Multiple independent transpositions of mitochondrial DNA control region sequences to the nucleus

Transpositions of mtDNA sequences to the nuclear genome have been documented in a wide variety of individual taxa, but little is known about their taxonomic frequency or patterns of variation. We provide evidence of nuclear sequences homologous to the mtDNA control region in seven species of diving ducks (tribe Aythyini). Phylogenetic analysis places each nuclear sequence as a close relative of the mtDNA haplotypes of the specie(s) in which it occurs, indicating that they derive from six independent transposition events, all occurring within the last ≈1.5 million years. Relative-rate tests and comparison of intraspecific variation in nuclear and mtDNA sequences confirm the expectation of a greatly reduced rate of evolution in the nuclear copies. By representing mtDNA haplotypes from ancestral populations, nuclear insertions may be valuable in some phylogenetic analyses, but they also confound the accurate determination of mtDNA sequences. In particular, our data suggest that the presumably nonfunctional but more slowly evolving nuclear sequences often will not be identifiable by changes incompatible with function and may be preferentially amplified by PCR primers based on mtDNA sequences from related taxa.

Synthesis of mitochondrial DNA in permeabilised human cultured cells

The mechanisms that underlie the maintenance of and increase in mutant mitochondrial DNA (mtDNA) are central to our understanding of mitochondrial disease. We have therefore developed a technique based on saponin permeabilisation that allows the study of mtDNA synthesis in intact cells. Permeabilisation of cells has been extensively used in an established method both for studying transcription and DNA replication in the nucleus and for measuring respiratory chain activities in mitochondria. We have quantitatively studied incorporation of radiolabelled DNA precursors into mtDNA in human cell lines derived from controls and from patients with mitochondrial DNA disease. Total cell DNA is extracted, restriction digested and Southern blotted, newly synthesised mtDNA being proportional to the label incorporated in each restriction band. A rate of synthesis can then be derived by estimating the relative steady-state mtDNA after probing with full-length mtDNA. Where co-existing mutant and wild-type mtDNA (heteroplasmy) can be distinguished using restriction digestion, their rates of synthesis can be compared within a single cell line. This will be particularly useful in elucidating the pathophysiology of mtDNA diseases in which the distribution of mutant and wild-type mtDNA in cell lines in patient tissues may evolve with time.

Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA.

We have analyzed the level of intraindividual sequence variability (heteroplasmy) of mtDNA in human brain by denaturing gradient gel electrophoresis and sequencing. Single base substitutions, as well as insertions or deletions of single bases, were numerous in the noncoding control region (D-loop), and 35-45% of the molecules from a single tissue showed sequence differences. By contrast, heteroplasmy in coding regions was not detected. The lower level of heteroplasmy in the coding regions is indicative of selection against deleterious mutations. Similar levels of heteroplasmy were found in two brain regions from the same individual, while no heteroplasmy was detected in blood. Thus, heteroplasmy seems to be more frequent in nonmitotic tissues. We observed a 7.7-fold increase in the frequency of deletions/insertions and a 2.2-fold increase in the overall frequency of heteroplasmic mutations in two individuals aged 96 and 99, relative to an individual aged 28. Our results show that intraindividual sequence variability occurs at a high frequency in the noncoding regions of normal human brain and indicate that small insertions and deletions might accumulate with age at a lower rate than large rearrangements.

Direct observation of iron-induced conformational changes of mitochondrial DNA by high-resolution field-emission in-lens scanning electron microscopy.

When respiring rat liver mitochondria are incubated in the presence of Fe(III) gluconate, their DNA (mtDNA) relaxes from the supercoiled to the open circular form dependent on the iron dose. Anaerobiosis or antioxidants fail to completely inhibit the unwinding. High-resolution field-emission in-lens scanning electron microscopy imaging, in concert with backscattered electron detection, pinpoints nanometer-range iron colloids bound to mtDNA isolated from iron-exposed mitochondria. High-resolution field-emission in-lens scanning electron microscopy with backscattered electron detection imaging permits simultaneous detailed visual analysis of DNA topology, iron dose-dependent mtDNA unwinding, and assessment of iron colloid formation on mtDNA strands.

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.

Philopatry of male marine turtles inferred from mitochondrial DNA markers

Recent studies of mitochondrial DNA (mtDNA) variation among marine turtle populations are consistent with the hypothesis that females return to beaches in their natal region to nest as adults. In contrast, less is known about breeding migrations of male marine turtles and whether they too are philopatric to natal regions. Studies of geographic structuring of restriction fragment and microsatellite polymorphisms at anonymous nuclear loci in green turtle (Chelonia mydas) populations indicate that nuclear gene flow is higher than estimates from mtDNA analyses. Regional populations from the northern and southern Great Barrier Reef were distinct for mtDNA but indistinguishable at nuclear loci, whereas the Gulf of Carpentaria (northern Australia) population was distinct for both types of marker. To assess whether this result was due to reduced philopatry of males across the Great Barrier Reef, we determined the mtDNA haplotypes of breeding males at courtship areas for comparison with breeding females from the same three locations. We used a PCR-restriction fragment length polymorphism approach to determine control region haplotypes and designed mismatch primers for the identification of specific haplotypes. The mtDNA haplotype frequencies were not significantly different between males and females at any of the three areas and estimates of Fst among the regions were similar for males and females (Fst = 0.78 and 0.73, respectively). We conclude that breeding males, like females, are philopatric to courtship areas within their natal region. Nuclear gene flow between populations is most likely occurring through matings during migrations of both males and females through nonnatal courtship areas.

Neuronal type information encoded in the basic-helix–loop–helix domain of proneural genes

The proneural genes encode basic-helix–loop–helix (bHLH) proteins and promote the formation of distinct types of sensory organs. In Drosophila, two sets of proneural genes, atonal (ato) and members of the achaete–scute complex (ASC), are required for the formation of chordotonal (ch) organs and external sensory (es) organs, respectively. We assayed the production of sensory organs in transgenic flies expressing chimeric genes of ato and scute (sc), a member of ASC, and found that the information that specifies ch organs resides in the bHLH domain of ato; chimeras containing the b domain of ato and the HLH domain of sc also induced ch organ formation, but to a lesser extent than those containing the bHLH domain of ato. The b domains of ato and sc differ in seven residues. Mutations of these seven residues in the b domain of ato suggest that most or perhaps all of these residues are required for induction of ch organs. None of these seven residues is predicted to contact DNA directly by computer simulation using the structure of the myogenic factor MyoD as a model, implying that interaction of ato with other cofactors is likely to be involved in neuronal type specification.

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function

Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce nonhuman ape mtDNA into human cells harboring either no mtDNA or mutated mtDNAs (partial deletion and tRNA gene point mutation). Unexpectedly, only cells containing no mtDNA could be repopulated with nonhuman ape mtDNA. Cells containing a defective human mtDNA did not incorporate or maintain ape mtDNA and therefore died under selection for oxidative phosphorylation function. On the other hand, foreign human mtDNA was readily incorporated and maintained in these cells. The suicidal preference for self-mtDNA showed that functional parameters associated with oxidative phosphorylation are less relevant to mtDNA maintenance and copy number control than recognition of mtDNA self-determinants. Non–self-mtDNA could not be maintained into cells with mtDNA even if no selection for oxidative phosphorylation was applied. The repopulation kinetics of several mtDNA forms after severe depletion by ethidium bromide treatment showed that replication and maintenance of mtDNA in human cells are highly dependent on molecular features, because partially deleted mtDNA molecules repopulated cells significantly faster than full-length mtDNA. Taken together, our results suggest that mtDNA copy number may be controlled by competition for limiting levels of trans-acting factors that recognize primarily mtDNA molecular features. In agreement with this hypothesis, marked variations in mtDNA levels did not affect the transcription of nuclear-coded factors involved in mtDNA replication.

Mitochondrial DNA ligase function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Δdnl4). The Escherichia coli EcoRI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant EcoRI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Δdnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.

Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium–PNA (ph–PNA) conjugates of 3.4–4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph–PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph–PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph–PNA uptake. The ph–PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease ‘myoclonic epilepsy and ragged red fibres’ (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph–PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph–PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

Does behavior reflect phylogeny in swiftlets (Aves: Apodidae)? A test using cytochrome b mitochondrial DNA sequences.

Swiftlets are small insectivorous birds, many of which nest in caves and are known to echolocate. Due to a lack of distinguishing morphological characters, the taxonomy of swiftlets is primarily based on the presence or absence of echolocating ability, together with nest characters. To test the reliability of these behavioral characters, we constructed an independent phylogeny using cytochrome b mitochondrial DNA sequences from swiftlets and their relatives. This phylogeny is broadly consistent with the higher classification of swifts but does not support the monophyly of swiftlets. Echolocating swiftlets (Aerodramus) and the nonecholocating "giant swiftlet" (Hydrochous gigas) group together, but the remaining nonecholocating swiftlets belonging to Collocalia are not sister taxa to these swiftlets. While echolocation may be a synapomorphy of Aerodramus (perhaps secondarily lost in Hydrochous), no character of Aerodramus nests showed a statistically significant fit to the molecular phylogeny, indicating that nest characters are not phylogenetically reliable in this group.

The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Zα. Here we report the solution structure of free Zα and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Zα)2/Z-DNA complex shows that most Z-DNA contacting residues in free Zα are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (α+β)helix–turn–helix/B-DNA complexes suggests that binding of Zα to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B- and Z-DNA.

Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells

Cytochrome b-type NAD(P)H oxidoreductases are involved in many physiological processes, including iron uptake in yeast, the respiratory burst, and perhaps oxygen sensing in mammals. We have identified a cytosolic cytochrome b-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) containing cytochrome b5 (b5) and b5 reductase (b5R) domains. A genetic approach, using blast searches against dbest for FAD-, NAD(P)H-binding sequences followed by reverse transcription–PCR, was used to clone the complete cDNA sequence of human b5+b5R from the hepatoma cell line Hep 3B. Compared with the classical single-domain b5 and b5R proteins localized on endoplasmic reticulum membrane, b5+b5R also has binding motifs for heme, FAD, and NAD(P)H prosthetic groups but no membrane anchor. The human b5+b5R transcript was expressed at similar levels in all tissues and cell lines that were tested. The two functional domains b5* and b5R* are linked by an approximately 100-aa-long hinge bearing no sequence homology to any known proteins. When human b5+b5R was expressed as c-myc adduct in COS-7 cells, confocal microscopy revealed a cytosolic localization at the perinuclear space. The recombinant b5+b5R protein can be reduced by NAD(P)H, generating spectrum typical of reduced cytochrome b with alpha, beta, and Soret peaks at 557, 527, and 425 nm, respectively. Human b5+b5R flavohemoprotein is a NAD(P)H oxidoreductase, demonstrated by superoxide production in the presence of air and excess NAD(P)H and by cytochrome c reduction in vitro. The properties of this protein make it a plausible candidate oxygen sensor.

Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes.

Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II [succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1] are conspicuously lacking in mitochondrial genomes so far characterized. Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate). These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD. In E. coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane. Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes. The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage.