ABSTRACT
Macrophages are multipurpose phagocytes and are considered to be irreplaceable during the early host innate immune response against microbial and parasitic pathogens. However, no report has investigated the novel anti-parasitic mechanism of macrophage-derived extracellular traps (ETs) against the abortive apicomplexan parasite Neospora caninum (N. caninum) in cattle. Scanning electron microscopy (SEM) was used to visualize and characterize N. caninum tachyzoite-induced macrophage-triggered ETs in exposed bovine macrophages. Fluorescence confocal microscopy was used to confirm the classical backbone structure of DNA embedded with histone 3 (H3) and myeloperoxidase (MPO) in N. caninum tachyzoite-induced macrophage-derived ETs. Furthermore, the lactate dehydrogenase (LDH) levels in the supernatants of parasite-exposed macrophages were detected by a LDH Cytotoxicity Assay® kit. The results clearly demonstrated that N. caninum tachyzoites triggered bovine macrophage-derived ET-like structures. Inhibiting assays revealed that N. caninum tachyzoite-induced macrophage-mediated ET formation may be an ERK 1/2- and p38 MAPK-dependent cell death process. In conclusion, the present study is the first report on the formation of ETs in bovine macrophages against N. caninum tachyzoites and adds new data on the possible role of macrophages in vivo infection by capturing invasive stages and exposing them to other leukocytes.
Subject(s)
Coccidiosis/veterinary , Extracellular Traps/immunology , Host-Parasite Interactions , Macrophages/immunology , Neospora/immunology , Animals , Cattle , Coccidiosis/immunology , Coccidiosis/parasitology , Culture Media/analysis , DNA, Bacterial/ultrastructure , DNA, Protozoan/ultrastructure , Extracellular Signal-Regulated MAP Kinases , Extracellular Traps/parasitology , Immunity, Innate , L-Lactate Dehydrogenase/analysis , Macrophages/parasitology , Macrophages/ultrastructure , Microscopy, Electron, Scanning , Neospora/geneticsABSTRACT
We investigate the self-assembly of short pieces of the Tetrahymena telomeric DNA sequence d[G4T2G4] in physiologically relevant aqueous solution using atomic force microscopy (AFM). Wire-like structures (G-wires) of 3.0 nm height with well-defined surface periodic features were observed. Analysis of high-resolution AFM images allowed their classification based on the periodicity of these features. A major species is identified with periodic features of 4.3 nm displaying left-handed ridges or zigzag features on the molecular surface. A minor species shows primarily left-handed periodic features of 2.2 nm. In addition to 4.3 and 2.2 nm ridges, background features with periodicity of 0.9 nm are also observed. Using molecular modeling and simulation, we identify a molecular structure that can explain both the periodicity and handedness of the major G-wire species. Our results demonstrate the potential structural diversity of G-wire formation and provide valuable insight into the structure of higher-order intermolecular G-quadruplexes. Our results also demonstrate how AFM can be combined with simulation to gain insight into biomolecular structure.
Subject(s)
DNA, Protozoan/ultrastructure , G-Quadruplexes , DNA, Protozoan/chemistry , Microscopy, Atomic Force/methods , Molecular Dynamics Simulation , Molecular Imaging/methods , Telomere/geneticsABSTRACT
Pulsed-field gel electrophoresis (PFGE) was applied to analyze the molecular karyotype of the ciliate Didinium nasutum. The data obtained indicate that D. nasutum belongs to the ciliate species with subchromosomal macronuclear genome organization. No short "gene-sized" DNA molecules were detected. Macronuclear DNAs formed a continuous spectrum from 50 kbp to approximately 1,000 kbp in size with a peak plateau between 250 and 400 kbp. The macronuclear DNA molecules were packed into chromatin bodies of 80-265 nm in size. Comparison of the PFGE and electron microscopic data shows that most if not all chromatin bodies contain more than one DNA molecule.
Subject(s)
Chromatin/ultrastructure , Ciliophora/genetics , DNA, Protozoan/ultrastructure , Macronucleus/genetics , Electrophoresis, Gel, Pulsed-Field , Karyotyping , Microscopy, ElectronABSTRACT
Liquid crystalline phases of DNA and nucleosome core particles can be formed in vitro, indicating the crucial roles of these phases in the maintenance and compaction of genomes in vivo. In the present study, sequential levels of liquid crystalline decompaction were identified in highly purified nuclei of Karenia papilionacea in response to the gradual chelation of divalent counterions by ethylenediaminetetraacetic acid (EDTA); the decompaction was observed using polarizing light microscopy, confocal laser scanning microscopy, and transmission electron microscopy and further confirmed utilizing microcalorimetry. Nested fibrous coils in 150 nm arc-like bands of chromatin were observed in the early stages of chromosomal decompaction. The microcalorimetry spectra of isolated nuclei revealed that the dynamic processes of nuclear decompaction occurred in a nonlinear manner; in addition, an EDTA-sensitive thermal transition between 60°C-70°C, corresponding to a liquid-crystalline-phase transition of chromosomes, was found. The results suggested that nested coils of fibrous chromatin filaments are responsible for the establishment and stabilization of the liquid crystalline and birefringence features of the chromosomes of dinoflagellates. The results also indicated that positively charged divalent counterions play significant roles in modulating liquid crystalline phases to compact the chromosomes of dinoflagellates.
Subject(s)
Chromatin/chemistry , Chromosomes/ultrastructure , Dinoflagellida/genetics , Liquid Crystals/chemistry , Birefringence , Cell Nucleus/genetics , Chromatin/genetics , Chromosomes/genetics , Crystallization , DNA, Protozoan/genetics , DNA, Protozoan/ultrastructure , Dinoflagellida/chemistry , Edetic Acid/chemistry , Microscopy, Confocal , Microscopy, Electron, Transmission , Microscopy, PolarizationABSTRACT
DNA is the biopolymer most studied by scanning probe methods, and it is now possible to obtain reliable and reproducible images of DNA using atomic force microscopy (AFM). AFM has been extensively used to elucidate morphological changes to DNA structure, such as the formation of knots, nicks, supercoiling and bends. The mitochondrial or kinetoplast DNA (kDNA) of trypanosomatids is the most unusual DNA found in nature, being unique in organization and replication. The kDNA is composed of thousands of topologically interlocked DNA circles that form a giant network. To understand the biological significance of the kinetoplast DNA, it is necessary to learn more about its structure. In the present work, we used two procedures to prepare kDNA networks of Crithidia fasciculata for observation by AFM. Because AFM allows for the examination of kDNA at high resolution, we were able to identify regions of overlapping kDNA molecules and sites where several molecules cross. This found support the earlier described kDNA structural organization as composed by interlocked circles. We also observed an intricate high-density height pattern around the periphery of the network of C. fasciculata, which appears to be a bundle of DNA fibers that organizes the border of the network. Our present data confirm that AFM is a powerful tool to study the structural organization of biological samples, including complex arrays of DNA such as kDNA, and can be useful in revealing new details of structures previously visualized by other means.
Subject(s)
Crithidia fasciculata/ultrastructure , DNA, Kinetoplast/ultrastructure , Microscopy, Atomic Force/methods , Crithidia fasciculata/chemistry , DNA, Kinetoplast/chemistry , DNA, Protozoan/ultrastructure , Microscopy, ElectronABSTRACT
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.
Subject(s)
Cytoskeleton/metabolism , DNA, Kinetoplast/metabolism , DNA, Protozoan/metabolism , Morphogenesis , Trypanosoma brucei brucei/growth & development , Cell Cycle , Cytoskeleton/ultrastructure , DNA Replication , DNA, Kinetoplast/ultrastructure , DNA, Protozoan/ultrastructure , Electron Microscope Tomography , Flagella/metabolism , Flagella/ultrastructure , Trypanosoma brucei brucei/metabolism , Trypanosoma brucei brucei/ultrastructureABSTRACT
DNA can self-assemble in vitro into several liquid crystalline phases at high concentrations. The largest known genomes are encoded by the cholesteric liquid crystalline chromosomes (LCCs) of the dinoflagellates, a diverse group of protists related to the malarial parasites. Very little is known about how the liquid crystalline packaging strategy is employed to organize these genomes, the largest among living eukaryotes-up to 80 times the size of the human genome. Comparative measurements using a semiautomatic polarizing microscope demonstrated that there is a large variation in the birefringence, an optical property of anisotropic materials, of the chromosomes from different dinoflagellate species, despite their apparently similar ultrastructural patterns of bands and arches. There is a large variation in the chromosomal arrangements in the nuclei and individual karyotypes. Our data suggest that both macroscopic and ultrastructural arrangements affect the apparent birefringence of the liquid crystalline chromosomes. Positive correlations are demonstrated for the first time between the level of absolute retardance and both the DNA content and the observed helical pitch measured from transmission electron microscopy (TEM) photomicrographs. Experiments that induced disassembly of the chromosomes revealed multiple orders of organization in the dinoflagellate chromosomes. With the low protein-to-DNA ratio, we propose that a highly regulated use of entropy-driven force must be involved in the assembly of these LCCs. Knowledge of the mechanism of packaging and arranging these largest known DNAs into different shapes and different formats in the nuclei would be of great value in the use of DNA as nanostructural material.
Subject(s)
Birefringence , Chromosomes/ultrastructure , DNA, Protozoan/ultrastructure , Dinoflagellida/genetics , Liquid Crystals , Animals , Cell Nucleus/genetics , Chromosomes/genetics , Crystallization , DNA, Protozoan/genetics , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Nucleic Acid ConformationABSTRACT
Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 2-3 kb in size, carrying from one to three genes. The non-coding regions (NCRs) of these minicircles share a conserved core region (250-500 bp) that are AT-rich and have several inverted or direct repeats. Southern blot analysis using an NCR probe, after resolving a dinoflagellate whole DNA extract in pulsed-field gel electrophoresis (PFGE), revealed additional positive bands (APBs) of 6-8 kb in size. APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2-3 kb minicircles, implicating they are not large minicircles. The APBs are also exonuclease III-sensitive, implicating the presence of linear DNA. These properties and the migration pattern of the APBs in a 2D-gel electrophoresis were in agreement with a rolling circle type of replication, rather than the bubble-forming type. Atomic force microscopy of 6-8 kb DNA separated by PFGE revealed DNA intermediates with rolling circle shapes. Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.
Subject(s)
DNA Replication , DNA, Circular/biosynthesis , DNA, Protozoan/biosynthesis , Dinoflagellida/genetics , Genome, Plastid , Animals , Aphidicolin/pharmacology , DNA, Catenated/chemistry , DNA, Circular/chemistry , DNA, Circular/ultrastructure , DNA, Protozoan/chemistry , DNA, Protozoan/ultrastructure , Electrophoresis, Gel, Pulsed-Field , Enzyme Inhibitors/pharmacology , Microscopy, Atomic ForceABSTRACT
From a variety of analytical electron microscopy experiments, the chromosomes of dinoflagellates are known to contain sizeable amounts of cations, the latter thought to contribute to the neutralization of the negative charge carried by the phosphate groups in the DNA backbone. From previous Ca and Mg chelation experiments, it is also known that these cations are necessary for the compaction and preservation of the chromosome architecture. Similar conclusions have been recently presented by our group concerning mammalian mitotic chromosomes, in studies based on secondary ion mass spectrometry (SIMS) carried out with the University of Chicago high-resolution scanning ion microprobe (UC-SIM). We have now applied this instrument to image the distribution of DNA-bound Ca(2+) and Mg(2+) in dinoflagellate chromosomes, a goal that could not be attained earlier by analytical electron microscopy. Analyzed quantitatively and imaged here by SIMS for the first time, through their cation content, are the chromosomes of the dinoflagellates Prorocentrum micans, Gymnodinium mikimotoi and Gymnodinium dorsum. The cell nuclei were isolated and prepared for SIMS analysis with a minimal protocol (mechanical fractionation in culture medium followed by ethanol drying), which did not expose the samples to artifact-creating, alien chemical agents. By this approach, we have confirmed the earlier findings by several authors, and contributed new structural information provided by our ion probe capability to erode the sample surface layer by layer (SIMS tomography). Dinoflagellates, due to the absence of histones, represent an ideal model system where cations may bind directly with DNA, allowing comparisons to be made with recently reported X-ray crystallography results at atomic resolution. Such comparisons yielded quantitative confirmation that the Ca(2+)+Mg(2+) concentrations found for e.g. P. micans are consistent with those anticipated to provide complete charge neutralization of naked DNA by cations, also resulting in maximal DNA compaction.
Subject(s)
Calcium/metabolism , Chromatin/metabolism , Chromosomes/metabolism , Dinoflagellida/genetics , Magnesium/metabolism , Spectrometry, Mass, Secondary Ion/methods , Animals , Cell Nucleus/chemistry , Chromosomes/ultrastructure , DNA, Protozoan/metabolism , DNA, Protozoan/ultrastructureABSTRACT
Apicomplexans, including the pathogens Plasmodium and Toxoplasma, carry a nonphotosynthetic plastid of secondary endosymbiotic origin called the apicoplast. The P. falciparum apicoplast contains a 35 kb, circular DNA genome with limited coding capacity that lacks genes encoding proteins for DNA organization and replication. We report identification of a nuclear-encoded bacterial histone-like protein (PfHU) involved in DNA compaction in the apicoplast. PfHU is associated with apicoplast DNA and is expressed throughout the parasite's intra-erythocytic cycle. The protein binds DNA in a sequence nonspecific manner with a minimum binding site length of approximately 27 bp and a K(d) of approximately 63 nM and displays a preference for supercoiled DNA. PfHU is capable of condensing Escherichia coli nucleoids in vivo indicating its role in DNA compaction. The unique 42 aa C-terminal extension of PfHU influences its DNA condensation properties. In contrast to bacterial HUs that bend DNA, PfHU promotes concatenation of linear DNA and inhibits DNA circularization. Atomic Force Microscopic study of PfHU-DNA complexes shows protein concentration-dependent DNA stiffening, intermolecular bundling and formation of DNA bridges followed by assembly of condensed DNA networks. Our results provide the first functional characterization of an apicomplexan HU protein and provide additional evidence for red algal ancestry of the apicoplast.
Subject(s)
DNA, Protozoan/chemistry , DNA-Binding Proteins/metabolism , Plasmodium falciparum/genetics , Plastids/genetics , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , DNA, Concatenated/chemistry , DNA, Protozoan/metabolism , DNA, Protozoan/ultrastructure , DNA-Binding Proteins/chemistry , Microscopy, Atomic Force , Models, Molecular , Molecular Sequence Data , Protein Folding , Protozoan Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
The endosymbiont-bearing trypanosomatids present a typical kDNA arrangement, which is not well characterized. In the majority of trypanosomatids, the kinetoplast forms a bar-like structure containing tightly packed kDNA fibers. On the contrary, in trypanosomatids that harbor an endosymbiotic bacterium, the kDNA fibers are disposed in a looser arrangement that fills the kinetoplast matrix. In order to shed light on the kinetoplast structural organization in these protozoa, we used cytochemical and immunocytological approaches. Our results showed that in endosymbiont-containing species, DNA and basic proteins are distributed not only in the kDNA network, but also in the kinetoflagellar zone (KFZ), which corresponds to the region between the kDNA and the inner mitochondrial membrane nearest the flagellum. The presence of DNA in the KFZ is in accordance with the actual model of kDNA replication, whereas the detection of basic proteins in this region may be related to the basic character of the intramitochondrial filaments found in this area, which are part of the complex that connects the kDNA to the basal body. The kinetoplast structural organization of Bodo sp. was also analyzed, since this protozoan lacks the highly ordered kDNA-packaging characteristic of trypanosomatid and represents an evolutionary ancestral of the Trypanosomatidae family.
Subject(s)
DNA, Kinetoplast/ultrastructure , DNA, Protozoan/ultrastructure , Nucleic Acid Conformation , Symbiosis , Trypanosomatina/ultrastructure , Animals , Crithidia/microbiology , Crithidia/ultrastructure , Evolution, Molecular , Flagella/microbiology , Flagella/ultrastructure , Freeze Etching , Histocytochemistry , Immunohistochemistry , Microscopy, Electron, Transmission , Mitochondrial Membranes/microbiology , Mitochondrial Membranes/ultrastructure , Trypanosomatina/microbiologyABSTRACT
Mitochondrial DNA of Kinetoplastea is composed of different chromosomes, the maxicircle (bearing 'regular' genes) and numerous minicircles (specifying guide RNAs involved in RNA editing). In trypanosomes [Kinetoplastea], DNA circles are compacted into a single dense body, the kinetoplast. This report addresses the question whether multi-chromosome mitochondrial genomes and compacted chromosome organization are restricted to Kinetoplastea or rather occur throughout Euglenozoa, i.e., Kinetoplastea, Euglenida and Diplonemea. To this end, we investigated the diplonemid Rhynchopus euleeides and the euglenids Petalomonas cantuscygni, Peranema trichophorum and Entosiphon sulcatum, using light and electron microscopy and molecular techniques. Our findings together with previously published data show that multi-chromosome mitochondrial genomes prevail across Euglenozoa, while kinetoplast-like mtDNA packaging is confined to trypanosomes.
Subject(s)
DNA, Mitochondrial/genetics , Euglenida/genetics , Mitochondria/genetics , Animals , DNA, Circular/genetics , DNA, Circular/isolation & purification , DNA, Circular/ultrastructure , DNA, Kinetoplast/genetics , DNA, Kinetoplast/isolation & purification , DNA, Kinetoplast/ultrastructure , DNA, Mitochondrial/isolation & purification , DNA, Mitochondrial/ultrastructure , DNA, Protozoan/genetics , DNA, Protozoan/isolation & purification , DNA, Protozoan/ultrastructure , Euglenida/ultrastructure , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Mitochondria/diagnostic imaging , UltrasonographyABSTRACT
Dinoflagellates are eukaryotic microalgae with distinct chromosomes throughout the cell cycle which lack histones and nucleosomes. The molecular organization of these chromosomes is still poorly understood. We have analysed the presence of telomeres in two evolutionarily distant and heterogeneous dinoflagellate species (Prorocentrum micans and Amphidinium carterae) by FISH with a probe containing the Arabidopsis consensus telomeric sequence. Telomere structures were identified at the chromosome ends of both species during interphase and mitosis and were frequently associated with the nuclear envelope. These results identify for the first time telomere structures in dinoflagellate chromosomes, which are formed in the absence of histones. The presence of telomeres supports the linear nature of dinoflagellate chromosomes.
Subject(s)
DNA, Protozoan/metabolism , Dinoflagellida/cytology , Dinoflagellida/genetics , Evolution, Molecular , Telomere/genetics , Telomere/metabolism , Animals , Arabidopsis , DNA, Protozoan/analysis , DNA, Protozoan/ultrastructure , Dinoflagellida/ultrastructure , In Situ Hybridization, Fluorescence , Interphase , Telomere/ultrastructureABSTRACT
We have analyzed by transmission electron microscopy the mitotic process of Entamoeba histolytica trophozoites in an asynchronous population of axenically cultured parasites. Our observations showed that nuclear microtubules, initially located at random in the karyosome during prophase, formed in subsequent stages a mitotic spindle closely related to the nuclear membrane at the polar regions of dividing nuclei. In late prophase and in anaphase, chromosomes appeared as dense bodies 0.1-0.5 microm. At least 15 chromosomes appeared in favorable planes of section, arranged as an incomplete elliptical circle, in close contact with microtubules. There was no morphological evidence of structures resembling the kinetochores of higher eukaryotes. When cut in cross-section, the mitotic spindle was made of 28-35 microtubular rosette assemblies. The latter probably correspond to a similar number of chromosomes, as has been shown by others with pulse-field electrophoresis and fluorescence microscopy of trophozoite spreads. In turn, each microtubular rosette was constituted by 7-12 parallel microtubules. In later stages of the metaphase, two sets of chromosomes were disposed forming a pair of elliptical circles. An additional finding in the dividing nuclei of E. histolytica trophozoites was the presence of compact conglomerates of numerous particles 50 nm in diameter, of similar electron density, shape, and size, probably corresponding to RNA episomes.
Subject(s)
Chromosomes/ultrastructure , Entamoeba histolytica/ultrastructure , Spindle Apparatus/ultrastructure , Animals , DNA, Protozoan/ultrastructure , Entamoeba histolytica/physiology , Microscopy, Electron, Transmission , Microtubules/ultrastructure , Mitosis/physiologyABSTRACT
Kinetoplast DNA (kDNA), the mitochondrial DNA of flagellated protozoa of the order Kinetoplastida, is unique in its structure, function and mode of replication. It consists of few dozen maxicircles, encoding typical mitochondrial proteins and ribosomal RNA, and several thousands minicircles, encoding guide RNA molecules that function in the editing of maxicircles mRNA transcripts. kDNA minicircles and maxicircles in the parasitic species of the family Trypanosomatidae are topologically linked, forming a two dimensional fishnet-type DNA catenane. Studies of early branching free-living and parasitic species of the Bodonidae family revealed various other forms of this remarkable DNA structure and suggested the evolution of kDNA from unlinked DNA circles and covalently-linked concatamers into a giant topological catenane. The replication of kDNA occurs during nuclear S phase and includes the duplication of free detached minicircles and catenated maxicircle and the generation of two progeny kDNA networks that segregate upon cell division. Recent reports of sequence elements and specific proteins that regulate the periodic expression of replication proteins advanced our understanding of the mechanisms that regulate the temporal link between mitochondrial and nuclear DNA synthesis in trypanosomatids. Studies on kDNA replication enzymes and binding proteins revealed their remarkable organization in clusters at defined sites flanking the kDNA disk, in correlation with the progress in the cell cycle and the process of kDNA replication. In this review I describe the recent advances in the study of kDNA and discuss some of the major challenges in deciphering the structure, replication and segregation of this remarkable DNA structure.
Subject(s)
DNA Replication , DNA, Circular/genetics , DNA, Kinetoplast/genetics , Kinetoplastida/chemistry , Trypanosomatina/genetics , Animals , Cell Nucleus/physiology , Cell Nucleus/ultrastructure , DNA, Catenated , DNA, Circular/chemistry , DNA, Circular/isolation & purification , DNA, Circular/ultrastructure , DNA, Kinetoplast/chemistry , DNA, Kinetoplast/ultrastructure , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , DNA, Protozoan/chemistry , DNA, Protozoan/genetics , DNA, Protozoan/ultrastructure , DNA-Binding Proteins/metabolism , Kinetoplastida/enzymology , Kinetoplastida/ultrastructure , Models, Biological , Protozoan Proteins/metabolism , S Phase , Trypanosomatina/ultrastructureABSTRACT
The dicyemid mesozoans are simple multicellular parasites with a long cylindrical axial cell surrounded by a single outer layer of 20 to 30 ciliated peripheral somatic cells. Their larval development proceeds within the axial cell. Here we demonstrate the appearance of extrachromosomal circular DNAs and their fate during early embryogenesis in Dicyema japonicum. These DNAs are highly heterogeneous in sequence, suggesting that they consist of unique--not repetitive--elements. Potential open reading frames were not evident in the elements, so these DNAs are unlikely to have a protein-encoding function. In situ hybridization revealed that the circular DNA elements were restricted to the early embryonic larvae and gradually faded out as larvae approached maturity. Furthermore Southern blot analysis and polymerase chain reaction analysis using a high molecular weight DNA as a template provided evidence that the extrachromosomal DNA circles are originally present in chromosomes. These observations suggest DNA elimination--or selective replication--of the elements from chromosomes during early embryogenesis in dicyemid mesozoans.
Subject(s)
DNA, Circular/biosynthesis , DNA, Protozoan/biosynthesis , Invertebrates/embryology , Invertebrates/genetics , Animals , Chromosomes/genetics , DNA Replication , DNA, Circular/chemistry , DNA, Circular/ultrastructure , DNA, Protozoan/chemistry , DNA, Protozoan/ultrastructure , Invertebrates/growth & development , Larva/genetics , Larva/growth & development , Microscopy, Electron, Scanning , Molecular Sequence Data , Molecular Weight , Octopodiformes/parasitology , Time FactorsABSTRACT
Supramolecular chromatin organization of the somatic nucleus (macronucleus--Ma) was studied in a free-living unicellular eukaryotic organism, the ciliate Bursaria ovata Beers 1952, at two late successive stages of its encystment (in the state of preparation to cryptobiosis). A modified Miller's method (Sergejeva et al., 1987) and the same technique in combination with high resolution DNA autoradiography were used. In chromatin spread preparations of Ma, not labeled by 3H-thymidine, numerous electron dense structures (rounded, stick-like and polygonal) were revealed, along with rarely occurring typical supramolecular chromatin structures, such as nucleosomic and not nucleosomic threads, nucleomeres, chromomeres, rosette-like looping chromatin, and electron dense chromonemes (Fig. 1). For DNA visualizing in the revealed polygonal structures, the vegetative cells (trophonts) of B. ovata were inoculated into the culture medium, containing 3H-thymidine and food (ciliates Paramecium caudatum). Here, the ciliates passed through 3-4 successive cell division cycles, thus progressively accumulating the radioactive DNA precursor in Ma. After washing the ciliates in 3H-thymidine-free culture medium, the process of their encystment was induced, and Ma were isolated by hand from the ciliates being at two late successive stages of encystment. Isolated Ma were dispersed in the low ionic solutions, as described elsewhere (Sergejeva et al., 1987). The carbon shadowed electron grids, that contained spread Ma preparations, were individually coated with photographic emulsion, according to the loop interference method (Angelier et al., 1976a; Bouteille, 1976). After a 6 month exposure at 4 degrees C, thymidine incorporation was revealed in fibril crowds, rosette-like structures (Fig. 2), and crystal-like plates of different size and morphology (Fig. 3). In all our experiments, non-specific localization of radioactive DNA precursor was not observed. The above data confirm undoubtedly our earlier assumption (Sergejeva et al., 1987; Sergejeva, Bobyleva, 1988) that the Ma chromatin of Bursaria may undergo crystallization during encystment, i.e. in the state of preparation to cryptobiosis. The present data enable us first to suggest that the looping rosette-like chromatin may be transformed into crystal-like structures ("exotic liquid crystal structures") by means of a peculiar loop packing within the limits of an individual resette (Fig. 2-4), these structural transformations taking place without any visible loop destruction. In this paper, we first describe new morphological types of polygonal plates, differing from those earlier reported elsewhere for the Ma of Bursaria (Sergejeva, Bobylova, 1988), and also from the plates earlier described in studies on liquid crystals both in vivo and in vitro (see: Gianonni et al., 1969; Lerman, 1974; Livolant, 1991: Leforestier et al., 1993, 1997, 1999). The technical approaches used in the present work enabled us to obtain, for the first time, a direct evidence of the presence of DNA in the crystallized structures of somatic nuclei of ciliates during their preparation to cryptobiosis, the DNA-containing polygonal structures being highly morphologically diverse. Further studies into the reasons of this diversity are needed.
Subject(s)
Ciliophora/ultrastructure , DNA, Protozoan/ultrastructure , Adaptation, Physiological , Animals , Autoradiography , Chromatin/ultrastructure , Ciliophora/physiologyABSTRACT
In common with other apicomplexan parasites, Plasmodium falciparum, a causative organism of human malaria, harbours a residual plastid derived from an ancient secondary endosymbiotic acquisition of an alga. The function of the 35 kb plastid genome is unknown, but its evolutionary origin and genetic content make it a likely target for chemotherapy. Pulsed field gel electrophoresis and ionizing radiation have shown that essentially all the plastid DNA comprises covalently closed circular monomers, together with a tiny minority of linear 35 kb molecules. Using two-dimensional gels and electron microscopy, two replication mechanisms have been revealed. One, sensitive to the topoisomerase inhibitor ciprofloxacin, appears to initiate at twin D-loops located in a large inverted repeat carrying duplicated rRNA and tRNA genes, whereas the second, less drug sensitive, probably involves rolling circles that initiate outside the inverted repeat.
Subject(s)
DNA Replication , DNA, Circular/biosynthesis , DNA, Protozoan/biosynthesis , Plasmodium falciparum/genetics , Plastids/genetics , Animals , Anti-Infective Agents/pharmacology , Ciprofloxacin/pharmacology , DNA Replication/drug effects , DNA, Circular/genetics , DNA, Circular/ultrastructure , DNA, Protozoan/genetics , DNA, Protozoan/ultrastructure , Electrophoresis, Gel, Pulsed-Field , Electrophoresis, Gel, Two-Dimensional , Microscopy, Electron , Models, Genetic , Plasmodium falciparum/drug effects , Plasmodium falciparum/ultrastructure , Plastids/drug effects , Plastids/ultrastructure , Protozoan Proteins/antagonists & inhibitors , Topoisomerase II InhibitorsABSTRACT
We studied the function of a Trypanosoma brucei topoisomerase II using RNA interference (RNAi). Expression of a topoisomerase II double-stranded RNA as a stem-loop caused specific degradation of mRNA followed by loss of protein. After 6 days of RNAi, the parasites' growth rate declined and the cells subsequently died. The most striking phenotype upon induction of RNAi was the loss of kinetoplast DNA (kDNA), the cell's catenated mitochondrial DNA network. The loss of kDNA was preceded by gradual shrinkage of the network and accumulation of gapped free minicircle replication intermediates. These facts, together with the localization of the enzyme in two antipodal sites flanking the kDNA, show that a function of this topoisomerase II is to attach free minicircles to the network periphery following their replication.
Subject(s)
DNA Topoisomerases, Type II/metabolism , DNA, Kinetoplast/genetics , DNA, Mitochondrial/genetics , RNA, Protozoan/genetics , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/genetics , Animals , DNA Replication , DNA Topoisomerases, Type II/genetics , DNA, Kinetoplast/metabolism , DNA, Kinetoplast/ultrastructure , DNA, Mitochondrial/metabolism , DNA, Mitochondrial/ultrastructure , DNA, Protozoan/genetics , DNA, Protozoan/metabolism , DNA, Protozoan/ultrastructure , Kinetics , Phenotype , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Time Factors , Trypanosoma brucei brucei/growth & developmentABSTRACT
The Phylum Apicomplexa comprises thousands of obligate intracellular parasites, some of which cause serious disease in man and other animals. Though not photosynthetic, some of them, including the malaria parasites (Plasmodium spp.) and the causative organism of Toxoplasmosis, Toxoplasma gondii, possess a remnant plastid partially determined by a highly derived residual genome encoded in 35 kb DNA. The genetic maps of the plastid genomes of these two organisms are extremely similar in nucleotide sequence, gene function and gene order. However, a study using pulsed field gel electrophoresis and electron microscopy has shown that in contrast to the malarial version, only a minority of the plastid DNA of Toxoplasma occurs as circular 35 kb molecules. The majority consists of a precise oligomeric series of linear tandem arrays of the genome, each oligomer terminating at the same site in the genetic map, i.e. in the centre of a large inverted repeat (IR) which encodes duplicated tRNA and rRNA genes. This overall topology strongly suggests that replication occurs by a rolling circle mechanism initiating at the centre of the IR, which is also the site at which the linear tails of the rolling circles are processed to yield the oligomers. A model is proposed which accounts for the quantitative structure of the molecular population. It is relevant that a somewhat similar structure has been reported for at least three land plant chloroplast genomes.