P.J. Weijers

The localization of mitochondrial creatine kinase, and its use for the determination of the sidedness of submitochondrial particles

Abstract 1. 1. Creatine kinase (EC 2.7.3.2) is extracted from heart mitochondria by suspension in salt-containing media. 2. 2. Digitonin treatment of isolated heart mitochondria causes rupture of the outer membranes and release of adenylate kinase (EC 2.7.4.3), while creatine kinase remains bound to the mitochondrial membranes. Although the outer membranes are ruptured by the action of digitonin, they can not be obtained in reasonable yield by differential centrifugation, indicating that most of the ruptured outer membranes are still connected to the inner membranes, which could be confirmed by electron microscopy. 3. 3. The addition of the detergent Lubrol WX to intact heart mitochondria has no effect upon the activity of the creatine kinase, indicating that this enzyme is not latent. 4. 4. In submitochondrial particles, obtained by ultrasonic treatment of heart mitochondria and removal of intact mitochondria by centrifugation, creatine kinase becomes partially latent. This indicates that in some of the submitochondrial particles the enzyme is localized inside the enveloping inner membrane fragment. Evidence is also presented for the existence of submitochondrial particles with the same orientation of the enveloping membrane as intact mitochondria, as indicated by the latency of aspartate aminotransferase (EC 2.6.1.1), a matrix enzyme that is firmly bound to the inside of the inner membrane. 5. 5. From Paragraphs 2–4 it is concluded that mitochondrial creatine kinase is bound to the outside of the inner mitochondrial membrane, in contrast to mitochondrial adenylate kinase, which is not bound to either of the mitochondrial membranes.

Characterization of DNA from Trypanosoma brucei and related trypanosomes by restriction endonuclease digestion.

Abstract We have digested non-kinetoplast DNA from various Trypanosoma strains with restriction endonucleases and analysed the fragment distribution by one-dimensional agarose gel electrophoresis. Visual inspection of ethidium-stained gels shows differences in banding pattern between DNAs from Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum and even between different T. brucei strains, but not between three different antigenic variants derived from the same T. brucei cell. By blotting the DNA on to nitrocellulose strips the restriction endonuclease recognition sites around genes available in cloned form can be analysed by molecular hybridization and we demonstrate this for the gene which codes for one of the variant surface glycoproteins of T. brucei. The use of this method for strain classification is discussed. Renaturation analysis of T. brucei non-kinetoplast DNA shows that about 68% of this is present as single-copy DNA with a complexity of 2.5 × 107 base pairs, whereas the remainder consists of intermediate repetitive and highly repetitive DNA. The latter fraction contains a DNA which is cut by AluI into fragments of 180 base pairs, bands in CsCl at 1.690 g/cm3 and contains duplex circles heterogeneous in size and circles with duplex tails.

DNA circles with cruciforms from Isospora (Toxoplasma) gondii

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.

The structure of kinetoplast DNA: I. Properties of the intact multi-circular complex from Crithidia luciliae

We have developed a modified isolation procedure that yields kinetoplast DNA networks containing more than 90% closed circular DNA, as judged by two criteria: (a) In 0.15 M NaCl/0.015 M sodium citrate (pH 7.0), less than 10% of the intact kinetoplast DNA melts in the temperature region of sonicated kinetoplast DNA. In 7.2 M NaCl04 the kinetoplast DNA melts with a Tm 26 degrees C higher than sonicated kinetoplast DNA. Even after complete melting in 7.2 M NaClO4 at 90 degrees C, the network remains intact, as judged by regain of hypochromicity on cooling and analysis in CsCl containing propidium dixodide. (b) In alkaline sucrose gradients more than 90% of the kinetoplast DNA sediments in a single peak. 2. In CsCl gradients containing ethidium bromide of propidium diiodide intact kinetoplast DNA gives a single uni-modal band showing an extremely restricted dye uptake. From the position of the bank relative to the bands of PM2 DNA, the superhelix density of these networks is calculated to be +3.9 twists per 1000 base pairs. The superhelix density of closed mini-circles, efficiently liberated from the networks by shear in a French press, is -0.5 twists per 1000 base pairs. We attribute the high superhelix density (the highest yet observed in any DNA) of intact networks to their compact, highly catenated structure, leading to an additional constraint on dye uptake, superimposed on the restriction due to closed circularity.

Properties of mitochondrial DNA from Kluyveromyces lactis

Abstract 1. We have isolated a closed circular DNA fraction from mitochondria purified from Kluyveromyces lactis by centrifuging a mitochondrial lysate to equilibrium in CsCl containing ethidium bromide. Electron micrographs of appropriate gradient fractions show predominantly circular duplex DNA with an average contour length ( ± S.D. ) of 11.4 (± 0.5) μ m . The circular DNA has the same buoyant density as mtDNA in NaI gradients, but represented only up to 6% of total mtDNA. 2. Denatured total mtDNA renatures with a kinetic complexity of about 20 · 106, a value consistent with the length observed in the electron microscope. 3. mtDNA from Kluyveromyces lactis hybridizes about twice as much mitochondrial rRNA isolated from Saccharomyces carlsbergensis than the equal amount of Saccharomyces carlsbergensis mtDNA (contour length 25 μm). 4. We conclude that intact mtDNA of Kluyveromyces lactis consists of a circular molecule with a contour length of 11.4 μm and a complexity equivalent to this size.

Kinetoplast DNA from Trypanosoma vivax and T. congolense

We have analysed kinetoplast DNA (kDNA) of the African trypanosomes Trypanosoma vivax and T. congolense. The maxi-circles from these organisms resemble those of T. brucei in size, but only to a limited extent in sequence as judged from restriction enzyme digests and DNA X DNA hybridization. The kDNA networks of T. vivax have three distinguishing features: they contain the highest maxi-circle concentration of any kDNA (at least twice that of T. brucei); they contain the smallest mini-circles (465 bp) yet found thus far and the width of the kDNA nucleoid in thin sections is correspondingly small (55 nm against 91 nm for T. brucei); they contain a substantial fraction of mini-circle dimers.

Analysis by electron microscopy of the variable segment in the maxi-circle of kinetoplast DNA from Trypanosoma brucei

Abstract The 20.5-kbp maxi-circle from the kinetoplast DNA of Trypanosoma brucei contains a 5-kbp segment which is not cut by most restriction endonucleases and which varies in size in closely-related trypanosome strains (Borst, P., Fase-Fowler, F., Hoeijmakers, J.H.J. and Frasch, A.C.C. (1980) Biochim. Biophys. Acta 610, 197–210). We have now analysed partial denaturation maps of the linearized maxi-circles by electron microscopy and find that the variable segment is not more AT-rich than the remainder of the maxi-circle. Early denaturation begins at two separate regions of the maxi-circle outside the variable region and one of these corresponds with the position of the gene for the large (12 S) ribosomal RNA. Denaturation-renaturation of maxi-circles leads to the formation of partially mismatched duplexes that look like underwound loops in electron micrographs. These loops are only found in the variable region and they vary in size and appearance. Under our renaturation conditions single-stranded maxi-circle DNA is devoid of secondary structure and this suggests that the underwound loops arise by misalignment of straight tandem repeats in the DNA. We have also analysed heteroduplexes between maxi-circles from two closely related T. brucei strains that differ by 1 kbp in the size of their variable segment. Most molecules had no underwound loops and contained mismatched regions in the variable segment only. The appearance of these regions is diverse, varying from fully duplex with two single-stranded loops to molecules with a heterogeneous array of smaller loops. The total size of single-stranded DNA in the heteroduplexes may be as high as 1.2 μm, i.e., a factor 4 higher than the size difference between the heteroduplex partners. We conclude that the variable region consists of imperfect tandem repeats of a sequence that evolves rapidly. This region might contain the origin of maxi-circle replication.

The adenine nucleotide translocator and the nucleotide specificity of oxidative phosphorylation

Abstract By studying P/O ratios, proton translocation and morphology of different mitochondrial preparations, it was shown, in agreement with previous work but in disagreement with a recent report, that the nucleotide specificity of the oxidative phosphorylation resides at the level of the adenine nucleotide translocator.

Intermediates in the replication of the mitochondrial DNA of Tetrahymena pyriformis

Abstract 1. 1. Pulse labelling of exponentially growing Tetrahymena pyriformis has been used to identify intermediates in the replication of its mtDNA. 2. 2. Pulse-labelled mtDNA sediments more rapidly than normal Tetrahymena mtDNA: its buoyant density in neutral CsCl and its chromatographic properties in benzoylated-naphthoylated-DEAE-cellulose indicate that it consists of duplex molecules having only short stretches of single-stranded DNA. Alkaline sucrose sedimentation velocity analysis demonstrates that the pulse label is incorporated into DNA varying in size from short pieces to full-length single strands. Longer molecules were not observed. 3. 3. Comparative electron microscopy of mtDNA sedimenting faster than the bulk of Tetrahymena mtDNA and of total mtDNA shows that the former is enriched in molecules containing one symmetrical, centrally positioned, predominantly duplex replication bubble (an “eye”). Oligomeric molecules (circular or linear) were absent from all preparations. 4. 4. The results indicate that the “eyed” molecules are intermediates in replication rather than in repair or recombination.