除核DNA、RNA和蛋白质等杂质, 获得高质量的mtDNA用于PCR反应和各种遗传学分析。研究还发现, 通过调整线粒体裂解温度(先50℃裂解1 h, 再37℃裂解1 h), 亦可大幅度提高mtDNA的产率。     

Quantitative determination of deleted mitochondrial DNA relative to normal DNA in parkinsonian striatum by a kinetic PCR analysis

Deleted mitochondrial DNA (mtDNA) was accumulated in the parkinsonian striatum, but the same deleted mtDNA was also detectable in the control striatum when cycles of polymerase chain reaction were increased. To discriminate between these pathological and physiological conditions, we quantitatively analyzed the proportion of deleted mtDNA to normal mtDNA by measuring the incorporation of alpha-[32P]deoxycytosine triphosphate into mtDNA fragments by using a laser image analyzer. To estimate the molar ratio of the deleted mtDNA to normal mtDNA, the radioactivity was normalized by each fragment size. By plotting logarithms of normalized radioactivities against PCR amplification cycles, straight lines were obtained with different slopes. By extrapolation of the line to the zero amplification, the proportion of mutant mtDNA to normal mtDNA in the original sample from the parkinsonian striatum was estimated to be ca. 5%, which was at least ten times higher than the proportion of ca. 0.3% in the control striatum. These results indicate that phenotype of the mutant mtDNA as Parkinson's disease is expressed when the proportion of deleted mtDNA to normal mtDNA exceeds a threshold of ten times higher value than in the normal subject.     

海南产桶形芋螺线粒体基因组DNA提取条件的优化

提取海南产桶形芋螺线粒体基因组完整DNA (mtDNA),并对提取条件进行优化。以桶形芋螺腹足肌肉、毒腺和肝胰脏三个不同组织为材料,分别采用改进高盐沉淀法、细胞器/磁珠法和试剂盒提取三种方法,提取桶形芋螺mtDNA,并利用琼脂糖凝胶电泳和紫外分光光度计对提取mtDNA的纯度和浓度进行测定。以coxⅠ-rRNA小亚基基因和α-芋螺毒素基因设计引物,通过PCR反应来确证所提取的DNA确实是mtDNA。试剂盒法提取肝胰脏、高盐沉淀法提取肝胰脏和腹足肌肉组织这三种方法的产率很高,分别为44.4μg/mg、43.3μg/mg和32.6μg/mg。A260/280比值表明,改进高盐沉淀法提取毒腺和腹足肌肉组织,细胞器磁珠法提取腹足肌肉组织的mtDNA纯度很高。综合比较,采用改进高盐沉淀法,利用桶形芋螺腹足肌肉组织所提取的mtDNA产率高、质量好、纯度高。高质量芋螺mtDNA的获取为利用分子生物学方法对芋螺进行遗传进化分析和系统分类提供了基础。     

Replicating animal mitochondrial DNA

The field of mitochondrial DNA (mtDNA) replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s) used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark) has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading- and lagging-strand synthesis (resembling bacterial genome replication) and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS). The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase γ, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase). Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.     

Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies.

When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g WGA using gDNA from two paired cytobrushes (cytobush 'A' kept in a cell lysis buffer, and 'B' dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed > 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n =20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.     

Nondestructive DNA extraction method for mitochondrial DNA analyses of museum specimens

Museum specimens have provided the material for a large proportion of ancient DNA studies conducted during the last 20 years. However, a major drawback of the genetic analyses is that the specimens investigated are usually damaged, as parts of skin, bone, or a tooth have to be removed for DNA extraction. To get around these limitations, we have developed a nondestructive extraction method for bone, tooth, and skin samples. We found that it is possible to amplify mitochondrial DNA (mtDNA) sequences up to at least 414 bp long from samples up to 164 years old. Using this method, almost 90% (35 of 40) of the investigated samples yielded amplifiable mtDNA. Moreover, we found that repeated extractions of the same samples allowed amplifications of the expected length for all samples at least three times and for some samples up to at least five times. Thus this method opens up the possibility to repeatedly use museum collections for mtDNA analyses without damaging the specimens and thus without reducing the value of irreplaceable collections for morphological analyses.     

Mitochondrial DNA diversity in Siberian Tatars of the Tobol-Irtysh basin

Data on the variation of the nucleotide sequence of hypervariable segment I (HVSI) and restriction fragment length polymorphism (RFLP) of the coding region of mitochondrial DNA (mtDNA) have been used to characterize the mitochondrial gene pool of Siberian Tatars of the Tobol-Irtysh basin (N = 218), one of three geographic/linguistic groups of Siberian Tatars. The gene pool of Siberian Tatars has been shown to contain both Asian and European mtDNA lineages at a ratio of 1 : 1.5. The mtDNA diversity of Siberian Tatars is substantially higher than that of other Turkic-speaking populations of North and Central Asia. The position of the mitochondrial gene pool of Tatars of the Tobol-Irtysh basin in the genetic space of northern Eurasia populations has been determined.     

Mitochondrial DNA synthesis in synchronous cultures of the yeast,Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) has been investigated by three independent methods of analysis during consecutive synchronous cell cycles in the yeast, Saccharomyces cerevisiae. The rates of pulse-label incorporation indicate maximal [³H]adenine uptake into mtDNA at the time of nuclear DNA synthesis. In contrast, the relative concentrations of mtDNA as determined by both the ratio of mtDNA to total cellular DNA and by the kinetics of isotope dilution analysis were found to increase continuously during synchronous growth. We conclude that whereas nuclear DNA replicates discontinuously during the cell cycle, mitochondrial DNA is synthesized continuously during this time. The discontinuous pattern of pulse-label incorporation into mtDNA is not considered to reflect its true mode of replication during the cell cycle.     

qPCR-based mitochondrial DNA quantification: influence of template DNA fragmentation on accuracy

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.     

Mitochondrial DNA diversity in Siberian Tatars of the Tobol-Irtysh basin

Data on the variation of the nucleotide sequence of hypervariable segment I (HVSI) and restriction fragment length polymorphism (RFLP) of the coding region of mitochondrial DNA (mtDNA) have been used to characterize the mitochondrial gene pool of Siberian Tatars of the Tobol-Irtysh basin (N = 218), one of three geographic/linguistic groups of Siberian Tatars. The gene pool of Siberian Tatars has been shown to contain both Asian and European mtDNA lineages at a ratio of 1.0 : 1.5. The mtDNA diversity of Siberian Tatars is substantially higher than that of other Turkic-speaking populations of North and Central Asia. The position of the mitochondrial gene pool of Tatars of the Tobol-Irtysh basin in the genetic space of northern Eurasia populations has been determined.