Absence of DNA in peroxisomes of Candida tropicalis.

Yeast peroxisomes were purified to near homogeneity from cells of Candida tropicalis grown on oleic acid for the purpose of examining the possible presence of DNA in this organelle. The purification procedure includes the effective conversion of cells to spheroplasts with Zymolyase and sodium sulfite and the separation of the organelles at extremely low ionic strength. The mitochondrial contamination was less than 1%, based on several criteria, and the yield of peroxisomes was about 40%. The purified peroxisomal fraction contained a very small amount of DNA, which yielded restriction fragments indistinguishable from those of mitochondrial DNA. The absence of DNA in peroxisomes was also supported by cesium chloride density gradient centrifugation of the organelles lysed with a detergent, staining of the organelles with a fluorescent dye specific to DNA, and labeling of the DNA with [3H]adenine.     

Complementation of mutant and wild-type human mitochondrial DNAs coexisting since the mutation event and lack of complementation of DNAs introduced separately into a cell within distinct organelles.

The rules that govern complementation of mutant and wild-type mitochondrial genomes in human cells were investigated under different experimental conditions. Among mitochondrial transformants derived from an individual affected by the MERRF (myoclonus epilepsy associated with ragged red fibers) encephalomyopathy and carrying in heteroplasmic form the mitochondrial tRNA(Lys) mutation associated with that syndrome, normal protein synthesis and respiration was observed when the wild-type mitochondrial DNA exceeded 10% of the total complement. In these transformants, the protective effect of wild-type mitochondrial DNA was shown to involve interactions of the mutant and wild-type gene products. Very different results were obtained in experiments in which two mitochondrial DNAs carrying nonallelic disease-causing mutations were sequentially introduced within distinct organelles into the same human mitochondrial DNA-less (rho 0) cell. In transformants exhibiting different ratios of the two genomes, no evidence of cooperation between their products was observed, even 3 months after the introduction of the second mutation. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which mitochondria carrying a chloramphenicol resistance-inducing mitochondrial DNA mutation were introduced into chloramphenicol-sensitive cells. A plausible interpretation of the different results obtained in the latter two sets of experiments, compared with the complementation behavior observed in the heteroplasmic MERRF transformants, is that in the latter, the mutant and wild-type genomes coexisted in the same organelles from the time of the mutation. This would imply that the way in which mitochondrial DNA is sorted among different organelles plays a fundamental role in determining the oxidative-phosphorylation phenotype in mammalian cells. These results have significant implications for mitochondrial genetics and for studies on the transmission and therapy of mitochondrial DNA-linked diseases.     

Transfection of plant mitochondria and in organello gene integration

Investigation and manipulation of mitochondrial genetics in animal and plant cells remains restricted by the lack of an efficient in vivo transformation methodology. Mitochondrial transfection in whole cells and maintenance of the transfected DNA are main issues on this track. We showed earlier that isolated mitochondria from different organisms can import DNA. Exploiting this mechanism, we assessed the possibility to maintain exogenous DNA in plant organelles. Whereas homologous recombination is scarce in the higher plant nuclear compartment, recombination between large repeats generates the multipartite structure of the plant mitochondrial genome. These processes are under strict surveillance to avoid extensive genomic rearrangements. Nevertheless, following transfection of isolated organelles with constructs composed of a partial gfp gene flanked by fragments of mitochondrial DNA, we demonstrated in organello homologous recombination of the imported DNA with the resident DNA and integration of the reporter gene. Recombination yielded insertion of a continuous exogenous DNA fragment including the gfp sequence and at least 0.5 kb of flanking sequence on each side. According to our observations, transfection constructs carrying multiple sequences homologous to the mitochondrial DNA should be suitable and targeting of most regions in the organelle genome should be feasible, making the approach of general interest.     

Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes

Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes.     

Single-nucleotide patch base excision repair of uracil in DNA by mitochondrial protein extracts.

Mammalian mitochondria contain several 16.5 kb circular DNAs (mtDNA) encoding electron transport chain proteins. Reactive oxygen species formed as byproducts from oxidative phosphorylation in these organelles can cause oxidative deamination of cytosine and lead to uracil in mtDNA. Upon mtDNA replication, these lesions, if unrepaired, can lead to mutations. Until recently, it was thought that there was no DNA repair in mitochondria, but lately there is evidence that some lesions are efficiently repaired in these organelles. In the study of nuclear DNA repair, the in vitro repair measurements in cell extracts have provided major insights into the mechanisms. The use of whole-cell extract based DNA repair methods has revealed that mammalian nuclear base excision repair (BER) diverges into two pathways: the single-nucleotide replacement and long patch repair mechanisms. Similar in vitro methods have not been available for the study of mitochondrial BER. We have established an in vitro DNA repair system supported by rat liver mitochondrial protein extract and DNA substrates containing a single uracil opposite to a guanine. Using this approach, we examined the repair pathways and the identity of the DNA polymerase involved in mitochondrial BER (mtBER). Employing restriction analysis of in vitro repaired DNA to map the repair patch size, we demonstrate that only one nucleotide is incorporated during the repair process. Thus, in contrast to BER in the nucleus, mtBER of uracil in DNA is solely accomplished by single-nucleotide replacement.     

Male infertility and mitochondrial DNA

The mitochondrial machinery plays a key role in the energy production and maintenance of spermatozoa motility. In this paper 200 idiopathic oligo-asthenozoospermic patients were classified on the basis of rapid progressive motility ("a") and sperm concentration. Mitochondrial enzymatic activity was studied and correlated to the viability of sperm cells. Mitochondrial DNA purified from both motile and non-motile sperm of the same individuals was amplificated using PCR. Results suggested that only motile sperm have organelles functional in oxygen consumption, unequivocally demonstrating that motility depends on the mitochondrial activity. Mitochondrial DNA of oligo-asthenozoospermic patients seemed to present some defects that made DNA unavailable for amplification.     

Plant mitochondria actively import DNA via the permeability transition pore complex

Plant mitochondria are remarkable with respect to their content in foreign, alien and plasmid-like DNA, raising the question of the transfer of this information into the organelles. We demonstrate the existence of an active, transmembrane potential-dependent mechanism of DNA uptake into plant mitochondria. The process is restricted to double-strand DNA, but has no obvious sequence specificity. It is most efficient with linear fragments up to a few kilobase pairs. When containing appropriate information, imported sequences are transcribed within the organelles. The uptake likely involves the voltage-dependent anion channel and the adenine nucleotide translocator, i.e. the core components of the mitochondrial permeability transition pore complex in animal cells, but it does not rely on known mitochondrial membrane permeabilization processes. We conclude that DNA import into plant mitochondria might represent a physiological phenomenon with some functional relevance.     

Cell cycle-regulated, microtubule-independent organelle division in Cyanidioschyzon merolae

Mitochondrial and chloroplast division controls the number and morphology of organelles, but how cells regulate organelle division remains to be clarified. Here, we show that each step of mitochondrial and chloroplast division is closely associated with the cell cycle in Cyanidioschyzon merolae. Electron microscopy revealed direct associations between the spindle pole bodies and mitochondria, suggesting that mitochondrial distribution is physically coupled with mitosis. Interconnected organelles were fractionated under microtubule-stabilizing condition. Immunoblotting analysis revealed that the protein levels required for organelle division increased before microtubule changes upon cell division, indicating that regulation of protein expression for organelle division is distinct from that of cytokinesis. At the mitochondrial division site, dynamin stuck to one of the divided mitochondria and was spatially associated with the tip of a microtubule stretching from the other one. Inhibition of microtubule organization, proteasome activity or DNA synthesis, respectively, induced arrested cells with divided but shrunk mitochondria, with divided and segregated mitochondria, or with incomplete mitochondrial division restrained at the final severance, and repetitive chloroplast division. The results indicated that mitochondrial morphology and segregation but not division depend on microtubules and implied that the division processes of the two organelles are regulated at distinct checkpoints.     

Binding of DNA with Abf2p increases efficiency of DNA uptake by isolated mitochondria

Mutations in mitochondrial DNA often lead to severe hereditary diseases that are virtually resistant to symptomatic treatment. During the recent decades, many efforts were made to develop gene therapy approaches for treatment of such diseases using nucleic acid delivery into the organelles. The possibility of DNA import into mitochondria has been shown, but this process has low efficiency. In the present work, we demonstrate that the efficiency of DNA import can be significantly increased by preforming its complex with a mitochondria-targeted protein nonspecifically binding with DNA. As a model protein, we used the yeast protein Abf2p. In addition, we measured the length of the DNA site for binding this protein and the dissociation constant of the corresponding DNA–protein complex. Our data can serve as a basis for development of novel, highly efficient approaches for suppressing mutations in the mitochondrial genome.     

Repair of mitochondrial DNA in aging and carcinogenesis.

Mitochondria are responsible for the generation of energy in the form of adenosine triphosphate. These organelles contain their own genetic material, mitochondrial (mt) DNA. This mtDNA has been hypothesized to play a role in the processes of aging and carcinogenesis. Initial reports have shown that there is no repair of cyclobutylpyrimidine dimers (CPD). More recent reports indicate however, that the mitochondrion contains several defence mechanisms against endogenous or exogenous damaging agents such as ultraviolet radiation or oxidative damage. The role of these defence mechanisms in the removal of mitochondrial DNA damage and the link to aging and carcinogenesis-associated processes are discussed in this review.     

Alternative excision repair pathway of UV-damaged DNA in Schizosaccharomyces pombe operates both in nucleus and in mitochondria

The fission yeast, Schizosaccharomyces pombe, possesses a UV-damaged DNA endonuclease-dependent excision repair (UVER) pathway in addition to nucleotide excision repair pathway for UV-induced DNA damage. We examined cyclobutane pyrimidine dimer removal from the myo2 locus on the nuclear genome and the coI locus on the mitochondrial genome by the two repair pathways. While nucleotide excision repair repairs damage only on the nuclear genome, UVER efficiently removes cyclobutane pyrimidine dimers on both nuclear and mitochondrial genomes. The ectopically expressed wild type UV-damaged DNA endonuclease was localized to both nucleus and mitochondria, while modifications of N-terminal methionine codons restricted its localization to either of two organelles, suggesting an alternative usage of multiple translation initiation sites for targeting the protein to different organelles. By introducing the same mutations into the chromosomal copy of the uvde(+) gene, we selectively inactivated UVER in either the nucleus or the mitochondria. The results of UV survival experiments indicate that although UVER efficiently removes damage on the mitochondrial genome, UVER in the mitochondria hardly contributes to UV resistance of S. pombe cells. We suggest a possible UVER function in mitochondria as a backup system for other UV damage tolerance mechanisms.     

The gene encoding translation initiation factor 3 is highly conserved in gram-negative bacteria

A 1.1-kb Hp alpha I fragment of the Escherichia coli chromosome containing the gene for translation initiation factor 3 was employed as a probe in heterologous hybridization to chromosomal DNA from a variety of other procaryotes. Positive hybridization was observed to DNA derived from all gram-negative bacteria tested. In contrast, no hybridization to DNA from gram-positive bacteria was detected. In addition, homologous sequences were found in Euglena gracilis chloroplast DNA, while this was not the case with Saccharomyces cerevisiae mitochondrial DNA. These results are discussed in light of existing data on the components and mechanism of translation initiation in the various organisms and organelles employed in this study.     

Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method

The restriction patterns of two chloroplast fragments and one mitochondrial DNA fragment, amplified by PCR with universal primers, were studied to determine the mode of inheritance of these organelles in 143 progeny of five intraspecific crosses in pedunculate oak (Quercus robur L.). The results indicate that both genomes are maternally inherited, an observation which agrees with the commonly observed pattern of inheritance in angiosperms. They confirm that both chloroplast DNA and mitochondrial DNA can be used as a source of seed-specific markers for the study of the geographic structure of oaks. This is the first report of organelle inheritance within the Fagaceae, an important and widespread tree family.     

线粒体蛋白质组学研究进展

线粒体是真核生物中重要的细胞器,其包含的全部蛋白质称为线粒体蛋白质组。人类线粒体大约包含1500多种蛋白质,由核基因和线粒体基因共同编码。线粒体是细胞能量合成和物质代谢的中心,其功能障碍将直接或问接引起许多疾病。目前线粒体蛋白质组学正是系统性地研究线粒体在生理、病理过程中的功能变化以及研究疾病发生机制的重要方法。将线粒体蛋白质组的研究方法、研究进展、线粒体蛋白质组的性质及其在相关疾病研究中的作用进行综述,并对线粒体蛋白质组学在疾病发生机制和诊断治疗中的发展前景进行展望。     

Targeting nucleic acids into mitochondria: Progress and prospects

Given the essential functions of these organelles in cell homeostasis, their involvement in incurable diseases and their potential in biotechnological applications, genetic transformation of mitochondria has been a long pursued goal that has only been reached in a couple of unicellular organisms. The challenge led scientists to explore a wealth of different strategies for mitochondrial delivery of DNA or RNA in living cells. These are the subject of the present review. Targeting DNA into the organelles currently shows promise but remarkably a number of alternative approaches based on RNA trafficking were also established and will bring as well major contributions.     

Mitochondrial fragmentation in neurodegeneration

Mitochondria are remarkably dynamic organelles that migrate, divide and fuse. Cycles of mitochondrial fission and fusion ensure metabolite and mitochondrial DNA mixing and dictate organelle shape, number and bioenergetic functionality. There is mounting evidence that mitochondrial dysfunction is an early and causal event in neurodegeneration. Mutations in the mitochondrial fusion GTPases mitofusin 2 and optic atrophy 1, neurotoxins and oxidative stress all disrupt the cable-like morphology of functional mitochondria. This results in impaired bioenergetics and mitochondrial migration, and can trigger neurodegeneration. These findings suggest potential new treatment avenues for neurodegenerative diseases.     

Nuclear genes that encode mitochondrial proteins for DNA and RNA metabolism are clustered in the Arabidopsis genome

The plant mitochondrial genome is complex in structure, owing to a high degree of recombination activity that subdivides the genome and increases genetic variation. The replication activity of various portions of the mitochondrial genome appears to be nonuniform, providing the plant with an ability to modulate its mitochondrial genotype during development. These and other interesting features of the plant mitochondrial genome suggest that adaptive changes have occurred in DNA maintenance and transmission that will provide insight into unique aspects of plant mitochondrial biology and mitochondrial-chloroplast coevolution. A search in the Arabidopsis genome for genes involved in the regulation of mitochondrial DNA metabolism revealed a region of chromosome III that is unusually rich in genes for mitochondrial DNA and RNA maintenance. An apparently similar genetic linkage was observed in the rice genome. Several of the genes identified within the chromosome III interval appear to target the plastid or to be targeted dually to the mitochondria and the plastid, suggesting that the process of endosymbiosis likely is accompanied by an intimate coevolution of these two organelles for their genome maintenance functions.