A.M. Kroon

Mitochondrial DNA: physicochemical properties, replication, and genetic function.

Publisher Summary This chapter discusses the physicochemical properties, replication, and genetic function of mitochondrial DNA. The biosynthesis of functional mitochondria requires the cooperation of two genetic systems: the nuclear system, involving nuclear mRNAs, probably translated on extramitochondrial ribosomes, and a mitochondrial system localized in the mitochondrial matrix space. The genetic continuity and expression of the mitochondrial system appear to be ensured by the presence within the mitochondrial inner membrane of the enzymes required for DNA and RNA synthesis and the complete machinery required for protein synthesis. Since several vital parts of this machinery differ from their extramitochondrial counterparts (e.g., the ribosomes), the mitochondrial system for the synthesis of macromolecules is in part unique and not merely a copy of the extramitochondrial system stationed in the mitochondrial matrix space for the benefit of translating the information present in mitochondrial DNA. The chapter also discusses several aspects of mitochondrial biogenesis.

Mitochondrial DNA. I. Preparation and properties of mitochondrial DNA from chick liver.

Abstract 1. A convenient procedure for the large-scale preparation of mitochondria from chick liver is described, which is suitable for the preparation of mitochondrial DNA. 2. For the extraction of mitochondrial DNA, the mitochondria were preincubated with deoxyribonuclease (EC 3.1.4.5) and lysed in 0.15 M NaCl, 0.1 M sodium EDTA (pH 9.0) with 2 % sodium dodecyl sulfate. After deproteinization with 1 M NaClO4 and chloroform, and treatment with ribonuclease (EC 2.7.7.16) the DNA was purified by chromatography on a column of methylated albumin adsorbed on kieselguhr. The maximal yield was 0.2 μg mitochondrial DNA of high molecular weight ( s 20,w ≧ 24 S ) per mg mitochondrial protein. 3. Mitochondrial DNA of chick liver prepared by this procedure has an equilibrium density in CsCl of 1.708 g/cm3 (nuclear DNA, 1.701 g/cm3); it melts sharply with a Tm of 90.0° in 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0 (nuclear DNA, 87.5°). After alkaline denaturation the equilibrium density of mitochondrial DNA in CsCl increased by 0.018 g/cm3. 4. Under optimal conditions, rapid renaturation of denatured mitochondrial DNA was shown to occur both by band shift in a CsCl equilibrium gradient and by spectrophotometric hypochromicity measurements at 260 mμ. Renaturation followed second-order kinetics which proves that the rapid renaturation is due to the homogeneity in base sequence of mitochondrial DNA and not to the inability of the complementary strands to completely separate under denaturing conditions. 5. The second-order renaturation constant of mitochondrial DNA is compared with the renaturation constants reported by others for bacterial and viral DNAs under standard conditions. It is shown that the renaturation rate of mitochondrial DNA is in good agreement with our earlier suggestion that the total genetic information in the mitochondrial population of chick liver is that contained in a double-stranded DNA molecule with a molecular weight of 10·106–11·106.

Protein synthesis in mitochondria III. On the effects of inhibitors on the incorporation of amino acids into protein by intact mitochondria and digitonin fractions

Abstract 1. 1. Submitochondrial fragments obtained by digitonin treatment of mitochondria from beef heart and rat liver incorporate amino acids into protein at a rate comparable with microsomes from rat liver. 2. Chloramphenicol and actinomycin D inhibit this incorporation. These inhibitions and the linear course of the incorporation for at least 3 h suggest that a functionally active DNA is present within the mitochondria. 3. The mean DNA content of beef-heart mitochondria is 2.4 μg per mg protein calculated from the absorption at 260 mμ and 2.1 μg per mg protein using the diphenylamine reaction. For rat-liver mitochondria these values are 2.2 and 1.8 μg per mg protein, respectively. 4. Mitochondrial preparations from beef heart contain about 7 μg RNA per mg protein, those from rat liver about 13 μg RNA per mg protein. 5. In the digitonin fragments the activity of amino acid incorporation rises simultaneously with the nucleic acid content and the specific activity of several typically mitochondrial enzymes. This is brought forward as evidence against contamination with nucleic acids from nuclear or microsomal origin.

Protein synthesis in heart mitochondria: I. Amino acid incorporation into the protein of isolated beef-heart mitochondria and fractions derived from them by sonic oscillation

Abstract 1. 1. The results of investigations on the incorporation of radioactive amino acids into the protein of isolated beef-heart mitochondria are presented. It is shown that a specific activity can be obtained many times higher than that reported for liver mitochondria. 2. 2. Judged by the RNA content, the DPN nucleosidase (EC 3.2.2.6) activity and the carboxyl esterase (EC 3.1.1.1) activity the mitochondrial fraction used was not appreciably contaminated with microsomes. 3. 3. The specific incorporation activity can be increased by preparing submitochondrial particulate fractions by sonic oscillation. 4. 4. The incorporation activity is dependent on energy-rich phosphate (ATP). Intramitochondrial-generated ATP (for example by oxidative phosphorylation) can serve this purpose best. However, optimal conditions for oxidative phosphorylation are by no means necessary for a considerable incorporation activity.

Mitochondrial DNA. II. Sedimentation analysis and electron microscopy of mitochondrial DNA from chick liver.

Abstract 1. 1. The physiochemical properties of pure mitochondrial DNA from chick liver were studied by band sedimentation in the analytical ultracentrifuge and by electron microscopy. 2. 2. Up to 80 % of native chick-liver mitochondrial DNA sedimented in a homogeneous band with an s20,w = 39 S, the remainder of the high-molecular-weight DNA sedimenting in a homogeneous band with an s20,w = 27 S. In some preparations a minor third component with an s20,w = 24 S was present. The 39-S component was not affected by the peptide hydrolase pronase, but it was converted into the 27-S component by treatment with pancreatic deoxyribonuclease (EC 3.1.4.5) or hydroquinone, or by “ageing”. 3. 3. Electron micrographs of all preparations of chick-liver mitochondrial DNA, spread according to the Kleinschmidt protein-monolayer technique, showed predominantly molecules in which no free ends could be distinguished. No branched molecules were seen in any preparation. 4. 4. Micrographs of 39-S DNA, prepared by preparative sucrose-gradient centrifugation, contained 84 % highly twisted circles, 14 % open or half-open circles and 1 % linear molecules. Micrographs of pure 27-S DNA contained only 4 % twisted circles, 77 % half-open or open circular molecules and 19 % linear molecules. 5. 5. The mean circumference of 63 more or less open molecules was 5.35 μ with 90 % of all values falling between 4.85 and 5.85 μ. 6. 6. In mitochondrial DNA denatured in 12 % formaldehyde, 3 major and 3 minor components were found in analytical band-sedimentation studies using 3 M CsCl containing 2 % formaldehyde as bulk solution. The major components were tentatively identified as the formaldehyde double-stranded cyclic coil (s20,w = 83 S), the single-stranded ring (s20,w = 32 S) and the single-stranded broken ring (s20,w = 28 S). 7. 7. In alkali, mitochondrial DNA sedimented as a heterogeneous collection of fragments. 8. 8. We conclude that chick-liver mitochondrial DNA in situ is a double-stranded circular molecule with a molecular weight of the sodium salt of 10·106–11·106. Both strands are covalently continuous and, on extraction, the DNA is obtained in a twisted circular form (s20,w = 39 S) which is converted into an open circular form (s20,w = 27 S) after cleavage of at least one phospho-diester bond. The 24-S component is tentatively identified as the linear form of mitochondrial DNA. 9. 9. The close similarity of the physicochemical properties of mitochondrial DNA and the circular viral DNA molecules is stressed.

Mitochondrial DNA: III. Electron microscopy of DNA released from mitochondria by osmotic shock

Abstract 1. 1. Isolated mitochondria were subjected to osmotic shock and the mitochondrial DNA released was studied by electron microscopy using the protein-monolayer technique. 2. 2. In shocked preparations of freshly isolated chick liver mitochondria, about 85 % of the long DNA visible was present as highly twisted circular molecules (average contour length ±S.D. = 5.2±0.4 μ ), the remainder as open or half-open circles (5.6±0.4 μ ). Storage of the mitochondria for up to 1 week prior to osmotic shock led to a decrease in the percentage of twisted circles with a concomitant increase in the percentage of open circles and linear molecules. 3. 3. The average number of cross-overs (±S.D.) in twisted circles at 20–22° was 33 (±7) for chick liver mitochondrial DNA (mol. wt. = 10–11·10 6 dalton) spread on H 2 O by osmotic shock, 35 (±6) for purified chick-liver mitochondrial DNA spread on 0.1 M ammonium acetate, and 13 (±2) for purified replicative form DNA of phage ΦX (mol. wt. = 3.4·10 6 dalton) spread on salt. 4. 4. Osmotic shock also released predominantly circular DNA from mitochondrial preparations of rat liver (average contour length 5.4 μ), carp liver (5.4 μ) and housefly flight muscle (5.2 μ). 5. 5. Heterogeneous linear DNA was obtained from Saccharomyces carlsbergensis mitochondria both by standard purification procedures and by osmotic shock. The conclusion is drawn that the intact mitochondrial DNA of yeast is substantially larger than 5 μ.

PROTEIN SYNTHESIS IN MITOCHONDRIA. II. A COMPARISON OF MITOCHONDRIA FROM LIVER AND HEART WITH SPECIAL REFERENCE TO THE ROLE OF OXIDATIVE PHOSPHORYLATION.

Abstract 1. 1. A procedure for preparing rat-liver mitochondria including a pre-incubation with RNAase (EC 2.7.7.16) to inactivate the microsomal system for amino acid incorporation is described. 2. 2. The inhibition of the incorporation of amino acids into mitochondrial protein by adding substrate is correlated with a lowering of the NADP + /NADPH ratio. 3. 3. It is shown that ammonia can relieve the inhibition by α-oxoglutarate or isocitrate of the incorporation into liver mitochondria, that this effect is not due to the glutamate formed, and that this is correlated with oxidation of NADPH to NADP + . 4. 4. The role of endogenous substrate is discussed. It is concluded that in the case of rat-liver mitochondria α-oxoglutarate plus ammonia give the best conditions for providing energy for incorporation, since a high NADP + /NADPH ratio is also assured. 5. 5. Judged by the effects of oligomycin and 2,4-dinitrophenol on the amino acid incorporation into protein by mitochondria, the possibility is discussed whether and in how far the high-energy intermediates of oxidative phosphorylation can meet the energy requirements for the incorporation process.