Purification of Entamoeba histolytica DNA Containing Organelles (EkhOs): A Further Characterization (original) (raw)
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A novel cytoplasmic structure containing DNA networks in Entamoeba histolytica trophozoites
Molecular and General Genetics, 1997
We report here the presence of cytoplasmic DNA arranged in networks in the trophozoites of the human parasite Entamoeba histolytica. Cytoplasmic DNA was detected in live trophozoites in a structure that we called EhkO, using the fluorescent dye acridine orange, and by in situ hybridization to trophozoites with a rDNA probe. The EhkO was found in the axenically grown clones A, L6 (strain HM1:IMSS) and MAVax (strain MAV) and in the polyxenically grown clone MAVpx (strain MAV). Bacteria present in MAVpx did not cross hybridize with the DNA probe neither in in situ hybridization or in Southern blot experiments. Autoradiography of metabolically [ 3 H]thymidine-labeled trophozoites showed the presence of EhkO, and an EhkO-enriched fraction, purified from a nuclei-free extract and examined by light microscopy, exhibited [ 3 H]thymidine incorporation into this structure. DNA was purified from the EhkO and enriched nuclear fractions and analyzed by transmission electron microscopy. The EhkO fraction contained DNA networks resembling those of trypanosome kDNA, whereas nuclear DNA was present mainly as linear molecules and some circles. Our findings imply that E. histolytica may be taxonomically more closely related to the Trypanosomatidae than previously suspected.
Entamoeba histolytica: DNA carrier vesicles in nuclei and kinetoplast-like organelles (EhkOs)
Molecular Genetics and Genomics, 2002
Entamoeba histolytica, the protozoan responsible for human amoebiasis, has a complex genome, whose linear chromosomes and DNA circles have so far eluded detailed analysis. We report the detection by transmission electron microscopy of nuclear vesicles (0.05-0.3 lm in diameter) carrying DNA in E. histolytica trophozoites. In late anaphase many of these nuclear vesicles were found to be organized in structures of 2.5·1 lm, in association with chromosomes and microtubules. In glutaraldehyde-fixed and detergenttreated trophozoites, nuclear vesicles displayed a nonmembranous envelope. Binding of phosphotungstate stain and recognition by serum from patients with systemic lupus erythematosus indicated that these vesicles contain DNA. Similar DNA carrier vesicles were found in the cytoplasm and in the E. histolytica kinetoplast-like organelle (EhkO). By Feulgen staining, we detected DNA carrier vesicles entering or leaving the nuclei, suggesting a structural relationship between the nuclear vesicles and the vesicles present in the EhkOs.
Entamoeba histolytica: ultrastructure of the chromosomes and the mitotic spindle
Experimental parasitology, 2006
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 micros...
Behavior of DNA-lacking mitochondria in Entamoeba histolytica revealed by organelle transplant
Scientific reports, 2017
The anaerobic protozoan parasite Entamoeba histolytica has mitosomes that are mitochondria lacking some canonical functions and organelle DNA. Mitosomes play an important role in the life cycle of the parasite. The distribution of proteins in mitosomes is not uniform, and how mitosomes are maintained and retained is unknown. To answer these questions, we developed a transplant method for mitosomes with hemagglutinin-tagged protein into recipient cells containing mitosomes with Myc-tagged protein. Immunofluorescence staining showed that the two protein tags colocalized in single mitosomes in some recipient cells. These results suggest that our transplant method can be used in anaerobic protozoa and that donor mitosomes may obtain recipient proteins through fusion with other mitosomes or through de novo synthesis of proteins in recipient cells.
Circular and linear DNA molecules in the Entamoeba histolytica complex molecular karyotype
European Biophysics Journal, 2000
Entamoeba histolytica genome was analysed by pulsed ®eld gel electrophoresis under conditions to separate linear chromosomes in the 170±1400 kb range. We identi®ed linear DNA molecules of 227, 366, 631, 850, 1112 and 1361 kb (mean sizes obtained by three dierent methods) and we estimated their reorientation times and migration velocities at various experimental conditions. DNA shift mobility assays, using ethidium bromide, suggested that bands migrating at 227 and 631 kb contain linear and circular DNA, whereas a band at 436 kb has only circular DNA. We obtained a regression equation relating sizes of supercoiled DNA molecules with their migration velocities during a pulse at constant electric ®eld and temperature. We also developed a computer program (EHPATTERNS) that predicts the migration per pulse and the resolution order of circular and linear E. histolytica DNA at dierent pulse times and constant driving and frictional forces. The simulation showed that linear DNA molecules frequently co-migrate with circular molecules, but circular molecules change when the pulse time varies. This molecular mixture generates broad bands and diculties in the interpretation of the molecular karyotype of E. histolytica.
Microbiology-sgm, 2004
The existence of mitochondrion-related relict organelles (mitosomes) in the amitochondrial human pathogen Entamoeba histolytica and the detection of extranuclear DNA-containing cytoplasmic structures (EhKOs) has led to the suggestion that a remnant genome from the original mitochondrial endosymbiont might have been retained in this organism. This study reports on the mutually exclusive distribution of Cpn60 and extranuclear DNA in E. histolytica and on the distribution of Cpn60-containing mitosomes in this parasite. In situ nick-translation coupled to immunofluorescence microscopy failed to detect the presence of DNA in mitosomes, either in fixed parasite trophozoites or in partially purified organellar fractions. These results indicate that a remnant organellar genome has not been retained in E. histolytica mitosomes and demonstrate unequivocally that EhKOs and mitosomes are distinct and unrelated cellular structures.
Electron microscopy of native DNA in sea urchin cells
Journal of Ultrastructure Research, 1967
A method is described for the extraction of native DNA from cells and its spreading with chymotrypsin. No evidence of discontinuities in the fibrils was recorded either after staining or shadowing. Evidence is presented for the DNA nature of the strands. The DNA from sea urchin sperm and sea urchin embryo nuclei appears as strands longer than 100 #. The height of the strands is about 21-25 •. Proteases do not affect the dimensions of the strands, bat DNase and shearing rapidly degrade them.