Mitochondrial DNA Copy Research Articles

Direct regulation of mitochondrial RNA synthesis by thyroid hormone.

We have analyzed the influence of in vivo treatment and in vitro addition of thyroid hormone on in organello mitochondrial DNA (mtDNA) transcription and, in parallel, on the in organello footprinting patterns at the mtDNA regions involved in the regulation of transcription. We found that thyroid hormone modulates mitochondrial RNA levels and the mRNA/rRNA ratio by influencing the transcriptional rate. In addition, we found conspicuous differences between the mtDNA dimethyl sulfate footprinting patterns of mitochondria derived from euthyroid and hypothyroid rats at the transcription initiation sites but not at the mitochondrial transcription termination factor (mTERF) binding region. Furthermore, direct addition of thyroid hormone to the incubation medium of mitochondria isolated from hypothyroid rats restored the mRNA/rRNA ratio found in euthyroid rats as well as the mtDNA footprinting patterns at the transcription initiation area. Therefore, we conclude that the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription machinery. Particularly, the influence on the mRNA/rRNA ratio is achieved by selective modulation of the alternative H-strand transcription initiation sites and does not require the previous activation of nuclear genes. These results provide the first functional demonstration that regulatory signals, such as thyroid hormone, that modify the expression of nuclear genes can also act as primary signals for the transcriptional apparatus of mitochondria.

Zidovudine (AZT) induced alterations in mitochondrial biogenesis in rat striated muscles.

Zidovudine (AZT) and didanosine (ddI), two drugs used in the treatment of AIDS, are also known to cause mitochondrial abnormalities. We investigated the physiological relevance of the mitochondrial defects by measuring in situ skeletal muscle performance and cytochrome c oxidase (CYTOX) enzyme activity in heart muscle, red highoxidative (RG) and white low-oxidative (WG) portions of the gastrocnemius muscle of control (n = 17), AZT-(n = 14), or ddI-treated (n = 11) rats for 28 days. We also evaluated the hypothesis that AZT treatment could alter the expression of the mitochondrial transcription factor A (mtTFA), a key molecule involved in mitochondrial DNA (mtDNA) replication and transcription. AZT had a pronounced effect on blood pressure and skeletal muscle performance, which were significantly decreased during contractile activity at 2 and 5 Hz, compared with control. A significant decrease in CYTOX activity in heart and RG, but not WG muscles, was also evident. In the heart, this was accompanied by an apparent compensatory increase in mtTFA mRNA level that could not be attributed to enhanced transcriptional activation mediated by nuclear respiratory factor 1 (NRF-1). In contrast with AZT, no effect of ddI was found on the extent of fatigue or muscle enzyme activity. These results indicate that AZT induces mitochondrial defects primarily in muscles with the highest oxidative capacities (heart and RG). The long-term effects of AZT on mitochondrial biogenesis have the potential to reduce muscle performance, but the effects on performance in this short-term study were likely due to an inability of the AZT-treated animals to maintain blood pressure during contractile activity.

Zidovudine (AZT) induced alterations in mitochondrial biogenesis in rat striated muscles

Zidovudine (AZT) and didanosine (ddI), two drugs used in the treatment of AIDS, are also known to cause mitochondrial abnormalities. We investigated the physiological relevance of the mitochondrial defects by measuring in situ skeletal muscle performance and cytochrome c oxidase (CYTOX) enzyme activity in heart muscle, red high-oxidative (RG) and white low-oxidative (WG) portions of the gastrocnemius muscle of control (n = 17), AZT- (n = 14), or ddI-treated (n = 11) rats for 28 days. We also evaluated the hypothesis that AZT treatment could alter the expression of the mitochondrial transcription factor A (mtTFA), a key molecule involved in mitochondrial DNA (mtDNA) replication and transcription. AZT had a pronounced effect on blood pressure and skeletal muscle performance, which were significantly decreased during contractile activity at 2 and 5 Hz, compared with control. A significant decrease in CYTOX activity in heart and RG, but not WG muscles, was also evident. In the heart, this was accompanied by an apparent compensatory increase in mtTFA mRNA level that could not be attributed to enhanced transcriptional activation mediated by nuclear respiratory factor 1 (NRF-1). In contrast with AZT, no effect of ddI was found on the extent of fatigue or muscle enzyme activity. These results indicate that AZT induces mitochondrial defects primarily in muscles with the highest oxidative capacities (heart and RG). The long-term effects of AZT on mitochondrial biogenesis have the potential to reduce muscle performance, but the effects on performance in this short-term study were likely due to an inability of the AZT-treated animals to maintain blood pressure during contractile activity.Key words: mitochondrial myopathy, endurance performance, cytochrome c oxidase, mitochondrial transcription factor A, zidovudine, didanosine.

Autonomous regulation in mammalian mitochondrial DNA transcription.

The regulation of the oxidative phosphorylation system (OXPHOS) biogenesis in eukaryotic cells is unique since it involves the expression of two genomes, the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA). The considerable effort done in collecting information on the factors that influence the expression of the genes encoded in mtDNA and nDNA has revealed that a multiplicity of regulatory options are available in mammalian cells to perform this task. Thus, at least three archetypal situations can be distinguished: mitochondrial proliferation, mitochondrial differentiation, and mitochondrial local tuning (MLT). Each of them seems to be predominantly under the control of specific strategies of regulation, although the description of the detailed molecular mechanisms involved is still in its beginnings. In the present review, we focus on the evidence supporting the existence of mechanisms for autonomous regulation of mtDNA transcription and its role in the integrated regulation of the OXPHOS system biogenesis.

Reduced Mitochondrial DNA Transcription in Senescent Rat Heart

We studied the effect of senescence on mitochondrial DNA (mtDNA) transcription with anin organellosystem using intact isolated rat heart mitochondria. A comparison of the electrophoretic patterns of mtDNA transcription products in young, adult and senescent rats showed an age-related reduction in newly-synthesized mitochondrial RNAs that reflects a decrease in the synthesis rate. These results correlate with the enzyme activities of the oxidative phosphorylation complexes I and IV, that are partially encoded by the mitochondrial genome. In addition, an age-related increase in the protein carbonyl content of the mitochondrial membranes was observed in senescent mitochondria suggesting an accumulation of mitochondrial oxidative damage. This reduction in the mtDNA transcriptional rate in the heart of senescent animals suggests that this could be one of the molecular bases underlying senescence of the myocardium.

Increases in the Mitochondrial DNA Replication and Transcription in the Remnant Liver of Rats

We studied mitochondrial DNA (mtDNA) replication and transcription in association with major hepatectomy. Changes in nuclear respiratory factor 1 (NRF-1) mRNA and RNA moiety of mitochondrial RNA-processing endoribonuclease (MRP-RNase RNA) were followed at 0, 3, 6, 12, and 24 h after hepatectomy. Contents of mtDNA, cytochromebmRNA, and β-actin mRNA were also measured. Contents of NRF-1 mRNA and MRP-RNase RNA increased to maximum or sub-maximum level 1 h after hepatectomy. mtDNA and cytochromebmRNA increased to maximum level 3 h after. On the contrary, content of β-actin mRNA increased to maximum level 12 h after. From these results, mtDNA replication and transcription occurred in the initial phase of liver regeneration, which might lead to an increase in mitochondrial respiratory function in the remnant liver, and cytoskeleton proteins such as β-actin were followed thereafter.

Human mitochondrial function during cardiac growth and development.

Little information is presently available concerning mitochondrial respiratory and oxidative phosphorylation function in the normal human heart during growth and development. We investigated the levels of specific mitochondrial enzyme activities and content during cardiac growth and development from the early neonatal period (10-20 days) to adulthood (67 years). Biochemical analysis of enzyme specific activities and content and mitochondrial DNA (mtDNA) copy number was performed with left ventricular tissues derived from 30 control individuals. The levels of cytochrome c oxidase (COX) and complex V specific activity, mtDNA copy number and COX subunit II content remained unchanged in contrast to increased citrate synthase (CS) activity and content. The developmental increase in CS activity paralleled increasing CS polypeptide content, but was neither related to overall increases in mitochondrial number nor coordinately regulated with mitochondrial respiratory enzyme activities. Our findings of unchanged levels of cardiac mitochondrial respiratory enzyme activity during the progression from early childhood to older adult contrasts with the age-specific regulation found with CS, a Krebs cycle mitochondrial enzyme.

Artemia Mitochondrial Genome: Molecular Biology and Evolutive Considerations

During the last two decades an increasing amount of information has been accumulated regarding the gene structure and organization of the mitochondrial genome from various organisms. Many studies carried out mainly in mammals, have contributed to the knowledge of the basic elements involved in the replication and transcription of mitochondrial DNA. However, very little is known about these processes in invertebrates. In this review we discuss our current knowledge of the animal mitochondrial genetic system and briefly summarize the structure of the Artemia mitochondrial genome, the characteristics of its transcriptional machinery and how its expression is controlled during early development, in relation with what is known in other organisms. Artemia is the only crustacean where the mtDNA has been studied at this level of detail up to date.

Alloplasmic wheats with Aegilops crassa cytoplasm which express photoperiod-sensitive homeotic transformations of anthers, show alterations in mitochondrial DNA structure and transcription.

Alloplasmic wheat. Triticum aestivum cv. Norin 26, with Aegilops crassa cytoplasm, shows photoperiod-sensitive cytoplasmic male sterility (PCMS). This alloplasmic line expresses pistillody of anthers only when grown in long-day conditions (> 15 h light). To assess the molecular basis of the PCMS, we carried out Southern and Northern hybridization analyses on mitochondrial DNAs and RNAs isolated from seedlings of alloplasmic lines showing various PCMS phenotypes using probes for twelve mitochondrial genes. All RFLP patterns of mitochondrial DNA from alloplasmic lines greatly differed from those of common wheat, and were slightly changed from those of the parental species, i.e., Ae. crassa. This indicates that nuclear substitutions between related plant species induce structural alterations in the mitochondrial genome. Furthermore, RFLP patterns of (cr)-N61 and FR-mutant probed with coxIII and orf25 were identical with each other, but different from those of the other alloplasmic lines, indicating that the nuclei of N61 and FR-mutant harbor some gene(s) that induces structural alterations of the mitochondrial genome in the coxIII and orf25 regions. The transcription patterns of atp6 and cob in Ae. crassa type were different from those of T. aestivum type. Furthermore, the orf25 transcript in alloplasmic wheats was about 300 nucleotides longer than that of euplasmic lines, including the Ae. crassa pure line, suggesting that transcription patterns of orf25 are associated with recovery from the PCMS phenomenon. These data clearly show the mutual cross-talk between the nuclear genome and chondriome. These observations raise the possibility that the dysfunction of mitochondria caused by the failure of a cooperative control of mitochondrial gene(s) expression influences the pathway of flower morphogenesis, especially in the process that determines organ identity.

Mitochondrial disorders.

Mitochondria, the organelles devoted to energy production, have unique genetic features. They possess their own genome encoding several subunits of the respiratory chain, the majority of which are encoded by nuclear DNA, as well as factors involved in replication, transcription and translation of mitochondrial DNA. In the past few years, molecular lesions of mitochondrial DNA have been reported with increasing frequency as a source of human disorders. Several mutations of mitochondrial DNA, either as sporadic large scale rearrangements (deletions, duplications) or maternally-inherited point mutations, have been associated with well defined clinical syndromes. Furthermore, because of the nuclear DNA contribution to the synthesis of respiratory chain enzymes, phenotypes transmitted as Mendelian traits have also been identified and associated with qualitative (multiple deletions) and quantitative (depletion) lesions of the mitochondrial genome. The clinical manifestations of mitochondrial DNA mutations are extremely heterogeneous, ranging from myopathies, encephalomyopathies, cardiopathies, to complex multisystem syndromes. Clinical, morphological, biochemical and molecular genetic data are necessary for diagnosis. The recent advances in genetic studies provide both diagnostic tools and new pathogenetic insights into this rapidly expanding area of human pathology.

Impaired mitochondrial function in microvesicular steatosis effects of drugs, ethanol, hormones and cytokines

Microvesicular steatosis occurs in conditions characterized by severe impairment of the mitochondrial β-oxidation process, due to genetic and/or acquired causes. Drugs and some endogenous compounds can sequester coenzyme A (aspirin, valproic acid), inhibit mitochondrial β-oxidation enzymes (tetracyclines, several 2-arylpropionate anti-inflammatory drugs, amineptine and tianeptine), or inhibit both mitochondrial β-oxidation and oxidative phosphorylation (endogenous bile acids, amiodarone, perhexiline and diethylaminoethoxyhexestrol), while female sex hormones have complex, but moderate, effects on mitochondrial structure and function. Other substances impair mitochondrial DNA transcription (interferon-α) or mitochondrial DNA replication (dideoxynucleosides), while alcohol abuse might accelerate the normal oxidative aging of mitochondrial DNA. When β-oxidation is severely impaired, fatty acids, which are poorly oxidized by mitochondria, are mainly esterified into triglycerides, but there is a residual increase in non-esterified fatty acids. Triglycerides (possibly emulsified by a rim of non-esterified fatty acids) accumulate as small vesicles. Impairment of energy production, and the mitochondrial and general toxicity of both non-esterified fatty acids and dicarboxylic acids, may contribute to liver failure, coma and death in severe forms. Although milder forms of microvesicular steatosis have a good short-term prognosis, they can lead to chronic lipid peroxidation and the development of steatohepatitis lesions. Investigational molecules with a carboxylic group or a protonatable amine, or those which might interfere with mitochondrial DNA, should be screened for possible mitochondrial effects.

Nuclear Respiratory Factors 1 and 2 Utilize Similar Glutamine-Containing Clusters of Hydrophobic Residues To Activate Transcription

Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) are ubiquitous transcription factors that have been implicated in the control of nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication. Recently, both factors have been found to be major transcriptional determinants for a subset of these genes that define a class of simple promoters involved in respiratory chain expression. Here, functional domains required for transactivation by NRF-1 have been defined. An atypical nuclear localization signal resides in a conserved amino-terminal region adjacent to the DNA binding domain and consists of functionally redundant clusters of basic residues. A second domain in the carboxy-terminal half of the molecule is necessary for transcriptional activation. The activation domains of both NRF-1 and NRF-2 were extensively characterized by both deletion and alanine substitution mutagenesis. The results show that these domains do not fall into known classes defined by a preponderance of amino acid residues, including glutamines, prolines, or isoleucines, as found in other eukaryotic activators. Rather, in both factors, a series of tandemly arranged clusters of hydrophobic amino acids were required for activation. Although all of the functional clusters contain glutamines, the glutamines differ from the hydrophobic residues in that they are inconsequential for activation. Unlike the NRF-2 domain, which contains its essential hydrophobic motifs within 40 residues, the NRF-1 domain spans about 40% of the molecule and appears to have a bipartite structure. The findings indicate that NRF-1 and NRF-2 utilize similar hydrophobic structural motifs for activating transcription.

Regulation and function of the mitochondrial genome.

Molecular changes in human mitochondrial DNA play a significant role in causing certain human diseases. Mitochondrial DNA mutations range from single base pair changes in the 16.5 kilobase pair genome up to large deletions and rearrangements. This report summarizes the current overall understanding of the mode and mechanism of mitochondrial DNA replication and transcription, and how this relates to mitochondrial gene expression, which is essential for cellular energy production and organelle biogenesis. Special attention is given to recent findings that bear on early steps in the process of transcription and, in turn, the consequences for initiating DNA replication.

Mitochondrial DNA polymerase gamma is expressed and translated in the absence of mitochondrial DNA maintenance and replication.

Mitochondria are essential organelles in all eukaryotic cells where cellular ATP is generated through the process of oxidative phosphorylation. Protein components of the respiratory assembly are gene products of both mitochondrial and nuclear genes. The mitochondrial genome itself encodes several protein and nucleic acid components required for such oxidative phosphorylative processes, but the vast majority of genes encoding respiratory chain components are nuclear. Similarly, the processes of replication and transcription of mitochondrial DNA rely exclusively upon RNA and protein species encoded by nuclear genes. We have analyzed two key nuclear-encoded proteins involved in mitochondrial DNA replication and transcription as a function of the presence or absence of mitochondrial DNA. Mitochondrial DNA polymerase (DNA polymerase gamma), the nuclear-encoded enzyme which synthesizes mtDNA, is expressed and translated in cells devoid of mitochondrial DNA itself. In contrast, mitochondrial transcription factor A protein levels are tightly linked to the mtDNA status of the cell. These results demonstrate that the DNA polymerase gamma protein is stable in the absence of mitochondrial DNA, and that there appears to be no regulatory mechanism present in these cells to alter levels of this protein in the complete absence of mitochondrial DNA. Alternatively, it is possible that this enzyme plays an additional, as yet undefined, role in the cell, thereby mandating its continued production.

A single mouse gene encodes the mitochondrial transcription factor A and a testis–specific nuclear HMG-box protein

Mitochondrial transcription factor A (mtTFA) is a key regulator of mammalian mitochondrial DNA transcription. We report here that a testis-specific isoform of mouse mtTFA lacks the mitochondrial targeting sequence and is present in the nucleus of spermatocytes and elongating spermatids, thus representing the first reported mammalian gene encoding protein isoforms targeted for the mitochondria or the nucleus. The presence of the mitochondrial transcriptional activator in the nucleus raises the possibility of a role for this protein in both genetic systems. Mutations in the nuclear mtTFA gene may therefore exhibit phenotypic consequences due to altered function in either or both genetic compartments.

Human liver mitochondrial RNA synthesized in isolated organelles

The study of alterations of mitochondrial DNA transcription could be of fundamental interest to understand the molecular mechanisms underlying the impairment of mitochondrial function in liver diseases related to hepatocyte energy production. We show that isolated normal human liver mitochondria, when incubated in the presence of [α- 32P]UTP in an appropriate incubation medium, in which the energy requirements are provided by exogenous ADP in the presence of oxidizable substrates, are able to support RNA synthesis in a way faithfully resembling the in vivo process. The autoradiographic pattern in agarosemethylmercury hydroxide gels of the newly synthesized RNA shows the presence of all the previously described RNAs coded in the mitochondrial genome (ribosomal, messenger and transfer RNAs). We conclude that this system could be of great value to investigate the role of the mitochondrial genome on human liver pathology related to mitochondrial damage.

The Synthesis of mRNA in Isolated Mitochondria can be Maintained for Several Hours and is Inhibited by High Levels of ATP

The dependence for the maintenance of the synthesis and maturation of mitochondrial RNA on the supply of nucleo-cytoplasmic factors has been investigated by a novel in organello RNA synthesis system. We found that mitochondrial DNA transcription can be maintained for several hours in isolated mitochondria. Analysis of the individual mitochondrial RNA species revealed that: the processing of the rRNA precursors and the stability of the mature rRNAs, but not the transcription itself, is severely impaired after short periods of incubation, indicating that these processes are strongly dependent on the mitochondrial interaction with the nucleo-cytoplasmic compartment; the events that lead to the synthesis, processing and turnover of the mitochondrial mRNAs do not require the continuous supply of nucleo-cytoplasmic factors, that are accumulated in excess by mitochondria. Furthermore, we present evidence indicating an inhibition of high ATP levels on the mitochondrial RNA polymerase activity, both in organello and in vitro. Consequently, it is proposed that mitochondrial mRNA synthesis can be regulated in response to changes in intramitochondrial ATP levels. This regulation of mitochondrial mRNA synthesis together with their very rapid turnover described here and elsewhere [Gelfand, R. & Attardi, G. (1981) Mol. Cell Biol. 1, 497-511], could represent a mechanism that would allow each individual mitochondrion to adjust its optimal levels of mRNA, and hence its translation capacity, in response to local energetic demands.

Differential expression of ornithine decarboxylase, poly(ADP)ribose polymerase, and mitochondrial mRNAs following testosterone administration to hypophysectomized rats.

The mRNAs of the nuclear encoded genes, ornithine decarboxylase (ODCase) and poly(ADP)ribose polymerase (PADPRP), and the mitochondrial encoded genes, cytochrome oxidase I and II (COI and COII) and ATPase 6, are differentially expressed during spermatogenesis (Alcivar et al., 1989: Biol Reprod 41:1133; 1989: Dev Biol 135:263; 1991: Biol Reprod 46:201). In this study, we use Northern blotting to examine the steady state levels of ODCase, PADPRP, COI, COII, and ATPase 6 mRNAs in testes of hypophysectomized male rats following testosterone administration. Four weeks after hypophysectomy, rats received 24 cm subcutaneous implants of testosterone-filled polydimethylsiloxane (PDS) and were killed at 3, 7, 14, 28, and 56 days thereafter. After hypophysectomy, the steady state levels for the PADPRP, COI, COII, and ATPase 6 mRNAs were not significantly different from controls, although hypophysectomy caused a 44% loss of preleptotene spermatocytes and an 88% loss of pachytene spermatocytes, the testicular cell types expressing the highest levels of these mRNAs. In contrast, the levels of the two ODCase mRNAs were greatly decreased after hypophysectomy and mirrored the number of germinal cells present in the testis. After testosterone treatment, ODCase mRNA levels remained low 3 days after treatment and gradually increased at days 14, 28, and 56. No major hybridization signal changes in PADPRP, COI, COII, and ATPase mRNA were observed after testosterone treatment. We conclude that the steady state mRNA levels for the housekeeping ODCase gene respond differently after hypophysectomy and testosterone treatment of male rats than the PADPRP and mitochondrial DNA transcripts.

5] In Organello RNA synthesis system from HeLa cells

Publisher Summary Alterations in mitochondrial gene expression occur in response to energetic conditions within the cell and possibly cytoplasmic and nuclear control factors. Hence, to simplify investigations of the potential control provided by the energetic states of the cell and cytoplasmic factors on mitochondrial DNA (mtDNA) transcription and processing of the mtRNA, a set of experimental protocols is developed that physically separates the mitochondria from the remaining cellular constituents. This separation is achieved by mechanically lysing HeLa cells, isolating the intact mitochondria by centrifugation, and removing the nonmitochondrial RNA through enzyme treatment and washings. The isolated mitochondria can then be assayed for transcriptional capacity and processing fidelity by analyzing the incorporation of radioactive nucleotides into their RNA using gel electrophoretic techniques. As described in the chapter, it is possible to isolate mitochondria easily and quickly that are competent in transcription events and the processing of RNA, comparable to that found in the in vivo situation. Therefore, it is possible to study the effects of changes in the energetic or metabolic environments and maturation of the mtRNA. The molecular analysis of these effects is possible because of the preexisting knowledge about HeLa cell mitochondrial DNA and its in vivo transcription products.

12] Mitochondrial DNA transcription initiation and termination using mitochondrial lysates from cultured human cells

Publisher Summary This chapter discusses mitochondrial DNA transcription initiation and termination using mitochondrial lysates from cultured human cells. In human mitochondria, the main initiation sites for transcription of the heavy (H) and light (L) strands of the DNA have been identified in vivo and in vitro , the discrete transcription products have been mapped, and their structural and metabolic properties have been characterized. One mitochondrial transcription factor—mtTFA—necessary for high levels of specific initiation of transcription at the L-strand promoter and rRNA-specific H-strand promoter, has also been identified and cloned. A different DNA-binding factor, mTERF—involved in termination of transcription at the 16 S rRNA/tRNA Leu(UUR) boundary—has been extensively characterized in the laboratory. On the other hand, nothing is known about the mtRNA polymerase itself and the probable existence of other factors involved in transcription initiation at the downstream, whole H-strand specific promoter. The chapter reports an adaptation of the mobility shift assay for the semiquantitative detection of mitochondrial transcription termination factor (mTERF) in the S-13 fraction from small amounts of cells, which allows an easy comparison of its relative content in different cell lines and under different growth conditions.