Mitochondrial DNA Rearrangements Research Articles

An alteration in transcription patterns of mitochondrial genes in alloplasmic lines of Brassica rapa.

The accumulation of transcripts of mitochondrial genes in young buds was examined in euplasmic and alloplasmic lines of Brassica rapa for the `mur' system of cytoplasmic male sterility (CMS). Southern blotting analysis revealed the general absence of restriction fragment length polymorphism between Diplotaxis muralis, the donor of cytoplasm, and each of three sterile alloplasmic lines, indicating that the rearrangement of mitochondrial DNA might not be caused by the nucleus of B. rapa. However, different Northern hybridization patterns were detected when coxI and nad3 were used as probes. Production of the 2.5-kb transcript of coxI and a reduction in levels of the transcript of nad3 in the sterile alloplasmic lines were clearly under the control of the nucleus of B. rapa. No such differences in patterns of transcripts were detected in the leaves. This observation suggests that some factor(s) or gene(s) in the nucleus of B. rapa acts in an organ-specific manner to alter the size of transcripts in the mitochondria of the donor cytoplasm without any structural changes in the mitochondrial genome. Further studies are required to clarify whether or not the unique transcripts in the young buds induce a CMS event.

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea.

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5' upstream region of the grisea ORF. One mutation is a G --> A transition at the 5' splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

Cardiomyopathies in disorders of oxidative metabolism.

Primary cardiomyopathy is an important cause of mortality in children and adults. Apart from inherited disorders of myocardial contractile and structural proteins, several defects of energy metabolism may cause cardiomyopathy. Most of the energy required for myocardial contraction is derived from aerobic metabolism. Faulty aerobic metabolism involving the heart may be due to defects of mitochondrial oxidative phosphorylation or to defects of fatty acid oxidation. Considerable advances have been made in the last 10 years in understanding the biochemical and molecular characteristics of mitochondrial disorders. Several point mutations or large-scale re-arrangements of mitochondrial DNA have been identified in patients with cardiomyopathy, either as part of complex multisystem syndromes or as the main clinical feature. Inborn errors of fatty acid oxidation are reported with increasing frequency as a cause of metabolic dysfunction, myopathy, cardiomyopathy, and sudden death in childhood. Advances in biochemical and molecular genetic techniques have considerably improved our understanding of the metabolic disorders causing cardiomyopathy, providing new tools for classification and diagnosis of candidate patients. The present review focuses on defects of mitochondrial oxidative metabolism associated with cardiomyopathy.

Male infertility associated with multiple mitochondrial DNA rearrangements

Male sterility results from a number of characterized exogeneous or genetic dysfunctions preventing normal differentiation into mobile spermatozoa. This may now be overcome by intra cytoplasmic sperm injection (ICSI). This practice does not require mobile, or even mature spermatozoa for in vitro fecondation. However, a functional respiratory chain, partly encoded by the mitochondrial DNA (mtDNA), is required for the mobility of the spermatozoa. We report the case of an infertile patient who wished to procreate. ICSI was proposed but he displayed multiple mtDNA deletions of possible nuclear origin in the spermatozoa and in the deltoid muscle. Even though mtDNA is maternally inherited, the possibility of a nuclear-driven mutation affecting the integrity of the mtDNA should be taken into account when ICSI is to be performed. Together with recent genetic in vitro manipulations in mammals, our data point to the importance of studying the mtDNA structure in human spermatozoa, and the potential risks of these non-natural practices for procreation.

Multiple hepatic mitochondrial DNA deletions suggest premature oxidative aging in alcoholic patients

Background/Aims: A 4977-base pair deletion has been detected in the hepatic mitochondrial DNA of alcoholic patients with microvesicular steatosis, a lesion ascribed to impaired mitochondrial β-oxidation. However, only a single deletion had been looked for in this previous study, and it could not be determined whether the deletion was preexisting or acquired. Alcohol abuse increases the formation of reactive oxygen species in hepatic mitochondria. If this effect accelerates the oxidative aging of mitochondrial DNA, several other mutations would be expected.Methods: The mtDNA region extending from nucleotide 8167 to nucleotide 14246 was screened for the presence of large mitochondrial DNA deletions in 58 alcoholic patients and 67 age-matched non-alcoholic controls. Hepatic DNA was subjected to polymerase chain reactions that amplified non-deleted and deleted mitochondrial DNA, respectively, and the boundaries of the mitochondrial DNA deletions were sequenced.Results: Only 3% of the non-alcoholic controls carried a mitochondrial DNA deletion, whereas 24% of all alcoholic patients and 85% of the 13 alcoholic patients with microvesicular steatosis exhibited either single or multiple 4977, 5385, 5039 and 5556-base pair mitochondrial DNA deletions. No deletion(s) were observed, however, in 13 patients with microvesicular steatosis due to other causes.Conclusions: Diverse mitochondrial DNA rearrangements are observed in alcoholic patients with microvesicular steatosis. We suggest that alcohol abuse leads to premature oxidative aging of mitochondrial DNA. Hypothetically, oxidative damage to mitochondrial constituents (DNA, proteins and lipids) may favor microvesicular fat deposition.

Multi-organ characterization of mitochondrial genomic rearrangements in ad libitum and caloric restricted mice show striking somatic mitochondrial DNA rearrangements with age.

Mitochondrial DNA (mtDNA) rearrangements have been shown to accumulate with age in the post-mitotic tissues of a variety of animals and have been hypothesized to result in the age-related decline of mitochondrial bioenergetics leading to tissue and organ failure. Caloric restriction in rodents has been shown to extend life span supporting an association between bioenergetics and senescence. In the present study, we use full length mtDNA amplification by long-extension polymerase chain reaction (LX-PCR) to demonstrate that mice accumulate a wide variety of mtDNA rearrangements with age in post mitotic tissues. Similarly, using an alternative PCR strategy, we have found that 2-4 kb minicircles containing the origin of heavy-strand replication accumulate with age in heart but not brain. Analysis of mtDNA structure and conformation by Southern blots of unrestricted DNA resolved by field inversion gel electrophoresis have revealed that the brain mtDNAs of young animals contain the traditional linear, nicked, and supercoiled mtDNAs while old animals accumulate substantial levels of a slower migrating species we designate age-specific mtDNAs. In old caloric restricted animals, a wide variety of rearranged mtDNAs can be detected by LX-PCR in post mitotic tissues, but Southern blots of unrestricted DNA reveals a marked reduction in the levels of the age- specific mtDNA species. These observations confirm that mtDNA mutations accumulate with age in mice and suggest that caloric restriction impedes this progress.

Cytosolic ribosomal mutations that abolish accumulation of circular intron in the mitochondria without preventing senescence of Podospora anserina.

The filamentous fungus Podospora anserina presents a degeneration syndrome called Senescence associated with mitochondrial DNA modifications. We show that mutations affecting the two different and interacting cytosolic ribosomal proteins (S7 and S19) systematically and specifically prevent the accumulation of senDNA alpha (a circular double-stranded DNA plasmid derived from the first intron of the mitochondrial cox1 gene or intron alpha) without abolishing Senescence nor affecting the accumulation of other usually observed mitochondrial DNA rearrangements. One of the mutant proteins is homologous to the Escherichia coli S4 and Saccharomyces cerevisiae S13 ribosomal proteins, known to be involved in accuracy control of cytosolic translation. The lack of accumulation of senDNA alpha seems to result from a nontrivial ribosomal alteration unrelated to accuracy control, indicating that S7 and S19 proteins have an additional function. The results strongly suggest that modified expression of nucleus-encoded proteins contributes to Senescence in P. anserina. These data do not fit well with some current models, which propose that intron alpha plays the role of the cytoplasmic and infectious Determinant of Senescence that was defined in early studies.

Detection and Analysis of Mitochondrial DNA Deletions by Whole Genome PCR

Southern blot analysis has been the best method available for the screening and detection of mitochondrial DNA (mtDNA) rearrangements. Recent developments in polymerase chain reaction (PCR) technology allowed the amplification of the whole mitochondrial genome (16.6 kb), making PCR a potentially useful technique for the detection of mtDNA deletions. We tested the usefulness of whole mitochondrial genome PCR by studying skeletal muscle DNA from seven patients with single and multiple deletions and controls from ages 3 to 91 years old. Specific patterns for single and multiple deletions were obtained with whole genome PCR, which were confirmed by the Southern analysis with probes hybridizing to mtDNA sequences. Amplifications from young controls (3 to 23 years old) yielded only one band (16.5 kb) while amplification from older controls revealed one or more additional smaller bands. The amplification from the 91-year-old control showed a pattern similar to amplifications from patients with multiple mtDNA deletions. Although single and multiple mtDNA deletions could be readily detected from patient samples, the high sensitivity of this method can lead to false positive results due to the presence of age-related deletions in old control samples. Despite its limitations, whole mitochondrial genome PCR can be useful for the detection of single deletions in muscle samples obtained from young individuals since the levels of age-related deletions are too low to be amplified.

Characterization of the radish mitochondrial nad3/rps12 locus: analysis of recombination repeats and RNA editing.

In order to further investigate sequences that are responsible for low-frequency recombination in plant mitochondrial DNAs and RNA editing in radish mitochondria, the nad3/rps12 locus has been isolated and characterized from a normal cultivar of radish and the male-sterile Ogura cytoplasm. A repeated sequence that has been implicated in other radish mitochondrial DNA rearrangements was identified at the breakpoint between the two loci indicating that it was also involved in the nad3/rps12 rearrangement. Similar to some other radish mitochondrial genes, nad3/rps12 genomic sequences already contain several, but not all, of the bases that are typically edited in plant mitochondrial nad3 and rps12 genes. Analysis of nad3/rps12 cDNAs indicated that the mRNAs are not edited. One partially edited transcript was identified out of the twenty two that were examined. This finding, along with the observation that nad3/rps12 RNAs are present at very low levels, raises the possibility that radish mitochondria may not encode functional copies of these genes. Consistent with this hypothesis, DNA-blot analysis detects nad3/rps12 sequences in the nucleus.

Corrigendum

Marked Increase in the Number and Variety of Mitochondrial DNA Rearrangements in Aging Human Skeletal Muscle

Rearrangements of mitochondrial DNA and the mitochondrial fusion-promoting plasmid (mF) are associated with defective mitochondrial fusion in Physarum polycephalum

Rearrangements of mitochondrial DNA and the mitochondrial fusion-promoting plasmid (mF) are associated with defective mitochondrial fusion in Physarum polycephalum

Rearrangements of mitochondrial DNA and the mitochondrial fusion-promoting plasmid (mF) are associated with defective mitochondrial fusion in Physarum polycephalum.

A specific linear mitochondrial plasmid (mF) is genetically associated with the fusion of mitochondria in the true slime mould, Physarum polycephalum. In matings between mF+ and mF- strains, which respectively carry and do not carry the mF plasmid, mitochondrial fusion occurs in the zygote. Mitochondrial fusion induces recombination between specific sites in the mitochondrial DNA (mtDNA) and in the mF plasmid. To detect a region which is associated with the mitochondrial fusion in the mF plasmid, we isolated, by fluorescence microscopy, strains which showed defective mitochondrial fusion (delta mif-) from those which showed normal mitochondrial fusion (mif+). Analysis of the mitochondrial genomes of delta mif- strains showed only mtDNA which recombined with the mF plasmid in mitochondria. Comparison of this recombinant mtDNA in one delta mif- strain (NG 15) with that of a mif+ strain showed that a 2.2-kbp region, which included the integration site of the mF plasmid, was deleted in the delta mif- strain by recombination between the main mtDNA and the mF plasmid. In other strains, in addition to this deletion, a 6-kbp region which included both termini was deleted by recombination at six repeats of AAT sequences in the mF plasmid. Moreover, transcripts of the mF plasmid were not detected in NG15 by slot hybridization.

ミトコンドリア脳筋症例に対する人工内耳

Kearns-Sayre syndrome (KSS) is one of a mitochondrial disease characterized by progressive eye disorders, such as pigmentary retinopathy, and complete heart blockage. A recent study revealed that the etiology of this syndrome involves sporadic large scale rearrangements of mitochondrial DNA. Other neurologic symptoms are frequently associated with this syndrome, and hearing loss is the other main symptom. At the age of 37 years, this female patient began to suffer progressive hearing loss, and eventually became completely deaf. MRI imaging showed diffuse, abnormally high signals in the brain white matter, in T2-weighted images. Further neurological examination revealed eye abnormalities, and a genetic analysis confirmed a diagnosis of KSS. In this case, no sensory agnosia or other focal signs of disorder in the brain cortex were found, and the electrophysical criteria for cochlear implant were fulfilled. Cochlear implantation was performed on May 19, 1995. Post-operatively, the patient underwent vowel and consonant recognition tests. The patients scored well with an accuracy of 75% for vowels and 36% for consonants. The accuracy of vowel and consonant recognition with lip reading was elevated to 100% and 72%, respectively. Sound recognition in this patient seemed to be quite high compared to that reported in other implanted patients. The excellent results obtained in this KSS patient suggest pure neural deafness, and raise the possibility of cochlear involvement in this condition.

Involvement of S2 episomal sequences in the generation of NCS4 deletion mutation in maize mitochondria.

Cytoplasmic male sterility (CMS) and the abnormal-growth, nochromosomal stripe (NCS) phenotypes are cytoplasmically determined traits in plants. Mitochondrial DNA rearrangements involving short repeats appear to be responsible for the production of CMS reversions to fertility and NCS mutations. NCS4, a new mutant of maize CMS-S, exhibits both abnormal growth and male fertility. This mutant is unique because both mutations occurred within the same plant. Free S1 and S2 episomes normally found in CMS-S mitochondria have been lost from NCS4 plants. An S2 sequence has recombined aberrantly with a ribosomal protein coding region, rps3/rpl16. One end of the S2 sequence and the 5' end of the rps3/rpl16 transcription unit are absent from the NCS4 recombinant genome. Loss of mitochondrial ribosomal protein function is lethal; therefore, NCS4 plants are heteroplasmic for the rps3 deletion. Loss of S sequences from CMS-S mitochondria is not lethal and plants regain pollen function. Thus, although NCS4 plants have very abnormal plant phenotypes, they are male-fertile.

Deducing the pattern of arthropod phylogeny from mitochondrial DNA rearrangements.

The origins of arthropods and the phylogenetic relationships among their three major living groups (atelocerates, crustaceans and chelicerates) are vigorously contended. To help resolve this, we determined mitochondrial gene arrangements for a chelicerate, a myriapod, two crustaceans, an onychophoran, a mollusc and an annelid, and compared them with published gene orders of other species. The result strongly supports the monophyly of Arthropoda and of Mandibulata (atelocerates plus crustaceans) and refutes the Uniramia (atelocerates plus onychophorans). Gene arrangement comparisons are emerging as a powerful new tool for resolving ancient phylogenetic relationships.

Brassica napus (+) B. tournefortii, a somatic hybrid containing traits of agronomic importance for rapeseed breeding

Somatic hybrids were produced between hypocotyl protoplasts of Brassica napus (rapeseed) and mesophyll protoplasts of B. tournefortii using protoplast fusion. Fusion products were selected by flow cytometry and cell sorting. Twenty-three plants regenerated from individual calli originating from 4 fusion experiments were successfully transferred to the greenhouse. Nineteen of these were characterized as hybrids according to analysis of morphological characteristics, isoenzyme and nuclear RFLP patterns. Analysis of nuclear DNA content revealed a variation in ploidy level among the hybrids, even though a large number (53%) had a DNA content corresponding to the sum of the parental nuclear DNA. Most of the hybrids were intermediate in morphology between B. napus and B. tournefortii. The flowers displayed a range of variation: from normal flowers to abnormal flowers with reduced number of anthers and petals. Fertility investigation showed that both fertile and sterile hybrids were obtained. Five were self fertile, while 10 were male sterile and 4 were completely sterile. Phoma lingam resistance was transferred from B. tournefortii to the hybrid plants. Out of the 16 hybrids examined 12 showed resistance to P. lingam. Chloroplast DNA (cpDNA) patterns of the hybrids were identified by hybridizing a cpDNA probe to total genomic DNA. Of 18 hybrids analysed, 11 had B. tournefortii and 7 B. napus cpDNA pattern. No recombinant cpDNA banding pattern was found. In contrast to the cpDNA, mitochondrial DNA (mtDNA) rearrangements were detected in the hybrids. Out of 18 analysed hybrids, 7 showed evidence of mitochondrial recombination, while 11 had mtDNA identical to either of the parental types.

Kearns-Sayre syndrome associated with mitochondrial DNA deletion or duplication: a molecular genetic and pathological study

The neuropathological findings in 2 patients with Kearns-Sayre syndrome and mitochondrial DNA (mtDNA) rearrangements, one a predominant deletion and the other a predominant duplication, were remarkably similar, showing diffuse vacuolation of white matter. There were some of the pathological features of Leigh's syndrome in the spinal cord of the patient with a duplication. In the patient with a predominant deletion, rearranged mtDNA was undetectable in blood, spleen, and testis, and present in highest amounts in muscle and the brain, but relatively low in cerebellum, reflecting the ratio seen, albeit in much smaller amounts, in normal aged brains. MtDNA rearrangements in this patient were largely deletions or deletion dimers; duplicated mtDNA was present in only trace amounts in some tissues and there was none in skeletal muscle. The patient with a predominant duplication of mtDNA had higher amounts of rearranged mtDNA in blood (mainly duplicated) than muscle (mainly deleted). Correlation of these data with tissue dysfunction is probably complicated by the replicative behaviour of deleted, duplicated and normal mtDNA.

Spectrum of mitochondrial DNA rearrangements in the Pearson marrow-pancreas syndrome.

The Pearson marrow-pancreas syndrome (MIM 557000) is a disorder involving the hematopoietic system and the exocrine pancreas in early infancy. We have previously shown that this disease results from a defect of oxidative phosphorylation associated with deletions of the mitochondrial DNA. We present here a series of 21 cases (including 15 unreported patients) with Pearson syndrome and describe mitochondrial DNA deletions as consistent features in this syndrome. Nine patients presented the same 4.9 kb deletion, while other patients presented different deletions ranging in size from 9 to 14 kb between tRNACyst and the D-loop. Direct repeats (4-13 bp) were consistently present in the wild-type mtDNA at the boundaries of the deletions. Deletion-dimers, deletion-multimers or duplications were observed in association with deletions. Duplications were identified both in patients who died of their Pearson syndrome and in the ones who survived and developed Kearns-Sayre syndrome, suggesting that no correlation could be made between the clinical severity and the type, size or location of the rearrangements.

Marked increase in the number and variety of mitochondrial DNA rearrangements in aging human skeletal muscle.

Several reports have shown that individual mitochondrial DNA (mtDNA) deletions accumulate with age. However, the overall extent of somatic mtDNA damage with age remains unclear. We have utilized full-length PCR to concurrently screen for multiple mtDNA rearrangements in total DNA extracted from skeletal muscle derived from physiologically normal individuals (n = 35). This revealed that both the number and variety of mtDNA rearrangements increases dramatically between young and old individuals (P < 0.0001). We further examined the mtDNA from both the younger and older subjects by Southern blot analysis and observed an age-related increase in mtDNA(s) comparable in size to mtDNA products unique to patients with known mtDNA deletions. These data imply that a wide spectrum of mtDNA rearrangements accumulate in old individuals, which correlates with the marked age related decrease in OXPHOS capacity observed in post-mitotic tissues.

Marked increase in the number and variety of mitochondrial DNA rearrangements in aging human skeletal muscle

Marked increase in the number and variety of mitochondrial DNA rearrangements in aging human skeletal muscle