Molecular & Biochemical Parasitology 116 (2001) 105–108

Short communication

Recombinational loss of a ribosomal DNA unit from the circularepisome of Entamoeba histolytica HM-1:IMSS

Soma Ghosh a, Mehreen Zaki b, C. Graham Clark b, Sudha Bhattacharya c,*a School of Life Sciences, Jawaharlal Nehru Uni�ersity, New Delhi 110 067, India

b Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, UKc School of En�ironmental Sciences, Jawaharlal Nehru Uni�ersity, New Delhi 110 067, India

Received 2 March 2001; accepted 17 May 2001

Keywords: Entamoeba histolytica; rDNA episome; Intramolecular recombination; Strain-specific markers; DNA repeats

www.parasitology-online.com.

The ribosomal RNA genes in Entamoeba histolyticaare located exclusively on high-copy-number circularDNA episomes [1]. In some strains, there are tworDNA units per circle, arranged as inverted repeats,while in others, there is a single rDNA unit per circle.Accordingly, depending on the strain, the rDNAepisome varies in size from 25 kb to 15 kb. The rDNAepisome in E. histolytica strain HM-1:IMSS (EhR1,shown in linear form in Fig. 1) has two rDNA units percircle. All strains with a single rDNA unit analyzed sofar (e.g. HK-9, Rahman) lack rDNAII and the regionupstream of it called HMg [2] (Fig. 1). This difference isthe basis of a method for strain identification in E.histolytica [3,4]. Here, we report results of a study withvariants of HM-1:IMSS that contain only one rDNAunit per circle and have lost rDNAII and the HMgregion. This region may be unsuitable as a strain-spe-cific marker if it is subject to frequent recombinationalloss.

The earlier analysis of the rDNA episome was car-ried out with E. histolytica HM-1:IMSS clone 6 cellsobtained from Dr. L.S. Diamond (N.I.H, USA). Someyears ago, we obtained cells of the same strain from Dr.W.A. Petri (University of Virginia, Charlottesville[UVA]) and subsequently also from Dr. J. Samuelson(Harvard School of Public Health [HSPH]). These cellswere obtained for a different purpose, but initial analy-

ses showed changes in the rDNA episome, and there-fore, we carried out a detailed study.

The organization of the rDNA episome was studiedby digesting the DNA with selected restriction enzymesand hybridizing Southern blots with probes spanningthe previously characterized rDNA circle of HM-1:IMSS (EhR1). The results are shown in Table 1.While the cells we were working with originally (fromNIH) gave the expected pattern (column A), cells fromUVA unexpectedly gave different patterns (column B).(The UVA patterns of restriction enzyme-digestedDNA were also obtained with cells from HSPH. Thesecells were not analyzed further.) From the Southernblot data in column B, the restriction map of the rDNAcircle was derived (Fig. 1). This circle was namedEhR2. It is clear that EhR2 contains only one rDNAunit per 14 kb circle. This corresponds to rDNAI ofEhR1. rDNAII along with the adjacent HMg region ismissing. The HMd region downstream of rDNAI,which contains the DraI and ScaI repeats, and theHMe region upstream of rDNAI are present in EhR2.

In order to confirm that the strain showing only onerDNA unit in the rDNA circle was in fact HM-1:IMSSand that these findings were not a result of contamina-tion, the DNA sample from UVA was compared withthe strain HM-1:IMSS clone 9 being maintained inLondon. The DNAs were amplified by PCR at eightpolymorphic loci and the product sizes compared. Alleight loci are marked by the presence of complex repeatunits ranging from 8 bp to 16 bp in length, which arearranged in tandem arrays [5]. The results (Fig. 2) show

* Corresponding author. Tel.: +91-11-610-7676x2308; fax: +91-11-6172-438.

E-mail address: sb@jnuniv.ernet.in (S. Bhattacharya).

0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 6 -6851 (01 )00305 -X

S. Ghosh et al. / Molecular & Biochemical Parasitology 116 (2001) 105–108106

Fig. 1. Sequence organization of the linear maps of circular rDNA episomes, EhR1 and EhR2. Restriction enzyme sites indicated are EcoRI (E),HindIII (H), BsaHI (B) and SacII (S). The rDNA units and their direction of transcription are shown. Different families of short tandem repeatsin the regions upstream and downstream of the rDNAs are marked as P�uI, ScaI, HinfI, A�aII, 74 bp and DraI. Arrows show their relativeorientation. S1, S2 and S3 are the spacer sequences that lie between the adjacent repeat families. EcoRI fragments from EhR1 were cloned intoplasmid vectors and named HMe (0–6.807 kb); HMd (11.668–16.006 kb); HMg (18.600–24.522 kb). The region Tr upstream of rDNAII codesfor a 0.7 kb transcript. The 361 bp region of identity immediately upstream of the rDNA is shown.

identical PCR product sizes and patterns at all eightloci. Based on these results, it may be concluded thatboth DNA samples are indeed HM-1:IMSS, as itsoverall pattern is unique among the c. 100 strainsanalyzed by this method to date ([5] and unpublishedresults).

If an intramolecular recombination between the re-peats in EhR1 generated the EhR2 episome, this eventwould be reflected in the orientation of repeats withrespect to each other in EhR2. To examine this, theScaI- and DraI- repeats in EhR2 were sequenced. TheEcoRI–HindIII fragment containing these repeats wascloned and sequenced from both ends. The repeatsequences were found to be identical to those in EhR1.Their orientation in EhR2 is shown by the arrows andis different from that in EhR1 (Fig. 1). This repeat

organization is similar to that seen in the HK-9 rDNAcircle (which also has one rDNA unit) with respect tothe arrangement and orientation of ScaI and DraIrepeats [2].

This result supports the proposal that EhR2 mayhave arisen from EhR1 as a result of two intramolecu-lar recombination events between repeats in EhR1. Inthe first event, the two inverted rDNA units in EhR1could undergo reciprocal homologous recombinationresulting in inversion of the region between them. Un-der such a scenario, the ScaI repeats in the upstreamand downstream spacers (HMg and HMd regions inEhR1) would be in direct orientation with respect toeach other. In the second event, recombination betweenthe ScaI repeats would generate two circles with onerDNA unit each. One of these would contain the HMg

Table 1Southern hybridization data with probesa from EhR1

Probe 4Probe 3Probe 2Probe 1Enzymes used

Bc B A B AA BAb

7.2 6.8, 5.9 7.2 4.4 4.35 5.9EcoRI No signal6.804.85 2.3, 5.9 2.3 2.9 2.8 5.9EcoRI+HindIII No signal4.5

No signal14.57.83.76.3HindIII 6.3,14.57.814.5N.D.BsaHI 24.5N.D.d 14.1 N.D. N.D. N.D. N.D.

N.D.SacII 14.110, 14.5N.D. N.D.N.D.N.D.N.D.

a Position of probes (in kb) with respect to EhR1: Probe 1 (from HMe): 1.02–2.2, Probe 2 (from HMe): 4.54–6.8, Probe 3 (from HMd):11.6–14.5, Probe 4 (from HMg): 20.8–24.5.

b A: HM-1:IMSS cells used in previous study (which contained two rDNA units per circle).c B: Cells of the same strain used in the present study.d N.D.: not done

S. Ghosh et al. / Molecular & Biochemical Parasitology 116 (2001) 105–108 107

Fig. 2. Electrophoretic comparison of PCR amplification products of E. histolytica strain HM-1:IMSS (LSHTM)(1) and strain HM-1:IMSS(UVA) (2) at eight different loci. Genomic DNA of E. histolytica strains from LSHTM and UVA was amplified as follows: 30 cycles of 1 minat 94 °C, 1 min at the primer-dependent annealing temperature and 2 min at 72 °C with a final extension of 5 min at 72 °C. Amplified productswere analyzed on 1.8% agarose gels. The 100 bp DNA ladder was used as marker.

region and the other the HMe region. The latter circleis equivalent to EhR2 and is retained by the cell whilethe former apparently is lost. From our previous resultswith rDNAI and rDNAII in EhR1 [6], the rRNAcoding regions in the two units are identical in se-quence. Therefore, the HMg-containing circle has anequal potential to code for functional rRNAs. Its pref-erential loss could be either due to inefficient transcrip-tion of rDNA or due to defects in replication and/orpartitioning of the plasmid. Transfection of HMg-con-taining plasmids into E. histolytica cells also failed togive stable transfectants [7], suggesting that the HMgfragment is unable to confer the ability to propagateindependently. The recombination events describedhere may be a relatively recent phenomenon as tworDNA units are also found in the HM-1:IMSS beingused for genome sequencing: http://www.tigr.org/tdb/

edb2/enta/htmls/). Whether the frequency of such re-combination is higher in cells maintained by continuoussubculture in artificial medium compared with naturalisolates remains to be studied. Almost all the rDNAcircles must have recombined in the EhR2-containingvariants as no evidence of the parental EhR1 moleculecould be found by Southern hybridization analysis.PCR analysis is required to detect a rare copy of EhR1that may persist in these cells.

Direct repeats mediating intramolecular recombina-tion can produce multiple products. The abundance ofthe products and the frequency of recombination anddeletion depend upon structural factors such as lengthof repeats and the distance between repeats [8]. Thegeneration of EhR2 from EhR1 by recombination be-tween direct tandem repeats is mechanistically simpleand feasible. The intriguing part is that EhR1 is not a

S. Ghosh et al. / Molecular & Biochemical Parasitology 116 (2001) 105–108108

simple dimer of EhR2 and the monomer cannot revertto the dimer because the HMg region appears to be lostfrom cells with the monomeric circle. If this is true,then the EhR1-type of rDNA circle should eventuallybe lost from all E. histolytica, especially since there isno evidence of a chromosomally integrated copy of therDNA episome [9]. Results so far do not show apredominance of EhR2 over EhR1. For example, of thefour E. histolytica strains studied previously (HM-1:IMSS, 200:NIH, HK-9 and Rahman), two (HM-1:IMSS and 200:NIH) contained the EhR1-type ofrDNA circle [2]. Similarly, of 18 E. histolytica isolatesanalyzed with the Tr portion of the HMg region, 15were positive [4]. This may indicate that there is aselective advantage to having two rDNA units in vivothat does not persist in axenic culture, as all threeHMg-lacking strains are long established in culture.Further experiments are needed to understand the dy-namics of this molecule. Meanwhile, in view of theobservation reported here, one should be cautious whileusing HMg region for strain identification in E. his-tolytica.

Acknowledgements

We thank Prof. Alok Bhattacharya for valuable ad-vice and critical reading of the manuscript. Financialsupport is gratefully acknowledged from the Depart-

ment of Science and Technology, Government of Indiaand University Grants Commission for research fellow-ship to S.G.

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