E.F.J. Van Bruggen

Mitochondrial DNA: V. A 25-μ closed circular duplex DNA molecule in wild-type yeast mitochondria. Structure and genetic complexity

Abstract 1. 1. Electron micrographs of DNA released by osmotic shock from isolated mitochondria of Saccharomyces carlsbergensis NCTC 74 and S. cerevisiae H1 contained closed circular duplex DNA molecules. 2. 2. The mean length of 6 closed circular molecules from S. carlsbergensis mitochondria was 25.6±0.9 μ and the mean length of fourteen molecules from S. cerevisiae mitochondria was 25.0±1.0 μ . The number of cross-overs for both yeast species was 6.5 per μ length. The majority of the linear molecules was longer than 15 μ, with lengths up to 27 μ. The DNA released from the mitochondria of S. cerevisiae H1 also contained three smaller closed circular molecules (3, 4 and 10 μ). 3. 3. All attempts to isolate the 25-μ circles intact were unsuccessful. DNA isolated from yeast mitochondria by a direct lysis method followed by CsCl equilibrium gradient centrifugation consisted exclusively of linear molecules of 2–24 μ with a modal length at 7–8 μ. 4. 4. A closed circular DNA fraction of nuclear density was present in the CsCl equilibrium gradients of yeast lysates in the amount of 1–4% of the nuclear DNA. The majority of these circles had an average contour length of 2 μ, but circles up to 7 μ were present. 5. 5. The renaturation of mitochondrial DNA of S. carlsbergensis and S. cerevisiae followed second-order kinetics with a renaturation constant about 1.1–1.6 times that of T 4 DNA, determined under identical conditions. 6. 6. We conclude that the intact mitochondrial DNA of S. carlsbergensis and S. cerevisiae consists of a circular molecule with a contour length of approx. 25 μ and a genetic complexity equivalent to this size.

Structure and properties of hemocyanins: I. Electron micrographs of hemocyanin and apohemocyanin from Helix pomatia at different pH values

Electron micrographs were made from Helix pomatia hemocyanin at different pH values with the negative-staining method. The pH-dependent dissociation on the alkaline side of the isoelectric point was especially studied. Our observations are in agreement with previously reported results, although they are not compatible with the model and mechanism of dissociation previously proposed. Our observations suggest that the Helix pomatia hemocyanin molecule is roughly cylindrical ; the diameter of the cylinder is about 300 A and the height is about 335 A. This cylinder has a fivefold axis; perpendicular to this axis it is built up from 6 parallel layers. The first dissociation step occurs perpendicular to the fivefold axis into halves. Further dissociation is supposed to be a splitting of these flat cylinders via submultiples into subunits. Divalent cations in concentrations of about 10 −4 molar or higher are required for the reversibility of this dissociation. Helix pomatia apohemocyanin shows a similar behaviour.

Subunit composition, X-ray diffraction, amino acid analysis and oxygen binding behaviour of Panulirus interruptus hemocyanin

The subunit composition of Panulirus interruptus hemocyanin has been studied. Equilibrium sedimentation, gel chromatography and sodium dodecyl sulphate-polyacrylamide electrophoresis reveal a hexameric structure for this hemocyanin, with a molecular weight of 450,000 for the undissociated protein and 75,000 for the subunits. Amino acid analysis data suggest homogeneity of the polypeptide chain. X-ray diffraction gives the cell parameters a = 119·8, b = 193·1 and c = 122·2 , β = 118·1 o . The asymmetric unit contains one molecule. Oxygen binding data show the undissociated protein to be co-operative while the dissociated protein binds oxygen non-co-operatively with a low oxygen affinity compared to that of whole molecules.

Structure and properties of hemocyanins. V. Binding of oxygen and copper in Helix pomatia hemocyanin

Abstract 1. 1. Reintroduction of monovalent copper into apo-α-hemocyanin of Helix pomatia leads to the complete return of the properties of native α-hemocyanin. 2. 2. The copper atoms in hemocyanin are required for the reassociation of tenth and twentieth molecules. 3. 3. In the reintroduction process, the copper atoms are randomly distributed among the empty binding sites of apo-α-hemocyanin. All copper-binding sites are equivalent and grouped in pairs; each pair forms a functional group. 4. 4. Upon storage for a few months, part of the copper atoms in every hemocyanin molecule are oxidized to Cu 2+ . This affects the oxygenation properties. 5. 5. The oxygenation curves of fresh α- and β-hemocyanin are nonhyperbolic at alkaline pH values in the presence of Ca 2+ and Mg 2+ . The influence of the pH on the oxygenation curves of α- and β-hemocyanin shows marked differences.

Electron microscopy of influenza virus: A comparison of negatively stained and ice-embedded particles☆

An electron microscopical study was made of the influenza virus, type B/Hong Kong, in the unstained, frozen, hydrated state after quench-freezing in cooled liquid ethane. The results are compared with data from negatively stained specimens. It is shown that cryo-electron microscopy confirms and extends the data obtained by conventional methods. In particular, the virus is shown to be circular in projection with no indication of icosahedral symmetry, the lipid membrane is clearly resolved as a bi-layer and it is demonstrated that the distribution of material within the bi-layer is non-uniform, with a shell of more electron dense material surrounding a less dense central region. Neuraminidase spikes are not clearly distinguished from haemaglutinin spikes. The diameter of the complete B/Hong Kong virus was estimated from cryo-micrographs as 1270(+/- 70) A. Some preliminary data for influenza virus type A/X31 are presented.

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.

The presence of DNA molecules with a displacement loop in standard mitochondrial DNA preparations.

Abstract In electron micrographs of standard closed circular duplex mtDNA preparations from chick liver, spread by the protein monolayer technique from solutions containing 40 or 76% formamide, up to 30% of the molecules contained a singled-stranded displacement loop, as depicted in Fig. 1. The average lengths of the loops and of the doublestranded sections between the attachment points were about the same and equivalent to 3.5% of the contour length of the mtDNA circle.

Structure and properties of hemocyanins. II. Electron micrographs of the hemocyanins of Sepia officinalis, Octopus vulgaris and Cancer pagurus.

Electron micrographs of the hemocyanins of Sepia officinalis, Octopus vulgaris and Cancer pagurus have been taken using the negative-staining technique. The results indicate that the molecules of Sepia and Octopus hemoeyanin at pH 7·8 are cylinders built of three layers perpendicular to a fivefold axis, about 140 A high and 300 A in diameter. They closely resemble half-molecules of Helix pomatia hemoeyanin. Cancer hemoeyanin, which is much smaller, is visualized as a pentalateral prism with ten subunits situated at the corners, resembling somewhat the dissociation products of the larger hemocyanins.

Circular DNA from mitochondria of Neurospora crassa.

Abstract Isolated mitochondria from two wild-type strains of Neurospora crassa were disrupted by osmotic shock in order to release mitochondrial DNA. In the electron microscope this DNA appeared to consist for a large part of circular molecules, most of which showed the typical supertwisted aspect, characteristic for closed circular duplex DNA. Strain 5256 contained circular molecules of 19 μm, one circle of 5.7 μm was found. Strain 5297 contained circular molecules of 19 μm as well as many small circles ranging in size from 0.5 to 7 μm. One circular molecule of 39 μm was found. The number of crossovers for molecules of both strains is about 7 per μm.

The unusual properties of mtDNA from a “low-density” petite mutant of yeast

Abstract 1. We have purified the mtDNA from the “low-density”, ethidium-induced petite mutant RD1A by repeated CsCl gradient centrifugation and hydroxylaptite chromatography. 2. The density in neutral CsCl of this DNA is 1.671 g/cm 3 and its T m in 0.2 M Na + is 69 °C. Base composition analysis gives A = T and G = C and G+C = 6 mole percent. 3. Quantitative renaturation studies show: (a) Denatured RD1A mtDNA renatures with second-order kinetics and contains no self-complementary single strands detectable by hydroxylapatite chromatography. (b) RD1A mtDNA must consist of a perfect repetition of a sequence of 300 nucleotides or less. (c) RD1A mtDNA is complementary to roughly 0.5 % of wild-type yeast mtDNA. 4. The behaviour of purified RD1A mtDNA on hydroxylapatite suggests that it largely consists of aggregates that are mechanically trapped on the column. Elution required a temperature near the T m of the DNA. 5. RD1A mtDNA sedimented through 3 M alkaline CsCl as heterogeneous linear DNA with a median molecular weight of about 1.7 · 10 6 . 6. In neutral CsCl gradients containing ethidium the bulk of RD1A mtDNA showed a restricted uptake of ethidium; this was abolished by treatment with pancreatic deoxyribonuclease I. 7. Electron micrographs of RD1A mtDNA spread by the protein monolayer technique contained very large networks and an occasional small circle; spreading of the DNA under denaturing conditions confirmed that less than 0.1 weight percent is circular. 8. We propose that most of the isolated RD1A mtDNA is present in large fishnet concatenanes; the unwinding restriction imposed on DNA of which the strands are plaited into a fishnet concatenate explains the restricted ethidium uptake.