Effects of a 300 mT static magnetic field on human umbilical vein endothelial cells

This study describes the effects of a static magnetic field (SMF) on cell growth and DNA integrity of human umbilical vein endothelial cells (HUVECs). Fast halo assay was used to investigate nuclear damage; quantitative polymerase chain reaction (QPCR), standard PCR, and real‐time PCR were used to evaluate mitochondrial DNA integrity, content, and gene expression. HUVECs were continually exposed to a 300 mT SMF for 4, 24, 48, and 72 h. Compared to control samples (unexposed cultures) the SMF‐exposed cells did not show a statistically significant change in their viability. Conversely, the static field was shown to be significant after 4 h of exposure, inducing damage on both the nuclear and mitochondrial levels, reducing mitochondrial content and increasing reactive oxygen species. Twenty‐four hours of exposure increased mitochondrial DNA content as well as expression of one of the main genes related to mitochondrial biogenesis. No significant differences between exposed and sham cultures were found after 48 and 72 h of exposure. The results suggest that a 300 mT SMF does not cause permanent DNA damage in HUVECs and stimulates a transient mitochondrial biogenesis. Bioelectromagnetics 31:630–639, 2010. © 2010 Wiley‐Liss, Inc.     

Development and characterization of continuous avian cell lines depleted of mitochondrial DNA

Summary Populations of quail and chicken cells were treated with ethidium bromide, an inhibitor of mitochondrial DNA replication. After long-term exposure to the drug, the cell populations were transferred to ethidium bromide (EtdBr)-free medium, and cloned. Clones HCF7 (quail) and DUS-3 (chicken) were propagated for more than a year, and then characterized. Analysis of total cellular DNA extracted from these cells revealed no characteristic mitochondrial DNA molecule by Southern blot hybridization of HindIII- or AvaI-digested total cellular DNA probed with cloned mitochondrial DNA fragments. Reconstruction experiments, where a small number of parental cells was mixed with HCF7 cells and DUS-3 cells before extraction of total cellular DNA, further strengthen the notion that the drug-treated cells are devoid of mitochondrial DNA molecules. The cell populations were found to proliferate at a moderately reduced growth rate as compared to their respective parents, to be auxotrophic for uridine, and to be stably resistant to the growth inhibitory effect of EtdBr and chloramphenicol. At the ultrastructural level, mitochondria were considerably enlarged and there was a severe reduction in the number of cristae within the organelles and loss of cristae orientation. Morphometric analysis revealed a fourfold increase of the mitochondrial profile area along with a twofold decrease of the numerical mitochondrial profiles. Analysis of biochemical parameters indicated that the cells grew with mitochondria devoid of a functional respiratory chain. The activity of the mitochondrial enzyme dihydroorotate dehydrogenase was decreased by 95% and presumably accounted for uridine auxotrophy. This work was supported by a grant from the Medical Research Council of Canada.     

Reactive oxygen species, mitochondria, apoptosis and aging

In this paper, we shall review various antioxygen defense systems of the cell paying particular attention to those that prevent superoxide formation rather than scavenge already formed superoxide and its products. The role of uncoupled, decoupled and non-coupled respiration, mitochondrial pore, mitochondrion-linked apoptosis will be considered. Mitochondrial theory of aging will be regarded in context of reactive oxygen species-induced damage of mitochondrial DNA. (Mol Cell Biochem 174: 305–319, 1997)     

Silver Carp, Hypophthalmichthys molitrix, in the Poyang Lake belong to the Ganjiang River Population Rather than the Changjiang River Population

The Poyang Lake is the largest lake and the main nursery area in the middle basin of the Changjiang (Yangtze) River. We compared molecular genetic markers of silver carp among populations of the Changjiang River, the Ganjiang River and the Poyang Lake using the ND5/6 region of mtDNA. Analysis of restriction fragment length polymorphisms (RFLPs) of this region revealed distinct variation between the Ganjiang River and the Changjiang River populations. The Poyang Lake is linked with the Ganjiang River and the Changjiang River. Shared RFLP fragments between the Ganjiang River population and the Poyang Lake population are as high as 61.4%. The value is 47.74% between the populations of the Changjiang River and that of the Poyang Lake. Frequencies of bands peculiar to the Ganjiang River population are the same as in the Poyang Lake population. We conclude that the Poyang Lake silver carp population consists mainly of the Ganjiang River population. The water level of the Poyang Lake outlet, which is higher than that of the Changjiang River in the silver carp spawning season, supports this conclusion.     

DNA and recent human evolution

The study of recent human evolution, or the origin of modern humans, is currently dominated by two theories. The recent African origin hypothesis holds that there was a single origin of modern humans in Africa about 100,000 years ago, after which these humans dispersed throughout the rest of the world, mixing little or not at all with nonmodern populations. The multiregional evolution hypothesis holds that there was no single origin of modern humans but, instead, that the mutations and other traits that led to modern humans were spread in concert throughout the old world by gene flow, leading to genetic continuity among old world populations during the past million years. Although both of these theories are based on observations stemming from the fossil record, much discussion and controversy during the past six years has focused on the application and interpretation of studies of DNA variation, particularly mitochondrial DNA (mtDNA). The past year, especially, has brought new data, interpretations, and controversies. Indeed, I initially resisted writing this review, on the grounds that new information would be likely to render it obsolete by the time it was published. However, now that the dust is starting to settle, it seems timely to review various investigations and interpretations and where they are likely to lead. While the focus of this review is the mtDNA story, brief mention is made of studies of nuclear DNA variation (both autosomal and Y-chromosome DNA) and the implications of the genetic data with regard to the fossil record and our understanding of recent human evolution.     

Recombinant DNA analysis indicates that the multiple chromosomes of maize mitochondria contain different sequences

Twenty-eight Bam H 1 restriction fragments were isolated from normal mitochondrial DNA of maize by recombinant DNA techniques to investigate the organization of the mitochondrial genome. Each cloned fragment was tested by molecular hybridization against a Bam digest of total mitochondrial DNA. Using Southern transfers, we identified the normal fragment of origin for d each clone. Twenty-three of the tested clones hybridized only to the fragment from which the clone was derived. In five cases, labeling of an additional band indicated some sequence repetition in the mitochondrial genome. Four clones from normal mitochondrial DNA were found which share sequences with the plasmid-like DNAs, S-1 and S-2, found in S male sterile cytoplasm. The total sequence complexity of the clones tested is 121×10⁶ d (daltons), which approximates two thirds of the total mitochondrial genome (estimated at 183×10⁶ d). Most fragments do not share homology with other fragments, and the total length of unique fragments exceeds that of the largest circular molecules observed. Therefore, the different size classes of circular molecules most likely represent genetically discrete chromosomes in a complex organelle genome. The variable abundance of different mitochondrial chromosomes is of special interest because it represents an unusual mechanism for the control of gene expression by regulation of gene copy number. This mechanism may play an important role in metabolism or biogenesis of mitochondria in the development of higher plants.     

线粒体DNA突变与相关人类疾病

在过去的20年里, 人们发现线粒体DNA(mitochondrial DNA, mtDNA)突变与多种人类疾病相关, 其致病范围从单器官组织损害到多系统受累。文章目的在于探讨mtDNA突变与人类疾病的关系。文章重点论述: (1)线粒体遗传学特征; (2) mtDNA突变与人类遗传性疾病; (3)体细胞mtDNA突变在衰老和肿瘤中的作用; (4)mtDNA疾病的诊断和治疗。     

线粒体转录终止因子蛋白家族研究进展

线粒体转录终止因子蛋白的作用是与线粒体DNA的特异位点结合,导致线粒体基因转录停止。近年来,随着人们对线粒体基因转录机制和人线粒体疾病的深入研究,线粒体转录终止因子的功能开始受到人们的关注。本文介绍了线粒体转录终止因子及其家族成员的研究进展和有待解决的一些问题。     

Mitochondrial DNA variation in Spanish populations of Ceratitis capitata (Wiedemann) (Tephritidae) and the colonization process

Abstract: Mitochondrial DNA variation in the Mediterranean fruit fly Ceratitis capitata (Wiedemann, Diptera: Tephritidae) was studied in three natural populations from southern, central and eastern Spain by means of restriction fragment length polymorphisms using 22 restriction endonucleases. Nine different haplotypes were found based upon the restriction patterns of the seven polymorphic endonucleases, providing a measure of discrimination between populations (N_ST = 0.2462, F_ST = 0.154). The observed distribution of haplotypes, corroborated by a parsimonious unrooted tree, suggests an ancient origin for haplotype VII, and a first step in the colonization of the Iberian Peninsula and subsequently the northern and eastern Mediterranean basin, through the Straits of Gibraltar. No relationship can be established between the colonization process for Europe and America.     

Prehistorical East–West admixture of maternal lineages in a 2,500‐year‐old population in Xinjiang

As an area of contact between Asia and Europe, Central Asia witnessed a scenario of complex cultural developments, extensive migratory movements, and biological admixture between West and East Eurasians. However, the detanglement of this complexity of diversity requires an understanding of prehistoric contacts of the people from the West and the East on the Eurasia continent. We demonstrated the presence of genetic admixture of West and East in a population of 35 inhabitants excavated in Gavaerk in southern Xinjiang and dated 2,800–2,100 years before present by analyzing their mitochondrial DNA variations. This result indicates that the initial contact of the East and the West Eurasians occurred further east than Central Asia as early as 2,500 years ago. Am J Phys Anthropol, 2010. © 2009 Wiley‐Liss, Inc.     

DNA ANALYSIS OF EUKARYOTIC ALGAL SPECIES1

Plastid genomes of algae resemble those of terrestrial plants in form, size, and rate of nucleotide sequence change. They are circular and range in size from 73 kilobases (kb) to over 400 kb. Their many copies per cell can compose >15% of total cell DNA. Mitochondrial genomes, like plastid genomes, are present in high copy number in preparations of total algal cell DNA. Almost all known algal mitochondrial DNA genomes are relatively small, <50 kb; in some species they are linear, whereas in others they are circular. One of the persistent perplexities for phycologists is the question of what relationship two clones or two groups of organisms bear to each other. Several relatively simple techniques can reveal whether or not two organisms belong to the same clone. Total mitochondrial genome size can be compared directly between isolates, although identity in size does not necessarily mean identity in sequence. Restriction endonuclease digestion combined with probing permits comparison of DNA fragment patterns to see if there is identity or near identity between two samples. This methodology can be applied both to organelle genomes and to nuclear genomes. So far, restriction endonucleases cleave plastid and mitochondrial DNA of organisms belonging to the same gene pool into nearly identical fragment patterns, whereas organisms nearly or totally incapable of interbreeding display patterns wherein ? 50% of restriction fragments differ in position on an agarose gel after electrophoresis. Thus, organelle genomes may be the first choice for comparing both total size and restriction endonuclease fragment patterns to obtain an indication of whether two organisms are closely related. This methodology can be applied both to organisms in which interbreeding is easy to test and to the many algae in which homothallism or lack of sexual clones has precluded standard breeding analyses. With further data on variability levels within and between fertile populations, it may be possible to state with confidence whether a sample of morphologically similar organisms shares a common gene pool, even if their breeding cannot be manipulated experimentally.     

Mitochondrial DNA as a genetic marker for brown trout, Salmo trutta L., populations

Mitochondrial DNA was examined in natural and hatchery-reared stocks of brown trout, using different methods of restriction analysis. The methods included the development of a brown trout mt DNA hybridization probe through cloning part of the brown trout mitochondrial genome. In addition, fragments were analysed by ethidium bromide staining and end-labelling. The relative merits of each of these methods in assessing levels of genetic relatedness between the natural and hatchery-reared brown trout stocks were evaluated. In addition, the study revealed a diagnostic mtDNA restriction pattern which could be used as a genetic marker for the discrimination of these two groups of brown trout.     

Mitochondrial DNA and RNA isolation from small amounts of potato tissue

We present a fast and simple protocol for purification of mitochondrial DNA and RNA from small amounts of potato tissue including tubers, leaves, flowers, and flower buds. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. The mitochondrial DNA was not contaminated by plastid DNA, was fully restrictable and was successfully used for PCR, cloning and Southern analyses. Similarly, the isolated mitochondrial RNA was not contaminated (flower buds) or only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for northern and RT-PCR analyses.     

Development of Mitochondrial Gene Replacement Therapy

Many "classic" mitochondrial diseases have been described that arise from single homoplasmic mutations in mitochondrial DNA (mtDNA). These diseases typically affect nonmitotic tissues (brain, retina, muscle), present with variable phenotypes, can appear sporadically, and are untreatable. Evolving evidence implicates mtDNA abnormalities in diseases such as Alzheimer's, Parkinson's, and type II diabetes, but specific causal mutations for these conditions remain to be defined. Understanding the mtDNA genotype-phenotype relationships and developing specific treatment for mtDNA-based diseases is hampered by inability to manipulate the mitochondrial genome. We present a novel protein transduction technology ("protofection") that allows insertion and expression of the human mitochondrial genome into mitochondria of living cells. With protofection, the mitochondrial genotype can be altered, or exogenous genes can be introduced to be expressed and either retained in mitochondria or be directed to other organelles. Protofection also delivers mtDNA in vivo, opening the way to rational development of mitochondrial gene replacement therapy of mtDNA-based diseases.     

Organization of mitochondrial DNA in normal and texas male sterile cytoplasms of maize

Recombinant DNA and hybridization techniques have been used to compare the organization of mitochondrial DNA (mtDNA) from normal (N) and Texas male sterile (T) cytoplasms of maize. Bam H1 restriction fragments of normal mtDNA were cloned and used in molecular hybridizations against Southern blots of Bam H1 digested N and T mtDNA. Fifteen of the 35 fragments were conserved in both N and T as indicated by hybridization to comigrating bands in their restriction patterns. Only three fragments produced autoradiographs whose differences could reasonably be attributed to single changes in the cleavage site of the enzyme while approximately half (17/35) of the clones resulted in more complicated differences between N and T. The autoradiographs produced by these 17 clones indicated multiple cleavage site changes and/or sequence rearrangements of the mtDNA. Patterns of six of these 17 clones indicated partial duplication of the sequence and two showed variation in the intensity of hybridization between N and T, which may be related to the molecular heterogeneity phenomenon found in maize mitochondrial genomes. The large proportion of changes observed between N and T mtDNA indicates that rearrangements may have played an important role in the evolution of the maize mitochondrial genome.