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Nucleic Acids Research, 2003, Vol. 31, No. 8 2035-2044
© 2003 Oxford University Press

Differential use of multiple replication origins in the ribosomal DNA episome of the protozoan parasite Entamoeba histolytica

Soma Ghosh1, S. Satish1, Sonika Tyagi2, Alok Bhattacharya1,2 and Sudha Bhattacharya3

1 School of Life Sciences, 2 Bioinformatics Centre and 3 School of Environmental Sciences, Jawaharlal Nehru University, New Delhi-110067, India

*To whom correspondence should be addressed. Tel: +91 11 26704308; Fax: +91 11 26172438; Email: sb{at}mail.jnu.ac.in

Received February 3, 2003; Revised and Accepted February 21, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The factors that control the initiation of eukaryotic DNA replication from defined origins (oris) on the chromosome remain incompletely resolved. Here we show that the circular rDNA episome of the human pathogen Entamoeba histolytica contains multiple potential oris, which are utilized in a differential manner. The primary ori in exponentially growing cells was mapped close to the promoter of rRNA genes in the upstream intergenic spacer (IGS) by two-dimensional gel electrophoresis. Replication initiated predominantly from the upstream IGS and terminated in the downstream IGS. However, when serum-starved cells were allowed to resume growth, the early oris which became activated were located in other parts of the molecule. Later the ori in the upstream IGS became activated, with concomitant silencing of the early oris. When the upstream IGS was located ectopically in an artificial plasmid, it again lost ori activity, while other parts of the rDNA episome could function as oris in this system. Therefore, the activation or silencing of the ori in this episome is context dependent, as is also the case with many eukaryotic replicons. This is the first replication origin to be mapped in this primitive protozoan and will provide an opportunity to define the factors involved in differential ori activity, and their comparison with metazoans.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies on initiation of eukaryotic DNA replication point to a high degree of conservation in the overall mechanism, across species (1–3). Homologues of initiator proteins required in this process have been reported in diverse organisms ranging from yeast to mammals, encouraging the belief that a common mechanism exists to ensure once per cell cycle initiation of DNA replication. The DNA sites to which the initiator proteins bind to form the replication complexes serve as replication origins (oris). While the sites are very well defined in terms of DNA sequence in some protozoa, e.g. Saccharomyces cerevisiae (4), this is not true in other protozoa such as Schizosaccharomyces pombe (5) and in most metazoan chromosomes (6). Thus an element of complexity is introduced in defining the eukaryotic ori. It is believed that oris, while generally not sequence specific, are rich in poly(dA/dT). Of all the potential oris that exist on the chromosome, only a fraction are used actively under a given set of growth conditions.

One of the most important unresolved issues is to understand the factors that control differential ori usage in eukaryotes. This question has been addressed with developing embryos of Xenopus and Drosophila (7,8) where a transition from random to specific oris takes place after the mid-blastula stage; and in the rDNA array of S.cerevisiae where clusters of active oris are interspersed with large gaps of inactive oris (9). Chromatin organisation and the transcription status of genes surrounding the ori are some of the factors implicated in differential activation of an ori.

Here we show that multiple oris in the rDNA episome (10,11) of the human pathogen Entamoeba histolytica are utilised differentially. Although the primary replication ori in this molecule maps near the rRNA gene promoter, other potential oris exist. The latter are transiently activated when serum-starved cells are allowed to resume growth, and are also active when cloned in an ectopic location. This system provides an opportunity to study the factors governing differential ori usage in this primitive protozoan, and to compare them with other eukaryotes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and cell growth
Entamoeba histolytica strain HM-1:IMSS clone 6 and transfected lines were maintained in TYI-S-33 medium (12) at 36°C with appropriate antibiotics.

DNA purification from E.histolytica trophozoites
Total DNA was purified from late-log phase-grown cells, as described (13). Briefly, cells were pooled from 40 tubes by chilling in ice water, and a cell pellet was obtained by centrifuging at 275 g for 7 min at 4°C. Cells (5–10 x 107) were resuspended in 5 ml of buffer (100 mM NaCl, 10 mM EDTA, 10 mM Tris–HCl pH 8.0) and lysed by addition of 0.25% SDS. DNA was purified by phenol–chloroform extraction followed by ethanol precipitation (14). At every step of isolation, proper precautions (pipetting up and down of resuspended pellet or supernatant was avoided and cut tips were used for transferring supernatant or suspensions) were taken to avoid shearing of DNA and breakage of bubbles. The ethanol pellet was resuspended in 5 ml of buffer (10 mM NaCl, 1 mM EDTA, 10 mM Tris–HCl pH 8.0) and treated with RNase A (100 µg/ml) followed by proteinase K (100 µg/ml). The suspension was again extracted with phenol–chloroform and ethanol precipitated. The DNA pellet was washed with 70% ethanol, air dried and dissolved in TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.0).

Analysis of replication intermediates by neutral/neutral two-dimensional (2-D) gel electrophoresis
Neutral/neutral 2-D gel electrophoresis was carried out by the method of Brewer and Fangman (15,16). DNA samples (2 µg) were digested with appropriate restriction enzymes (30–40 U in a 40 µl reaction mixture). Electrophoresis was carried out in the first dimension in 0.4% agarose (FMC) at 1 V/cm for 14–16 h. Samples were loaded in duplicate and one of the lanes was stained with ethidium bromide to locate the DNA bands. The unstained lane was cut out and used for second dimension electrophoresis. This was done in 1–1.2% agarose (depending on fragment size) at 5 V/cm for 3–5 h. The time of electrophoresis in each dimension was optimised for best separation of the different fragment sizes. After electrophoresis, the DNA in the gel was depurinated using 0.25 M HCl for 15 min and then blotted onto Nylon membrane (Gene Screen Plus, NEN) following the manufacturer’s protocol. Southern hybridisation with [{alpha}-32P]dATP-labelled DNA probes (17) was carried out as described (14).

Plasmids and transfection
The rDNA fragments of EhR2 were cloned at the unique HindIII site of pTCV1 (18) (Fig. 7) and the constructs were transfected into E.histolytica cells by electroporation (19). The cell pellet was washed twice in phosphate-buffered saline (PBS) (0.11% KH2PO4, 0.37% K2HPO4 and 0.95% NaCl pH 7.2) and thereafter in cytomix buffer (10 mM K2HPO4/KH2PO4 pH 7.6, 120 mM KCl, 0.15 mM CaCl2, 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2). For each transfection, 1 x 107 trophozoites and 200 µg of plasmid DNA were used along with 4 mM ATP and 10 mM glutathione. For stable transfection, two consecutive pulses were applied with an exponential discharge of 3000 V/cm (1.2 kV) and a capacitance of 25 µF (Gene Pulser II, Bio-Rad, USA), resulting in a time constant of 0.94 ms. Electroporated trophozoites were transferred into tissue culture tubes containing 12 ml of TYI-S-33, and were incubated in a slanted position at 36°C. Drug (neomycin) selection was started after 48 h of transfection at a lower concentration of 5 µg/ml for the first few subcultures until the transfected lines were stable. Subsequent drug selection was at 10 µg/ml. Stably transfected lines were used for 2-D gel analysis.



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  		Figure 7. (a) Restriction map of pTCV1 indicating the unique HindIII site in which three EhR2 fragments were cloned. The positions of the restriction enzymes EcoRI (E) and HindIII (H) are shown in the linear map of EhR2 above the pTCV1 map. The three EhR2 fragments that were cloned (filled bars) in pTCV1 are indicated. (b) Dot blot analysis of DNA samples isolated from E.histolytica cells transfected with the pTCV1 constructs shown in (a). Cells were grown in the absence of G418 selection. DNA samples for each construct were loaded in duplicate and hybridised with the neo probe. The intensity of each spot was quantified. DNA was loaded in the following order: column 1, normal transfected cells in the presence of neomycin; columns 2–5, cells grown in the absence of neomycin for: 2, 2 weeks; 3, 4 weeks; 4, 6 weeks; and 5, 8 weeks. DNA loaded on each spot was 100 ng (panel I) and 50 ng (panel II).

 
Serum starvation of E.histolytica cells
Mid-log phase cells growing in 15% adult bovine serum (Biological Industries, Israel) were collected by centrifugation and grown in medium containing 0.5% serum for 24 h. Normal serum was restored at the end of 24 h and DNA was isolated from cells at different time points for 2-D gel electrophoresis.

Measurement of cell count and [methyl-3H]thymidine incorporation in serum-starved cells
A total of 2–4 x 105 cells/ml were transferred to 5 ml glass tubes in a total volume of 3.5 ml of complete medium, and were incubated for the required time at 36°C. Cell number was determined at different time points by counting in a haemocytometer. For radioactive incorporation, 15 µCi of [methyl-3H]thymidine (specific activity 25.0 Ci/mmol; Amersham Life Sciences) was added to each tube. The cells were incubated at 36°C for 30 min, chilled in ice water, harvested by centrifugation and resuspended in 1 ml of ice-cold PBS. DNA was precipitated by the addition of 1 ml of 15% trichloroacetic acid (TCA) and kept on ice for 30 min. The precipitate was collected by filtration through GF/C filters. Free radioactivity was removed by repeated washing with 5% TCA and finally 95% ethanol. Filters were dried at 50°C for 30 min, and the radioactivity incorporated was measured in a Beckman scintillation counter.

Dot blot analysis
The DNA samples were denatured with 0.25 M NaOH for 10 min and spotted on Nylon membrane, pre-soaked in 0.4 M Tris–HCl pH 7.5, using a manifold apparatus. After loading the samples, the apparatus was kept undisturbed for 30 min at room temperature, after which gentle suction was applied. The blots were air dried and used directly for hybridisation.

GC skewness analysis
From the EhR2 nucleotide sequence the cumulative skew was calculated (20) as the sum of ([G] – [C])/([G] + [C]), where G and C are counts of the two complementary nucleotides in adjacent non-overlapping windows. Cumulative GC skew (100 bp windows with 3 bp displacement) was plotted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Replication of EhR2 initiates predominantly from the upstream intergenic spacer
The rRNA genes in E.histolytica are located on circular episomes (11,21). EhR2, an rDNA circle of E.histolytica [Fig. 1; (10)] is 14.1 kb in size. It contains a single rDNA transcription unit (5.9 kb), which encodes the 18S, 5.8S and 28S rRNAs. Several families of short tandem repeats are located in the intergenic spacers (IGSs) upstream and downstream of the rDNA. The promoter of rRNA genes has been mapped 2.6 kb upstream of the mature 18S rRNA (22), between the AvaII and HinfI repeats.



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  		Figure 1. Sequence organisation of the E.histolytica HM-1:IMSS rDNA plasmid EhR2. The EcoRI site at the 12 o’clock position is used as reference for numbering. The bold arrow shows the rDNA unit and its direction of transcription. Nucleotide positions (in kb) are indicated at each restriction enzyme site. B, BsaHI; E, EcoRI; H, HindIII; S, SacII. Different families of short tandem repeats in the regions upstream and downstream of the rDNA are marked as HinfI, AvaII, 74 bp, DraI and ScaI. Arrows show their relative orientation, where relevant. S1, S2 and S3 are the spacer sequences that lie between the adjacent repeat families. EcoRI fragments named HMe (0–7.2 kb) and HMd (9.8–14.1 kb), and HindIII fragment (4.8–11.2 kb) have been cloned into the plasmid vector pBlueScript.

 
Neutral/neutral 2-D gel electrophoresis (Fig. 2; (15)] was used to analyse replication intermediates of EhR2. DNA was digested with the appropriate restriction enzymes (Fig. 3a) to generate fragments (A–L) in the size range of 3–7 kb, which is optimal for analysis by this method. After 2-D gel electrophoresis, Southern blots were hybridised with DNA probes specific for each fragment. As seen in Figure 3b, all fragments from EhR2 contained prominent Y arcs indicative of replication bubbles traversing the entire length of the fragment. However, signals corresponding to replication bubbles were seen only in fragments A–D, which encompass the upstream IGS. Fragment E (0–2.9 kb) and fragment F (2.3–5.9 kb) did not show any bubble signal even after prolonged exposure. Since the upstream IGS is a repeat-rich region, there is a paucity of restriction enzyme sites. In this region, the shortest restriction fragment that gave a complete bubble arc was fragment D (1.1–4.5 kb). From these results, it may be concluded that replication initiation takes place in a zone centering around 2.5 kb from the 12 o’clock EcoRI site. Replication bubbles arising in this zone would rapidly turn into Ys in fragments E and F and would, therefore, not be detectable. Fragments G–J spanning the rDNA transcription unit failed to show any bubble signal, even after prolonged exposure. Fragments K and L spanning the downstream spacer also gave no bubble signal. Thus, EhR2 is replicated predominantly from bubbles initiating upstream of the rDNA. The observation that fragments A–D contain prominent Y signals, in addition to bubbles, is explained below in the Discussion.



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  		Figure 2. Replication intermediates as depicted by 2-D gel electrophoresis. The different types of replication intermediates are illustrated in the top panel. The expected migration of the resultant replication intermediates upon 2-D gel electrophoresis is shown below by bold lines. The dashed lines indicate the migration of linear double-stranded molecules in the 2-D gel. The positions of the unreplicated form (n) and fully replicated form (2n) are shown. The arc of simple Y is shown as dotted line for comparison. The figure is adapted from Friedman and Brewer (16).

 


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  		Figure 3. 2-D gel electrophoresis of replication intermediates in EhR2. (a) A linear map of the 14.1 kb plasmid cut at the 12 o’clock EcoRI site (as shown in Fig. 1) is shown. E, EcoRI; S, ScaI; N, NdeI; B, BsaHI; A, AvaII; H, HindIII; Sc, SacII; X, XbaI; Xh, XhoI. The DNA fragments analysed by 2-D gel electrophoresis are shown as filled bars below the EhR2 map. The restriction enzyme(s) used to generate each fragment and fragment sizes (in kb) are indicated above each bar. The various DNA probes used (probe 1 for fragments A, B, E; probe 2 for fragments C, D, F; probe 3 for fragment G; probe 4 for fragments H, I, J and probe 5 for fragments K, L) are shown as open bars above the linear map. (b) Autoradiograms of Southern blots of individual fragments. E.histolytica DNA was digested with indicated enzymes to generate fragments of interest. These were separated by neutral/neutral 2-D gel electrophoresis under conditions optimised for fragment size, as described in Materials and Methods.

 
GC skewness of EhR2
Additional support that the ori in EhR2 is located at ~2.5 kb from the 12 o’clock EcoRI site in the upstream IGS came from GC skewness analysis of this molecule. Strand compositional asymmetry between the two DNA strands has been observed in various bacterial and viral genomes (23). There is a preference for G over C in the leading strand compared with the lagging strand. A switch in the sign of cumulative GC skew at the ‘global minimum’ (20) is correlated very well with the experimentally observed location of the replication origin and terminus in many bacterial and viral genomes. The plot of cumulative GC skewness in EhR2 shifts distinctly at ~2.5 kb (Fig. 4) where the most commonly used ori of this molecule has been mapped by 2-D gel analysis.



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  		Figure 4. Cumulative GC skew (100 bp windows with 3 bp displacement) plotted for the EhR2 episome. The y-axis indicates the (G – C)/(G + C) values in sliding windows. The arrow indicates the ‘global minimum’.

 
Replication terminates in the downstream intergenic spacer
Replication forks, which converge in a termination zone, result in double Y molecules giving a typical migration pattern in 2-D gels (Fig. 2). Such a pattern was seen prominently in fragments K and L from the downstream spacer (Fig. 3b). Fragments G–J from the rDNA transcription unit showed no such pattern. Fragments A–D from the upstream spacer did not give a strong termination signal. However, a smear between the Y arc and the 2n signal was sometimes seen, which could correspond to asymmetric termination events. Therefore, replication of EhR2 terminates predominantly in the downstream spacer, although some termination may also occur in the upstream spacer.

DNA fragments on both sides of the downstream spacer show ‘spike’ signals indicative of recombined molecules
Joint DNA molecules of size 2n may be generated either by Holliday junctions between molecules undergoing homologous recombination, or by branch migration of daughter strands annealing to each other at stalled replication forks. In our analysis of replication intermediates, we observed prominent ‘spike’ signals arising from the 2n position, which correspond to joint DNA molecules of the same size, in which the junction between the two molecules can occur over the entire length (Fig. 3b). Fragments showing the most prominent ‘spike’ signals were located in the upstream IGS (Fig. 3b, A–D). The signal was almost absent or very faint in fragments derived from the midpoint of the rDNA circle (G and H). It was again visible in fragments containing the middle and 3' end of the rDNA unit (I and J), and was completely absent in fragments from the downstream spacer (K and L). The distribution of this signal in EhR2 indicates that joint DNA molecules are formed in regions on both sides of the downstream spacer, which contains the termination zone. By analogy with other systems (24), we conjecture that these structures may form as a consequence of recombination events that occur at replication forks stalling before they enter the termination zone.

Different oris are used transiently in cells recovering from serum starvation
The data presented so far are based on cells growing under normal conditions. We wanted to see whether the same initiation sites are utilised when DNA synthesis is reinitiated after perturbation of growth. Replication was arrested by subjecting the cells to serum starvation. Cells were restored to normal serum after 24 h of starvation. This treatment synchronised the cell cycle for at least one round of cell division (Fig. 5). DNA was purified from 24 h serum-starved cells and from cells restored to normal serum for different time periods, to map the ori in EhR2. The 24 h-starved cells did not show any replication intermediates [Fig. 6b(i)]. After restoration to normal serum, cells grown for 30 min showed a prominent bubble signal in the ScaI fragment from the rDNA transcription unit, while the NdeI–SacII fragment from the upstream IGS did not show any bubble signal [Fig. 6b(ii)]. This fragment did not give a well defined Y signal either. Since at later time points, normal replication intermediates were seen in this fragment, the lack of these signals at the 30 min time point shows unproductive replication through this region in cells recovering from serum starvation. Cells grown for 2 h in normal serum continued to give a bubble signal in the ScaI fragment (and also in the XbaI fragment), and a faint bubble signal now also appeared in the NdeI–SacII fragment [Fig. 6b(iii)]. Cells grown for 4 h in normal serum showed a bubble signal only in the NdeI–SacII fragment, while the signal could not be detected in the ScaI and XbaI fragments even after prolonged exposure [Fig. 6b(iv)]. The same pattern was observed in cells grown for 12 h [Fig. 6b(v)]. These data suggest that while the upstream IGS is the preferred ori in exponentially growing cells, other potential oris exist. The latter are probably recruited when the upstream ori is not in an active state, but become silenced when the upstream ori is activated.



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  		Figure 5. Synchronisation of cell growth by serum starvation. Mid-log phase cells growing in 15% serum were collected by centrifugation and grown in 0.5% serum for 24 h. Normal serum (15%) was restored at time zero. At different time points thereafter, samples were removed. Cell number was determined by counting in a haemocytometer, and DNA synthesis was measured by [methyl-3H]thymidine incorporation, as described in Materials and Methods. Duplicate samples were taken for each time point. Cell count is normalised with respect to the value at zero time (2.5 x 105 cells/ml). Cell count data are shown as the mean ± SD of four independent experiments.

 


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  		Figure 6. 2-D gel electrophoretic analysis of EhR2 after serum starvation and post-serum replenishment. (a) A linear map of EhR2 cut at the 12 o’clock EcoRI site is shown. E, EcoRI; S, ScaI; N, NdeI; B, BsaHI; H, HindIII; Sc, SacII; X, XbaI. The DNA fragments analysed by 2-D gel electrophoresis are shown as filled bars below the EhR2 map. The restriction enzyme(s) used to generate each fragment and the fragment sizes (in kb) are indicated above each bar. The various DNA probes used (probes 1, 2, 3 used for fragments A, B, C, respectively) are shown as open bars above the linear map. (b) Autoradiograms of Southern blots of the indicated fragments separated by 2-D gel electrophoresis as described before. Cells were grown in serum- deprived conditions for 24 h (i) as described in Materials and Methods. Serum was then restored and cells harvested after (ii) 30 min, (iii) 2 h, (iv) 4 h and (v) 12 h of growth. The bubble signals are indicated by an arrowhead.

 
Effect of cloned rDNA fragments of EhR2 on replication and maintenance of the shuttle vector pTCV1
We were interested to see whether the upstream IGS of EhR2 could function as an ori when placed in an ectopic location. Restriction fragments of EhR2 were cloned in the plasmid vector pTCV1. pTCV1 is derived from an Escherichia coli plasmid in which the neomycin resistance gene is expressed from E.histolytica regulatory sequences (18,25). Since EhR2 shows preferential replication initiation from the upstream IGS (Fig. 3), we decided to check if the same preference is exhibited when the cells are transfected with pTCV1 containing cloned fragments of EhR2. The E.histolytica cells were transfected with pTCV1 alone, and with three different constructs: pTCV1-Up (containing the upstream IGS), pTCV1-rDNA (containing the rDNA transcription unit) and pTCV1-Down (containing the downstream IGS). In each case, G418-resistant cells were selected and subsequently grown in the absence of selection pressure for 8 weeks. The transfected DNA remained episomal in these cells, as demonstrated by Southern hybridisation of total DNA from the cells (data not shown). DNA was purified at 2-week intervals and the level of pTCV1 was determined by dot blot hybridisation (Fig. 7). Cell lines with pTCV1 alone showed a progressive decline in plasmid levels, and by 8 weeks retained only ~3% of the plasmid. Cells transfected with pTCV1-rDNA showed the maximum retention of plasmid in the absence of selection pressure (~40% after 8 weeks), followed by cells with pTCV1-Up (~8% after 8 weeks). Cells with pTCV1-Down lost the plasmid very rapidly, indicating a possible negative effect of this fragment. The relative stability of pTCV1-rDNA could mean that the rDNA fragment serves as a more efficient replication origin in pTCV1, or it assists in better segregation of the plasmid to progeny cells, or both. The ability of this fragment to serve as a replication origin was tested by 2-D gel electrophoresis.

2-D gel electrophoresis of transfectants with pTCV1 constructs
DNA was purified from stable E.histolytica transfectants (pTCV1, pTCV1-Up and pTCV1-rDNA) grown with selective pressure, and analysed by 2-D gel electrophoresis. pTCV1 DNA was digested with the appropriate restriction enzymes to yield a fragment containing the bacterial plasmid sequence (Ib, Fig. 8A) and another containing the neomycin reporter gene and flanking amoebic sequence from the 3' region of the hgl1 gene (Ia, Fig. 8A). Two fragments each were analysed from pTCV1-Up (Fig. 8B) and pTCV1-rDNA (Fig. 8C). One contained the bacterial plasmid sequence and the other contained the cloned rDNA sequence. The enzymes used to release the latter fragments from pTCV1 were BamHI + PvuII (pTCV1-Up) and SacII + PvuII (pTCV1-rDNA). BamHI and PvuII do not have any sites in EhR2, and SacII has only one site in it. Therefore, the indigenous EhR2 would not interfere with the analysis of the cloned rDNA fragments. The results of 2-D gel analysis show that in pTCV1, the fragment containing the bacterial plasmid (Ib) served as an ori while the fragment with the neomycin gene (Ia) showed no bubble signal. On the other hand, in addition to a prominent Y, it showed a signal corresponding to doubleY molecules typical of a termination zone (Fig. 8A). In the pTCV1-Up construct, a bubble signal was clearly visible in the fragment containing the bacterial plasmid (IIb), but no such signal was seen in the upstream IGS fragment (IIa, Fig. 8B). Overexposure of the blot also did not show any bubble signal. In the pTCV1-rDNA construct, a prominent bubble signal was seen in the rDNA fragment as well as the fragment with the bacterial plasmid (Fig. 8C). Therefore, the better retention of pTCV1-rDNA by E.histolytica cells may be the result of enhanced replication of this molecule.



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  		Figure 8. 2-D gel analysis of DNA from cells transfected with plasmids pTCV1, pTCV1-Up and pTCV1-rDNA. Linear maps of (A) pTCV1, (B) pTCV1-Up and (C) pTCV1-rDNA. The fragments analysed are shown as thick lines below the map, and the sizes of the fragments are also indicated. The probes used are shown as open bars above the map. DNA was purified from stably transfected cells grown in the presence of G418. Autoradiograms of Southern blots of each fragment, separated by 2-D gel electrophoresis as described before, are shown in adjacent panels (Ia, Ib for pTCV1; IIa, IIb for pTCV1-Up; and IIIa, IIIb for pTCV1-rDNA). The bubble signals are indicated by an arrowhead.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the protozoan parasite E.histolytica, multiple potential oris exist in the rDNA episome, and a specific sequence may not define the replication origin. This has also been demonstrated for the rDNA episome, EhR1, of E.histolytica (26). Here we show that under normal growth conditions, the ori in the upstream IGS of the rDNA episome, EhR2, is utilised predominantly. Replication initiates from the upstream IGS and terminates in the downstream IGS. With the limited availability of restriction enzyme sites in this molecule, the origin in the upstream IGS could be mapped to a zone centering at ~2.5 kb from the 12 o’clock EcoRI site on the rDNA map (Fig. 3). Since all bubble-containing fragments (Fig. 3b, A–D) had complete bubble arcs, and none showed a partial bubble arc that progresses into a Y arc, we infer that replication initiates from a zone and not from a narrow location. The presence of complete Y arcs in these fragments is expected if initiation events take place from a relatively broad zone. Other factors could also contribute to the Y signals, e.g. breakage of bubbles during DNA purification, and replication of multimeric forms of the rDNA episomes. Replication originating from relatively broad zones, which may, in turn, be composed of closely spaced oris, is a common occurrence in metazoan chromosomes (2,6) but is rare in episomal replicons. That the ‘primary’ ori in EhR2 is located in the upstream IGS appears to be borne out by the GC skewness data (Fig. 4). GC skewness reflects a property of the genome resulting from a replication mechanism operating over evolutionary time (20). For EhR2, it probably means that the ori in the upstream IGS is the most commonly used ori in this episome under natural conditions in which the E.histolytica genome has evolved. Other potential oris are used much less commonly, or are recruited under special conditions.

Replication from oris other than those in the upstream IGS of EhR2 was particularly in evidence when normal serum was restored to serum-starved cells (Fig. 6). At early time points, replication initiations occurred in the rDNA transcription unit, and only later did the ori in the upstream IGS become activated. Progressively, this ori predominated and, at later time points, few, if any, initiations were seen in other regions. Several factors may contribute to the shift in ori usage. Interference of closely spaced oris has been observed in both S.cerevisiae (27) and S.pombe (28,29). Factors such as chromatin organisation (8), nucleosome positioning (30), DNA methylation (1) and transcriptional status (3) are all known to influence replication initiation. One or more of these may determine ori usage in our system. The proximity of the ‘primary’ ori in EhR2 with the transcription start point makes it likely that transcription factors may be involved. This will be the subject of future analysis. Physical association of promoters and replicators is a common occurrence (31–33) and, in a number of studies, transcription factors have been shown to influence the initiation of DNA replication (34–36). A direct link between transcription and ori usage has been reported in Xenopus oocytes, where random initiations are seen in the rDNA locus in early embryogenesis when cells divide rapidly without transcription. At mid-blastula, when the rDNA is transcriptionally active, the ori shifts to the IGS (7). Future studies in our laboratory will address the question of a link, if any, between the transcriptional status of the rDNA and choice of ori in EhR2.

The upstream IGS failed to function as an ori when it was placed in an ectopic location by cloning the fragment into a plasmid vector. In this system, the rDNA transcription unit functioned efficiently as an ori (Fig. 8). This was also observed with the rDNA episome, EhR1, of E.histolytica (26). Such context dependence of ori usage has also been demonstrated in other systems. The ori of the ß-globin locus is controlled by an upstream region called the locus control region (LCR). Deletion of the LCR represses transcription of the locus and also the activity of the ori (37). However, when the ß-globin ori is placed in an ectopic location, it can function without the LCR (38). Amongst other factors, differences in chromatin organisation of the same sequences in two different contexts may influence their ability to initiate replication.

An interesting observation in this study is the existence of joint DNA molecules where the cross-over point is seen predominantly in two parts of EhR2 (Fig. 3), namely the upstream IGS and the 3' end of the rRNA-coding region, which flank the termination zone. It appears unlikely that the joint DNA molecules are due to homologous recombination between fully replicated EhR2 circles, since these structures are absent in the termination zone in which frequent recombination occurs between the repeats (21). It is likely that these joint DNA molecules arise due to a replication-mediated recombination event. It is known that replication forks arrested near the termination site, or for other reasons, including single-strand interruptions in the template DNA, or the presence of DNA–protein complexes (24), are processed by homologous recombination enzymes, leading to replication restart (39,40). In EhR2, the prevalence of joint DNA molecules in regions flanking the termination zone could indicate that a recombination-mediated process is required to restore replication from forks that may be arrested near the termination zone.

The data presented herein constitute one of the more detailed analyses of replication initiation in a protozoan parasite. Apart from the maxicircle and minicircle replicons of trypanosomatids (41) and the plastid DNA of Plasmodium falciparum (42), the structure of an ori in these organisms is unknown. The EhR2 episome of E.histolytica provides an opportunity to dissect out the cis- and trans-factors that are necessary for ori function in this primitive eukaryote. The assemblage of proteins (ORCs, Cdc6, MCMs and Cdt1) that form the pre-replicative complex has been characterised extensively in S.cerevisiae and Xenopus (43,44). Although the six-subunit origin replication complex (45,46) is a conserved feature of chromosomal replication in all eukaryotes studied, the sequence of individual subunits is highly divergent across species (47). We have searched the E.histolytica genome database for homologues of these proteins. Homologues of Orc1p, Orc4p, Cdc6p, Mcm2-7p and Cdc45p (43) could be found by BLAST analysis of the database. With the availability of more E.histolytica sequences, the genes not found so far may also be detected. Thus, comparative genomics will aid in understanding the conserved and divergent features of the E.histolytica replication initiation machinery. The latter could provide useful targets for drug design.


   ACKNOWLEDGEMENTS
 
We thank Dr Anindya Dutta and Dr Suman K. Dhar for useful suggestions and critical reading of the manuscript. This work was supported by the Department of Science and Technology, Government of India. Financial support is acknowledged from the University Grants Commission for research fellowship to S.G., S.S. and S.T.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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