Effects of Ethidium Bromide and Several Acridine Dyes on the Kinetoplast DNA of Leishmania tropica*†

Whole cell DNA from Leishmania tropica has 2 peaks when banded by CsCl equilibrium density centrifugation. The main band has a buoyant density of 1.721 and the satellite band a buoyant density of 1.705, with Clostridium perfringens DNA (ρ= 1.6915) used as a reference. The satellite band has been identified as the kinetoplast DNA by purifying DNA from isolated kinetoplasts. L. tropica has the highest G + C content of both nuclear and kinetoplastic DNA thus far reported for trypanosomatids. The effects of ethidium bromide, acriflavin, proflavin, and 5-aminoacridine on the kinetoplast of L. tropica have been compared. Ethidium bromide and acriflavin, but not proflavin or 5-aminoacridine, induce dyskinetoplasty. L. tropica is one of the most sensitive trypanosomatids to ethidium bromide and acriflavin. Examination of the DNA from drug-treated cells in CsCl gradients revealed a loss of the satellite band after ethidium bromide or acriflavin treatment, but not after proflavin or 5-aminoacridine treatment. Cell division was required to produce these effects on the kinetoplast.     

Effect of hydroxystilbamidine on the kinetoplast of Trypanosoma gambiense in mice.

Hydroxystilbamidine (OHSA), an inhibitor of DNA synthesis, was shown to induce the dyskinetoplastic forms (akinetoplastic forms, AK forms) of Trypanosoma gambiense. The mode of appearance of the AK forms after injection of various doses of OHSA into infected mice was examined. The results suggested that production of the AK form is due to the selective inhibition of kinetoplast duplication of the drug without any effect on nuclear and cytoplasmic multiplication. When the parasites were treated with moderate doses of OHSA, segmenting forms without stainable kinetoplasts, were occasionally seen but attempts to establish a clone of akinetoplastic parasite were unsuccessful. Electron microscopy of parasites obtained after OHSA treatment showed not only irregular division of the kinetoplast but also the disorganization of kinetonucleus with disappearance of its envelope. Therefore, it was concluded that the AK forms were also produced by OHSA through disappearance of the kinetoplast.     

Multiplication of Trypanosoma pacifica (Euglenozoa: Kinetoplastea) in English sole, Parophrys vetulus, from Oregon coastal waters

Multiplication of Trypanosoma pacifica was common in the fish host from observations of live flagellates and Giemsa-stained blood smears. Multiplication began with the elongation of the kinetoplast, thickening of the posterior portion of the body, and appearance of a new flagellum near the kinetoplast. The new flagellum was very rigid when less than 3 microm in length, but it became flexible as it elongated. When the new flagellum was approximately 12 microm in length, cell division began and the kinetoplast also began to divide. The timing of nuclear division was variable. Generally, it did not occur until division of the kinetoplast had begun, but occasionally binucleate individuals were observed before cell or kinetoplast division was apparent. As division continued, 1 nucleus migrated past the dividing kinetoplast into the future daughter trypanosome. Finally, the kinetoplast completed division and the trypanosomes separated. Cell division was unequal, with the daughter trypanosome being smaller than the parent and with a more weakly developed undulating membrane.     

Synchronization of Leishmania tarentolae by Hydroxyurea

SYNOPSIS Leishmania tarentolae cells in brain-heart infusion medium were partially synchronized in terms of DNA synthesis and cell division by a 10 hour period of inhibition in 200 μg/ml hydroxyurea at 27 C. Nuclear and kinetoplast DNA synthesis commenced immediately upon removal of hydroxyurea, and kinetoplast and nuclear division occurred after about 5 hr. The Index of Synchronization (3) varied from 33-41%.
A moderate decay of the synchronicity was noted by the 2nd cell cycle. Hydroxyurea was selectively lethal to S-phase cells.     

Role of p38 in replication of Trypanosoma brucei kinetoplast DNA

Trypanosomes have an unusual mitochondrial genome, called kinetoplast DNA, that is a giant network containing thousands of interlocked minicircles. During kinetoplast DNA synthesis, minicircles are released from the network for replication as theta-structures, and then the free minicircle progeny reattach to the network. We report that a mitochondrial protein, which we term p38, functions in kinetoplast DNA replication. RNA interference (RNAi) of p38 resulted in loss of kinetoplast DNA and accumulation of a novel free minicircle species named fraction S. Fraction S minicircles are so underwound that on isolation they become highly negatively supertwisted and develop a region of Z-DNA. p38 binds to minicircle sequences within the replication origin. We conclude that cells with RNAi-induced loss of p38 cannot initiate minicircle replication, although they can extensively unwind free minicircles.     

Resistance to Purified Acriflavin in <Emphasis Type="Italic">Leishmania tarentolae</Emphasis>

THE mechanism by which purified acriflavin (3,6-diamino-N-methyl acridinium chloride) affects haemoflagellates has long been a matter of speculation. Available evidence indicates that the dye may interfere with replication of kinetoplast DNA^1–9, with mitochondrial function^{1, 9} and with certain cytoplasmic enzymes¹⁰. In preparations stained with Feulgen or Giemsa, the kinetoplast appears as the darkly stained granule near the base of the flagellum (Fig. 1). Electron microscopy and biochemical techniques have shown that it contains DNA surrounded by a double membrane that is continuous with the mitochondrial system^{4, 6–8, 11–17}. When acriflavin is added to cultures of Leishmania tarentolae, fluorescence from the dye appears in the kinetoplast¹. Several cell generations later, the kinetoplast disappears in many haemoflagellates, dividing in the presence of the dye (as judged by Giemsa or Feulgen staining^{1, 2, 5–7, 13, 18, 19}), leading to dyskinetoplasia⁶. The amount^1–3 and fine structure^{2, 6, 7, 9, 13} of kinetoplast DNA are, moreover, altered. Tritiated thymidine no longer appears in the kinetoplast region when it is examined by light¹ and electron microscope⁹ autoradiography.     

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.     

Effects of RNA interference of Trypanosoma brucei structure-specific endonuclease-I on kinetoplast DNA replication

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid protozoa, is a network containing several thousand topologically interlocked DNA minicircles. Kinetoplast DNA synthesis involves release of minicircles from the network, replication of the free minicircles, and reattachment of the progeny back onto the network. One enzyme involved in this process is structure-specific endonuclease-I. This enzyme, originally purified from Crithidia fasciculata, has been proposed to remove minicircle replication primers (Engel, M. L., and Ray, D. S. (1998) Nucleic Acids Res. 26, 4773-4778). We have studied the structure-specific endonuclease-I homolog from Trypanosoma brucei, showing it to be localized in the antipodal sites flanking the kinetoplast DNA disk, as previously shown in C. fasciculata. RNA interference of structure-specific endonuclease-I caused persistence of a single ribonucleotide at the 5' end of both the leading strand and at least the first Okazaki fragment in network minicircles, demonstrating that this enzyme in fact functions in primer removal. Probably because of the persistence of primers, RNA interference also impeded the reattachment of newly replicated free minicircles to the network and caused a delay in kinetoplast DNA segregation. These effects ultimately led to shrinkage and loss of the kinetoplast DNA network and cessation of growth of the cell.     

Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate

Subplastidic preparations from cotyledons of cucumber (Cucumis sativus L.) were tested for their ability to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Envelope or thylakoid membranes failed to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Stromal preparations synthesized a very low amount of protoporphyrin IX. In a reconstitution experiment using stroma + envelope membranes, protoporphyrin IX synthesis from 5-aminolevulinic acid was enhanced by 660% over that of stroma alone. However, when thylakoids were added to the stroma + envelope mixture, protoporphyrin IX synthesis from 5-aminolevulinic acid was completely inhibited. In the reconstituted stroma + envelope membrane mixture, the reducing agent dithiothreitol enhanced the protoporphyrin IX-synthesizing ability and completely abolished the inhibition of protoporphyrin IX synthesis by thylakoids. This suggested that the oxidizing agents usually associated with the thylakoid membranes inhibited protoporphyrin IX biosynthesis and the inhibition was alleviated by the reducing power of dithiothreitol. This study exposes the weakness of in vitro reconstitution experiments in mimicking the in vivo-conditions. Addition of ATP stimulated protoporphyrin IX synthesis by 50% in the supernatant fraction of chloroplast lysate. This ATP-induced stimulation of protoporphyrin IX synthesis was due to the enhancement of the activities of uroporphyrinogen decarboxylase and protoporphyrinogen oxidase, involved in tetrapyrrole biosynthesis. The ATP-induced stimulation of porphyrinogen oxidase activity was an energy-dependent reaction. Received: 21 March 2000 / Accepted: 9 May 2000     

Inhibition of plant protoporphyrinogen oxidase by the herbicide acifluorfen-methyl

The effect of acifluorfen-methyl on tetrapyrrole synthesis in greening chloroplasts of Cucumis sativus was examined. Formation of Mg-proto-porphyrin IX from δ-aminolevulinate was reduced 98% by 10 micromolar acifluorfen-methyl. Conversion of protoporphyrin IX to Mg-protoporphyrin IX was unaffected, but protoporphyrin IX synthesis from δ-aminolevulinate was blocked, indicating a site of inhibition prior to the Mg-chelatase. The enzymic oxidation of protoporphyrinogen IX to protoporphyrin IX was highly sensitive to acifluorfen-methyl, indicating that the site of action of the herbicide is the protoporphyrinogen oxidase. (© 1989 FMC Corporation. All rights reserved.)     

Bending and flexibility of kinetoplast DNA

We have evaluated the extent of bending at an anomalous locus in DNA restriction fragments from the kinetoplast body of Leishmania tarentolae using transient electric dichroism to measure the rate of rotational diffusion of DNA fragments in solution. We compare the rate of rotational diffusion of two fragments identical in sequence except for circular permutation, which places the bend near the center in one case and near one end of the molecule in the other. Hydrodynamic theory was used to conclude that the observed 20% difference in rotational relaxation times is a consequence of an overall average bending angle of 84 +/- 6 degrees between the end segments of the fragment that contains the bending locus near its center. If it is assumed that bending results from structural dislocations at the junctions between oligo(dA).oligo(dT) tracts and adjacent segments of B DNA, a bend angle of 9 +/- 0.5 degrees at each junction is required to explain the observations. The extent of bending is little affected by ionic conditions and is weakly dependent on temperature. Comparison of one of the anomalous fragments with an electrophoretically normal control fragment leads to the conclusion that they differ measurably in apparent stiffness, consistent with a significantly increased persistence length or contour length in the kinetoplast fragments.     

The kinetoplast duplication cycle in Trypanosoma brucei is orchestrated by cytoskeleton-mediated cell morphogenesis

The mitochondrial DNA of Trypanosoma brucei is organized in a complex structure called the kinetoplast. In this study, we define the complete kinetoplast duplication cycle in T. brucei based on three-dimensional reconstructions from serial-section electron micrographs. This structural model was enhanced by analyses of the replication process of DNA maxi- and minicircles. Novel insights were obtained about the earliest and latest stages of kinetoplast duplication. We show that kinetoplast S phase occurs concurrently with the repositioning of the new basal body from the anterior to the posterior side of the old flagellum. This emphasizes the role of basal body segregation in kinetoplast division and suggests a possible mechanism for driving the rotational movement of the kinetoplast during minicircle replication. Fluorescence in situ hybridization with minicircle- and maxicircle-specific probes showed that maxicircle DNA is stretched out between segregated minicircle networks, indicating that maxicircle segregation is a late event in the kinetoplast duplication cycle. This new view of the complexities of kinetoplast duplication emphasizes the dependencies between the dynamic remodelling of the cytoskeleton and the inheritance of the mitochondrial genome.     

A simple procedure for the preparation of pure kinetoplast DNA network free of nuclear DNA from the kinetoplast hemoflagellate Leishmania donovani

A simple, inexpensive procedure for preparing pure kinetoplast DNA network from Leishmania donovani is described. L. donovani promastigotes were lysed by incubating with pronase in presence of sodium dodecylsulfate. Crude kinetoplast DNA networks were obtained by centrifugation of the lysate through a 20% sucrose solution. The pellet containing kinetoplast DNA was deproteinized by phenol extraction. Contaminating nuclear DNAs were removed by denaturation with alkali, neutralization, and addition of polyethylene glycol-8000 to a concentration of 10% to facilitate precipitation of kinetoplast DNA. kDNA isolated after centrifugation was deproteinized several times with phenol and finally precipitated with ethanol. The average yield by this procedure is 30-50 micrograms of kDNA per gram of wet cells. By slot-blot hybridization with a nuclear DNA probe, no nuclear DNA contamination of the kDNA networks could be detected.     

LaRbp38: a Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.     

Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants

Protoporphyrin IX is the last common intermediate of tetrapyrrole biosynthesis. The chelation of a Mg2+ ion by magnesium chelatase and of a ferrous ion by ferrochelatase directs protoporphyrin IX towards the formation of chlorophyll and heme, respectively. A full length cDNA clone encoding a ferrochelatase was identified from a Nicotiana tabacum cDNA library. The encoded protein consists of 497 amino acid residues with a molecular weight of 55.4 kDa. In vitro import of the protein into chloroplasts and its location in stroma and thylakoids confirm its close relationship to the previously described Arabidopsis thaliana plastid-located ferrochelatase (FeChII). A 1700-bp tobacco FeCh cDNA sequence was expressed in Nicotiana tabacum cv. Samsun NN under the control of the CaMV 35S promoter in antisense orientation allowing investigation into the consequences of selective reduction of the plastidic ferrochelatase activity for protoporphyrin IX channeling in chloroplasts and for interactions between plastidic and mitochondrial heme synthesis. Leaves of several transformants showed a reduced chlorophyll content and, during development, a light intensity-dependent formation of necrotic leaf lesions. In comparison with wild-type plants the total ferrochelatase activity was decreased in transgenic lines leading to an accumulation of photosensitizing protoporphyrin IX. Ferrochelatase activity was reduced only in plastids but not in mitochondria of transgenic plants. By means of the specifically diminished ferrochelatase activity consequences of the selective inhibition of protoheme formation for the intracellular supply of heme can be investigated in the future.     

Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.     

Structural requirements for porphyrin inhibition of the hemin-controlled protein kinase and maintenance of protein synthesis in reticulocytes

The role of hemin in the maintenance of protein synthesis in reticulocyte lysates was examined by comparing the effects of various porphyrins and metalloporphyrins on the protein kinase activity of the hemin-controlled repressor and on protein synthesis. The porphyrin requirements for maintenance of protein synthesis were relatively specific. Iron and cobalt metalloporphyrins sustained protein synthesis whereas other metalloporphyrins, metal-deficient porphyrins, and non-porphyrin precursor and degradation products of protoporphyrin IX were ineffective. These same compounds were examined for their effectiveness in inhibiting the protein kinase activity of the hemin-controlled repressor with initiation factor 2 (eIF-2). Most of the metalloporphyrins and porphyrins tested were inhibitory. The presence of the iron atom in the porphyrin was not essential for inhibition, but the maintenance of the integrity of the porphyrin ring was imperative. The porphyrins which inhibited the hemin-regulated protein kinase contained vinyl groups or ethyl groups, or were protonated in the 2- and 4-positions of the porphyrin ring, whereas those with bulky or acidic groups in these positions were ineffective. Precursor and degradation products of protoporphyrin IX and synthetic porphyrins modified at other positions had no effect on the enzyme. Both hemin and protoporphyrin IX inhibited phosphorylation of eIF-2 exogenously added to a reticulocyte lysate; however, hemin sustained protein synthesis in the lysate, whereas protoporphyrin IX did not. These results suggest that regulation of the protein kinase phosphorylating the alpha subunit of eIF-2 is not the only point at which hemin modulates protein synthesis in reticulocytes and reticulocyte lysates, since a correlation between inhibition of protein synthesis, inhibition of protein kinase activity, and phosphorylation of eIF-2 is not observed with all porphyrins.