Transkinetoplastidy—A Novel Phenomenon Involving Bulk Alterations of Mitochondrion-Kinetoplast DNA of a Trypanosomatid Protozoan1,2

ABSTRACT. Dramatic and consistent changes of mitochondrial or kinetoplast DNA (kDNA) were observed in certain variants of Leishmania amazonensis (A variants) selected in vitro for arsenite-resistance. This was found initially by comparing different lots of wild-type cells and their respective A variants resistant to 30 μM arsenite. The kDNAs isolated from these two groups had different restriction patterns and hybridized poorly to each other, whereas those from different lots within each of the two groups were identical. Hybridization data showed an overall identity of less than 10^?3 between total kDNAs of the two groups. This difference was further examined in three independent series of variants, which were selected from three different clones for resistance to graded concentrations of arsenite (5–30 μM). In all three series, their kDNAs were found to change abruptly in an identical pattern at a late step of the selection process, i.e. A variants resistant to 15 μM or 30 μM arsenite. There was no apparent loss of kDNA in the process. Most of the changes observed appear to involve a shift in either the dominance or the copy number of different minicircle subclasses. Surprisingly, the kDNAs of tunicamycin-resistant variants (T variants) were also found to undergo similar changes. Genetic changes previously described in both A and T variants are limited to their nuclei. Namely, different chromosomal regions are amplified to produce large DNA circles which are responsible for the drug-resistant phenotypes. Interestingly, other arsenite-resistant clones without such chromosomal DNA amplification (A'variants) had kDNA of the wild-type pattern. The profound changes of kDNA observed are unprecedented. We propose the term “transkinetoplastidy” for this phenomenon to distinguish it from dyskinetoplastidy or the loss of kDNA described previously in trypanosomatid protozoa. This phenomenon is discussed with respect to the possible mechanisms of its generation, regulation and relation to the drug-resistant phenotypes.     

DNA amplification in arsenite-resistant Leishmania

Arsenite-resistant variants of a trypanosomatid protozoan, Leishmania mexicana amazonensis, were selected in vitro by stepwise increases of sodium arsenite concentrations up to 30 microM in the culture medium. These variants were found to contain amplified DNA as extrachromosomal supercoiled molecules of about 69 kb. They originate from a single chromosome in the wild-type cells. There is evidence of chromosomal changes in these cells associated with the selection for arsenite resistance. The apparent absence of these circular molecules in the wild type and their loss from the drug-sensitive revertants suggest amplification of chromosomal DNA into these extrachromosomal circles as the mechanism of arsenite resistance.     

Application of polymerase chain reaction with specific and arbitrary primers to identification and differentiation of Leishmania parasites

Abstract Two oligonucleotide primers Lsmc1 and Lsmv1 derived from the conserved and the variable region of a major class kinetoplast DNA (kDNA) minicircle (pLURkE3) of Leishmania strain UR6 were used for the polymerase chain reaction (PCR) in order to amplify a 461-bp fragment from the kDNAs of different Leishmania species. These primers amplify the specific fragment from the kDNAs of cutaneous species only. The cutaneous species can further be distinguished by randomly amplified polymorphic DNA (RAPD) analysis of the kDNAs of these organisms using arbitrarily chosen oligonucleotides. The arbitrary primers also generate polymorphic DNA fingerprints at the genomic level with different L. donovani isolates. The results indicate that the PCR and arbitrarily primed PCR (AP-PCR) may be extremely useful approaches for identifying and distinguishing Leishmania parasites.     

Changes in Trypanosoma cruzi Kinetoplast DNA Minicircles Induced by Environmental Conditions and Subcloning

ABSTRACT. Reversible changes in kinetoplast DNA (kDNA) minicircles sequences were observed in clones of Trypanosoma cruzi strain Y, following a number of passages during exponential growth phase or after subcloning in blood-free medium. kDNA restriction patterns of clones were similar to those of the original uncloned strain, while subclones presented distinct kDNA restriction patterns. Homology experiments demonstrated strong hybridization between kDNA with the same electrophoretic mobility patterns while only weak signals were observed with kDNA of different patterns. The changes observed, which are unprecedented in T. cruzi clones, characterize transkinetoplastidy, and seem to be associated with similarly reversible changes both in zymodeme and in infectivity.     

Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana.

Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite- or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-type minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type- or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.     

Development of a genus specific primer set for detection of Leishmania parasites by polymerase chain reaction

Abstract We have compared the sequences of a major class of kinetoplast DNA (kDNA) minicircle (pLURkE3) of Leishmania strain UR6 with other minicircle sequences from different Leishmania species. Alignment of these sequences allowed the selection of a pair of oligonucleotides suitable as primers in polymerase chain reaction (PCR) which is specific for Leishmania parasites. PCR with this genus-specific primer set is capable of detecting 1 femtogram of kDNA. These primers have been tested with kDNAs from both old world and new world Leishmania species. The results indicate that the primers may be suitable for detection of any kind of leishmaniasis.     

Specific inhibition of bovine viral diarrhea virus replicase

Compound-1453 was identified and characterized as a specific inhibitor of bovine viral diarrhea virus (BVDV). The concentration of compound-1453 which results in 50% protection from virus-induced cytopathic effect is approximately 2.2 microM, with a therapeutic index of 60, and it is not active against a panel of RNA and DNA viruses. A time-of-addition experiment suggested that compound-1453 targets a stage of the viral life cycle after viral entry. To determine the target of compound-1453, resistant virus was generated. Resistant variants grew efficiently in the presence or absence of 33 micro M compound-1453 and exhibited replication efficiency in the presence of compound-1453 approximately 1,000-fold higher than that of the wild-type (wt) virus. Functional mapping and sequence analysis of resistant cDNAs revealed a single amino acid substitution (Glu to Gly) at residue 291 in the NS5B polymerase in all eight independently generated cDNA clones. Recombinant virus containing this single mutation retained the resistance phenotype and a replication efficiency similar to that of the original isolated resistant virus. Since compound-1453 did not inhibit BVDV polymerase activity in vitro (50% inhibitory concentration > 300 microM), we developed a membrane-based assay that consisted of a BVDV RNA replicase complex isolated from virus-infected cells. Compound-1453 inhibited the activity of the wt, but not the drug-resistant, replicase in the membrane assay at concentrations similar to those observed in the viral infection assay. This work presents a novel inhibitor of a viral RNA-dependent RNA replicase.     

Resistance to Microtubule-Stabilizing Drugs Involves Two Eventse: β-Tubulin Mutation in One Allele Followed by Loss of the Second Allele

Resistance to paclitaxel (PTX) or the epothilones (Epo) occurs via the acquisition of point mutations in β-tubulin residues important for drug-tubulin binding. We have isolated four drug-resistant clones selected with PTX or Epo A which harbor distinct β-tubulin mutations. During the development of a stable drug-resistant phenotype, early clones expressing both wild-type (wt) and mutant β-tubulin sequences exhibited a 10-fold drug resistance, while more advanced clones expressing only the mutant β-tubulin sequence exhibited 30 to 50-fold drug resistance. The drug-sensitive parental 1A9 ovarian carcinoma cell line and the drug resistant clones (1A9-A8, 1A9-PTX10 and 1A9-PTX22) were evaluated for loss of heterozygosity (LOH) for β-tubulin (6p25) by single nucleotide polymorphism (SNP) and fluorescent in situ hybridization (FISH) analyses. Functional assays such as drug-induced tubulin polymerization, cell cycle analysis by FACS, DNA sequencing for β-tubulin and mitotic index by immunofluorescence were performed to correlate the β-tubulin LOH status with drug response in the early- and late-step drug-resistant clones. Late-step drug resistant clones revealed LOH in one allele for wt b-tubulin in addition to a β-tubulin mutation in the other allele leading to increased levels of drug resistance, while the early-step clones that contained both a wt and a mutant b-tubulin allele were considerably less drug resistant. The LOH and functional assays revealed cell response that was proportional to the tubulin gene and heterozygosity status. Acquired tubulin mutations in conjunction with LOH for the wt tubulin resulted in a highly resistant phenotype, revealing a new mechanism for taxane resistance.     

Detection and identification of Leishmania parasites by in situ hybridization with total and recombinant DNA probes.

In situ hybridization on cultured promastigotes and sandfly smears were performed with nonradioactively labeled total DNA and recombinant DNA probes containing minicircle kinetoplast DNA (kDNA) or nuclear DNA inserts. Total DNA probes lack specificity whereas recombinant nuclear DNA probes work only if they contain repetitive sequences. Minicircle kDNAs of five Leishmania isolates, representative of five Leishmania taxa found in Kenya, were sequenced. Comparison of the sequences showed a 150-bp region with around 80% homology, whereas the rest of the minicircles had about 50% homology. Nevertheless, application of these probes in in situ hybridization assays as tested on Leishmania promastigotes in the vector gave good specificity and hybridization signal. Two types of labeling were tested: incorporation of biotin-labeled dUTP or directly horseradish peroxidase (HRP)-labeled nucleotides. Both techniques provided good sensitivity and signal-to-noise ratio on cultured promastigotes. Hybridization with HRP-labeled kDNA probes gave a superior signal-to-noise ratio if tested on sandfly preparations. This method provided a reliable and fast identification and facilitated the detection of promastigotes in sandflies. The technique presented here may be helpful in rapid identification of Leishmania promastigotes, and thus make epidemiological studies easier and less time consuming.     

Physicochemical properties of kinetoplast DNA from Crithidia acanthocephali. Crithidia luciliae, and Trypanosoma lewisi

The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.     

Low-level self-tolerance to arsenite in human HepG2 cells is associated with a depressed induction of micronuclei

Arsenic (As) is one of the most important global environmental toxicants. There is evidence that humans may develop tolerance to As's toxicity. For instance, it is known that uptake of small amounts of As leads to an acquisition of elevated resistance to the element's acute toxicity. Moreover, it was suggested that As-exposed native Andean females of Atacame?o ethnicity may have acquired resistance to skin cancer. It is not known how such adaptation could be mechanistically conferred. In this context, the biological selection and cloning of human cells tolerant to As provides a valuable approach to investigate this question. By the means of a 12 weeks culture with increasing doses of As, three different As-resistant clones of the human hepatoma cell line HepG2 were selected. These three clones were similarly and roughly two-fold resistant to the acute toxicity of arsenite (50% reduction of neutral red (NR) uptake at 65 microM versus 115 microM; HepG2 control versus clones HepG2 K1, HepG2 K11 and HepG2 K14, respectively). Moreover, in the cytokinesis-block micronucleus test, these clones showed a significantly reduced induction of micronuclei (MNi) indicating elevated resistance to As genotoxicity as well (e.g. mean MNi rates at a concentration of 25 microM arsenite: 28.5 (control) versus 21.6 (HepG2 K1), 18 (HepG2 K11), and 16 (HepG2 K14), respectively, each P<0.05). The tolerance was neither associated with mRNA induction of putatively As-extruding membrane transporters multidrug resistance-associated protein 1 (MRP1), 2, or 3 nor to mRNA induction of the ubiquitously expressed mammalian ABC half-transporter UMAT (ABCB6). Changes in the metabolic methylation of As could not be detected. There were no differences in the cellular levels of GSH when comparing the clones and the parental line. Taken together the data showed that low-level tolerance to As-mediated cytotoxicity in human HepG2 cells was associated with enhanced resistance to As-induced DNA damage as well.     

Chinese hamster ovary cell variants resistant to monensin, an ionophoric antibiotic. I. Isolation and altered endocytosis of ricin

Chinese hamster ovary (CHO) cell variants resistant to a carboxylic ionophore, monensin, have been isolated. Two monensin-resistant variants (MonR-31 and MonR-32) showed a three- to fourfold higher resistance to monensin than did CHO. These MonR clones also showed fourfold higher resistance to another carboxylic ionophore, nigericin, and twofold higher resistance to valinomycin. They were also slightly more resistant to other unrelated drugs such as adriamycin, colchicine, bleomycin, and chloroquine, and in particular, they showed about threefold higher resistance to ricin, a toxin of Ricinus communis. MonR clones were found to retain a normal level of [125I]ricin binding, but internalization of [125I]ricin into the MonR clones was one-half or less than with CHO. Present data suggest that drug-resistant clones selected in culture may provide a way to isolate cells with altered response to various bioactive molecules.     

Schizodeme analysis of Trypanosoma cruzi Colombian strain clones isolated from the acute phase of murine infection

Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom. Characterization by their biological and biochemical behavior showed an identity among the several clones and their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA (kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA minicircle by the analysis of restriction fragment length polymorphism for the isolated clones, their parental strain, and to the clones isolated at two different phases of the infection. Results suggest the predominance of a "principal clone", in the composition of the Colombian strain, responsible for the biological and biochemical behavior. However, no relationship was detected between the molecular profile of kDNA and the degree of virulence presented by the several clones.     

Heterogeneity in sensitivity to cleavage by the restriction endonucleases ECORI and HindIII of circular kinetoplast DNA molecules of Crithidia acanthocephali

Kinetoplast DNA (kDNA) of the protozoan Crithidia acanthocephali consists mainly of an association of approximately 27,000 covalently closed, 0.8-micron (1.58 X 10(6) daltons) circular molecules apparently held together in a particular structural configuration by topological interlocking. The sensitivities of circular kDNA molecules to the restriction endonucleases EcoRI and HindIII have been studied using agarose gel electrophoresis and electron microscopy. Digestion with EcoRI or HindIII of collections of single circular molecules obtained from sonicated kDNA associations resulted in a single cleavage of 9.3 and 12% of the molecules, respectively. Digestion of intact kDNA associations with EcoRI or HindIII resulted in cleavage of 9.2 and 10.4%, respectively, of the component circular molecules, but not in detectable disruption of the characteristic structure of the associations. Analysis of the products of sequential digestion of kDNA with the two enzymes indicated that approximately 8% of the circular molecules each contain a single site sensitive to EcoRI and a single site sensitive to HindIII; 1.5-3% contain only an EcoRI-sensitive site; 3-4% contain only a HindIII-sensitive site; and the remainder (approximately 86%) are insensitive to either enzyme. Further, data obtained from sequential digestion experiments and from studies of the partial denaturation products of the circular molecules digested with EcoRI or HindIII indicated that when they occur the EcoRI site and the HindIII site are each at a unique position in all molecules, 10-13% of the circular contour length apart. Similar digestion products were found for kDNAs from different cloned organisms, suggesting that the four different kinds of circular molecules, in regard to EcoRI and HindIII sensitivity, are found in similar proportions in the kDNA association of different organisms.     

Plasmid loss and changes within the chromosomal DNA of Streptomyces reticuli.

The sporulating wild-type strain of Streptomyces reticuli, which produces a melanin pigment and the macrolide leucomycin, contains plasmid DNA of 48 to 49 megadaltons. Plasmidless variants had an altered secondary metabolism and a changed antibiotic resistance pattern. By using a new colony hybridization technique developed for streptomycetes, it could be shown that plasmidless variants could be transformed with the wild-type plasmid DNA, which, however, is quickly lost from regenerated mycelium. In contrast to the wild-type strain, the plasmidless variants contain amplified nucleotide sequences within the chromosomal DNA. The number and size of these sequences vary with the strain tested. Hybridization studies revealed that the reiterated sequences are neither amplified ribosomal nor plasmid genes, but are present in small concentrations within the wild-type chromosome. Some of them share extensive homologies with each other and are located at different positions within the chromosome. It is assumed that alterations in secondary metabolism are due to changes within both the chromosomal and the extrachromosomal DNAs of S. reticuli.     

Identification of Trypanosoma cruzi Zymodemes by Kinetoplast DNA Probes

Analysis of zymograms of extracts of Trypanosoma cruzi isolated from different hosts in Argentina allowed characterization of 12 zymodemes or "isozymic strains," only six of which were found in human patients. Two of these six zymodemes (Z1 and Z12) were widely distributed and found in more than 80% of human patients. These two "major natural clones" differed significantly in pathogenic activity. Because the groupings obtained by studying enzymes and kinetoplast DNA (kDNA) were similar, it is possible to identify the zymodeme by analyzing kDNA. A 290-bp fragment was amplified by PCR using primers for the sequences flanking the hypervariable regions of kDNA minicircles. Labeled probes for this fragment, prepared from Z1 and Z12 reference stocks, hybridized specifically with PCR-amplified kDNA from parasite stocks, allowing identification of zymodemes.