Mutants of Chlamydomonas reinhardtii deficient in mitochondrial complex I: characterization of two mutations affecting the nd1 coding sequence.

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 30 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components of complex I are coded for by mitochondrial genes. Three mutants deprived of complex I activity and displaying slow growth in the dark were isolated after mutagenic treatment with acriflavine. A genetical analysis demonstrated that two mutations (dum20 and dum25) affect the mitochondrial genome whereas the third mutation (dn26) is of nuclear origin. Recombinational analyses showed that dum20 and dum25 are closely linked on the genetic map of the mitochondrial genome and could affect the nd1 gene. A sequencing analysis confirmed this conclusion: dum20 is a deletion of one T at codon 243 of nd1; dum25 corresponds to a 6-bp deletion that eliminates two amino acids located in a very conserved hydrophilic segment of the protein.     

Normal Mitochondrial Genome in Brain from Patients with Parkinson''s Disease and Complex I Defect

The mitochondrial genome codes for 13 proteins which are located in the respiratory chain. In postmortem brain of patients with Parkinson's disease, decreased activity of complex I of the respiratory chain could be demonstrated. Because seven subunits of complex I are coded by the mitochondrial genome, we analyzed the mitochondrial DNA of human postmortem substantia nigra, putamen, and frontal cortex by the Southern blot technique. No deletions of the mitochondrial genome could be demonstrated, thus indicating that either subunits which are encoded by the nuclear genome are decreased or enzyme activity is diminished by metabolites, toxins, or increase of Fe3+.     

ND3 and ND4L subunits of mitochondrial complex I, both nucleus encoded in Chlamydomonas reinhardtii, are required for activity and assembly of the enzyme

Made of more than 40 subunits, the rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. In vascular plants, fungi, and animals, at least seven complex I subunits (ND1, -2, -3, -4, -4L, -5, and -6; ND is NADH dehydrogenase) are coded by mitochondrial genes. The role of these highly hydrophobic subunits in the enzyme activity and assembly is still poorly understood. In the unicellular green alga Chlamydomonas reinhardtii, the ND3 and ND4L subunits are encoded in the nuclear genome, and we show here that the corresponding genes, called NUO3 and NUO11, respectively, display features that facilitate their expression and allow the proper import of the corresponding proteins into mitochondria. In particular, both polypeptides show lower hydrophobicity compared to their mitochondrion-encoded counterparts. The expression of the NUO3 and NUO11 genes has been suppressed by RNA interference. We demonstrate that the absence of ND3 or ND4L polypeptides prevents the assembly of the 950-kDa whole complex I and suppresses the enzyme activity. The putative role of hydrophobic ND subunits is discussed in relation to the structure of the complex I enzyme. A model for the assembly pathway of the Chlamydomonas enzyme is proposed.     

Gene flow between Drosophila yakuba and Drosophila santomea in subunit V of cytochrome c oxidase: A potential case of cytonuclear cointrogression

Introgression is the effective exchange of genetic information between species through natural hybridization. Previous genetic analyses of the Drosophila yakuba—D. santomea hybrid zone showed that the mitochondrial genome of D. yakuba had introgressed into D. santomea and completely replaced its native form. Since mitochondrial proteins work intimately with nuclear‐encoded proteins in the oxidative phosphorylation (OXPHOS) pathway, we hypothesized that some nuclear genes in OXPHOS cointrogressed along with the mitochondrial genome. We analyzed nucleotide variation in the 12 nuclear genes that form cytochrome c oxidase (COX) in 33 Drosophila lines. COX is an OXPHOS enzyme composed of both nuclear‐ and mitochondrial‐encoded proteins and shows evidence of cytonuclear coadaptation in some species. Using maximum‐likelihood methods, we detected significant gene flow from D. yakuba to D. santomea for the entire COX complex. Interestingly, the signal of introgression is concentrated in the three nuclear genes composing subunit V, which shows population migration rates significantly greater than the background level of introgression in these species. The detection of introgression in three proteins that work together, interact directly with the mitochondrial‐encoded core, and are critical for early COX assembly suggests this could be a case of cytonuclear cointrogression.     

Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 35 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components (ND1, ND2, ND4, ND5 and ND6) are coded for by the mitochondrial genome. Here, we characterize two mitochondrial mutants (dum5 and dum17) showing strong reduction or inactivation of complex I activity: dum5 is a 1T deletion in the 3' UTR of nd5 whereas dum17 is a 1T deletion in the coding sequence of nd6. The impact of these mutations and of mutations affecting nd1, nd4 and nd4/nd5 genes on the assembly of complex I is investigated. After separation of the respiratory complexes by blue native (BN)-PAGE or sucrose gradient centrifugation, we demonstrate that the absence of intact ND1 or ND6 subunit prevents the assembly of the 850 kDa whole complex, whereas the loss of ND4 or ND4/ND5 leads to the formation of a subcomplex of 650 kDa present in reduced amount. The implications of our findings for the possible role of these ND subunits on the activity of complex I and for the structural organization of the membrane arm of the enzyme are discussed. In mitochondria from all the strains analyzed, we moreover detected a 160-210 kDa fragment comprising the hydrophilic 49 kDa and 76 kDa subunits of the complex I peripheral arm and showing NADH dehydrogenase activity.     

Comparative analysis of mitochondrial and nuclear DNA topoisomerase I from maize

We conducted a comparative study of the properties of topoisomerase I isolated from maize nuclei and mitochondria. We found that nuclear and mitochondrial enzymes possess different ability to bind single stranded DNA. Study of the enzyme activity dependence on Mg2+ demonstrated an absolute dependence of the mitochondrial topoisomerase activity. Contrary, nuclear enzyme activity was not absolutely dependent but stimulated by the magnesium cation. Mitochondrial topoisomerase formed covalent bond with the 5'-end of the cleaved DNA what is unique property of prokaryotic topoisomerase I. Nuclear enzyme bound covalently to the 3'-end like all eukaryotic topoisomerases I. The search through databases revealed genes which could encode mitochondrial topoisomerase I in the genomes of higher plants. Using both cDNA sequencing and in silico methods we demonstrated an existence of the ortholog gene in the maize genome. This gene shares significant homology with prokaryotic topoisomerase I genes that may explain differences in the properties of the mitochondrial and nuclear enzyme. Data obtained is of a significant interest both from the point of view of plant organelle evolution and mitochondrial genome expression mechanisms study.     

Mutations in human nuclear genes encoding for subunits of mitochondrial respiratory complex I: the NDUFS4 gene

Among the mitochondrial disorders, complex I deficiencies are encountered frequently. Although some complex I deficiencies have been associated with mitochondrial DNA mutations, in the majority of the complex I-deficient patients mutations of nuclear genes are expected. This review attempts to summarize genetic defects affecting nuclear encoded subunits of complex I reported to date focusing on those found in the NDUFS4 gene. NDUFS4 product is 18 kDa protein which appears to have a dual role in complex I, at least: cAMP-dependent phosphorylation activates the complex; non-sense mutation of NDUFS4 prevents normal assembly of a functional complex in the inner mitochondrial membrane.     

Clinical and molecular findings in children with complex I deficiency

Isolated complex I deficiency, the most frequent OXPHOS disorder in infants and children, is genetically heterogeneous. Mutations have been found in seven mitochondrial DNA (mtDNA) and eight nuclear DNA encoded subunits, respectively, but in most of the cases the genetic basis of the biochemical defect is unknown. We analyzed the entire mtDNA and 11 nuclear encoded complex I subunits in 23 isolated complex I-deficient children, classified into five clinical groups: Leigh syndrome, progressive leukoencephalopathy, neonatal cardiomyopathy, severe infantile lactic acidosis, and a miscellaneous group of unspecified encephalomyopathies. A genetic definition was reached in eight patients (35%). Mutations in mtDNA were found in six out of eight children with Leigh syndrome, indicating a prevalent association between this phenotype and abnormalities in ND genes. In two patients with leukoencephalopathy, homozygous mutations were detected in two different nuclear-encoded complex I genes, including a novel transition in NDUFS1 subunit. In addition to these, a child affected by mitochondrial encephalomyopathy had heterozygous mutations in NDUFA8 and NDUFS2 genes, while another child with neonatal cardiomyopathy had a complex rearrangement in a single NDUFS7 allele. The latter cases suggest the possibility of unconventional patterns of inheritance in complex I defects.     

Metazoan OXPHOS gene families: evolutionary forces at the level of mitochondrial and nuclear genomes

Mitochondrial and nuclear DNAs contribute to encode the whole mitochondrial protein complement. The two genomes possess highly divergent features and properties, but the forces influencing their evolution, even if different, require strong coordination. The gene content of mitochondrial genome in all Metazoa is in a frozen state with only few exceptions and thus mitochondrial genome plasticity especially concerns some molecular features, i.e. base composition, codon usage, evolutionary rates. In contrast the high plasticity of nuclear genomes is particularly evident at the macroscopic level, since its redundancy represents the main feature able to introduce genetic material for evolutionary innovations. In this context, genes involved in oxidative phosphorylation (OXPHOS) represent a classical example of the different evolutionary behaviour of mitochondrial and nuclear genomes. The simple DNA sequence of Cytochrome c oxidase I (encoded by the mitochondrial genome) seems to be able to distinguish intra- and inter-species relations between organisms (DNA Barcode). Some OXPHOS subunits (cytochrome c, subunit c of ATP synthase and MLRQ) are encoded by several nuclear duplicated genes which still represent the trace of an ancient segmental/genome duplication event at the origin of vertebrates.     

Comparative analysis of nuclear and mitochondrial DNA topoisomerase I from <Emphasis Type="Italic">Zea mays</Emphasis>

The properties were compared for maize nuclear and mitochondrial DNA topoisomerases I (topo I). Some differences in their ability to bind to single-stranded DNA were revealed. Mitochondrial topo I was active only in the presence of Mg²⁺, whereas the activity of the nuclear enzyme did not completely depend on Mg²⁺, although being essentially stimulated in the presence of Mg²⁺. The mitochondrial enzyme covalently bound to the 5′ DNA end, as unique to prokaryotic topo I. The nuclear enzyme, like all eukaryotic topo I, covalently bound to the 3′ DNA end. A search for homologous sequences in several databases revealed genes probably encoding mitochondrial topo I in other higher plants. Using cDNA sequencing and in silico analysis, an orthologous gene was revealed in the maize genome. The gene was strongly homologous to the genes encoding prokaryotic topo I, which could explain the differences in properties between mitochondrial and nuclear topo I from maize. The presence of prokaryotic topo I in mitochondria of higher plants is interesting and important for studying the evolution of these plant organelles and the mechanisms of mitochondrial genome expression.     

Next generation molecular diagnosis of mitochondrial disorders

Mitochondrial disorders are by far the most genetically heterogeneous group of diseases, involving two genomes, the 16.6 kb mitochondrial genome and ~ 1500 genes encoded in the nuclear genome. For maternally inherited mitochondrial DNA disorders, a complete molecular diagnosis requires several different methods for the detection and quantification of mtDNA point mutations and large deletions. For mitochondrial disorders caused by autosomal recessive, dominant, and X-linked nuclear genes, the diagnosis has relied on clinical, biochemical, and molecular studies to point to a group of candidate genes followed by stepwise Sanger sequencing of the candidate genes one-by-one. The development of Next Generation Sequencing (NGS) has revolutionized the diagnostic approach. Using massively parallel sequencing (MPS) analysis of the entire mitochondrial genome, mtDNA point mutations and deletions can be detected and quantified in one single step. The NGS approach also allows simultaneous analyses of a group of genes or the whole exome, thus, the mutations in causative gene(s) can be identified in one-step. New approaches make genetic analyses much faster and more efficient. Huge amounts of sequencing data produced by the new technologies brought new challenges to bioinformatics, analytical pipelines, and interpretation of numerous novel variants. This article reviews the clinical utility of next generation sequencing for the molecular diagnoses of complex dual genome mitochondrial disorders.     

Genetic Diversity,Heteroplasmy, and Recombination in Mitochondrial Genomes of Daphnia pulex,Daphnia pulicaria,and Daphnia obtusa

Genetic variants of mitochondrial DNA at the individual (heteroplasmy) and population (polymorphism) levels provide insight into their roles in multiple cellular and evolutionary processes. However, owing to the paucity of genome-wide data at the within-individual and population levels, the broad patterns of these two forms of variation remain poorly understood. Here, we analyze 1,804 complete mitochondrial genome sequences from Daphnia pulex, Daphnia pulicaria, and Daphnia obtusa. Extensive heteroplasmy is observed in D. obtusa, where the high level of intraclonal divergence must have resulted from a biparental-inheritance event, and recombination in the mitochondrial genome is apparent, although perhaps not widespread. Global samples of D. pulex reveal remarkably low mitochondrial effective population sizes, <3% of those for the nuclear genome. In addition, levels of population diversity in mitochondrial and nuclear genomes are uncorrelated across populations, suggesting an idiosyncratic evolutionary history of mitochondria in D. pulex. These population-genetic features appear to be a consequence of background selection associated with highly deleterious mutations arising in the strongly linked mitochondrial genome, which is consistent with polymorphism and divergence data suggesting a predominance of strong purifying selection. Nonetheless, the fixation of mildly deleterious mutations in the mitochondrial genome also appears to be driving positive selection on genes encoded in the nuclear genome whose products are deployed in the mitochondrion.     

Cytoplasmic transfer of platelet mtDNA from elderly patients with Parkinson's disease to mtDNA-less HeLa cells restores complete mitochondrial respiratory function

For determination of whether platelet mtDNA in patients with Parkinson's disease (PD) possesses some lesions to reduce respiratory enzyme activities, platelet mtDNA was transferred into mtDNA-less (rho0) HeLa cells from aged PD patients and age-matched normal subjects, since their activities were controlled by both mitochondrial and nuclear genomes. The resultant mtDNA-repopulated cybrid clones containing the HeLa nuclear genome as a common background were used for comparison of respiratory enzyme activities. Remarkable variations of the enzyme activities were observed in the cybrid clones, irrespective of whether their mtDNA was transferred from normal subjects or PD patients, and some of them showed 20% reduction of average activities. Thus, the mtDNA mutations responsible for inducing 20% reduction should be polymorphic rather than pathogenic. On the other hand, pathogenic control cybrid clones possessing mtDNA mutations from patients with mitochondrial disorders showed significant and specific decline of respiratory enzyme complex I activity beyond the normal range of the variations. These observations warrant reassessment of the conventional concept that complex I activity in platelets of PD patients is defective due to mtDNA mutations.     

Investigation of the role of mitochondrial DNA in multiple sclerosis susceptibility

Several lines of evidence suggest that mitochondrial genetic factors may influence susceptibility to multiple sclerosis. To explore this hypothesis further, we re-sequenced the mitochondrial genome (mtDNA) from 159 patients with multiple sclerosis and completed a haplogroup analysis including a further 835 patients and 1,506 controls. A trend towards over-representation of super-haplogroup U was the only evidence for association with mtDNA that we identified in these samples. In a parallel analysis of nuclear encoded mitochondrial genes, we also found a trend towards association with the complex I gene, NDUFS2. These results add to the evidence suggesting that variation in mtDNA and nuclear encoded mitochondrial genes may contribute to disease susceptibility in multiple sclerosis.     

The 28.5-kDa iron-sulfur protein of mitochondrial complex I is encoded in the nucleus in plants

 The intrinsic 28.5-kDa iron-sulfur protein of complex I in the mitochondrial respiratory chain is encoded in the nucleus in animals and fungi, but specified by a mitochondrial gene in trypanosomes. In plants, the homologous protein is now found to be encoded by a single-copy nuclear gene in Arabidopsis thaliana and by two nuclear genes in potato. The cysteine motifs involved in binding two iron-sulfur clusters are conserved in the plant protein sequences. The locations of the seven introns, with sizes between 60 and 1700 nucleotides, are identical in the A. thaliana and the two potato genes, while their primary sequences diverge considerably. The A+T contents of the intron sequences range between 61% and 73%, as is characteristic for dicot plants, but are in some instances not higher than in the adjacent exons. Here, differences in T content may instead serve to discriminate exons and introns. In potato, both genes are expressed, with the highest levels found in flowers. Sequence similarities between the homologous nuclear and mitochondrial genes indicate that the nuclear forms in animals and plants originate from the endosymbiont genome. Received: 28 May 1996 / Accepted: 22 August 1996