LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis

DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.     

A 'Southern Cross' method for the analysis of genome organization and the localization of transcription units

A 'Southern Cross' hybridization method is described which permits the rapid restriction mapping of DNA molecules, up to 40 kb in size, for at least ten enzymes in a single operation. The procedure allows the full set of 32P-end-labelled fragments derived from one restriction enzyme digest to intersect and attempt to hybridize to the gel-separated fragments of as many as ten unlabelled digests immobilized on parallel sheets of filter paper. A two-dimensional array of hybridization spots is revealed on each recipient paper, indicating which radioactive and non-radioactive DNA fragments have sequences in common. A restriction map can then be directly and simply deduced from the matrix of hybridization spots in each cross-blot. The method affords advantages over other procedures for obtaining restriction maps in terms of the time required, the number of restriction enzymes that can be mapped, and the potential for eliminating ambiguity. It is also sufficiently sensitive to detect DNA rearrangements and restriction-site polymorphisms in moderately complex genomes. Furthermore, the procedure is applicable to other aspects of the study of genome organization: for example, the exon and intron areas of a segment of cloned genomic DNA can be identified by cross-hybridizing a set of radioactive restriction fragments from the genomic clone against immobilized RNA from a cell type of interest.     

A method for estimating penetrance of pathogenic mutations in a mitochondrial genome

Variation in the manifestation age is typical of many mitochondrial diseases. The estimation of penetrance of pathogenic mutations causing such diseases is usually conducted on samples of individuals whose age exceeds the maximum age of the disease manifestation. In the case of rare diseases, samples of sufficient size sometimes cannot be formed. In this study, we propose a method for estimating penetrance involving individuals of any age. The efficiency of the method is demonstrated using Leber hereditary optic neuropathy as an example. It is shown that the method provides an unbiased estimate of penetrance and considerably reduces the error of this estimate in comparison with a sample including individuals whose age exceeds the maximum age of disease manifestation.     

Identification of chaetognaths as protostomes is supported by the analysis of their mitochondrial genome

Determining the phylogenetic position of enigmatic phyla such as Chaetognatha is a longstanding challenge for biologists. Chaetognaths (or arrow worms) are small, bilaterally symmetrical metazoans. In the past decades, their relationships within the metazoans have been strongly debated because of embryological and morphological features shared with the two main branches of Bilateria: the deuterostomes and protostomes. Despite recent attempts based on molecular data, the Chaetognatha affinities have not yet been convincingly defined. To answer this fundamental question, we determined the complete mitochondrial DNA genome of Spadella cephaloptera. We report three unique features: it is the smallest metazoan mitochondrial genome known and lacks both atp8 and atp6 and all tRNA genes. Furthermore phylogenetic reconstructions show that Chaetognatha belongs to protostomes. This implies that some embryological characters observed in chaetognaths, such as a gut with a mouth not arising from blastopore (deuterostomy) and a mesoderm derived from archenteron (enterocoely), could be ancestral features (plesiomorphies) of bilaterians.     

土蜂科线粒体基因组序列测定和分析

【目的】本研究旨在通过针尾部(Aculeata)昆虫线粒体基因组系统发育分析认知土蜂科(Scoliidae)的单系性及系统发育位置。【方法】利用Illumina Hiseq2500二代测序技术测序土蜂科3属5种的线粒体基因组,并进行注释和分析;基于针尾部昆虫36个线粒体基因组13个蛋白质编码基因(protein-coding genes, PCGs)和2个rRNA基因序列采用最大似然法(maximum likelihood, ML)和贝叶斯法(Bayesian inference, BI)法构建系统发育树。【结果】新测序的土蜂科5个线粒体基因组为五带波壁土蜂Colpa quinquecincta线粒体基因组(GenBank登录号：OM103696),齿石波壁土蜂Colpa tartara线粒体基因组(GenBank登录号：OM103697),厚大长腹土蜂Megacampsomeris grossa线粒体基因组(GenBank登录号：OM103796),台湾大长腹土蜂Megacampsomeris formosensis线粒体基因组(GenBank登录号：OM142776)和斯式土蜂Sc...     

Nuclear pseudogenes of mitochondrial DNA as a variable part of the human genome

INTRODUCTIONNuclearpseudogenesofmitochondrial(mt)DNAwereinitiallydiscoveredintheearly80's[1--6].However,mechanismsforthegenerationofmtDNApseudogenesarestillnotclearandmayvaryindifferentcases.BothRNA--[7--8]andDNAmediated[9--11]processeshavebeensugges...     

Use of single-point genome signature tags as a universal tagging method for microbial genome surveys

We developed single-point genome signature tags (SP-GSTs), a generally applicable, high-throughput sequencing-based method that targets specific genes to generate identifier tags from well-defined points in a genome. The technique yields identifier tags that can distinguish between closely related bacterial strains and allow for the identification of microbial community members. SP-GSTs are determined by three parameters: (i) the primer designed to recognize a conserved gene sequence, (ii) the anchoring enzyme recognition sequence, and (iii) the type IIS restriction enzyme which defines the tag length. We evaluated the SP-GST method in silico for bacterial identification using the genes rpoC, uvrB, and recA and the 16S rRNA gene. The best distinguishing tags were obtained with the restriction enzyme Csp6I upstream of the 16S rRNA gene, which discriminated all organisms in our data set to at least the genus level and most organisms to the species level. The method was successfully used to generate Csp6I-based tags upstream of the 16S rRNA gene and allowed us to discriminate between closely related strains of Bacillus cereus and Bacillus anthracis. This concept was further used successfully to identify the individual members of a defined microbial community.     

Expression of the mammalian mitochondrial genome. Stability of mitochondrial translation products as a function of membrane potential

Polypeptide decay has been measured as a function of membrane potential. Mitochondrial translation products were pulse-labeled in vitro with [35S]methionine using isolated rat heart mitochondria in the presence of an energy-generating system. The relative rate of protein degradation was estimated from the specific activity (counts/min/mg of protein) of the labeled translation products following the addition of unlabeled methionine (chase). To modulate membrane potential, inhibitors of oxidative phosphorylation were used singly or in combination; their effect was monitored by following uptake of the nonmetabolizable lipophilic cation triphenylmethylphosphonium. When the potential was dissipated, the rate of polypeptide decay increased and vice versa. These results suggest that the stability of mitochondrial translation products is linked to a process(es) that is dependent upon delta psi; likely candidates include synthesis and/or assembly of mitochondrial gene products.     

The mitochondrial genome of the brown alga Laminaria digitata: a comparative analysis

We report here the complete sequence of the mitochondrial genome of the brown alga Laminaria digitata (Hudson) J.V. Lamouroux. L. digitata mtDNA is a circular molecule of 38,007?bp (64.9% A?+?T), encoding 63 genes and 3 ORFs and with only 6.7% of non-coding sequences. Based on gene content and order, its overall organization is very similar to that of the mitochondrial genome of Pylaiella littoralis, another brown alga belonging to a different sublineage of the Phaeophyceae. In particular, the two genomes share unusual features, which hence could be unique to brown algae among the heterokont lineage, namely the presence of a rn5 gene, a short nad11 gene, a cox2 gene with a large in-frame insertion and α-proteobacterial-like promoter sequences. On the other hand, L. digitata lacks the sequences which are thought to have been transmitted horizontally to the P. littoralis genome, that is, the group-II introns in the rnl and cox1 genes, and it features only traces of an ancestral T7-like RNA polymerase. Distance phylogenetic trees inferred from concatenated mitochondrial genes confirm that speciation of brown algae occurred recently compared to other heterokonts.