Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation

Mitochondrial DNA (mtDNA) is packaged into DNA-protein assemblies called nucleoids, but the mode of mtDNA propagation via the nucleoid remains controversial. Two mechanisms have been proposed: nucleoids may consistently maintain their mtDNA content faithfully, or nucleoids may exchange mtDNAs dynamically. To test these models directly, two cell lines were fused, each homoplasmic for a partially deleted mtDNA in which the deletions were nonoverlapping and each deficient in mitochondrial protein synthesis, thus allowing the first unequivocal visualization of two mtDNAs at the nucleoid level. The two mtDNAs transcomplemented to restore mitochondrial protein synthesis but were consistently maintained in discrete nucleoids that did not intermix stably. These results indicate that mitochondrial nucleoids tightly regulate their genetic content rather than freely exchanging mtDNAs. This genetic autonomy provides a molecular mechanism to explain patterns of mitochondrial genetic inheritance, in addition to facilitating therapeutic methods to eliminate deleterious mtDNA mutations.     

Pearson syndrome and mitochondrial encephalomyopathy in a patient with a deletion of mtDNA.

A patient is described who has features of Pearson syndrome and who presented in the neonatal period with a hypoplastic anemia. He later developed hepatic, renal, and exocrine pancreatic dysfunction. At the age of 5 years he developed visual impairment, tremor, ataxia, proximal muscle weakness, external ophthalmoplegia, and a pigmentary retinopathy (Kearns-Sayre syndrome). Muscle biopsy confirmed the diagnosis of mitochondrial myopathy. Analysis of mtDNA from leukocytes and muscle showed mtDNA heteroplasmy in both tissues, with one population of mtDNA deleted by 4.9 kb. The deleted region was bridged by a 13-nucleotide sequence occurring as a direct repeat in normal mtDNA. Both Pearson syndrome and Kearns-Sayre syndrome have been noted to be associated with deletions of mtDNA; they have not previously been described in the same patient. These observations indicate that the two disorders have the same molecular basis; the different phenotypes are probably determined by the initial proportion of deleted mtDNAs and modified by selection against them in different tissues.     

Maintenance of human rearranged mitochondrial DNAs in long-term cultured transmitochondrial cell lines

Large-scale rearrangements of mitochondrial DNA (mtDNA; i.e., partial duplications [dup-mtDNAs] and deletions [Delta-mtDNAs]) coexist in tissues in a subset of patients with sporadic mitochondrial disorders. In order to study the dynamic relationship among rearranged and wild-type mtDNA (wt-mtDNA) species, we created transmitochondrial cell lines harboring various proportions of wt-, Delta-, and dup-mtDNAs from two patients. After prolonged culture in nonselective media, cells that contained initially 100% dup-mtDNAs became heteroplasmic, containing both wild-type and rearranged mtDNAs, likely generated via intramolecular recombination events. However, in cells that contained initially a mixture of both wt- and Delta-mtDNAs, we did not observe any dup-mtDNAs or other new forms of rearranged mtDNAs, perhaps because the two species were physically separated and were therefore unable to recombine. The ratio of wt-mtDNA to Delta-mtDNAs remained stable in all cells examined, suggesting that there was no replicative advantage for the smaller deleted molecules. Finally, in cells containing a mixture of monomeric and dimeric forms of a specific Delta-mtDNA, we found that the mtDNA population shifted towards homoplasmic dimers, suggesting that there may be circumstances under which the cells favor molecules with multiple replication origins, independent of the size of the molecule.     

Rearranged mitochondrial genomes are present in human oocytes.

Using quantitative PCR, we have determined that a human oocyte contains approximately 100,000 mitochondrial genomes (mtDNAs). We have also found that some oocytes harbor measurable levels (up to 0.1%) of the so-called common deletion, an mtDNA molecule containing a 4,977-bp rearrangement that is present in high amounts in many patients with "sporadic" Kearns-Sayre syndrome (KSS) and progressive external ophthalmoplegia (PEO). This is the first demonstration that rearranged mtDNAs are present in human oocytes, and it provides experimental support for the supposition that pathogenic deletions associated with the ontogeny of sporadic KSS and PEO can be transmitted in the female germ line, from mother to child. The relevance of these finding to the accumulation of extremely low levels of deleted mtDNAs in both somatic and germ-line tissues during normal human aging is also discussed.     

Significant existence of deleted mitochondrial DNA in cirrhotic liver surrounding hepatic tumor.

To understand the role of mitochondria in carcinogenesis, we compared the amount of deleted mtDNAs between human hepatic tumors and surrounding cirrhotic portion of the liver of ten patients by using polymerase chain reaction (PCR). Multiple mtDNA deletions were detected in cirrhotic portion, but no deletions were detected in the tumor portion. Direct sequencing of the fragments revealed a 7,079-bp deletion (nucleotide position 8,992-16,072) involving no direct repeated sequences and a 7,436-bp deletion (position 8,649-16,084) involving a 12-bp directly repeated sequence of 5'-CATCAACAACCG-3' exists in both the ATP6 gene and the D-loop region. These mtDNA mutations could be one of the endogenous factors that induce somatic mutations in nuclear genome and etiologically contribute to human carcinogenesis.     

Maternal inheritance of deleted mitochondrial DNA in a family with mitochondrial myopathy

Skeletal muscles from a mother and her daughter both with chronic progressive ophthalmoplegia were analyzed. Histological and biochemical analyses of their muscle samples showed typical features of this type of mitochondrial myopathy. Southern blot analysis revealed that, in both patients, there were two species of mitochondrial DNA (mtDNA): normal one and partially deleted one. The sizes of the deletion were different; the mutant mtDNAs from the mother and the daughter had about 2.5- and 5-kilobase deletions, respectively. The two mutant mtDNAs shared a common deleted region of 1.2-kilobase. However, both the start and the end of deletion were different between them, implying a novel mode of inheritance. This is the first report that the mutant mtDNA is responsible for the maternal inheritance of a human disease.     

Mitochondrial DNA diseases: Histological and cellular studies

Large-scale deletions and tRNA point mutations in mitochondrial DNA (mtDNA) are associated with a variety of different mitochondrial encephalomyopathies. Skeletal muscle in these patients shows a typical pathology, characterized by the focal accumulation of large numbers of morphologically and biochemically abnormal mitochondria (ragged-red fibers). Both mtDNA deletions and tRNA point mutations impair mitochondrial translation and produce deficiencies in oxidative phosphorylation. However, mutant and wild-type mtDNAs co-exist (mtDNA heteroplasmy) and the translation defect is not expressed until the ratio of mutant: wild-type mtDNAs exceeds a specific threshold. Below the threshold the phenotype can be rescued by intramitochondrial genetic complementation. The mosaic expression of the skeletal muscle pathology is thus determined by both the cellular and organellar distribution of mtDNA mutants.     

Nuclear-mitochondrial epistasis and drosophila aging: introgression of Drosophila simulans mtDNA modifies longevity in D. melanogaster nuclear backgrounds

Under the mitochondrial theory of aging, physiological decline with age results from the accumulated cellular damage produced by reactive oxygen species generated during electron transport in the mitochondrion. A large body of literature has documented age-specific declines in mitochondrial function that are consistent with this theory, but relatively few studies have been able to distinguish cause from consequence in the association between mitochondrial function and aging. Since mitochondrial function is jointly encoded by mitochondrial (mtDNA) and nuclear genes, the mitochondrial genetics of aging should be controlled by variation in (1) mtDNA, (2) nuclear genes, or (3) nuclear-mtDNA interactions. The goal of this study was to assess the relative contributions of these factors in causing variation in Drosophila longevity. We compared strains of flies carrying mtDNAs with varying levels of divergence: two strains from Zimbabwe (<20 bp substitutions between mtDNAs), strains from Crete and the United States (approximately 20-40 bp substitutions between mtDNAs), and introgression strains of Drosophila melanogaster carrying mtDNA from Drosophila simulans in a D. melanogaster Oregon-R chromosomal background (>500 silent and 80 amino acid substitutions between these mtDNAs). Longevity was studied in reciprocal cross genotypes between pairs of these strains to test for cytoplasmic (mtDNA) factors affecting aging. The intrapopulation crosses between Zimbabwe strains show no difference in longevity between mtDNAs; the interpopulation crosses between Crete and the United States show subtle but significant differences in longevity; and the interspecific introgression lines showed very significant differences between mtDNAs. However, the genotypes carrying the D. simulans mtDNA were not consistently short-lived, as might be predicted from the disruption of nuclear-mitochondrial coadaptation. Rather, the interspecific mtDNA strains showed a wide range of variation that flanked the longevities seen between intraspecific mtDNAs, resulting in very significant nuclear x mtDNA epistatic interaction effects. These results suggest that even "defective" mtDNA haplotypes could extend longevity in different nuclear allelic backgrounds, which could account for the variable effects attributable to mtDNA haplogroups in human aging.     

Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA

Autosomal dominant and/or recessive progressive external ophthalmoplegia (ad/arPEO) is associated with mtDNA mutagenesis. It can be caused by mutations in three nuclear genes, encoding the adenine nucleotide translocator 1, the mitochondrial helicase Twinkle or DNA polymerase γ (POLG). How mutations in these genes result in progressive accumulation of multiple mtDNA deletions in post- mitotic tissues is still unclear. A recent hypothesis suggested that mtDNA replication infidelity could promote slipped mispairing, thereby stimulating deletion formation. This hypothesis predicts that mtDNA of ad/arPEO patients will contain frequent mutations throughout; in fact, our analysis of muscle from ad/arPEO patients revealed an age-dependent, enhanced accumulation of point mutations in addition to deletions, but specifically in the mtDNA control region. Both deleted and non-deleted mtDNA molecules showed increased point mutation levels, as did mtDNAs of patients with a single mtDNA deletion, suggesting that point mutations do not cause multiple deletions. Deletion breakpoint analysis showed frequent breakpoints around homopolymeric runs, which could be a signature of replication stalling. Therefore, we propose replication stalling as the principal cause of deletion formation.     

Linked oligodeoxynucleotides show binding cooperativity and can selectively impair replication of deleted mitochondrial DNA templates

Mutations in mitochondrial DNA (mtDNA) cause a spectrum of human pathologies, which predominantly affect skeletal muscle and the central nervous system. In patients, mutated and wild-type mtDNAs often co-exist in the same cell (mtDNA heteroplasmy). In the absence of pharmacological therapy, a genetic strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative to the single-stranded replication intermediate, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Previous studies have shown the efficacy of this anti-genomic approach in vitro, targeting pathogenic mtDNA templates with only a single point mutation. Pathogenic molecules harbouring deletions, however, present a more difficult problem. Deletions often occur at the site of two short repeat sequences (4–13 residues), only one of which is retained in the deleted molecule. With the more common larger repeats it is therefore difficult to design an anti-genomic molecule that will bind selectively across the breakpoint of the deleted mtDNA. To address this problem, we have used linker-substituted oligodeoxynucleotides to bridge the repeated residues. We show that molecules can be designed to bind more tightly to the deleted as compared to the wild-type mtDNA template, consistent with the nucleotide sequence on either side of the linker co-operating to increase binding affinity. Furthermore, these bridging molecules are capable of sequence-dependent partial inhibition of replication in vitro.     

Novel large-range mitochondrial DNA deletions and fatal multisystemic disorder with prominent hepatopathy

Hepatic involvement in mitochondrial cytopathies rarely manifests in adulthood, but is a common feature in children. Multiple OXPHOS enzyme defects in children with liver involvement are often associated with dramatically reduced amounts of mtDNA. We investigated two novel large scale deletions in two infants with a multisystem disorder and prominent hepatopathy. Amount of mtDNA deletions and protein content were measured in different post-mortem tissues. The highest levels of deleted mtDNA were in liver, kidney, pancreas of both patients. Moreover, mtDNA deletions were detected in cultured skin fibroblasts in both patients and in blood of one during life. Biochemical analysis showed impairment of mainly complex I enzyme activity. Patients manifesting multisystem disorders in childhood may harbour rare mtDNA deletions in multiple tissues. For these patients, less invasive blood specimens or cultured fibroblasts can be used for molecular diagnosis. Our data further expand the array of deletions in the mitochondrial genomes in association with liver failure. Thus analysis of mtDNA should be considered in the diagnosis of childhood-onset hepatopathies.     

Accumulation of mitochondrial DNA deletions in human oral tissues -- effects of betel quid chewing and oral cancer

Accumulation of mitochondrial DNA (mtDNA) mutations in human tissues has been associated with intrinsic aging and environmental insult. Recently, mtDNA mutations have been detected in various tumors, including head and neck tumors. However, the factors affecting the occurrence and accumulation of mtDNA deletions in tumor tissues are poorly understood. In Taiwan, betel quid chewing is a major risk factor for oral cancer. Using polymerase chain reaction (PCR) techniques, we examined large-scale deletions of mtDNA in 53 pairs of tumor and non-tumor oral tissues from the patients with or without betel quid chewing history. The results revealed that irrespective of the history of betel quid chewing, the incidences of the 4977bp deletion and other deletions of mtDNA were lower in the tumor portion as compared with the non-tumor portion. The average proportions of the 4977bp deleted mtDNA in the tumor tissues of the betel quid chewers and non-betel quid chewers were 13- and 5-fold, respectively, lower than those in the corresponding non-tumor tissues. Moreover, the average proportion of 4977bp deleted mtDNA was significantly higher (P<0.05) in the non-tumor oral tissues of the patients with betel quid chewing history than that of the patients without the history of betel quid chewing. These results suggest that betel quid chewing may increase mtDNA mutation in human oral tissues and that accumulation of mtDNA deletions and subsequent cytoplasmic segregation of these mutations during cell division could be an important contributor to the early phase of oral carcinogenesis.     

Mitochondrial DNA and genetic disease.

Since the human mitochondrial genome was characterised and sequenced in 1981, it has been viewed as the likely site of genetic diseases showing a maternal inheritance pattern and associated with defects of the respiratory chain, such as the mitochondrial myopathies (MMs). The properties that make it a candidate for the source of such conditions are that it encodes polypeptides involved in electron transport and that it is maternally inherited. However, several of the mtDNA diseases only fulfill one or other of these criteria: the first group of mtDNA diseases showed only sporadic deletions, and the first point mutation in Leber's Hereditary Optic Neuropathy (LHON) is not associated with a clear biochemical defect. Furthermore, it is now clear that both autosomal dominant and probably recessive nuclear genes can cause abnormalities of mtDNA. Each of these major groups will be considered in turn.     

Differential accumulations of 4,977 bp deletion in mitochondrial DNA of various tissues in human ageing

Several types of deletions in mitochondrial DNA (mtDNA) have been recetly identified in various tissues of old humans. In order to determine whether there are differences in the incidence and proportion of deleted mtDNAs in different tissues during human ageing, we examined tha 4,977 bp deletion in mtDNA of various tissues from subjects of different ages. Total DNA was extracted from each of the biopsied tissues and was serially diluted by two-fold with distilled water. A 533 bp DNA fragment was amplified by PCR from total mtDNA using a pair of primers L3304-3323 and H3817-3836, and another 524 bp PCR product was amplified from 4,977 bp deleted mtDNA by identical conditions using another pair of primers L8150-8166 and H13631-13650. The maximum dilution fold of each sample that still allowed the ethidium bromide-stained PCR product (533 bp or 524 bp) in the agarose gel to be visible under UV light illumination was taken as the relative abundance of the mtDNA (wild-type or mutant) in the original sample. By this method, we were able to determine the proportion of deleted mtDNA in human tissues. We found that the 4,977 bp deletion started to appear in the second and third decades of life in human muscle and liver tissues. But the deletion was not detectable in the testis until the age of 60 years. Moreover, the proportion of deleted mtDNA varied greatly in different tissues. Among the tissues examined, muscle was found to harbor higher proportin of deleted mtDNA than the other tissues. The average proportion of the 4,977 bp depleted mtDNA of the muscle from subjects over 70 years old was approximately 0.06%, and that of the liver and the testis was 0.0076% and 0.05%, respectively. These findings suggest that the frequency and proportion of the deleted mtDNA in human tissues increase with age and that the mtDNA deletions occur more frequently and abundantly in high energy-demanding tissues during the ageing process of the human.     

Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA. II. The organization of sequences in petite mitochondrial DNAs carrying genetic markers from the 21S region.

We have investigated the organization of sequences in ten rho- petite mtDNAs by restriction enzyme analysis and electron microscopy. From the comparison of the physical maps of the petite mtDNAs with the physical map of the mtDNA of the parental rho+ strain we conclude that there are at least three different classes of petite mtDNAs: I. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA. II. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA with a terminal inverted duplication. III. Mixed repeats of an (almost) continuous rho+ mtDNA segment. In out petite mtDNAs of the second type, the inverted duplications do not cover the entire conserved rho+ mtDNA segment. We have found that the petite mtDNAs of the third type contain a local inverted duplication at the site where repeating units can insert in two orientations. At least in one case this local inverted duplication must have arisen by mutation. The rearrangements that we have found in the petite mtDNAs do not cluster at specific sites on the rho+ mtDNA map. Large rearrangements or deletions within the conserved rho+ mtDNA segment seem to contribute to the suppressiveness of a petite strain. There is also a positive correlation between the retention of certain segments of the rho+ mtDNA and the suppressiveness of a petite strain. We found no correlation between the suppressiveness of a petite strain and its genetic complexity. The relevance of these findings for the mechanism of petite induction and the usefulness of petite strains for the physical mapping of mitochondrial genetic markers and for DNA sequence analysis are discussed.