An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region

Deletions of muscle mitochondrial DNA (mtDNA) have recently been found in patients with mitochondrial myopathy. However, as most of the described cases were sporadic, and individual deletions involved different portions of mtDNA, the mechanism(s) producing the molecular lesions, as well as their mode of transmission, remain unclear. By studying families with mtDNA heteroplasmy, valuable information can be obtained about the role of inheritable factors in the pathogenesis of these disorders. We have studied four members of a family with autosomal dominant mitochondrial myopathy. Multiple deletions, involving the same portion of muscle mtDNA, were identified in all patients. Sequence analysis of the mutant mtDNAs, performed after DNA amplification by the polymerase-chain reaction showed that all the deletions start within a 12-nucleotide stretch at the 5' end of the D-loop region, a site of active communication between the nucleus and the mtDNA. The data indicate that a mutation of a nuclear-coded protein can destroy the integrity of the mitochondrial genome in a specific, heritable way.     

Paternal inheritance of mitochondrial DNA in mice.

For nearly 20 years it has been assumed on the basis of low-resolution experiments that mitochondrial (mt)DNA, in contrast to the genes in the nucleus, has an exclusively maternal mode of inheritance in animals. Using the polymerase chain reaction, paternally inherited mtDNA molecules have now been detected in mice at a frequency of 10(-4), relative to the maternal contributions. These mice were hybrids between two inbred strains (C57BL/6J and Mus spretus) whose mtDNAs can be distinguished easily. This new mode of inheritance provides a mechanism for generating heteroplasmy and may explain mitochondrial disorders exhibiting biparental transmission.     

Evidence from mitochondrial DNA that African honey bees spread as continuous maternal lineages

African honey bees have populated much of South and Central America and will soon enter the United States. The mechanism by which they have spread is controversial. Africanization may be largely the result of paternal gene flow into extant European populations or, alternatively, of maternal migration of feral swarms that have maintained an African genetic integrity. We have been using both mitochondrial and nuclear DNA restriction fragment length polymorphisms to follow the population dynamics between European and African bees. In earlier reports, we suggested that if African honey bees had distinctive mitochondrial (mt) DNA, then it could potentially distinguish the relative contributions of swarming and mating to the Africanization process. Because mtDNA is maternally inherited, it would not be transmitted by mating drones and only transported by queens accompanying swarms. Furthermore, the presence of African mtDNA would reflect unbroken maternal lineages from the original bees introduced from Africa. The value of mtDNA for population studies in general has been reviewed recently. Here we report that 19 feral swarms, randomly caught in Mexico, all carried African mtDNA. Thus, the migrating force of the African honey bee in the American tropics consists of continuous African maternal lineages spreading as swarms. The mating of African drones to European queens seems to contribute little to African bee migration.     

Nucleotide sequence evidence for rapid genotypic shifts in the bovine mitochondrial DNA D-loop

Mitochondrial DNA (mtDNA) is unusual in its rapid rate of evolution and high level of intraspecies sequence variation. The latter is thought to be related to the strict maternal inheritance of mtDNA, which effectively isolates within a species mitochondrial gene pools that accumulate mutations and vary independently. A fundamental and as yet unexplained aspect of this process is how, in the face of somatic and germ-line mtDNA ploidy of 10(3) to 10(5) (refs 4, 5), individual variant mtDNA molecules resulting from mutational events can come to dominate the large intracellular mtDNA population so rapidly. To help answer this question, we have determined here the nucleotide sequence of all or part of the D-loop region in 14 maternally related Holstein cows. Four different D-loop sequences can be distinguished in the mtDNA of these animals. One explanation is that multiple mitochondrial genotypes existed in the maternal germ line and that expansion or segregation of one of these genotypes during oogenesis or early development led to the rapid genotypic shifts observed.     

Direct evidence for extensive paternal mitochondrial DNA inheritance in the marine mussel Mytilus.

Inheritance of mitochondrial DNA in animals was thought to be strictly maternal. Recently, evidence for incidental paternal mtDNA leakage was obtained in hybrid crosses of Drosophila and mice. In mice, the frequency of paternal mtDNA contributions was estimated at 10(-4), compared with maternal contributions. The common occurrence in the marine mussel Mytilus of heteroplasmic individuals with two or more types of highly diverged mtDNA molecules was interpreted as strong evidence for biparental mtDNA inheritance by some, but not by others. We report here results from pair-matings involving two species of mussels, Mytilus edulis and Mytilus trossulus. Extensive contribution of paternal mtDNA, amounting to several orders of magnitude higher than that inferred for Drosophila or mice, was observed in both intra- and interspecific crosses.     

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson's disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson's disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson's disease have been identified, including PTEN-induced kinase 1 (PINK1; PARK6) and parkin (PARK2), which are also associated with sporadic forms of Parkinson's disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1-parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson's disease pathogenesis.     

Premature ageing in mice expressing defective mitochondrial DNA polymerase

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in a variety of tissues during ageing in humans, monkeys and rodents. These mutations are unevenly distributed and can accumulate clonally in certain cells, causing a mosaic pattern of respiratory chain deficiency in tissues such as heart, skeletal muscle and brain. In terms of the ageing process, their possible causative effects have been intensely debated because of their low abundance and purely correlative connection with ageing. We have now addressed this question experimentally by creating homozygous knock-in mice that express a proof-reading-deficient version of PolgA, the nucleus-encoded catalytic subunit of mtDNA polymerase. Here we show that the knock-in mice develop an mtDNA mutator phenotype with a threefold to fivefold increase in the levels of point mutations, as well as increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations is associated with reduced lifespan and premature onset of ageing-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anaemia, reduced fertility and heart enlargement. Our results thus provide a causative link between mtDNA mutations and ageing phenotypes in mammals.     

A family of mitochondrial proteins involved in bioenergetICS and biogenesis

The respiratory chain complexes of mitochondria consist of many different subunits, of which only a few partake directly in electron transport. The functions of the subunits that do not contain prosthetic groups are largely unknown. The cytochrome reductase complex of Neurospora crassa, for examine, consists of nine different subunits, of which the peripheral membrane proteins I and II (ref.3) that are located on the matrix side of the mitochondrial inner membrane are the largest subunits devoid of redox centres. Significantly, a cytochrome reductase fraction lacking these two subunits was inactive in electron transfer, and in yeast mutants with defective genes for either of the two subunits, assembly of the reductase is disrupted. Most mitochondrial proteins are imported into the mitochondrion as precursor proteins, and two proteins are necessary for cleaving their presequences, namely the matrix processing peptidase (MPP) and the processing enhancing protein (PEP), the latter strongly stimulating the activity of the former. Temperature-sensitive yeast mutants, which are affected in PEP or MPP, accumulate precursors at the nonpermissive temperature. We report here that subunit I of the cytochrome reductase can be grouped as members of the same protein family.     

Independent transfer of mitochondrial plasmids in Neurospora crassa

In the ascomycete fungus Neurospora, the distribution of homologous mitochondrial plasmid DNAs in different species and among mitochondrial types of N. crassa suggests that these molecules have moved between lineages of clonally propagated mtDNA. Here we report direct evidence for independent inheritance of mitochondrial plasmids by sexual reproduction which may help explain the distribution of these molecules among mitochondrial lineages.     

Conservation and rearrangement of mitochondrial structural gene sequences

Mitochondria contain the simplest DNA molecules that are present in eukaryotes. Mitochondrial DNA (mtDNA) is easily purified, and is an important model system for studying eukaryote gene structure and basic molecular processes. The protein sequences of mitochondrial gene products have been shown to be conserved from yeast to man, and there are definite similarities at the DNA sequence level. In contrast, the overall organization of the mitochondrial genome is drastically different in these organisms. To understand this, we need to extend work on mtDNA to a wider range of species. We have chosen to study the mtDNA of Aspergillus nidulans because a particularly comprehensive analysis of this system can be achieved using genetics as well as biochemistry, and like most eukaryotes it is an obligate aerobe, whereas Saccharomyces cerevisiae is not. We have investigated whether defined pieces of particular yeast mitochondrial genes show enough homology to Aspergillus mtDNA fragments to enable the corresponding Aspergillus genes to be located on the physical map. The results reported here show that this is the case for all five genes tested, and present the first data on the physical organization of the structural genes in the mitochondrial genome of A. nidulans.     

Mitochondrial genome variation and the origin of modern humans

The analysis of mitochondrial DNA (mtDNA) has been a potent tool in our understanding of human evolution, owing to characteristics such as high copy number, apparent lack of recombination, high substitution rate and maternal mode of inheritance. However, almost all studies of human evolution based on mtDNA sequencing have been confined to the control region, which constitutes less than 7% of the mitochondrial genome. These studies are complicated by the extreme variation in substitution rate between sites, and the consequence of parallel mutations causing difficulties in the estimation of genetic distance and making phylogenetic inferences questionable. Most comprehensive studies of the human mitochondrial molecule have been carried out through restriction-fragment length polymorphism analysis, providing data that are ill suited to estimations of mutation rate and therefore the timing of evolutionary events. Here, to improve the information obtained from the mitochondrial molecule for studies of human evolution, we describe the global mtDNA diversity in humans based on analyses of the complete mtDNA sequence of 53 humans of diverse origins. Our mtDNA data, in comparison with those of a parallel study of the Xq13.3 region in the same individuals, provide a concurrent view on human evolution with respect to the age of modern humans.     

Conformational mutations in human mitochondrial DNA

Variation in the human mitochondrial DNA (mtDNA) sequence has been extensively analysed using restriction fragment length polymorphisms (RFLPs). MtDNA RFLPs have previously been attributed to nucleotide changes within restriction endonuclease recognition sites or to small insertion-deletion mutations. We now report that RFLPs detected by polyacrylamide gel electrophoresis can also result from single nucleotide substitutions which alter the mobility of small- to medium-sized restriction fragments that incorporate the sequence. We have defined the mutation responsible at two loci and have identified several possible additional loci. When screening human mtDNAs with multiple restriction endonucleases, such mutations can be misidentified as insertion-deletion mutations or counted as multiple polymorphic restriction sites. This can lead to errors in constructing restriction maps and estimating sequence diversity.     

线粒体病的分子生物学机制

线粒体病是一种少见的能量代谢病,病情复杂多样,从单一组织损伤或无明显临床症状到多系统发病乃致患者早期死亡,在临床上容易误诊或漏诊,甚至延误治疗.由于线粒体的结构与功能受核基闪组(nDNA)与线粒体基冈组(mtDNA)双重调控,其中大多数线粒体酶、结构蛋白和各种蛋白因子由nDNA编码,因而多数原发性线粒体病是nDNA突变...     

In vitro suppression of UGA codons in a mitochondrial mRNA

Although both prokaryotic and eukaryotic messenger RNAs can be easily translated in heterologous protein-synthesizing systems, attempts to achieve correct synthesis of mitochondrial proteins by translation of mitochondrial mRNAs in such systems have failed. In general, the products of synthesis are of low molecular weight and presumably represent fragments of mitochondrial proteins. These fragments display a strong tendency to aggregate. Explanations have included the use by mitochondria of codons requiring a specialized tRNA population and the fortuitous occurrence within genes of purine-rich sequences resembling bacterial ribosome binding sites. In addition, the long 5'-leader sequences present in many mitochondrial (mt) RNAs may also contribute to difficulties in mRNA recognition by heterologous ribosomes. Recent sequence analysis of human mtDNA suggests that the genetic code used by mammalian mitochondria deviates in a number of respects from the 'universal' code, the most striking of these being the use of the UGA termination codon to specify tryptophan. That this may also apply in yeast mitochondria has been shown by Fox and Macino et al., thus providing an obvious and easily testable explanation for the inability of heterologous systems to synthesize full-length mitochondrial proteins. We confirm this explanation and describe here the in vitro synthesis of a full-length subunit II of yeast cytochrome c oxidase in a wheat-germ extract supplemented with a partially purified mitochondrial mRNA for this protein and a UGA-suppressor tRNA from Schizosaccharomyces pombe.     

A mutation in the tRNA(Leu)(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies

Mitochondrial encephalomyopathies are usually divided into three distinct clinical subgroups: (1) mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS); (2) myoclonus epilepsy associated with ragged-red fibres (MERRF); and (3) chronic progressive external ophthalmoplegia (CPEO) including Kearns-Sayre syndrome. Large deletions of human mitochondrial DNA and a transition mutation at the mitochondrial transfer RNALys gene give rise to CPEO including Kearns-Sayre syndrome and MERRF, respectively. Here we report an A-to-G transition mutation at nucleotide pair 3,243 in the dihydrouridine loop of mitochondrial tRNA(Leu)(UUR) that is specific to patients with MELAS. Because this mutation creates an ApaI restriction site, we could perform a simple molecular diagnostic test for the disease. The mutation was present in 26 out of 31 independent MELAS patients and 1 out of 29 CPEO patients, but absent in the 5 MERRF and 50 controls tested. Southern blot analysis confirmed that the mutant DNA always coexists with the wild-type DNA (heteroplasmy).     

Evolutionary genetics. Clonal inheritance of avian mitochondrial DNA.

We have taken a new approach to test the commonly accepted, but recently questioned, principle of clonal inheritance of vertebrate mitochondrial DNA (mtDNA) by relating its inheritance to a female-specific marker of nuclear DNA. Whereas this is impossible in organisms with male heterogamy (such as mammals), we show here that genealogies of mtDNA and the female-specific W chromosome of a bird species are completely concordant. Our results indicate that inheritance of mtDNA is free of detectable recombination effects over an evolutionary timescale.     

DNA sequences from the quagga, an extinct member of the horse family

To determine whether DNA survives and can be recovered from the remains of extinct creatures, we have examined dried muscle from a museum specimen of the quagga, a zebra-like species (Equus quagga) that became extinct in 1883 (ref. 1). We report that DNA was extracted from this tissue in amounts approaching 1% of that expected from fresh muscle, and that the DNA was of relatively low molecular weight. Among the many clones obtained from the quagga DNA, two containing pieces of mitochondrial DNA (mtDNA) were sequenced. These sequences, comprising 229 nucleotide pairs, differ by 12 base substitutions from the corresponding sequences of mtDNA from a mountain zebra, an extant member of the genus Equus. The number, nature and locations of the substitutions imply that there has been little or no postmortem modification of the quagga DNA sequences, and that the two species had a common ancestor 3-4 Myr ago, consistent with fossil evidence concerning the age of the genus Equus.     

Antiquity of clonal salamander lineages revealed by mitochondrial DNA.

The existence of clonally reproducing vertebrates has often served as a foil in attempts to explain the near-ubiquity of sexual reproduction in eukaryotes, but the absence of recombination, with its attendant limitation of new genotypes to those produced through mutations, restricts the adaptive ability of clonal organisms. It has been argued, therefore, that clonal vertebrate taxa have short lifespans. Variation in mitochondrial DNA (mtDNA) within clonal populations is interpreted instead as reflecting multiple, although limited, independent hybridization events. On the basis of an analysis of an average of 373 nucleotide pairs, we report here that the mtDNA of clonal, hybrid, gynogenetic mole salamanders (Ambystoma, Ambystomatidae) differs by 5% or more from mtDNA of their closest possible sexual relatives (A. jeffersonianum, A. laterale and A. texanum). Assuming usual rates of mtDNA divergence, these lineages have persisted for about 5 million years, far longer than estimated for other clonal vertebrate populations. The low mtDNA variability in the clonal lineages suggests that they have undergone population reductions during the Pleistocene.