Abstract We report here the presence of cytoplasmic. DNA arranged in networks in the trophozoites of the human parasite Entamoeba histolytica. Cytoplasmic.
Springer-Verlag 1997
Mol Gen Genet (1997) 254:250 – 257
ORIGINAL PAPER
E. Orozco · R. Gharaibeh · A. M. Rivero´n D. M. Delgadillo · M. Mercado · T. Sa´nchez E. Go´mez Conde · M. A. Vargas · R. Lo´pez-Revilla
A novel cytoplasmic structure containing DNA networks in Entamoeba histolytica trophozoites
Received: 7 August 1996 / Accepted: 7 October 1996
Abstract 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 [3H]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 [3H]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
Communicated by W. Goebel E. Orozco · D. M. Delgadillo · E. Go´mez Conde M. A. Vargas · R. Lo´pez-Revilla Program of Molecular Biomedicine, CINVESTAV-IPN, A.P. 14-470, Me´xico 07300 D.F. R. Lo´pez-Revilla Department of Cell Biology, CINVESTAV-IPN A.P. 14-740, Me´xico 07300 D.F. A. M. Rivero´n National Center for Scientific Research (CNIC) P.O. Box 6990, Cubanacan, Playa, Havana, Cuba E. Orozco (&) · R. Gharaibeh · M. Mercado · T. Sa´nchez Department of Experimental Pathology, CINVESTAV-IPN, A.P. 14-470, Me´xico 07300 D.F.
circles. Our findings imply that E. histolytica may be taxonomically more closely related to the Trypanosomatidae than previously suspected. Key words DNA networks · Entamoeba histolytica · DNA organization
Introduction Entamoeba histolytica, the microaerophilic protozoan species responsible for human amebiasis, belongs to the phylum Sarcomastigophora, like the aerobic Trypanosomatidae. A mitochondrion with kinetoplast DNA (kDNA) formed by maxicircles and minicircles catenated into a network characterizes Trypanosomatidae (Simpson and Silva 1971; Borst 1991; Cozzarelli 1993; Ferguson et al. 1994) and places them in the Order Kinetoplastidae, formed by organisms which are known to contain kDNA networks in their mitochondria (Borst 1991). Some 25 years ago Albach et al. (1969) suggested the presence of cytoplasmic DNA in E. histolytica. However, Albach et al. (1980) later on explained these findings as being due to bacterial contamination. Therefore, it is currently accepted that E. histolytica lacks extranuclear DNA and mitochondria (Martı´nez Palomo 1986; Clark and Roger 1995). Phylogenetic studies of the chaperonin 60 protein (cpn60) place E. histolytica between Trypanosoma cruzi and Rickettsia tsutsugamushi, suggesting that it may form the first branch of amitochondrial organisms (Clark and Roger 1995). In contrast, studies of the rDNA small subunit sequence locate E. histolytica on a phylogenetic branch that emerged after the appearance of organisms with typical eukaryotic organelles (Cavalier-Smith 1993; Hasegawa et al. 1993). Ubiquinone (CoQ) has been identified (Ellis et al. 1994) and several housekeeping mitochondrial genes have been cloned from this parasite (Clark and Roger 1995; Ellis et al. 1994); their presence has been explained as deriving from the nuclear remnant of a mitochondrial primordium, which was secondarily
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lost (Clark and Roger 1995). However, the presence of cytoplasmic DNA as a remnant of this mitochondrial primordium has not been totally excluded. We have carried out a number of experiments to elucidate some aspects of the unique DNA organization in this parasite. Our recent results demonstrate the presence of extranuclear DNA, and rosette-like structures were observed in total DNA (Ba´ez-Camargo et al., in press). In this paper we report the identification, isolation and initial characterization of a novel E. histolytica cytoplasmic structure, designated the EhkO, that contains DNA networks (EhkDNA), similar in some respects to the kDNA of Trypanosomatidae (Simpson and Silva 1971; Borst 1991; Cozzarelli 1991; Ferguson et al. 1994; Robinson and Gull 1994; Pe´rez-Morga and England 1993). These findings place E. histolytica probably much closer to the Trypanosomatidae than expected; additionally, they open the possibility that the EhkO corresponds to an organelle, the functions of which are currently under study.
presence of [35S]dUTP (Ausubel et al. 1993) from the Bluescript phagemid in which it was cloned. Hybridization was carried out in the presence of 50% formamide at 42° C and washing steps were done at 65° C in 0.1 × SSC (1 × SSC is 0.15 M NaCl, 15 mM sodium citrate, pH 7.0) (Ausubel et al. 1993). Slides were covered with an emulsion film (Kodak) and developed after 5–7 days of exposure in the dark, stained with Giemsa and observed with the immersion objective of an optical microscope at 100 × (Olympus). As a control for the absence of bacteria in the axenic cultures we plated one ml of the trophozoite cultures in Luria and Terrific broth media (Ausubel et al. 1993). We also separated high molecular weight DNA by transverse alternating field electro-phoresis (TAFE) (Ba´ez-Camargo et al., in press) using 3 × 106 trophozoite of clones A, MAVax, MAVmx (grown in TY1-S-33 medium with Fusobacterium simbiosum) and MAVpx, and approximately 108 bacteria from the intestinal flora that was used to culture the MAVpx clone (Vargas and Orozco 1993). Then, DNA was transferred to Nylon membranes and hybridized with the 500 bp probe for the16S rDNA gene as described by Ba´ez-Camargo et al. (in press). In vivo [3H]thymidine labeling and isolation of the structures that incorporated [3H]thymidine
Trophozoites of clones A, L6 (strain HM1:IMSS) (Orozco et al. 1983) and MAVax (Vargas and Orozco 1993) were axenically cultured in TY1-S-33 medium as described before (Diamond et al. 1978), while clone MAVpx was cultured in Robinson medium in the presence of its original intestinal flora (Vargas and Orozco 1993). All trophozoites were harvested in logarithmic phase from rich culture media.
Trophozoites were grown for 14 h in TYI-S-33 medium supplemented with 10 lCi [3H]thymidine/ml, washed with PBS pH 6.8 and resuspended in a buffer containing 0.3 M sucrose, 1 mM EDTA and 10 mM TRIS-HCl, pH 8, before being mechanically disrupted in a Dounce homogenizer. The homogenate was centrifuged three times at 2,500 rpm (Beckman TJ-C) at 4° C for 10 min to pellet the nuclei; the postnuclear supernatant was retained. The integrity of the nuclei was checked by phase-contrast microscopy and by autoradiography of the labeled nuclei. The postnuclear supernatant, containing the EhkOs, was centrifuged again for 15 min at 8 500 rpm (Beckman JA-20 rotor) and 4° C, and the pellet was resuspended in 4% paraformaldehyde, spread on slides, covered with autoradiographic film emulsion (Kodak), exposed for 5 days in the dark, developed, stained with Giemsa, and observed with the immersion objective of an optical microscope at 100 x.
Acridine orange staining of live and paraformaldehyde-fixed trophozoites
Transmission electron microscopy (TEM) of DNA from EhkO and nuclear fractions prepared from clone A trophozoites
Trophozoites in logarithmic phase were chilled on ice, centrifuged for 5 min at 1,200 rpm and resuspended in PBS at 106 trophozoites per ml. Aliquots of 100 ll were spotted on coverslips that were placed in 50 ml conical tubes with TYS-1–33 medium, and horizontally incubated for 30 min at 37° C. Then, the coverslips were taken out with forceps, rinsed twice with phosphate-buffered saline PBS pH 6, prewarmed at 37° C, and incubated for 10 min at 37° C in a humid chamber with 0.1% acridine orange prepared in PBS pH 6. Trophozoites were washed twice with PBS at 37° C and the coverslips were placed on slides with a drop of PBS prewarmed at 37° C and immediately observed with a fluorescence microscope (Olympus). In parallel experiments, trophozoites were fixed on the slides with 4% paraformaldehyde before incubation with the acridine orange dye. As a control, the trophozoites on the coverslips were treated with 50 U DNase for 1–6 h at 37° C before adding the acridine orange.
To perform TEM analysis, the EhkO and enriched nuclear fractions were treated for 1 h with 5 mg/ml proteinase K at 37° C, extracted once with phenol, and incubated for 1 h with 50 lg/ml RNase before extracting again with phenol:chloroform and precipitating the DNA with ethanol. A hyperphase solution with 100 ng of EhkO or nuclear DNA was prepared in a buffer containing 200 mM TRIS-HCl (pH 7.5), 20 mM EDTA, 50% (v/v) formamide and 0.01 mg/ml cytochrome c. The solution was spread on a hypophase formed by 17% formamide, 100 mM TRIS-HCl (pH 8.0) and 1 mM EDTA, and samples were picked up on collodioncoated grids, and stained with 0.5 mM uranyl acetate. The grids were rotary shadowed with platinum/palladium (80:20) at a 6 ° angle (Davis et al. 1971). Circular pBR322 was used as a control for DNA integrity and for the absence of artifactual structures in the samples. Samples were observed through a JEOL Temscan electron microscope.
In situ hybridization of E. histolytica trophozoites with the ribosomal DNA EhVR1 probe
Results
Materials and methods Culture of E. histolytica trophozoites
Trophozoites of the axenically growing clones A, L6 and MAVax, and of the clone MAVpx growing with intestinal flora, were fixed with 4% paraformaldehyde, placed on slides at 37° C for 1 h and incubated for 3 h with 50 U RNase/ml at 37° C. A 500 bp DNA probe derived from the EhVR1 fragment (Orozco et al. 1993) of the 5′ end of the 16S rDNA gene was labeled by random priming (Ausubel et al. 1993) using [35S]dATP. For control experiments, the single-stranded sense DNA probe was transcribed in vitro in the
Localization of cytoplasmic DNA in acridine orange-stained live and paraformaldehyde-fixed trophozoites Most of the live and paraformaldehyde-fixed trophozoites treated with acridine orange showed green fluorescence in the central part of the nuclei, corresponding
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to DNA (Fig. 1A–C), in agreement with the Feulgen stain experiments carried out by Pan and Geiman (1955) and by Albach et al. (1980), who found [3H]thymidinelabeled DNA in the endosome. Green fluorescence in the nuclei was surrounded by red or orange fluorescence corresponding to RNA, in agreement to the results of the same authors. RNA was also very abundant in the cytoplasm (Fig. 1A–C). In 90% of the live and motile, as well as the fixed trophozoites, green fluorescence was also detected in a cytoplasmic structure of a polymorphic shape. The 50% of trophozoites with cytoplasmic fluorescence exhibited a green spot approximately 0.5– 2.5 lm in diameter (Fig. 1A, B), brighter and smaller than the nuclei; we called this structure the EhkO. Differences in fluorescence intensity between the nucleus and the EhKO may be due to differences in DNA content or structure. The other 50% of the trophozoites presented one or more fluorescent spots that were smaller than 0.5 lm in diameter. Paraformaldehydefixed trophozoites displayed fluorescent patterns similar to those seen in live trophozoites (Fig. 1C). Trophozoites that had been treated for 6 h with DNase did not show green fluorescence (Fig. 1D).
Fig. 1A–D Acridine orange staining of trophozoites. A, B. Live trophozoites. C Paraformaldehyde-fixed trophozoites. D Trophozoites fixed with 2% paraformaldehyde and treated with 50 U DNase for 6 h. A and D show whole trophozoites, while B and C show the nucleus and EhkO area. N, nucleus Bars, 5 lm
Labeling of EhkO by in situ hybridization of the trophozoites of clone A with a 500 bp probe from the E. histolytica 16S rDNA gene In Fig. 2 we present details of the results obtained with trophozoites of clone A by in situ hybridization with a 500 bp fragment from the 5′ end of the 16S rDNA gene. In agreement with our previous results (Ba´ez-Camargo et al., in press) the in situ hybridization experiments carried out here showed the DNA to be distributed between the nucleus and the cytoplasm (Fig. 2A–D, F). As was found in the acridine orange assays (Fig. 1), in paraformaldehyde-fixed trophozoites the ribosomal probe detected a faint signal in the nucleus and a strong signal in the cytoplasm (Fig. 2A–D). In most trophozoites the cytoplasmic hybridization was seen as a 0.5–2.5 lm long, compact V-shaped structure (Fig. 2A) and in 10–20% of the positive trophozoites we found a duplicate structure which may correspond to a dividing EhkO (Fig. 2B). To prove that the hybridization signal indeed comes from DNA, we carried out several controls. (i) The trophozoites of clone A were also hybridized with a 1-kb fragment of the EhVR1 variable region located upstream
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Fig. 2A–I In situ hybridization of trophozoites with DNA probes contained in the EhVR1 fragment of the 16S rDNA gene. Paraformaldehyde-fixed trophozoites (4%) were treated with 60 U RNase for 3 h and hybridized in situ with a denatured 500 bp doublestranded probe containing the 5′ end of the 16S rDNA gene (A–C) or with the single-stranded sense transcript (E, F) or with a 1000 bp probe located upstream of the 5′ end of the 16S rDNA gene (D). In some experiments the trophozoites were incubated with 50 U DNase at 37° C for 3 h incubated (C, D, F), or 6 h (E). Trophozoites were
also grown in the presence of 10 lCi/ml [3H]uridine for 24 h (G, H) and untreated (G) or treated (H) with 60 U RNase for 3 h. Trophozoites hybridized with the random primer labeled Bluescript phagemid in which the probes were cloned are shown in I. Slides were exposed for five days in the dark (A–F, I) or for three weeks in the dark (G, H) before developing and observing in the light microscope. Panels and H, I were photographed at 40 × and panels A–G at 100 × magnification. N, nucleus. Bars: A–E = 6 lm, H, I = 10 lm
of the 5′ end of the 16S gene (Orozco et al. 1993). Hybridization with this fragment yielded the same results as were obtained with the 5′ end of the 16S gene (Fig. 2D). (ii) The sensitivity of the EhkO signal to DNase was tested by incubating the trophozoites with 50 U of the enzyme for different times. Untreated trophozoites showed the 0.5–2.5 lm EhkO (Fig. 2A, B), after 3 h incubation they showed more diffuse labeling of the EhkO structure (Fig. 2C, D), and this disappeared completely after 6 h incubation (Fig. 2E). (iii) The trophozoites were hybridized with the single-stranded sense transcript. No hybridization signal was detected in DNase treated trophozoites (Fig. 2E), while the untreated trophozoites display the EhkO (Fig. 2F), strongly suggesting that the material in this structure is DNA. Results from these experiments suggest that the DNase acts first by relaxing the DNA and then digesting it. This assumption is supported by analogy with Trypanosomatidae, which have small histone-like proteins that protect kDNA from nuclease attack (Tittawella et al. 1993). (iv) The trophozoites were grown in the presence of [3H]uridine and submitted to RNase digestion for the same time as used for cells on slides. The
untreated trophozoites (Fig. 2G) showed labeled RNA distributed throughout the cytoplasm, whereas the RNase treated cells showed no radioactive label (Fig. 2H). To ensure that no labeled RNA remained the slides were overexposed for three weeks. No RNA was detected after the RNase treatment, corroborating that the ribosomal probe hybridized specifically with DNA in the RNase treated trophozoites used in our experiments. (v) The trophozoites were in situ hybridized with the Bluescript phagemid in which the probes were cloned. The phagemid did not give any radioactive signal, ruling out the possibility of unspecific hybridization (Fig. 2I). Hybridization of trophozoites from the axenic clones L6 and MAVax and the polyxenic clone MAVpx with ribosomal DNA probes In order to determine if EhkO was present in other E. histolytica clones and strains, we carried out the in situ hybridization experiments with axenically grown trophozoites of clones MAVax and L6, and with the polyxenically grown MAVpx trophozoites. EhkO was
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with our published results (Orozco et al. 1993). The DNA of clone MAVpx gave a fainter hybridization signal than the DNA of the other clones; we believe that this is because the amount of E. histolytica DNA was overestimated due to the presence of bacteria grown together with the trophozoites. These experiments confirmed that the hybridization signals associated with the EhkO in the in situ hybridization experiments are not due to bacterial contamination and that the EhkO is present in the cytoplasm of all four clones tested. In vivo [3H]thymidine labeling of trophozoites and isolation of an EhkO-enriched cell fraction
Fig. 3A–E Trophozoites of clones L6 (strain HM1:IMSS), MAVax and of the MAVpx (strain MAV) hybridized in situ with the 500 bp probe from the 16S rDNA gene. In situ hybridization of the axenically grown clones L6 (A) and MAV ax (B) and polyxenically grown clone MAVpx (C) was done as in Fig. 2. In C bacteria (b) ingested by, and outside of, the trophozoites are shown. D Total DNA of the clones A (lane 1), MAVpx (lane 2), MAVmx (lane 3), MAVax (lane 4) and bacteria (lane 5) (bacterial flora including: Escherichia coli, Clostridium symbiosum, Pseudomonas aeruginosa and Streptococcus fecalis) fractinated by tranverse alternating field electrophoresis, stained with ethidium bromide (D). TAFE was carried out in two steps, at 280 mA for 48 h with 3-min pulses, and then at 400 mA for 24 h with 8-min pulses DNA from TAFE gels was transferred to nylon membranes and hybridized (E) with the complete EhVR1 fragment including the 16S rDNA and the variable region. Final washings were carried out at 50° C in 0.1 × SSC and 0.1% SDS. M, Saccharomyces cerevisiae molecular weight markers
detected in all clones tested (Fig. 3A–C). Shape and size of the EhkO in axenically growing clones L6 and MAVax presented similar characteristics to those exhibited by the EhkO detected in clone A. Although hybridization signals in the polyxenically growing MAVpx trophozoites were larger (Fig. 3C), no cross hybridization with bacterial DNA was observed. Albach et al. (1980) reported that the cytoplasmic signal obtained in metabolically [3H]thymidine labeled trophozoites was due to bacterial contamination; however, none of our axenically growing cultures showed any evidence of bacterial growth when plated in rich media. To confirm the absence of cross hybridization of the ribosomal probes with bacteria we hybridized TAFE fractionated total DNA of clones A, MAVpx, MAVmx and MAVax, and DNA from intestinal bacteria (Fig. 3D), with a 3.5 kb ribosomal probe containing the16S rDNA gene of E. histolytica and the variable region at the 5′ end of this gene. Figure 3E shows that under the stringency conditions used in these experiments E. histolytica rDNA did not cross-hybridize with bacterial DNA, in agreement
About 90% of trophozoites grown in the presence of [3H]thymidine showed autoradiographically detectable cytoplasmic label and half of them contained a labeled EhkO larger than 0.5 lm in diameter, and of variable shape (Fig. 4A). We also observed differences in labeling intensity between the nucleus and the EhKO, probably due to different levels of thymidine incorporation during the cell cycle. We isolated an EhkO-enriched fraction from nuclei-free lysates of trophozoites that had been grown for 14 h in the presence of [3H]thymidine. These experiments were performed under the assumption that the DNA in EhkO would replicate once during the cell cycle, which lasts for 14–18 h in E. histolytica trophozoites (Orozco et al. 1988). EhkOs isolated by the procedure used here increased in size, probably due to DNA expansion, but retained their varied shapes. One hundred isolated EhkOs were analyzed and classified according to their shape and size; the one shown in Fig. 4B is 5.1 and 6.2 lm long in its horizontal (minor) and vertical (major) axes, respectively; 20% of the structures observed had similar shapes ranging from 4.2 to 6.2 lm and 5 to 6.8 lm in their minor and major axes, respectively. The structure in Fig. 4C measures 3.2 × 6.1 lm in its highly labeled portion; 60% of the structures present in the enriched fraction had a similar appearance. The EhkO in Fig. 4D measures 3.5 × 12 lm and appears to be disrupted; around 5% of the isolated EhkO had this appearance and showed labeled DNA forming small spots. Representative ‘‘replicating’’ EhkOs are presented in Fig. 4E and F. The ‘‘boomerang’’ structure in Fig. 4E (see also Fig. 2A) displays symmetrical wings extending about 12.3 lm from end to end. Two nearly symmetrical, 6.8 lm long EhkOs are shown in Fig. 4F; they may just have finished dividing. About 15% of the structures analyzed seemed to be dividing. In 10% of the isolated EhkOs only part of the DNA was labeled (Fig. 4B–D), suggesting that [3H]thymidine incorporation varies during the cell cycle. Nuclei pelleted by centrifugation of the disrupted labeled trophozoites at 2 500 rpm appeared to be intact; they did not expand as isolated EhkOs did, and ranged in length from 3 to 6 lm. In Fig. 4N we show two representative nuclei, the one on the left has an elongated shape typical of dividing nuclei (Solı´s and Barrios 1991), whereas the nucleus at the right is a typ-
255 Fig. 4A–F In vivo [3H]thymidine labeling of trophozoites and isolated EhkO fractions. The trophozoites were grown in the presence of 10 lCi [3H]thyimidine for 14 h, washed twice with PBS, spread on slides, covered with an emulsion film and developed after 5 days of exposure in the dark (A). EhkOs were isolated from a nucleus-free lysates, spread on slides, covered with emulsion and developed after 5 days (B–F). Nuclei were isolated from the same trophozoites (N). Arrows in D point at small spots of radiolabeled DNA. Bars = 10 lm
ical interphase nucleus (Orozco et al. 1988). These results confirm that E. histolytica has two different types of DNA-containing structures: the nucleus and the EhkO. Transmission electron microscopy (TEM) of the DNA contained in enriched EhkO and nuclear fractions
panosomatidae, and are contained in a structure called the EhkO by us. The following characteristics of the DNA-containing EhkO structure and of the EhkDNA suggest an unexpectedly closer phylogenetic relation between E. histolytica and Trypanosomatidae: (i) the EhkO contains DNA networks (EhkDNA), just as the
DNA was isolated from the EhkO and nuclear-enriched fractions and observed by TEM. Intact and fragmented DNA networks (EhkDNA) were observed in the EhkO fraction. A complete elongated network, approximately 14.5 × 4 lm in size, composed of thousands of 0.4–0.8 kb minicircles with some DNA loops (maxicircles?) around them, is shown in Fig. 5. Preparations incubated with DNase neither present any network nor DNA strands. Determination of the exact size and sequence of minicircles and maxicircles depends on their future isolation and characterization, which is presently being carried out in our laboratory. A magnification of a part of the network is also shown in Fig. 5. We also observed partially lysed EhKOs with networks of DNA attached to them (data not shown). In Fig. 6 we illustrate the differences between the EhkO and nuclear DNA structure. While the DNA from EhkO is mainly composed of free circles, heterogeneous in size, and by networks of connected circles with loops in the extremes (Fig. 6A, B), the nuclear DNA did not exhibit network structures (Fig. 6C, D), but linear strands of different sizes and some circles.
Discussion The results presented here show for the first time the presence of cytoplasmic DNA networks in E. histolytica trophozoites; the networks resemble the kDNA of Try-
Fig. 5 Electron microscopy of isolated an EhkDNA network. A complete DNA network found in DNA purified from nuclei depleted lysate of E. histolytica clone A is shown. The inset is a magnified view of the area in the square
256 Fig. 6A–D DNA isolated from enriched EhkO and nuclear fractions from clone A. A, B EhkO DNA. C, D Nuclear DNA fractions obtained from different preparations. Bars in A and B = 1 lm; in C and D = 0.2 lm
Trypanosomatidae mitochondrion contains kDNA; (ii) the expanded networks found are about 14 lm long (data not shown) and are formed by thousands of circles with sizes in the range described for Trypanosomatidae kDNA; (iii) the [3H]thymidine-labeled EhkO replication-division images resemble those of the Trypanosomatidae kinetoplast (Robinson and Gull 1994); (iv) the E. histolytica rDNA episome may be equivalent to Trypanosomatidae kDNA maxicircles because it carries rDNA genes and is located mainly in the EhkO. In addition, the 1.9 kb intergenic sequences of the EhVR1 fragment from the rDNA episome (Orozco et al. 1993) are up to 54% homologous with 1000 bp fragments from the genes for yeast transfer RNA (accession number: EMBL 36006), oxidoreductase 1 and 2 (EMBL L36097, J01472) and other yeast mitochondrial genes. However, the networks obtained from EhkO differ from others found in various Trypanosomatidae species, and many questions remain open; for instance, are the circles catenated in the way reported for Trypanosomatidae and are the observed DNA loops equivalent to the maxicircles. The EhkDNA has circles of different sizes and we also found circles in the nuclear DNA preparations; the presence of circles of different sizes in the nucleus and in
the cytoplasmic networks could explain the abundance and heterogeneity of DNA circles reported by others (Lioutas et al. 1995; Dhar et al. 1995), and the intriguing changes in DNA content previously found in E. histolytica trophozoites (Lo´pez-Revilla and Go´mez 1978). The diversity of EhkO morphology and size possibly depends on the trophozoite cell cycle. However, cytoplasmic DNA was detected in our cultures in 90% of the trophozoites, although 0.5–2.5 lm structures were clearly seen in only 40–50% of the trophozoites, using acridine orange staining, in situ hybridization or in vivo [3H]thymidine labeling, whereas about 40% of the parasites showed radioactive cytoplasmic spots smaller than 0.5 lm and no label was observed in the cytoplasm of 10% of them. These intriguing results suggest that EhkO might be assembled by the trophozoites under unknown physiological conditions, as occurs with certain Trypanosomatidae species (Borst 1991). The EhkO may also be formed during certain phases of the cell cycle, but its variation in size and shape also could be related to the changing metabolic needs of the parasite. The most dramatic adaptive change that the amoeba undergoes is its passage from trophozoite to the cyst that can survive outside the human host. Excystation in the intestine al-
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lows the parasite to invade the host tissues, where it must adapt to various oxygen concentrations, which differ in the intestine, liver and feces, the three main habitats of E. histolytica during its life cycle. The Krebs cycle and the respiratory chain have not been found to operate in E. histolytica trophozoites, which are known to have affinity for oxygen (Weinbach and Diamond 1974), and to possess CoQ (Ellis et al. 1994). In T. brucei bloodstream forms, ATP is not synthesized by the Krebs cycle and the respiratory chain is absent in the mitochondrion (Borst 1991). It is known that T. brucei lives for long periods without functional mitochondria and represses mitochondrial biogenesis surviving by glycolysis while in the bloodstream of the mammalian host (Borst 1991). The kinetoplast provides metabolic energy to the Trypanosomatidae (Simpson 1987), whose flagellae are located right next to the kDNA. E. histolytica trophozoites have no flagellae but move actively by forming pseudopodia; their relationship with the EhkO is unknown. Acknowledgements We thank Pedro Cha´vez and the CINVESTAV’s Electron Microscopy Unit and Department of Photography for excellent technical work. E. Orozco is an International Fellow of the Howard Hughes Medical Institute (USA). This work was also supported by CONACYT (Me´xico), and INSERM (France).
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