Entamoeba histolytica ribosomal RNA genes are carried on palindromic circular DNA molecules

Molecular and Biochemical Parasitology, 32 (1989) 285-296

285

Elsevier MBP 01085

Entamoeba histolytica ribosomal R N A genes are carried on palindromic circular D N A molecules Marion Huber 1, Barbara Koller 1,**, Carlos Gitler 2, David Mirelman 1, Michel ReveP, S h m u e l R o z e n b l a t t 3 a n d L e o n a r d G a r f i n k e l 2,* The Weizmann Institute of Science, Departments of 1Biophysics, 2Membrane Research and 3Virology, Rehovot, Israel (Received 1 August 1988; accepted 28 September 1988)

Highly abundant DNA fragments obtained after restriction enzyme digests of nuclear DNA of Entamoeba histolytica strain HMI:IMSS have been cloned and characterized. Northern blot hybridization to E. histolytica rRNA and sequence analysis identified the abundant DNAs as ribosomal DNA containing species. Several overlapping clones containing these abundant DNAs were isolated from 4 different genomic libraries of E. histolytica. Alignment of the restriction maps was consistent with a circular molecule, about 24.6 kilobase pairs (kb) in size. Nuclease BA131 digestion provided additional evidence for the circular nature of this DNA. The ribosomal DNA molecule contains two large inverted repeat-regions, each at least 5.2 kb in length. Sequence analysis of clone R715 revealed homology to the large rRNA units of various eukaryotic organisms. This clone was located in both inverted repeats, suggesting two rRNA cistrons per molecule. The inverted repeats are flanked by stretches of DNA which contain tandemly reiterated sequences. Southern blot analysis of E. histolytica nuclear DNA revealed the presence of two populations of molecules. These molecules have identical arrangements of restriction sites, but differ in size (0.7 kb) in a fragment containing tandemly reiterated sequences. Analysis of E. histolytica nuclear DNA by electron microscopy also revealed circular molecules. These molecules are about 26.6 kb - 0.5 kb in size and contain structural features predicted by the restriction map of the extrachromosomal ribosomal DNA of E. histolytica. Key words: Entamoeba histolytica; rDNA organization; Electron microscopy; Circular episome; 25S rDNA sequence

Introduction Trophozoites of the intestinal parasite Entamoeba histolytica are primitive eukaryotic cells with a peculiar biology [1,2]. The cells contain a nucleus but lack typical eukaryotic organelles,

*Correspondence (present) address: L. Garfinkel, Biotechnology General Corp., Kiryat Weizmann, 76100 Rehovot, Israel **Present address: Swedish University of Agricultural Sciences SLU, Department of Plant Breeding, 26800 Svalov, Sweden. Abbreviations:

SSC, standard citrate saline; SDS, sodium

dodecyl sulfate.

Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M Data Bank with the accession number J04003.

such as Golgi apparatus and mitochondria. The nucleus appears different from that of other eukaryotes. An endosome comprised of fibrogranular material including some DNA [3] is found approximately in the centre of the nucleus. It has been speculated that this endosome might be a site of DNA condensation prior to segregation. A spindle apparatus, responsible for segregation of chromosomes during cell division seems to be absent, and there is no evidence for chromosomes in E. histolytica. Nucleoli that could serve as sites of rRNA synthesis seem to be absent in E. histolytica. However, the inner periphery of the nuclear wall of E. histolytica is lined by clusters of 'peripheral chromatin', rich in RNA and including some DNA [3]. It has been suggested that these structures might be involved in rRNA synthesis and may functionally resemble nucleoli. The genes coding for rRNAs in eukaryotes have to meet physiological and developmental require-

0166-6851/89/$03.50 ~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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ments for active protein synthesis and are, therefore, present in multiple copies per cell. The rRNA genes of metazoans are generally organized as several hundred tandemly repeated copies clustered on chromosomes [4]. In several protozoan species, r R N A genes are extrachromosomal, existing as linear palindromic or 'genesized' molecules or are present in circular form [4-6]. Extrachromosomal rRNA genes replicate autonomously during vegetative growth, and have the capacity to supply the cell with rRNAs in high abundance. In Tetrahymena, the well characterized extrachromosomal rRNA genes are derived from a single chromosomally integrated copy, and are amplified during formation of a new macronucleus after conjugation [7]. In order to gain more insight in the D N A organization of E. histolytica, we have investigated the nature of highly abundant DNAs of this organism. Some of these DNAs are part of a molecule encoding rRNAs. Detailed restriction mapping of this molecule is consistent with a structure of episomal circles, containing large inverted repeats. Electron microscopy of nuclear DNA of E. histolytica confirmed the presence of such circular molecules with structural features closely resembling those predicted from the restriction mapping data. Material and Methods

Entamoeba strains. Trophozoites of E. histolytica strain HM-I:IMSS/clone 6 (obtained from Dr. L. Diamond, NIH, Bethesda) were grown axenically in TYI-S-33 medium [8]. Cells were harvested by chilling on ice for 5 min and subsequent centrifugation at 300 × g for 5 min. Isolation of nucleic acids. Nuclear D N A was isolated from exponentially growing E. histolytica cells essentially as described before [9], and was analyzed on agarose gels according to standard procedures [10]. Total R N A was purified from exponentially growing E. histolytica cells by extraction with guanidinium thiocyanate [11]. Poly(A)- R N A was obtained as an eluate after passage of total R N A over an oligo(dT)12-18 cellulose (Collaborative Research, Lexington, MA) column [12]. Isolated R N A was extracted with a

mixture of phenol/chloroform (1:1) and was subsequently precipitated with ethanol.

Cloning of rDNAs and flanking regions. Four different genomic libraries were prepared from E. histolytica nuclear DNA, digested by BgllI, EcoRI, HindlII and XhoI, respectively, and subsequently ligated in the bacterial plasmids pUC8 or pUC18. EcoRI clones containing ribosomal DNA (rDNA) sequences were identified by hybridization with a radioactive labeled cDNA probe, prepared from E. histolytica poly(A)RNA, using sonicated calf thymus D N A for random priming [10]. These clones were subsequently nick-translated [13] and used to identify overlapping rDNAs and flanking sequences from the above libraries. Hybridizations were carried out under stringent conditions, essentially as described before [9]. Northern blot hybridization. Poly(A)- R N A was separated under denaturing conditions on agarose gels containing formaldehyde [10]. RNA was transferred to a nylon membrane (BioRad) in 10 mM Tris, 5 mM sodium-acetate, 0.5 mM E D T A , pH 7.8 (blotting-buffer). The gel was presoaked twice for 10 min in 5 x blotting-buffer and then for 10 min in 1 x blotting-buffer before transfer. The filter was air dried and then baked for 2 h at 80°C. Hybridization was carried out with nicktranslated rDNA clone H6.6 as described before [9].

DNA sequence analysis. Overlapping restriction fragments of rDNA clone R715, were subcloned into the phage vector M13 mp18 [14]. Single stranded DNA was prepared from recombinant phage, and the D N A sequence of each insert was determined by the dideoxy chain termination method of Sanger et al. [15]. Sequence data were analyzed using the MicroGenie program (Beckmann) and the Fast P program of Lipman and Pearson [16], and were compared against the E M B L and GenBank data bases.

DNA mobility shift assay. Opposite polarities of recombinant M13 mpl8 phage DNAs of clone R715 were detected by mobility shift of a self-hybrid of these DNAs in Tris-borate buffered [10]

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agarose gels. Self hybridization of each 500 ng DNA from two M13 clones was in 250 mM NaC1, 25 mM Tris and 4 mM EDTA, pH 7.5 for 1 h at 55°C in a total volume of 60 Ixl. 20 ixl were subsequently loaded on a gel. The polarity of the rDNA genes was determined by testing complementary recombinant phage DNAs of known sequence for mobility shift in agarose gels, after hybridization to 20 ixg poly(A)- RNA of E.

histolytica. Restriction mapping of E. histolytica rDNA. Clones described above were analysed by restriction enzyme digests using 6 different enzymes (BgllI, EcoRI, HindlII, PvuI, SaclI and XhoI). Restriction maps were established by successive complete digestions, using varying combinations of the above enzymes for double and triple digests [17]. A physical map of the E. histolytica rDNA molecule was obtained by aligning the overlapping restriction maps of these clones. For analysis of highly repetitive sequences, clones H16, B4 and R21 were partially digested with PvuI and clone H4.4 was partially digested with DraI.

Southern blot analysis. Nuclear DNA of E. histolytica was digested with different restriction enzymes, separated on an agarose gel, and was subsequently transferred to a nylon membrane (BioRad) in 1 × blotting-buffer, as recommended by BioRad. The filter was air dried, baked, and then hybridized with nick-translated clones B14, B4, B22 and B20 as described before [9]. After exposure, hybridization signals were removed in 0.4 M NaOH for 30 min at 42°C. Neutralization was performed in 0.1 x sodium citrate-standard saline (SSC) [10], 0.5% sodium dodecyl sulfate (SDS), 0.2 M Tris, pH 7.5 for 30 min at 42°C. The filter was subsequently rehybridized with nicktranslated nuclear DNA of E. histolytica.

Nuclease Ba131 digestion. A mixture of nuclear DNA of E. histolytica (2 Ixg) and h DNA digested with HindlII (3 ~g) was treated by nuclease Bal31 (2.5 units) Biolabs) at 30°C in a total volume of 80 ~1 of 600 mM NaCI, 12 mM CaCI2, 0.2 mM EDTA and 20 mM Tris, pH 8.0. Aliquots of 10 ~1 were removed at 30 min time in-

tervals, added to an equal volume of 1% SDS and 20 mM EDTA, and the reaction was stopped by incubation for 20 min at 65°C. DNA aliquots were subsequently size fractionated on a 0.7% agarose gel in a buffer containing 40 mM Tris, 5 mM sodium acetate, 1 mM EDTA and 1.5 ml 1-1 acetic acid glacial.

Electron microscopy of E. histolytica nuclear DNA. The DNA at a concentration of about 10 ~zg ml -~ in a volume of 40 Ixl, was made 1% with respect to SDS, and passed over a Sepharose CL2B column, which was equilibrated with 10 mM Tris, pH 7.5. 5 I~1 of this DNA were spread in a 50 txl spreading mixture which contained 50% formamide, 100 mM Tris, pH 8.5, 1 mM EDTA, and 0.1 mg ml -l cytochrome C (Sigma type V), on a hypophase of water containing 0.005% octylglucopyranoside (Sigma). The DNA of a sequenced plasmid was used as internal length standard. To analyze the DNA in the singlestranded form, the spreading mixture was heated for 1 min in a boiling water bath, without the cytochrome, then incubated for 15 min at 37°C. The denatured, self annealed DNA was then spread with cytochrome as before. Electron micrographs were taken with a Philips EM 400, at a magnification of 5400 ×. Length measurements were done on the negatives with a digitizer (Bruehl, Ntirnberg) connected to a Crommenco microcomputer. Results

Cloning of E. histolytica highly abundant DNAs. Electrophoresis of undigested nuclear DNA of Entamoeba histolytica on agarose gels revealed that a major DNA component comigrated with a 23 kilobase pair (kb) marker band (Fig. 1, lane 1). This fraction presumably represents a heterologous population of DNA which has not resolved by size, and therefore might contain species with a higher molecular weight. No additional, low molecular weight DNA bands were visible in the DNA of this organism, and Southern blot hybridizations confirmed this finding (see below). EcoRI restriction enzyme digest of nuclear DNA released several highly abundant DNA bands (Fig. 1, lane 2).

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Fig. 1. Agarose gel electroplaoresls of E. histol,vtica DNA. Nuclear DNA of E. histolytica was separated on a tris-borate buffered 1% agarose gel, lane (1), undigested and (2), after EcoRI restriction enzyme digest. HindIII digested hDNA was used as size marker. To characterize the nature of this highly abundant D N A , a genomic library was p r e p a r e d from E c o R I digested nuclear D N A of E. histolytica, and screened with randomly primed, radioactively labeled c D N A of E. histolytica total p o l y ( A ) - R N A . Five clones (R20, R2.3, R706, R705 and R715) were found to contain inserts from abundant D N A species which corresponded in size to some of the ethidium bromide stained D N A bands described above. Several overlapping clones were isolated from three different genomic libraries of E. histolytica nuclear D N A , digested with H i n d I I I , BglII and XhoI, respectively. Using these clones as probes, we identified two additional E c o R I clones (R21 and R13), three H i n d I I I clones (H16, H6.6 and H4.4), four BglII clones (B14, B4, B22 and B20) and one XhoI clone (X13).

A b u n d a n t D N A s contain r D N A sequences. P o l y ( A ) - R N A from E. histolytica revealed three highly abundant R N A species, migrating as 18S

or faster (Fig. 2A). These R N A s correspond to the parasite r R N A , two of the species probably being processing products of a single high molecular weight r R N A molecule [18]. E. histolytica p o l y ( A ) - R N A , therefore, was used for Northern blot hybridization using the abundant D N A clones as probes to analyze whether any correspond to r D N A . Clone H6.6 showed strong hybridization to the 3 highly abundant R N A species described above (Fig. 2B). To further confirm whether clone H6.6 contains sequences complementary to r R N A , a small E c o R I D N A fragment (R715) contained in clone H6.6 was partially sequenced (Fig. 3A). The sequence (Fig. 3B) showed homology to the large r R N A sub-units of various organisms, indicating that clone R715 contains part of the E. histolytica 25S r R N A [18]. High degrees of similarity to the genes of human 28S r R N A (76.5%) and chimpanzee 28S r R N A (76.5%) [19], yeast 25S r R N A (75.9%) [20], rice 25S r R N A (73.2%) [21] and mouse 28S r R N A (71.1%) [22] were obtained by comparison with the 3' terminal sequence of r D N A clone R715. The homologous regions of human, chimpanzee and mouse 28S r R N A correspond to similar locations within the respective r R N A sequences.

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Fig. 2. Northern blot analysisof rDNA clone H6.6. Poly(A) RNA of E. histolytica lanes (1) 2 I,g; (2) 5 p,g and (3) 10 I~g were separated under denaturing conditions on an agarose gel containing formaldehyde, (A) three rRNA species (discussed in the text) are visible by ethidium bromide staining, rRNA marker bands of 28S and 18S are indicated by arrows. (B) RNAs were blotted to a nylon membrane, and hybridization was carried out with nick-translated rDNA clone H6.6.

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The 25S rRNA-units of yeast and rice are considerably smaller, yet the homologous regions occupy similar relative locations. The sequences from the three mammalian species map at distances about 30% of the length of the molecule from the 5' end, while the yeast and rice sequences map at about 25% from the 5' end. Comparison of the 5' terminal sequence of R715 revealed lower degrees of similarity to the yeast 25S rRNA (56.9%) and rice 25S rRNA (51.2%) genes, and no similarity was obtained to the other species. The 5' to 3' direction of the rDNA sequence of clone R715 was determined by a mobility shift of single stranded recombinant M13 mpl8 DNA in situ after hybridization to poly(A)-

RNA. This was confirmed by comparison of the sequence with those of yeast and rice 25S rRNAs.

Organization of rDNA and flanking regions. The organization of E. histolytica rDNA was determined by the construction of restriction maps from the entire set of cross-hybridizing clones described above (Fig. 4). Alignment of the overlapping restriction maps revealed the presence of large inverted repeats, each repeat at least 5.2 kb long (Fig. 4, arrows), rDNA clone R715 is located within these inverted repeats. The clone contains a single asymmetrically located HpaI site. This allowed orientation of clone R715 with respect to clone B22 (Fig. 4, asterisk in clones R715

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Fig. 4. Restriction maps of rDNA clones and flanking regions. Restriction maps were established from the set of overlapping clones indicated, using 5 different restriction enzymes: B, BglII; E, EcoRI; H, HindIII; S, SacII; X, XhoI. A cluster of about 15 PvnI 140 bp repeats is indicated by a thick bar. The region of highly reiterated DraI restriction sites in clones H4.4, R13, X]3 and B14 is indicated by a thin bar. Dotted lines in clones X13 and B14 represent length variations in the large EcoRI/HindIII fragment, and are introduced to align this fragment with clone R13. Asterisks (*) represent a HpaI restriction site mapped in clones B22 and R715 in the 3' half of the rDNA sequence (see Fig. 3). Arrows represent the minimal length of an inverted repeat (5.2 kb), and the direction of arrows indicates the 5' to 3' transcription directions of the 2 putative rRNA cistrons, contained in the inverted repeats. and B22) and determines the direction of transcription with respect to the r D N A restriction map (Fig. 4, directions of arrows). A cluster of about 15 tandemly reiterated PvuIfragments, each about 140 base-pairs (bp) in size, was identified in the region between the inverted repeats (Fig. 4, thick boxes). These repetitive fragments are located upstream from the two putative rRNA cistrons. Sequence analysis of one 140 bp unit from the PvuI-cluster revealed no similarity to known nucleotide sequences (Garfinkel et al., in preparation). Tandemly reiterated 180 bp sequences were also detected in the large E c o R I - H i n d I I I fragment of clone H4.4, which is located downstream of the r D N A containing inverted repeat. This E c o R I HindIII fragment contained reiterated DraI restriction sites. Interestingly, the length of this region was variable among the various overlapping clones (Fig. 4, dotted lines). This might be due, in part, to elimination of repetitive units by homologous recombination. Indeed, clone H4.4 was

unstable in bacterial plasmids resulting in a ladder of HindIII fragments after release of the insert by HindIII restriction enzyme digest. These fragments differed by about 180 bp in size (data not shown). The most striking feature of the overlapping restriction maps described above (Fig. 4, compare clones X13 and H16) is that they are indicative of a circular molecule, 24.55 kb in size (Fig. 5). Episomal circular r D N A molecules have been described for various organisms, such as Xenopus, the water beetle Dityscus, the cricket Acheta, several yeast species and recently for the free-living soil amoeba Naegleria gruberi and the alga Euglena gracilis [4-6, 23, 24].

Multiple abundant DNA species in E. histolytica DNA. To test for the presence of additional highly abundant E. histolytica D N A s , identical Southern blots were successively probed with a mixture of clones spanning the entire r D N A molecule, and with total labeled nuclear D N A of E. histolytica

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(Fig. 6). Hybridization patterns obtained with both probes confirmed the general pattern predicted by the r D N A restriction map (Fig. 6A). However, we detected an additional EcoRI and HindlII band, respectively, which migrated in the 4.4 kb marker region (compare lanes 3 and 4 in Fig. 6A, B and C). To analyze the origin of these bands, the Southern blot was re-hybridized with clone X13 (Fig. 4). In the r D N A map clone X13 contains EcoRI fragments R13 and HindlII fragment H4.4, both about 4.4 kb in size. In the total E. histolytica D N A , however, clone X13 hybridized simultaneously to two similar sized EcoRI (4.5 kb and 5.2 kb) and HindlII bands (3.8 kb and 4.5 kb) (data not shown). These two hybridizing EcoRI and HindlII bands differ in size by about 0.7 kb, which suggests the presence of two populations of r D N A molecules, identical in their overall restriction map, but different in size by about 0.7 kb in this region (Fig. 6D). This region

was also heterogeneous in size in the cloned r D N A molecules (Fig. 4, dotted lines). Using nuclear D N A as probe, two additional highly abundant D N A fragments, present after HindlII digestion of E. histolytica D N A were detected by hybridization (Fig. 6C, filled arrow heads). Since neither the overlapping restriction maps described above (Fig. 4) nor the above Southern blot hybridization patterns allow the insertion of these fragments in the physical map of the r D N A molecule, it seems likely that these additional abundant DNAs are from a different origin.

Sensitivity of rDNA to nuclease Bal31. Nuclear D N A of E. histolytica, together with HindlII-cut h D N A as internal control were treated with nuclease Bal31. Aliquots were withdrawn at 30 min intervals, size fractionated on a 0.7% agarose gel, transferred to a nitrocellulose filter [10], and probed with r D N A clone R715. Fig. 7A shows successive degradation of h D N A by nuclease Bal31, which was used as a control for the activity of the enzyme. Fig. 7B demonstrates that, at high resolution, there are three hybridizing rDNA molecules (bands a, b and c) in undigested nuclear D N A of E. histolytica. After 30 min of digestion with Ba131, band 'b' disappeared completely, even after overexposure of the autoradiogram (Fig. 7C), whereas band 'a' disappeared partially, and band 'c' increased significantly in intensity. In addition, a decrease in size of band 'c' is evident, indicating that band 'c' is linear DNA. With longer periods of digestion, band 'a' decreases slowly in intensity but not in size. These data suggest that band 'b' may be supercoiled D N A with local single-stranded regions due to torsional stress, which is quickly converted to nicked circular DNA, and subsequently to linear D N A by the single-strand endonuclease activity of nuclease Bal31 [25]. Band 'a' is likely to be composed of a mixture of nicked and covalently closed circular DNA. The nicked circles would be rapidly converted to linear DNA, while the closed circular DNA would be resistant to digestion [25]. This would explain the increase in intensity of band 'c' after short periods of digestion. The remaining D N A in band 'a' is probably nicked and linearized gradually by contaminating nucleases.

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Fig. 6. Southern blot analysis of E. histolytica nuclear DNA. (A) A pattern of abundant DNA fragments from E. histolytica nuclear DNA was predicted from the physical map of rDNA (see Fig. 5) for lanes (1) undigested nuclear DNA; (2) nuclear DNA digested by BglII; (3) EcoRI; (4) HindlII; (5) PvuI; (6) SaclI; (7) XhoI and (8) SaclI and XhoI. (B) A Southern electrophoretic blot of E. histolytica nuclear DNA, size fractionated on a 1% agarose gel (undigested and digested DNA in lanes 1-8 as above) was probed with a mixture of nick-translated rDNA clones B14, B4, B22 and B20 (Fig. 4), spanning the entire rDNA molecule. (C) Hybridization signals were removed and the Southern blot was re-hybridized with nick-translated nuclear DNA as probe. Exposure was for 5 h at -70°C. Empty arrow heads indicate bands seen after longer exposure (1-2 days at -70°C). 'M' symbolizes a 140 bp monomer of the PvuI cluster described (Fig. 4). Filled arrow heads represent additional abundant DNAs not predicted or observed in (A) and (B), respectively. (D) Two populations of rDNA molecules, deduced from the above Southern blot hybridization data as described in the text. These molecules differ by about 0.7 kb in the EcoRI/HindIII fragment, marked with a broken line and a bar. Overlapping EcoRI restriction fragments (4.5 kb and 5.2 kb; see Fig. 4) are marked by a broken line, and overlapping HindlII fragments (3.8 kb and 4.5 kb) are indicated by a bar. The junctions of the two circular molecules are indicated by the bent arrows.

Electron microscopy of E. histolytica nuclear DNA. N a t i v e n u c l e a r D N A o f E. histolytica w a s prepared for electron microscopic analysis using the cytochrome spreading technique. The DNA consisted mainly of linear molecules of heterog e n o u s size, b u t a f r a c t i o n c o n s i s t i n g o f less t h a n 1 0 % h a d a c i r c u l a r s t r u c t u r e o f u n i f o r m size. A series of circular molecules was photographed.

Fig. 8 A s h o w s o n e d o u b l e - s t r a n d e d c i r c u l a r m o l e c u l e . T h e size o f t h e s e circles w a s m e a s u r e d as 26.6 ± 0.5 kb (13 m o l e c u l e s ) . T o d e t e c t i n v e r t e d r e p e a t s in t h e s e m o l e c u l e s , t h e D N A w a s d e n a t u r e d , a l l o w e d to self a n n e a l a n d t h e n s p r e a d with c y t o c h r o m e . A s i n g l e - s t r a n d e d self a n n e a l e d D N A m o l e c u l e is s h o w n in Fig. 8B. It c o n s i s t s o f a l o n g d o u b l e - s t r a n d e d r e g i o n , w h i c h is f o r m e d

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Fig. 7. Nuclease Bal31 digestion of E. histolytica rDNA. Southern electrophoretic blots of a mixture of undigested nuclear DNA of E. histolytica and HindIII digested hDNA as internal control, treated with nuclease Bal31 for 30 rain time periods. Lanes: (1) no nuclease; (2) treatment with nuclease Bal31 for 0.5 h; (3) 1 h; (4) 1.5 h; (5) 2 h; (6) 2.5 h and (7) 3.0 h. (A) Nick-translated kDNA served as hybridization probe. (B) Hybridization with nick-translated rDNA clone R715. Three differently migrating rDNA molecules (a, b and c) are marked by arrows according to their mobility in a low percentage (0.7%) agarose gel, containing 1.0 ~g m1-1 ethidium bromide. Exposure for panels A and B was 6 h at -70°C. (C) Longer exposure (3 days at -70°C) of (B).

by inverted repeats, and of several single-stranded loops. The sizes of the double-stranded and of the single-stranded regions were measured, and are given in the schematic drawing of Fig. 8C. The sum of these lengths of the different parts of the single-stranded circle added up to about 26 kb. A small percentage of the circles lacked the singlestranded loop of 1.18 kb, shown near the left side of Fig. 8C. This indicates that either the culture contains a heterogeneous population or, that the structure of the circular molecule varies within each cell. Discussion Total nuclear DNA of E. histolytica analysed by EcoRI restriction enzyme digest revealed several highly abundant DNA bands of different sizes. Similar results have recently been described by Bhattacharya et al., who suggested that part of these abundant DNAs might comprise ribosomal DNA (rDNA) [26]. To further characterize the nature of these abundant DNAs we have isolated a set of 15 overlapping clones from

four different genomic libraries of E. histolytica nuclear DNA. Several lines of evidence show that the clones of abundant DNA were derived from a rDNA containing molecule. By separation of poly(A)RNA of E. histolytica on agarose gels we found 3 highly abundant RNA species. As reported for Drosophila melanogaster [27,28] and several parasites, such as Leishmania donovani [29] and Schistosoma mansoni [30], the large ribosomal RNA unit contains a sequence-stretch designated as a gap, which is susceptible to processing or degradation. Such degradation, resulting in the appearance of 3 rRNA species, was also reported for E. histolytica [18]. Northern blot analysis revealed that genomic clone H6.6 hybridized to all 3 rRNA species of E. histolytica poly(A)- RNA, indicating that the DNA contains sequences of both 17S and 25S rRNA of E. histolytica. Partial sequence analysis of a small DNA fragment (R715) contained within clone H6.6 confirmed the hybridization data in that the insert showed homology (75% for the 3' terminal sequence and 54% for the 5' terminal sequence) to the large

294

..........

c ,,8

0.62 ~

1.36

0 48

7.52

I

.

0.13

. 13.

Fig. 8. Electron micrographs of cytochrome spreadings of nuclear DNA from E. histolytica. (A) A double stranded circular DNA molecule. (B) A denatured, self annealed circular DNA molecule. (C) A schematic drawing of the structure of the self annealed DNA and lengths measurements of the double-stranded and single-stranded parts of the molecule. The sizes are averages from 13 molecules. The standard deviations vary between -+ 0.2 and ± 0.5 kb.

rRNAs subunit from various eukaryotic origins. The organization of the r D N A containing molecule of E. histolytica was deduced from the overlapping restriction maps of the entire set of abundant D N A clones isolated. The most striking feature was that the overlap generated a circular molecule of about 24.6 kb in size. The alternative explanation that this represents a direct repeat of a highly abundant linear D N A unit, with a unit size of 24.6 kb, seems unlikely, since we have not observed a ladder of multimeric D N A fragments after limited restriction enzyme digests of nuclear DNA. It is known that circular DNAs might exist or convert, respectively, to supercoil, relaxed circle, and linear species. These structures migrate to a different position in a low percentage agarose gel, and are differentially sensitive to nuclease treatment. Analysis of nuclear D N A of E. histolytica showed three types of r D N A molecules migrating to different positions

in a low percentage agarose gel (Fig. 7, bands a, b and c). These molecules were, indeed, differentially sensitive to digestion by nuclease Ba131. The rapid disappearance of band 'b' and the persistence of band 'a' after treatment with nuclease Ba131 suggests that these molecules are supercoiled and closed circular forms, respectively. However, attempts to purify supercoiled D N A from E. histolytica by ethidium bromide/cesium chloride density-gradient centrifugation were unsuccessful. In addition, neither band 'a' or band 'b' were affected by treatment with Escherichia coli D N A gyrase or topoisomerase I, as would be expected for closed circular or supercoiled DNAs, respectively (unpublished data). On the other hand, circular molecules similar in size (about 26.6 kb) to that predicted from restriction mapping data and with similar structural features were found to be abundantly present by electron microscopy analysis of nuclear D N A of E. histolytica (see below). It seems unlikely that these ptasmid-like molecules could be derived from DNAviruses of E. histolytica, described by Diamond and Mattern [31], since it would be highly coincidental that these virus DNAs closely resemble structural features, predicted from the r D N A restriction map (see below). The restriction map of the E. histolytica r D N A molecule revealed several interesting structural features. Most apparent is the presence of large inverted repeats, with a repeat length of at least 5.2 kb. These repeats might be longer, but we have at present no restriction data adjacent to the 5.2 kb D N A regions. Interestingly, r D N A clone R715 is located in both halfs of the inverted repeat, suggesting two rRNA cistrons present in one r D N A molecule. It has been demonstrated in chloroplast D N A that the presence of inverted repeats confers stability to a molecule against recombinational events [32]. Inverted repeats therefore might be a structural feature of E. histolytica r D N A which ensures the maintenance of its function. A similar structure has been described for the linear episomal rDNAs of Tetrahymena [33], Physarum polycephalum [34] and Dictyostelium discoideum [35]. Restriction enzyme analysis revealed that the regions separating the inverted repeats contain highly repetitive DNAs. The region upstream of the putative

295 rRNA cistrons is characterized by the presence of a cluster of about 15 PvuI fragments, with a unit size of 140 bp. Interestingly, a cluster of about 15 tandemly reiterated BamHI fragments of 140 bp unit size was also found in a similar location on rDNA molecules of a different E. histolytica strain (Michael Giladi, personal communication). This finding, therefore, suggests that the location of these reiterated DNAs rather than their sequence may be of functional importance. This feature resembles type II-repeats, found upstream of the ciliate rRNA genes of Tetrahymena and Glaucoma [36]. Type II-repeats belong to a family of repeated sequence elements involved in transcription initiation and replication in these protozoan species. Repetitive elements, located in non-transcribed spacer regions of rDNA are common for several organisms studied to date, and often fulfil an important role in the regulation of rRNA transcription [37,38]. A stretch of repetitive DNA, about 4 kb in length, was also found in a region downstream of the rDNA sequence of E. histolytica, and contained repeated DraI restriction fragments, each about 180 bp in size. This 4 kb region has been found to be unstable in bacterial plasmids, and sequences contained in this DNA might be highly susceptible to recombination events. Southern blot hybridization studies of E. histolytica DNA revealed two distinct rDNA molecules. These molecules differ by about 0.7 kb in size in the 4 kb repetitive DNA region described above. It is likely that variation exists within the population of amoeba cells, and could have originated from prolonged in vitro culture. A possible explanation, therefore, could be that some of the 180 bp reiterated DraI-units were deleted, and a population of slightly shorter molecules stabilized. A second explanation could be that this region contains a transposable element, present in a subset of E. histolytica rDNA molecules. Analysis of nuclear DNA of E. histolytica by electron microscopy confirmed the presence of

circular molecules, similar in size to that suggested from the rDNA restriction map. In addition, renaturation of single-stranded circular DNA revealed a large snap-back, 7.5 kb in length, derived from self-renaturation of inverted repeats. Single-stranded loops were present on each end of the snap-back, consistent with the two DNA regions separating the inverted repeats within the rDNA restriction map. The restriction map shows that these two DNA regions differ in size by about 2.6 fold. By analogy, electron microscopy analysis of the self-renatured DNA circle revealed that the large single-stranded loop was about 2.5 fold larger than the region containing the two smaller sized loops on the opposite site of the snap-back. The large loop of the self-renatured DNA circle is characterized by the presence of two small palindromes. By analogy to the rDNA molecule, the location of these palindromes resembles the location of the reiterated PvuI sequences, found upstream of the inverted repeat. The nature of circular DNA molecules found in E. histolytica and their similarity to structural features predicted from the rDNA restriction map strongly suggests that part of the rDNA in E. histolytica is arranged in an episomal circular form. This finding suggests that these molecules might contain an independent origin of replication, which allows amplification of these molecules to high copy number. We currently are conducting experiments to analyze this function, which in the future might allow the development of a DNAmediated transformation system for E. histolytica.

Acknowledgements We thank Jeff Rothenberg for technical assistance, Ada Wexler for help in culturing amoeba cells and Irit Orr for assistance in performing computer analysis of sequences. This work was supported by a grant from the John D. and Catharine T. MacArthur Foundation.

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