Yolk platelets in artemia embryos: are they really storage sites of immature mitochondria?

We have used semi-quantitative polymerase chain reaction (PCR) technology to determine the mitochondrial DNA (mtDNA) content of yolk platelets isolated from embryos of the brine shrimp, Artemia franciscana, and ultrastructural analysis of yolk platelet formation to determine whether these organelles contain mitochondria as reported previously. Using six different isolation and purification protocols, we found one yolk platelet preparation to be devoid of mtDNA, while four yolk platelet preparations contained mtDNA ranging from 16.4 to 85 pg/10(6) yolk platelets. One preparation contained 600 pg mtDNA per 10(6) yolk platelets. Based on our PCR analyses, the mtDNA component of Artemia yolk platelets represented 0.16-4.5% of the total DNA isolated from the platelets. We calculated that Artemia yolk platelets contain, on average, approximately 1.78 molecules of mtDNA/platelet. Direct analysis of mtDNA in "free" mitochondria isolated from yolk platelet-free preparations of Artemia embryos and newly hatched larvae yielded 0.76-0.80 ng/animal. Based on these values, the mtDNA content of yolk platelets was approximately 0.2% of total mtDNA in Artemia embryos. Microscopic analysis of yolk platelet formation during oogenesis in Artemia failed to show the inclusion of mitochondria during the assemblage of yolk platelets. The "mitochondria-like" structures that appear in yolk platelets during their utilization lack the well defined inner and outer membranes characteristic of mitochondria making it unlikely that the yolk platelet inclusions are mitochondria. Our results from PCR technology and ultrastructure analysis demonstrate that mtDNA in yolk platelets of Artemia franciscana embryos is a minor component of the total mtDNA in the embryo, and they fail to support the notion that yolk platelets in Artemia are a major source of immature mitochondria for development.     

Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell.

Mitochondrial nucleoids (mt-nucleoids) of the A2780 line of cultured human cells were stained with DAPI and observed using an epifluorescence microscope. The mt-nucleoids appeared to be organized compactly in mitochondria. Numbers of mt-nucleoids per mitochondrion ranged from 1 to more than 10, and 70% were "multinucleated" mitochondria. Intensities of fluorescence of mt-nucleoids in each mitochondrion were measured by a video-intensified microscope system (VIM system) and copy numbers of mitochondrial DNA (mtDNA) in each mitochondria were determined. The copy numbers of mtDNA per mitochondrion ranged from 1 to 15, and the average was 4.6. Because the cells had 107 mitochondria on average, the copy number of mtDNA per cell was estimated to be about 500.     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system     

Distinct genomic copy number in mitochondria of different mammalian organs

This study shows that mitochondria in liver, kidney, heart, and brain of the mouse have a distinct mitochondrial density. It also demonstrates that the mtDNA copy number per mitochondrion is organ-specific. A reliable method of determining mitochondrial density per organ is by stereological analysis of tissue sections while mtDNA quantitation is by the use of radiolabelled mtDNA probe. This is the first study in which a comprehensive examination of mitochondrial density and quantitation of mitochondrial genomes in mouse organs have been done. In summary the variability is not only in mitochondrial density but also in genomic copy number in mitochondria of various tissues.     

Variance of ploidy in mitochondrial nucleus during spherulation in Physarum polycephalum

In Physarum polycephalum, microplasmodia differentiated into spherules when cultures were aged for 8–10 days. Respiration rates of the microplasmodia decreased rapidly with ageing to a 90% decrease in oxygen consumption over 9 days. We studied this phenomena by isolating and characterizing mitochondria from microplasmodia and spherules at different stages of spherulation. Oxygen uptake by the isolated mitochondria decreased with spherulation. Morphological and biochemical analyses showed that mitochondrial differentiation to inactive state was characterized by a decrease not only in dimension but also of content (DNA, RNA and protein). Diminutive mitochondria contained small particle-shaped mitochondrial nuclei. The DNA content, measured by microscopic fluorometry, was about 1.15 and 0.58 × 10⁻¹⁰ g, which corresponded to about 16 and 8 genome copies, respectively (e.g., 32 genome copies per mitochondrion at mitochondrial G1). Restriction endonuclease analysis showed that the physical structure and methylation pattern of the mtDNA had not changed although the DNA content per mitochondrion had decreased remarkably with spherulation. This showed that changes in the ploidy level of the mitochondrial nucleus during spherulation were due to reduction in the number of whole mitochondrial genomes.     

Behavior of mitochondria and their nuclei during amoebae cell proliferation inPhysarum polycephalum

Summary We investigated the manner of mitochondrial DNA (mtDNA) replication and distribution during the culture ofPhysarum polycephalum amoebae cells by microphotometry, anti-BrdU immunofluorescence microscopy, and quantitative hybridization analysis. In amoebae cells ofP. polycephalum, the number of mitochondria per cell and the shape of both mitochondria and mitochondrial nuclei (mt-nuclei) noticeably changed over the culture period. At the time of transfer, about 27 short ellipsoidal shaped mitochondria, which each contained a small amount of DNA, were observed in each cell. The number of mitochondria per cell decreased gradually, while the amount of mtDNA in an mt-nucleus and the length of mt-nuclei increased gradually. Midway through the middle logarithmic growth phase, the number of mitochondria per cell reached a minimum (about 10 mitochondria per cell), but most mtnuclei assumed an elongated shape and contained a large amount of mtDNA. During the late log- and stationary-growth phase, the number of mitochondria per cell increased gradually, while the amount of DNA in an mt-nucleus and mt-nuclei length decreased gradually. Upon completion of the stationary phase, the number and condition of mitochondria within cells returned to that first observed at the time of transfer. The total amount of mtDNA in a cell increased about 1.6-fold the first day, decreased immediately, then maintained a constant level ranging from 130 to 160 T. Except for the fact that mtDNA synthesis began earlier than synthesis of cell nuclei, the rate of increase in mtDNA paralleled that of cell-nuclear DNA throughout the culture. These results indicate that mtDNA is continuously replicated in pace with cell proliferation and the rate of mitochondrial division varies during culture; this mitochondrial division does not synchronize with either mtDNA replication or cell division. Furthermore, we observed the spatial distribution of DNA replication sites along mt-nuclei. Replication began at several sites scattered along an mt-nucleus, and the number of replication sites increased as the length of mt-nuclei increased. These results indicate that mtDNA replication progresses in adjacent replicons, which are collectively termed a mitochondrial replicon cluster.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system - BrdU 5-bromodeoxyuridine - FITC fluorescein isothiocyanate     

Detection of heteroplasmic mitochondrial DNA in single mitochondria

Background

Mitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Leber''s hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases.

Methodology

This work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrion''s DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion.

Significance

Molecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria.     

The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion

Plant cells exhibit a high rate of mitochondrial DNA (mtDNA) recombination. This implies that before cytokinesis, the different mitochondrial compartments must fuse to allow for mtDNA intermixing. When and how the conditions for mtDNA intermixing are established are largely unknown. We have investigated the cell cycle-dependent changes in mitochondrial architecture in different Arabidopsis (Arabidopsis thaliana) cell types using confocal microscopy, conventional, and three-dimensional electron microscopy techniques. Whereas mitochondria of cells from most plant organs are always small and dispersed, shoot apical and leaf primordial meristematic cells contain small, discrete mitochondria in the cell periphery and one large, mitochondrial mass in the perinuclear region. Serial thin-section reconstructions of high-pressure-frozen shoot apical meristem cells demonstrate that during G1 through S phase, the large, central mitochondrion has a tentaculate morphology and wraps around one nuclear pole. In G2, both types of mitochondria double their volume, and the large mitochondrion extends around the nucleus to establish a second sheet-like domain at the opposite nuclear pole. During mitosis, approximately 60% of the smaller mitochondria fuse with the large mitochondrion, whose volume increases to 80% of the total mitochondrial volume, and reorganizes into a cage-like structure encompassing first the mitotic spindle and then the entire cytokinetic apparatus. During cytokinesis, the cage-like mitochondrion divides into two independent tentacular mitochondria from which new, small mitochondria arise by fission. These cell cycle-dependent changes in mitochondrial architecture explain how these meristematic cells can achieve a high rate of mtDNA recombination and ensure the even partitioning of mitochondria between daughter cells.     

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

Sperm Mitochondria in Reproduction： Good or Bad and Where Do They Go？

The mitochondrion is the major energy provider to power sperm motility. In mammals, aside from the nuclear genome, mitochondrial DNA （mtDNA） also contributes to oxidative phosphorylation to impact production of ATP by coding 13 polypeptides. However, the role of sperm mitochondria in fertilization and its final fate after fertilization are still controversial. The viewpoints that sperm bearing more mtDNA will have a better fertilizing capability and that sperm mtDNA is actively eliminated during early embryogenesis are widely accepted. However, this may be not true for several mammalian species, including mice and humans. Here, we review the sperm mitochondria and their mtDNA in sperm functions, and the mechanisms of maternal mitochondrial inheritance in mammals.     

A computational model of mitochondrial deoxynucleotide metabolism and DNA replication

We present a computational model of mitochondrial deoxynucleotide metabolism and mitochondrial DNA (mtDNA) synthesis. The model includes the transport of deoxynucleosides and deoxynucleotides into the mitochondrial matrix space, as well as their phosphorylation and polymerization into mtDNA. Different simulated cell types (cancer, rapidly dividing, slowly dividing, and postmitotic cells) are represented in this model by different cytoplasmic deoxynucleotide concentrations. We calculated the changes in deoxynucleotide concentrations within the mitochondrion during the course of a mtDNA replication event and the time required for mtDNA replication in the different cell types. On the basis of the model, we define three steady states of mitochondrial deoxynucleotide metabolism: the phosphorylating state (the net import of deoxynucleosides and export of phosphorylated deoxynucleotides), the desphosphorylating state (the reverse of the phosphorylating state), and the efficient state (the net import of both deoxynucleosides and deoxynucleotides). We present five testable hypotheses based on this simulation. First, the deoxynucleotide pools within a mitochondrion are sufficient to support only a small fraction of even a single mtDNA replication event. Second, the mtDNA replication time in postmitotic cells is much longer than that in rapidly dividing cells. Third, mitochondria in dividing cells are net sinks of cytoplasmic deoxynucleotides, while mitochondria in postmitotic cells are net sources. Fourth, the deoxynucleotide carrier exerts the most control over the mtDNA replication rate in rapidly dividing cells, but in postmitotic cells, the NDPK and TK2 enzymes have the most control. Fifth, following from the previous hypothesis, rapidly dividing cells derive almost all of their mtDNA precursors from the cytoplasmic deoxynucleotides, not from phosphorylation within the mitochondrion. simulation; nucleotide phosphorylation; nucleoside transport; mitochondrial DNA     

Cell cycle dependent morphology changes and associated mitochondrial DNA redistribution in mitochondria of human cell lines

Mitochondria of osteosarcoma cells (143B) in culture have variable morphologies, classified according to the shape and size of the organelle as reticular, fragmented or intermediate. Synchronization and release from G0 has shown that the morphology of mitochondria oscillates between the reticular and fragmented state in a cell cycle dependent manner. Cells in G1 have reticular mitochondria while those in S phase have fragmented mitochondria. By using a novel method of fluorescence in situ hybridization, the morphology of mitochondria was correlated with mitochondrial DNA distribution. MtDNA molecules were seen in clusters of two to four along mitochondrial filaments. In the fully fragmented state, each mitochondrion contained at least one cluster. We discuss the importance of fission and fusion events in regulating the morphology of mitochondria, segregation of mtDNA and maintenance of the organelle's functional unity.     

Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells

A new biochemical method for estimating the virtual number of mitochondria (mt) per cell was developed and used together with a plasmid probe to measure mt DNA/mitochondrion and mt DNA/cell. These methods were used in five cell types from four mammalian species. Mt DNA/mitochondrion was essentially constant in all cell types (mean 2.6 +/- 0.30 SE mitochondrial DNA molecules/mt). Mt DNA molecules/cell encompassed an eight-fold range between various cell types (low 220 +/- 6.2; high 1,720 +/- 162 mt DNA molecules/cell). Virtual mt number/cell ranged from 83 +/- 17 to 677 +/- 80 (SE) mt/cell in various cell types. All five mammalian virtual mitochondria contained the same genomic mass. The number of virtual mitochondria per cell and amount of mt DNA per cell appear to be closely regulated within a given cell type but differ widely from cell type to cell type.     

Role of mitochondrial DNA in calcium signaling abnormality in bipolar disorder

Altered intracellular calcium levels are a consistent finding in studies of bipolar disorder, and recent studies point to the role of mitochondrial dysfunction, leading to the possibility that mitochondrial calcium dysregulation is involved in the pathophysiology of the disease. Although the mitochondrion is a key organelle for calcium accumulation, initial calcium signaling studies in bipolar disorder did not focus on the role of mitochondria. Later, neuroimaging and molecular genetic studies suggested the possibility that altered mitochondrial calcium regulation due to mitochondrial DNA (mtDNA) polymorphisms/mutations might be involved in the pathophysiology of bipolar disorder. Recent studies show that certain mtDNA polymorphisms alter mitochondrial calcium levels. Mutant mtDNA polymerase (Polg) transgenic mice carrying mtDNA mutations in forebrain cells show an increased calcium uptake rate in isolated mitochondria. This was found to be mediated by downregulation of cyclophilin D, a component of the mitochondrial permeability transition pore. In addition, agonist-stimulated calcium response is attenuated in hippocampal neurons of these transgenic mice. The finding that mtDNA polymorphisms and mutations affect mitochondrial calcium regulation supports the idea that mitochondrial calcium dysregulation may be involved in the pathophysiology of bipolar disorder. In this review, the history and recent findings of studies elucidating the role of mitochondrial calcium signaling in bipolar disorder are summarized.     

Elimination of paternal mitochondria through the lysosomal degradation pathway in C. elegans

In mammals, the inheritance of mitochondrion and its DNA (mtDNA) is strictly maternal, despite the fact that a sperm can inject up to 100 functional mitochondria into the oocyte during fertilization. The mechanisms responsible for the elimination of the paternal mitochondria remain largely unknown. We report here that this paternal mitochondrial elimination process is conserved in Caenorhabditis elegans, and that the lysosomal pathway actively participates in this process. Molecular and cell biological analyses indicate that in wild-type animals paternal mitochondria and mtDNA are destroyed within two hours after fertilization. In animals with compromised lysosomes, paternal mitochondria persist until late embryonic stages. Therefore, the lysosomal pathway plays an important role in degrading paternal mitochondria introduced into the oocyte during fertilization. Our study indicates that C. elegans is an excellent animal model for understanding and dissecting this conserved biological process critical for animal development and reproduction.     

Autophagy is not involved in the degradation of sperm mitochondria after fertilization in mice

In almost all animals, mitochondrial DNA (mtDNA) is transmitted only from the female, while the paternal mitochondria and mtDNA are thought to be eliminated during early embryogenesis. Autophagy is involved in the elimination of sperm mitochondria and mtDNA in early embryos in Caenorhabditis elegans; however, solid evidence is still lacking in mammals. Recently, we found that despite the fact that some autophagy-related proteins, such as SQSTM1 and LC3 could localize nearby sperm mitochondria before the 2-cell stage, autophagy did not participate in the elimination of sperm mitochondria and mtDNA. Instead, the pre-elimination of sperm mtDNA before fertilization and the restriction of sperm mitochondria in one blastomere before 4-cell stage embryos are the most important mechanisms of maternal mitochondrial inheritance in mice.     

Human mitochondria and mitochondrial genome function as a single dynamic cellular unit

rho 0 HeLa cells entirely lacking mitochondrial DNA (mtDNA) and mitochondrial transfection techniques were used to examine intermitochondrial interactions between mitochondria with and without mtDNA, and also between those with wild-type (wt) and mutant-type mtDNA in living human cells. First, unambiguous evidence was obtained that the DNA-binding dyes ethidium bromide (EtBr) and 4',6-diamidino-2- phenylindole (DAPI) exclusively stained mitochondria containing mtDNA in living human cells. Then, using EtBr or DAPI fluorescence as a probe, mtDNA was shown to spread rapidly to all rho 0 HeLa mitochondria when EtBr- or DAPI-stained HeLa mitochondria were introduced into rho 0 HeLa cells. Moreover, coexisting wt-mtDNA and mutant mtDNA with a large deletion (delta-mtDNA) were shown to mix homogeneously throughout mitochondria, not to remain segregated by use of electron microscopic analysis of cytochrome c oxidase activities of individual mitochondria as a probe to identify mitochondria with predominantly wt- or delta- mtDNA in single cells. This rapid diffusion of mtDNA and the resultant homogeneous distribution of the heteroplasmic wt- and delta-mtDNA molecules throughout mitochondria in a cell suggest that the mitochondria in living human cells have lost their individuality. Thus, the actual number of mitochondria per cell is not of crucial importance, and mitochondria in a cell should be considered as a virtually single dynamic unit.     

The incomplete natural history of mitochondria

Mitochondrial DNA (mtDNA) has been used to study molecular ecology and phylogeography for 25 years. Much important information has been gained in this way, but it is time to reflect on the biology of the mitochondrion itself and consider opportunities for evolutionary studies of the organelle itself and its ecology, biochemistry and physiology. This review has four sections. First, we review aspects of the natural history of mitochondria and their DNA to show that it is a unique molecule with specific characteristics that differ from nuclear DNA. We do not attempt to cover the plethora of differences between mitochondrial and nuclear DNA; rather we spotlight differences that can cause significant bias when inferring demographic properties of populations and/or the evolutionary history of species. We focus on recombination, effective population size and mutation rate. Second, we explore some of the difficulties in interpreting phylogeographical data from mtDNA data alone and suggest a broader use of multiple nuclear markers. We argue that mtDNA is not a sufficient marker for phylogeographical studies if the focus of the investigation is the species and not the organelle. We focus on the potential bias caused by introgression. Third, we show that it is not safe to assume a priori that mtDNA evolves as a strictly neutral marker because both direct and indirect selection influence mitochondria. We outline some of the statistical tests of neutrality that can, and should, be applied to mtDNA sequence data prior to making any global statements concerning the history of the organism. We conclude with a critical examination of the neglected biology of mitochondria and point out several surprising gaps in the state of our knowledge about this important organelle. Here we limelight mitochondrial ecology, sexually antagonistic selection, life-history evolution including ageing and disease, and the evolution of mitochondrial inheritance.     

Platelet-mediated transformation of mtDNA-less human cells: analysis of phenotypic variability among clones from normal individuals--and complementation behavior of the tRNALys mutation causing myoclonic epilepsy and ragged red fibers.

In the present work, we demonstrate the possibility of using human blood platelets as mitochondrial donors for the repopulation of mtDNA-less (rho 0) cells. The noninvasive nature of platelet isolation, combined with the prolonged viability of platelet mitochondria and the simplicity and efficiency of the mitochondria-transfer procedure, has substantially increased the applicability of the rho 0 cell transformation approach for mitochondrial genetic analysis and for the study of mtDNA-linked diseases. This approach has been applied to platelets from several normal human individuals and one individual affected by the myoclonic-epilepsy-and-ragged-red-fibers (MERRF) encephalomyopathy. A certain variability in respiratory capacity was observed among the platelet-derived rho 0 cell transformants from a given normal subject, and it was shown to be unrelated to their mtDNA content. The results of sequential transfer of mitochondria from selected transformants into a rho 0 cell line different from the first rho 0 acceptor strongly suggest that this variability reflected, at least in part, differences in nuclear gene content and/or activity among the original recipient cells. A much greater variability in respiratory capacity was observed among the transformants derived from the MERRF patient and was found to be related to the presence and amount of the mitochondrial tRNALys mutation associated with the MERRF syndrome. An analysis of the relationship between proportion of mtDNA carrying the MERRF mutation and degree of respiratory activity in various transformants derived from the MERRF patient revealed an unusual complementation behavior of the tRNALys mutation, possibly reflecting the distribution of mutant mtDNA among the platelet mitochondria.