Mitochondrial DNA Copy Research Articles

Investigations on the turnover of adrenocortical mitochondria. XIV. A correlated biochemical and stereological study of the effects of chronic treatment with ethidium bromide on the growth maintenance of the rat zona fasciculata mitochondria.

The effects of ethidium bromide (EB) on rat adrenocortical cells were investigated by biochemical and stereological methods. It was found that a treatment with ip. injections of 10 microgram/gm of EB every 12 hours induced a persistent inhibition of the incorporation of 3H-thymidine and 3H-uridine into the mitochondrial fraction, but not into the nuclear fraction. Chronic treatment with this dose of EB provoked in zona fasciculata cells of rats treated with maintenance doses of ACTH a noticeable decrease in the volume of the mitochondrial compartment (due to the decrease both in the number per cell and in the average volume of the organelles) and in the surface area of the mitochondrial cristae. These findings lend support to the hypothesis that the mechanism underlying the ACTH-induced maintenance of the growth of adrenocortical mitochondria involves mitochondrial DNA reduplication and transcription.

On the effect of inhibitors of mitochondrial macromolecular-synthesizing systems and respiration on the growth of cultured chick embryo cells.

We have found that chick embryo fibroblasts (DEF) cultivated in the presence of tryptose phosphate broth (TPB) are inherently resistant to the growth inhibitory effect of ethidium bromide (EB). As demonstrated by cytochrome oxidase activity and oxygen consumption measurements, analyses of reduced-minus-oxidized cytochrome spectra and electron microscopic observations, TPB did not seem to prevent the inhibitory effect of EB on mitochondrial DNA transcription. EB-treated chick cell populations cultivated in the presence of TPB behave essentially the same as populations treated with chloramphenicol (CAM) and grow with mitochondria devoid of a functional respiratory chain. In contrast to CAM-treated CEF populations, however, the respiratory activity of EB-treated cell populations did not reappear when the cells were shifted back to EB-free medium. Attempts to demonstrate that TPB confers resistance to the growth inhibitory effect of carbomycin and mikamycin, inhibitors of the mitochondrial protein-synthesizing system, have failed, the drugs being cytotoxic at doses where protein synthesis on mitoribosomes is not suppressed. On the other hand, the present results demonstrated that chick cell populations proliferate in the presence of the respiratory inhibitors rotenone, antimycin A, amytal and oligomycin whether or not TPB is present in the growth medium.

Molecular and genetic approaches to the analysis of the informational content of the mitochondrial genome in mammalian cells.

Our laboratory has been involved in the last few years in investigations aiming at analysing by molecular approaches the informational content of the mitochondrial genome in mammalian cells and the mechanisms and control of its expression, H eLa cells and other mammalian cell lines have been utilized for these studies. These investigations, as well as work carried out in other laboratories, have yielded a considerable amount of information concerning the mechanism, products and regulation of transcription of mitochondrial DNA (mit-DNA), the apparatus and products of mitochondria-specific protein synthesis in animal cells, and the number and topology of the sites on mit-DNA which code for the primary gene products identified so far. It is the purpose of the present report to summarize the latest observations in this area, as well as some recent results on the isolation and characterization of chloramphenicol-resistant variants of a human cell line. Reference is made to previous review articles 1,2,3 for the earlier work.

Native and imported transfer RNA in mitochondria.

Tetrahymena mitochondrial and cytoplasmic tRNAs for valine, lysine, arginine and phenylalanine produced identical or different isoacceptor patterns on reversed-phase chromatography. Positive bindings of individual isoacceptors to Escherichia coli ribosomes were demonstrated in response to trinucleoside diphosphates and polynucleotides. It was shown that different isoacceptors exhibited different but degenerate coding recognition patterns. One of two phenylalanyl-tRNA species in mitochondria, however, showed no recognition toward either phenylalanine codon triplets or poly(U). Under suitable annealing conditions, two phenylalanyl-tRNA species could hybridize with mitochondrial DNA. The other mitochondrial tRNAs for valine, lysine and arginine showed no hybridization with mitochondrial DNA but produced significant binding to nuclear DNA-immobilized filters. From these and other coding data presented here, it is postulated that there are two classes of tRNA in Tetrahymena pyriformis mitochondria; the one, “native” tRNA, is mitochondrial DNA-transcript whose coding specificity is unique to mitochondrial tRNA. The other, termed “imported” tRNA, is transcribed from the nuclear DNA and probably transported into mitochondria. This class of tRNA exhibits the same or similar coding specificity as the corresponding cytoplasmic tRNA.

Mitochondrial Protein Synthesis in Chlamydomonas reinhardtii y-l: POLYPEPTIDE PRODUCTS OF MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION IN VIVO AS REVEALED BY SELECTIVE LABELING WITH [3H]LEUCINE

Abstract The incorporation of [3H]leucine into polypeptides within intact cells of Chlamydomonas reinhardtii y-1 was analyzed by electrophoresis on polyacrylamide gels and was compared with the incorporation of [14C]arginine. Labeled arginine was a useful agent for tracing general protein synthesis in these cells, as the pattern of radioactivity that resulted from the incorporation of this amino acid was nearly identical with the pattern of protein stain. In contrast, the incorporation of [3H]leucine produced patterns of radioactivity that differed markedly from the pattern of protein stain. When the concentration of leucine in the medium was low (5 x 10-8 to 10-7 m), relatively few polypeptides became labeled with [3H]leucine. Synthesis of these polypeptides was not affected by cycloheximide, an inhibitor of cytoplasmic ribosomes, but was inhibited by chloramphenicol and other inhibitors of the ribosomes within mitochondria and the chloroplast of this alga. However, the following results demonstrated that the site of incorporation of [3H]leucine was on mitochondrial rather than on chloroplast ribosomes. (a) The large subunit of ribulose 1,5-diphosphate carboxylase, a marker of chloroplast protein synthesis, was not detectably labeled with [3H]leucine, although this polypeptide became prominently labeled with [14C]arginine. (b) Streptolydigin, acriflavine, and ethidium bromide prevented the incorporation of [3H]leucine, but these drugs had no specific effect on the incorporation of [14C]arginine into the bulk of the cellular polypeptides, including incorporation into the large subunit of ribulose 1,5-diphosphate carboxylase. Also, streptolydigin had no effect on the synthesis of chlorophyll. (c) When the concentration of leucine in the medium was increased from 10-7 m to 2 x 10-5 m, labeling of polypeptides synthesized on cytoplasmic ribosomes was observed in addition to those made on mitochondrial ribosomes, but labeling of the large subunit of ribulose 1,5-diphosphate carboxylase still was barely noticeable. These characteristics of the incorporation of [3H]leucine, apparently resulting from an unusual accessibility of this amino acid to the mitochondria, have permitted an analysis of the polypeptides synthesized specifically by this organelle within a plant cell. Furthermore, these results indicated that the synthesis of most if not all polypeptides on mitochondrial ribosomes in C. reinhardtii required continuous transcription of mitochondrial DNA.

Transcription of supercoiled mitochondrial DNA by bacterial RNA polymerase.

Mitochondrial DNA isolated from cysts of Artemia salina (brine shrimp) was found to have a closed‐circular structure 5.1 μm long and a buoyant density of 1.697 g/cm3. Escherichia coli RNA polymerase can transcribe this DNA. RNA synthesis is greatly stimulated by the σ subunit and is influenced by the presence of KCl. After formation of an initial complex between the enzyme and the mitochondrial DNA only a limited synthesis of RNA took place in the presence of rifampicin.The products obtained with holo enzyme and core enzyme in the presence of excess nucleoside triphosphates have a mean molecular weight of 1.5 × 106 and greater than 3 × 106 respectively, as determined by acrylamide‐agarose gel electrophoresis. Addition of the termination factor ϱ to the reaction mixture reduced the molecular weight of the products to 1.0 × 106 at a KCl concentration of 0.05 M but not at 0.1 M KCl.Under limiting UTP concentrations, a few discrete RNA bands of shorter size (about 8 × 105, 105 and 3 × 104 mol. wt) are synthesized. Addition of ϱ factor to the reaction mixture caused the disappearance of the higher‐molecular‐weight band (8 × 105) while the other RNA bands remained.Little self‐annealing was observed upon heating the [3H]RNA synthesized in vitro at 70 °C. On the other hand, when the [3H]RNA was heated in the presence of unlabeled mitochondrial rRNA, about 54% of the labeled product was resistant to degradation by a mixture of T1 and pancreatic RNase. In hybridization‐competition experiments, annealing of the [3H]RNA synthesized in vitro was reduced by 65% upon addition of unlabeled mitochondrial rRNA. RNA‐RNA annealing is thought to be the main cause for this competition reaction since similar results were obtained by simultaneous or step‐wise hybridization procedures. It is concluded that transcription in vitro of closed circular‐mitochondrial DNA from Artemia by E. coli RNA polymerase proceeds mainly on the anti‐rRNA strand.

In vivo transcription of mitochondrial DNA in some ϱ − mutants

Summary o (1) We have examined mitochondrial RNA produced in vivo in several ϱ − mutants. All of the mutants were found to transcribe their modified mitochondrial DNAs. (2) One of the mutants has conserved a large fraction of the region of mitochondrial DNA coding for mitochondrial ribosomal RNA, and this region was transcribed into an RNA which was similar or identical to intact 23S ribosomal RNA. No 16S ribosomal RNA was detected. (3) Another mutant which has a highly modified mitochondrial DNA also actively produced RNA transcribed from its mitochondrial DNA. The RNA was heterogeneous in size, hybridized extensively with mitochondrial DNA from the same mutant, but very poorly to wild type (ϱ + ) mitochondrial DNA. The base composition of the ϱ − RNA isolated from hybrids with mutant mitochondrial DNA reflected the very low guanine-cytosine content of the mitochondrial DNA. This mutant appeared to have lost practically all the DNA sequences coding for mitochondrial ribosomal RNA.

Letter: On the repression of mitochondrial DNA transcription by fructose 1.6-diphosphate and its derepression by cyclic adenosine 3',5'-monophosphate.

Letter: On the repression of mitochondrial DNA transcription by fructose 1.6-diphosphate and its derepression by cyclic adenosine 3',5'-monophosphate.

Post-transcriptional addition of polyadenylic acid to mitochondrial RNA by a cordycepin-insensitive process

An RNA fraction in HeLa cell mitochondria has previously been shown to contain a small polyadenylic acid sequence (56 nucleotides) and to behave like messenger RNA. This fraction can be resolved into approximately eight distinct species which may be the messages for the mitochondrial-specified proteins. The poly(A) sequence (4 S by electrophoresis) attached to mitochondrial-specific RNA is shown to be added post-transcriptionally. The kinetics of poly(A) addition in the presence of actinomycin indicate that the half-life of this process is approximately one hour. The enzymatic process that adds poly(A) to mitochondrial RNA appears to be distinct from the nuclear enzyme with similar function. Cordycepin, in contrast to its effect in the nucleus, is found to inhibit the transcription of mitochondrial DNA, but to have no direct effect on the synthesis and addition of mitochondrial poly(A). Little or no completely “free” poly(A) is apparent in these experiments, suggesting that the polymerization of the adenine residues takes place on existing RNA. In the presence of ethidium, poly(A) addition becomes aberrant and most of the newly synthesized poly(A) is attached to very small pieces of RNA.

Autonomy of mitochondria in Saccharomyces cerevisiae in their production of messenger RNA.

In ts(-)136, a temperature-sensitive mutant of Saccharomyces cerevisiae, nuclear and mitochondrial RNA production can be inhibited selectively by exposure to 36 degrees and ethidium bromide, respectively. Using the programming of mitochondrial polysomes, as measured by their ability to form nascent polypeptide chains, as an assay for functional messenger RNA, we have determined its response to temperature shifts and ethidium bromide. Only ethidium bromide produced a measurable effect; in contrast the cell-sap system responded exclusively to temperature shifts. We conclude that transcription of mitochondrial DNA is sufficient and that import of messenger RNA transcribed from nuclear chromosomes makes no measurable contribution to intramitochondrial protein synthesis.

Triiodothyronine action on RNA synthesis in rat-liver mitochondria.

Chronic treatment of thyroidectomized rats with physiological doses of triiodothyronine leads to a significant increase in the incorporation of [3H]UTP into RNA by isolated rat liver mitochondria after the 4th day of treatment. The increased activity of mitochondrial RNA polymerase is followed by a net increase in the RNA content of organelles which can be observed after the 5th day of chronic treatment. Single injection of triiodothyronine at a dose of 250 μg/kg body wt also causes great stimulation of mitochondrial RNA synthesis which increases three‐fold after 40 h. At the same DNA/RNA ratio, RNA labelled in vitro with [3H]UTP from liver mitochondria of thyroidectomized animals hybridizes with mitochondrial DNA less efficiently than the newly synthesized RNA from liver mitochondria of animals treated for 10 days with 5 μg/100 g body wt of the hormone. Hybridization‐competition experiments with unlabelled RNA extracted from mitochondria of treated and untreated animals seem to suggest that a change in the transcription of mitochondrial DNA under the influence of the hormone occurs also in vivo.

Cooperation of mitochondrial and nuclear genes specifying the mitochondrial genetic apparatus in Neurospora crassa.

Enzymes involved in the expression of the mitochondrial genome in Neurospora crassa are induced by chloramphenicol and ethidium bromide, which block transcription and translation of mitochondrial DNA. It is concluded that most, if not all, proteins of the mitochondrial genetic apparatus are coded by nuclear genes, synthesized on cytoplasmic ribosomes, and controlled by a repressor-like mitochondrial gene product. A model explaining the coordination of nuclear and mitochondrial division cycles by repressor control is discussed.

Transcription of Mitochondrial DNA in vitro from Neurospora crassa

Intact mitochondrial DNA from Neurospora crassa (linear molecules of contour length 25–26 μm) has been transcribed in vitro with RNA polymerase from Escherichia coli.At low concentrations (6 μg/ml) mitochondrial DNA is transcribed as efficiently as DNA from E. coli or calf thymus. The transcription product has the low G + C content characteristic of mitochondrial RNA.After formation of an initial complex between enzyme and mitochondrial DNA only a limited number of bound enzymes can start RNA synthesis in the presence of rifampicin. The sigma factor is required to confer rifampicin resistance.By comparing the initial rate of rifampicin resistant transcription of bacteriophage and mitochondrial DNA under saturating enzyme concentrations, the number of specific binding sites per mitochondrial genome (molecular weight 50 to 60 × 106) has been estimated to be 8 to 9. The transcription product synthesized in the presence of rifampicin has been characterized by competition‐hybridization and by RNAase digestion in solution. About 50% of the sequences hybridizing with mitochondrial DNA are homologous to RNA isolated from intact mitochondria; 35% of the product consists of rapidly renaturing double‐stranded regions characteristic of ribosomal RNA, and 5% of slowly renaturing double strands.25% of the sequences synthesized in vitro form double strands only after annealing for 16 h at 62 °C but do not contain sequences complementary to stable mitochondrial RNA synthesized in vivo.From these results it is concluded that bacterial RNA polymerase recognizes mitochondrial DNA sequences resembling bacteriophage initiation sites, and that enzymes bound to binding sites conferring rifampicin resistance transcribe asymmetrically cistrons coding for mitochondrial RNA.

Expression of the mitochondrial genome in HeLa cells: V. Transcription of mitochondrial DNA in relationship to the cell cycle

An investigation of the rate of incorporation of [5- 3H]ur dine into mitochondrial RNA in synchronized HeLa cells in different phases of the cell cycle has revealed a considerable acceleration of this incorporation in cells in S and especially in G 2 phase. An analysis of the labeling of the intramitochondrial UTP pool has shown that this acceleration reflects a true increase in the rate of synthesis of mitochondrial RNA: this increase is considerably greater than can be accounted for by the expected doubling of mit-DNA ‡ ‡ Abbreviation used: mit-DNA, mitochondrial DNA. templates during the S and G 2 phases.

Selective inhibition of the [formula omitted] transcription of mitochondrial DNA by ethidium bromide and by acriflavin

Acriflavin (10 −5M) as well as ethidium bromide (3 × 10 −5M) selectively inhibited the in vivo transcription of mitochondrial DNA in normal yeast.

Erroneous transcription of mitochondrial DNA by RNA polymerase from Escherichia coli

1. 1. We have studied the transcription of closed circular duplex mitochondrial DNA by DNA-dependent RNA polymerase (EC 2.7.7.6) from Escherichia coli. Under the conditions used for RNA synthesis the template remained covalently closed, showing that the polymerase was free of DNA endonuclease activity. 2. 2. The rate of RNA synthesis at a high enzyme to DNA ratios was approximately the same with intact mitochondrial DNA circles, broken circles or calf thymus DNA as template. Heat-inactivation experiments showed that the σ subunit of the enzyme, present in our polymerase preparations, was not required for the transcription of intact mitochondrial DNA circles. 3. 3. After self-annealing, up to 34 % of the RNA synthesized with intact circles as template showed the resistance to degradation by pancreatic ribonuclease (EC 2.7.7.16) of double-helical RNA. The remaining single-stranded RNA hybridized only with the “light strand” of mitochondrial DNA and not with the “heavy strand”, the strand shown previously to be the codogenic strand in vivo. 4. 4. These results show that RNA polymerase of E. coli can transcribe intact circular mitochondrial DNA, even when the σ factor is inactivated, but that transcription is in part symmetric and with a preference for the non-codogenic strand.

Transcriptional origin of RNA in a mitochondrial fraction of yeast and its bearing on the problem of sequence homology between mitochondrial and nuclear DNA.

RNA was isolated from mitochondrial fractions of various respiratory sufficient and deficient yeast strains. The RNA hybridizes in vitro with mitochondrial DNA as well as with nuclear DNA. The result is interpreted as showing that mitochondria contain both mitochondrial and nuclear DNA transcripts. RNA was separated from the hybrids, and by its re-hybridization with either nuclear or mitochondrial DNA, the sequence homology between these two classes of DNA was examined. A very low degree of homology was found which is at the limit of significance. Other reports on the sequence homology between nuclear and mitochondrial DNA are discussed critically.

Mitochondrial DNA IV. Interaction of ribopolynucleotides with the complementary strands of chick-liver mitochondrial DNA

1. 1. The complementary strands of chick-liver mitochondrial DNA have been separated in a preparative alkaline-CsCl gradient. The equilibrium densities of l-strand DNA were 1.746 and 1.716, and of H-strand DNA 1.788 and 1.732 g/cm 3 in alkaline and neutral CsCl, respectively ( Escherichia coli DNA, 1.772 and 1.710 g/cm 3). 2. 2. In the presence of a molar excess of poly (I, G) the density of the H-strand is 1.734 g/cm 3 and that of the L-strand > 1.79. In the presence of a molar excess of poly U the density of the H-strand is 1.736 and that of the L-strand > 1.77. 3. 3. If H- and L strand DNA are present in equal amounts in the same neutral-CsCl gradient, only one aggregate band is obtained at about 1.726 g/cm 3 (native density 1.708 g/cm 3). Complete aggregation is also observed between rat mitochondrial L-strand DNA (density 1.707 g/cm 3) and chick H-strand DNA, but not between chick H-strand DNA and denatured DNA of chick nuclei, bacteriophage T 4 or Micrococcus lysodeikticus. 4. 4. Aggregation is prevented by denaturing mitochondrial DNA in the presence of a molar excess of poly (I, G) but not by an excess of poly U. Denatured mitochondrial DNA, aggregated in CsCl, binds much less polyribonucleotide than the L-strand it contains. It is, possible therefore, that aggregation involves the sites in the DNA that bind polyribonucleotides. 5. 5. We conclude that the L-strand of chick mitochondrial DNA is rich in dA and dC, and in dC-rich clusters, whereas the H-strand is rich in dT-rich clusters. Since the H-strand acts as the messenger strand in rat liver it is possible that dT-rich clusters are involved in the transcription of mitochondrial DNA.