multiple replication origins within the inverted repeat region of the plasmodium falciparum...

Molecular & Biochemical Parasitology 139 (2005) 99–106

Multiple replication origins within the inverted repeat region of thePlasmodium falciparumapicoplast genome are differentially activated�

Divya Singh, Ambrish Kumar, E.V.S. Raghu Ram, Saman Habib∗

Division of Molecular and Structural Biology, Central Drug Research Institute, P.O. Box 173, Chattar Manzil,Mahatma Gandhi Marg, Lucknow 226001, India

Received 14 July 2004; received in revised form 7 August 2004; accepted 13 September 2004Available online 20 November 2004

Abstract

The 35 kb circular genome (plDNA) of thePlasmodium falciparumapicoplast replicates by the bidirectionalori/D-loop mechanism. PlDNAreplication was previously shown to initiate within the inverted repeat (IR) region of the apicoplast genome [Williamson DH, Preiser PR,Moore PW, McCready S, Strath M, Wilson RJM (Iain). The plastid DNA of the malaria parasitePlasmodium falciparumis replicated byti tow numberop©

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wo mechanisms. Mol Microbiol 2002;45:533–42; Singh D, Chaubey S, Habib S. Replication of thePlasmodium falciparumapicoplast DNAnitiates within the inverted repeat region. Mol Biochem Parasitol 2003;126:9–14.] and the presence of at least twoori within each segmenf the IR was postulated. Using 5′ end-labelled nascent DNA as probe, we now demonstrate the utilization of several putativeori locatedithin the IR for plDNA replication. Quantitation of signals obtained for different segments of the IR as well as determination of thef molecules emanating from twoori regions by competitive PCR analysis indicated differential strengths ofori during plDNA replicationrior to schizogony.2004 Elsevier B.V. All rights reserved.

eywords: Plasmodium falciparum; Apicoplast; DNA replication; Origin mapping; Competitive PCR

. Introduction

The plastid-like organelle (apicoplast) ofPlasmodium fal-iparumhas generated immense interest as a target for drugsgainst malaria[3,4]. The apicoplast is believed to be the site

or type II fatty acid biosynthesis[5,6], the non-mevalonateathway of isoprenoid biosynthesis[7,8], as well as synthesisf heme-intermediates within the parasite[9]. It contains a5 kb circular genome (plDNA) that carries genes for largend small subunit rRNAs as well as a minimal set of tRNAnd ribosomal genes that are likely to participate in transla-

ion of protein-encoding genes on the circle[10].TheP. falciparumapicoplast exists as a branched structure

n the late trophozoite-early schizont stages while multiple,

� CDRI communication number: 6600.∗ Corresponding author. Tel.: +91 522 2212411–18x4282;

ax: +91 522 2223938/9504/3405.E-mail address:[email protected] (S. Habib).

discrete, slightly rod-shaped apicoplasts are visible at cynesis[11]. PlDNA replication starts in late trophozoitesfew hours before the onset of schizogony, and seems tocide with total DNA synthesis in the parasite[12–14]. Eachsporozoan cell of the parasite carries a single apicoplasestimates of copy number of plDNA varying from 1 to[15,16]. Replication ofP. falciparumplDNA is inhibited byciprofloxacin that targets DNA gyrase[1,17]. Additionally,ciprofloxacin and clindamycin block plDNA replicationthe related apicomplexanToxoplasma gondiiand also inhibiparasite growth in culture[17].

The plDNA ofP. falciparumandT. gondiishare high sequence similarity but differ in their in vivo topologies[18].The former has a circular topology while the latter existsmarily as a precise oligomeric series of linear tandem aof the basic 35 kb genome[12,18]. This difference in structurhas important implications for the mode of plDNA replition. While T. gondii plDNA may replicate via the rollincircle mode[18], plDNA of P. falciparumis likely to follow

166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2004.09.011

100 D. Singh et al. / Molecular & Biochemical Parasitology 139 (2005) 99–106

the bidirectional replication mode reported for most chloro-plast genomes[19–22]. Reports by D.H. Williamson et al.[1] and our laboratory[2] have shown that replication ofP.falciparumplDNA initiates within the inverted repeat (IR)region that covers a∼10 kb segment and carries genes forlarge and small subunit rRNAs and several tRNAs[10]. Us-ing complementary approaches of electron microscopy andtwo-dimensional gel analysis, Williamson et al.[1] proposedthatP. falciparumplDNA replication proceeds via two mech-anisms. The first and predominant mechanism, also inferredby us[2], follows the theta mode with replication initiatingwithin the IR region while a minor population of plDNAmolecules may follow the rolling circle mode possibly util-ising as yet unidentified initiation sites outside the IR. Us-ing 5′ end-labelled nascent DNA to probe different segmentswithin the IR, we now demonstrate the presence of multi-ple ori within each IR sector. Measurement of potentialoriactivity by competitive PCR additionally suggests differen-tial utilization of initiation sites duringP. falciparumplDNAreplication.

2. Materials and methods

2.1. Parasite culture

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loaded onto a benzoylated naphthoylated DEAE-cellulose(BND-cellulose) column that had been pre-equilibrated with0.3 M NET8 [0.3 M NaCl, 10 mM Tris–HCl (pH 8), 1 mMEDTA]. The column was washed thrice with 0.3 M NET8followed by three washes with 0.8 M NET8 [0.8 M NaCl,10 mM Tris–HCl (pH 8), 1 mM EDTA]. This fraction con-tains linear double-stranded DNA and is termed ‘salt wash’.DNA enriched in replication intermediates was then eluted bywashing the column three times with 1 M NET8/1.8% (w/v)caffeine [1 M NaCl, 10 mM Tris–HCl (pH 8), 1 mM EDTA,1.8% (w/v) caffeine]. The ‘salt’ and ‘caffeine’ washes wereprecipitated with ethanol, washed with 70% ethanol, dried,and suspended in TE. DNA from the ‘salt wash’ and ‘caffeinewash’ were analysed for radioactivity (Cerenkov counting).The latter was denatured and used as probe in Southern hy-bridization.

2.3. Southern hybridization

PlDNA sequences cloned in plasmid constructs[2] weredigested with restriction enzymes, electrophoresed on 0.8or 1.4% agarose gels and blotted onto nylon membraneusing standard procedures[25]. 5′ end-labelled nascentDNA (‘caffeine wash’) was used as probe for hybridiza-tion. The membranes were washed and exposed to X-rayfi car-r amB

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P. falciparum(strain NF-54) was cultured in human RBaintained in RPMI-1640 supplemented with 0.5% AlaxII (Invitrogen). The parasites were synchronised

orbitol [23] and harvested at the late trophozoite-earlyzont stages.

.2. Preparation of plDNA and enrichment of 5′nd-labelled replication intermediates

PlDNA-enriched DNA was purified using the Qiagen pid mini preparation kit as described earlier[2]. Briefly, par-sites released from infected RBCs by saponin lysisashed with PBS, suspended in buffer P1, lysed with b2, and neutralised with buffer P3. After centrifugation,ample was treated with Proteinase K and passed throuquilibrated Qiagen tip-20 column. The column was wasith QC buffer and the extrachromosomal DNA was eluith QF buffer preheated to 65◦C. The DNA was precipitateith isopropanol, washed with 70% ethanol and suspe

n TE.Labelling of nascent DNA strands followed by enri

ent of replication intermediates was carried out as descy Kunnimalaiyaan and Nielsen[20], Little and Schildkrau

24], and Singh et al.[2]. Apicoplast DNA was dephosphylated using calf intestinal alkaline phosphatase (CIAP)reated with Proteinase K. After phenol extraction and ethrecipitation, the DNA sample was labelled with [�32P]-ATP100�Ci) in the presence of 10 U of polynucleotide kint 37◦C for 1 h. The end-labelled DNA was digested withtriction enzymes (NspI, ClaI–ScaI, or HincII–HindIII) and

lm for autoradiography. Densitometric analysis wasied out using ImageMaster 1D Elite software (Amershiosciences).

.4. Purification of nascent DNA for competitive PCR

Total P. falciparum DNA was isolated from culturerom 10 T75 flasks at∼10% parasitemia containing paites at the late trophozoite-early schizont stages. Totasite DNA was isolated by phenol/chloroform extrac

26].Isolation of nascent DNA was carried out by sucrose

ient fractionation followed by further size selection ofractionated DNA by agarose gel electrophoresis[27–29].otal parasite DNA was denatured by a 10 min incubatiooiling water and size separated on 16 ml of 5–30% coous neutral sucrose gradient (∼150�g of DNA per gradi-nt) for 18–20 h at 26,000 rpm in a Beckman SW28 roto5◦C. Sucrose gradients were prepared in 10 mM Tris–pH 8), 1 mM EDTA, and 0.3 M NaCl. The bottom of the tuas punctured and 500�l fractions were collected from ea

ube. Fractions containing 0.3–1.5 kb segments of naNA were pooled and dialyzed against Tris-EDTA [0.5ris (pH 8), 0.01 M EDTA] for at least 8 h. DNA was pripitated with sodium acetate and ethanol, rinsed withthanol, dried, and suspended in TE. Further size seleas performed by fractionating the nascent DNA on areparative agarose gel and eluting 0.3–1.5 kb segmeNA from the gel. After purification, the concentration

his DNA was determined and the preparation was useemplate in competitive PCRs.

D. Singh et al. / Molecular & Biochemical Parasitology 139 (2005) 99–106 101

Table 1Primer sequences and PCR product lengths ofP. falciparumplDNA ori and control regions

Fragment Primer coordinates (nt) Sequence of the primer with tag in bold Product length (bp)

I P1: 1635–1655 P1: 5′-CTGAGCTAGGATCAAACTCGC-3′ Genomic, 325P2: 1933–1959 (C) P2: 5′-CGAAATAGGTAAACGCACTAAATTTAG-3 ′ Competitor, 345P3: 1819–1838 (C) P3: 5′-ACCTGCAGGGATCCGTCGAC CGAATGCTTTAACCACTAAG-3′P4: 1839–1860 P4: 5′-GTCGACGGATCCCTGCAGGT GCTGTTAACCGAAATACACTAG-3′

II P1: 3974–3994 P1: 5′-GATTGACATCTGCCCAGTGC-3′ Genomic, 251P2: 4202–4224 (C) P2: 5′-GCTTCATAGGGTCTTTCTGTCC-3′ Competitor, 271P3: 4080–4099 (C) P3: 5′-ACGTCCAGCCATCGGTCGAG GACAAGGAATTTCGCTACC-3′P4: 4100–4120 P4: 5′-CTCGACCGATGGCTGGACGT GGGTAAGTTCCGACCTGCATG-3′

III P1: 14382–14411 P1: 5′-TTATAAAAAAAATGTTATCATGTATAAAGA-3 ′ Genomic, 332P2: 14686–14713 (C) P2: 5′-ATATCTATACCATCCATTACCAACAGG-3′

IV P1: 10213–10236 P1: 5′-CAGATTTGAACTGATAACACATGG-3′ Genomic, 317P2: 10506–10527 (C) P2: 5′-CATGTCCTAAAGGATTCGAACC-3′

P1 and P2 oligonucleotides represent external primers used in competitor construction as well as the final competitive PCRs. P3 and P4 oligonucleotides,carrying a 20-nt tail at the 5′ end (tail nts shown in bold) represent internal primers used for competitor construction. Primer coordinates of regions I, II, andIII are fromP. falciparumIRA EMBL accession no. X95275 while coordinates of region IV are from IRB accession no. X95276. The lengths of the plDNAtemplate and competitor DNA amplified by P1 and P2 are shown.

2.5. PCR amplification and competitor construction

Primers used for competitor construction and competitivePCR analysis of the 325 and 251 bpori regions as well asprimers used to amplify the 332 and 317 bp control non-oriregions are shown inTable 1. Competitor construction foreach of these regions was carried out as described by Divi-acco et al.[30], and Habib and Hasnain[29]. Four specificoligonucleotides (two external primers, P1 and P2, andtwo internal primers, P3 and P4) were synthesized for eachregion to be amplified. The external primers were designedto amplify DNA regions in the range of 150–350 bp. Thesequence of the upper (P1) and lower (P2) external primersis identical to the genomic region to be amplified. The upper(P4) and lower (P3) internal primers have 3′ ends identicalto contiguous sequences on the upper and lower genomicstrands, respectively, and their 5′ ends carry a 20 nt tag. The20 nt tags of the P4 and P3 primers are complementary toeach other and unrelated to the target sequence to be ampli-fied. Competitor DNA segments carrying the correspondinggenomic sequence with the addition of 20 extra bp in themiddle were constructed for each primer set in a three-stepprocess [29]. One or more subsequent reamplificationsteps of the full-length competitor were needed to enrichfor the competitor product and allow its quantificationb rec rmalc

nti-fii eti-t -do1 db d-

ucts were resolved on an 8% polyacrylamide gel, and the la-beled competitor band was eluted in 100�l of water. Five mi-crolitres of the eluted DNA was counted and the concentrationof the competitor (number of molecules per microlitre) wasdetermined from the final specific activity of [�−32P]dATPand the number of nucleotides incorporated. Dilutions of thisquantified competitor preparation were used as template incompetitive PCRs.

2.6. Competitive PCR experiments

Competitive PCR experiments were carried out for eachregion by first using a fixed amount of nascent DNA tem-plate with 10-fold serial dilutions of the corresponding com-petitor in the presence of primers P1 and P2. The rangewithin which the point of equivalence between competi-tor and template lay was thus determined. Further dilutionsof competitor within this range were then used in similarPCR reactions. Competitive PCR for the 325 bp (region I)and 251 bp (region II) segments was carried out in 30 cy-cles with the following conditions: denaturation, 94◦C, 30 s;annealing, 47◦C, 1 min; extension, 72◦C, 1 min. PCR con-ditions for amplification of the control non-ori regions IIIand IV (332 and 317 bp, respectively) were as above ex-cept that the annealing temperature for the 332 bp region was4 ◦

3

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nu-c en-r tes

y radioactive labelling. All amplification reactions wearried out in an advanced version of thermostarII theycler[31].

Competitor template for each DNA region was quaed by measuring the amount of incorporated [�−32P]dATPn a competitor reamplification PCR cycle. The compor PCR amplification mixture (50�l) contained the stanard amount of cold dATP (10 nmol) and 0.2�l (0.57 pmol)f [�−32P]dATP (Jonaki, India) (3500 Ci mmol−1 and0 mCi ml−1), corresponding to 1.34× 107 cpm as measurey Cerenkov counting in a�-counter. The amplification pro

1 C.

. Results and discussion

.1. Each segment of the IR contains at least threeeplication origins

The use of BND-cellulose, that binds single-strandedleic acids more strongly than double-stranded DNA, toich for DNA molecules containing replication intermedia


Fig. 1. Schematic representation of the location of A650, A1820, and A1988 in a sector of theP. falciparumplDNA inverted repeat. Restriction sites of relevantenzymes on plDNA as well as fragments generated by digestion of cloned plDNA IR sequences are indicated. Level of signal obtained in 5′ end-labellingexperiments is indicated by ‘+’ or ‘−’ signs. Speckled region in the plDNA marks the zone of replication initiation. The location of large subunit rRNA (LSU),small subunit rRNA (SSU) and tRNAs is shown.

(single-stranded D-loop regions as well as expanding repli-cation forks with small transient single-stranded DNA) hasbeen reported for many organisms includingPlasmodium fal-ciparum[2,24]. Studies in our laboratory using plDNA iso-lated at the late trophozoite-early schizont stages, labelled atthe 5′ end and enriched in nascent DNA by BND-cellulosechromatography to probe cloned apicoplast DNA fragmentshad previously indicated the presence of replication initia-tion sites within the IR region of theP. falciparumapicoplastgenome[2]. Two distinct regions (A1820 and A1988) con-tainingori sequences were identified within each IR sector. Inaddition, equally strong signals were obtained for the 650 bpregion[2]. The location of these segments within the IR isdepicted inFig. 1.

F dicates els d A19( lled DN ). Thes seen i hown in basp

In order to determine whether the signal obtained for A650was due to 5′ end-labelled nascent DNA molecules emanat-ing from the overlapping 1820 region, we digested labellednascent strands of plDNA withNspI and used these to probeA650 (previously cloned by us[2]) that had also been di-gested with the same enzyme (Fig. 2). NspI cuts at the edgeof the overlapping region of A650 and A1820 (Fig. 1) so thatmolecules emanating from A1820 and entering the rest ofA650 would be cleaved by the enzyme. Southern hybridiza-tion gave a signal for the 417 bp fragment of A650 while nosignal was detected for the 232 bp fragment (Fig. 2) indicat-ing that the 417 bp segment of A650 contains an independentori. The presence of at least threeori within each sector ofthe IR can thus be inferred.

ig. 2. Hybridisation withNspI-digested, end-labelled nascent DNA inhowing plDNA clones digested to release fragments A650, A1820 anii) Southern hybridization of the same gel withNspI-digested 5′ end-labeignal observed above this band is from undigested A650 that can beairs.

the presence of at least threeori within each sector of the IR. (i) Agarose g19 as well as A650 digested withNspI. A1919 is a control non-ori plDNA region.A gave a signal in the 417 bp fragment of A650 (indicated by arrow

n the ethidium bromide-stained gel as a faint band. Markers (M) are se


Fig. 3. Replication initiates at multiple sites within each IR sector. (A) Nascent DNA digested withScaI andClaI was used as probe. A1919 and A1288represent non-ori control regions (B) nascent DNA digested withHindIII andHincII was used as probe. Panel (i) in A and B shows ethidium bromide-stainedagarose gels containing plDNA fragments while panel (ii) shows the corresponding autoradiographs after Southern hybridization. Markers (M) are shown inbase pairs.

3.2. The IR serves as a replication initiation zone withmultiple origins exhibiting differential activity

To determine the location ofori sequences within theA1820 and A1988 segments we digested cloned 1988 and1820 segments as well as the 5′ end-labelled nascent DNAprobe with combinations of different restriction enzymes(Fig. 1). The signals obtained in Southern blots were analyzedto determine approximateori locations as well as relativeoriefficiencies.

When labelled nascent DNA digested withScaI andClaI(cleavage withScaI would prevent entry of molecules fromthe A1820 region whileClaI would cut within A1988)was used to probe A1988 digested withClaI, signalswere obtained for both the 1257 and 730 bp fragments(Fig. 3A). The relative intensity ratio of the signals was1:1.16 (A1257:A730) indicating the presence of initiationsites within both fragments of A1988. Control plDNA frag-ments A1919 and A1288 that have previously been shown tolack ori [2] did not give a signal. A1988 was also digestedwith HindIII and probed with 5′ end-labelled DNA digestedwith HindIII andHincII (Fig. 3B). A strong signal was ob-served for the middle 973 bp fragment while fainter signalswere seen for the flanking 536 and 478 bp fragments thatmigrated close together in the gel (Fig. 3B, panel ii). Thes ikelyt theua n).A ent

lacks anori [2] and is unlikely to generate a background sig-nal. These results indicate the presence of multipleori withinA1988 and suggest the possibility of differential utilizationof ori sequences lying within this region during replicationof the apicoplast genome.

Localization ofori sequences within the A1820 segmentwas carried out by first probing A1820 digested withHindIIIwith end-labelled nascent plDNA digested with bothHindIIIandHincII. A strong signal was observed only for the 1277 bpfragment while no signal was obtained for the 542 bp frag-ment of A1820 (Fig. 3 B, panel ii). The 1277 bp fragmentdigested withHincII gave signals in both the 740 and 537 bpfragments in the same Southern blot (Fig. 3B, panel ii). Therelative intensity of these signals was 1.57:1 (A740:A537).These results suggest the presence of differentially activeoriin both the 740 and 537 bp fragments while the 542 bp frag-ment of A1820 lacks anori. A portion of this 542 bp frag-ment overlaps with the 232 bp fragment generated by thedigestion of A650 withNspI that also did not give a signal(Fig. 2). Additionally, no signal was obtained in the 542 bpfragment generated by digestion of A1820 withHindIIIand probed with end-labelled plDNA digested withHindIIIandHhaI (data not shown). Unlike the rest of the IR, thisregion is deficient in sequences capable of forming stronghairpin loops, a feature of most replication origins. The pres-e 820id onalgH v-

ignal observed for the 536 and 478 bp fragments is unlo be a result of initiation from flanking sequences asse of 5′ end-labelled nascent DNA digested withHindIIIndScaI gave signals for both fragments (data not showdditionally, the region downstream of the 478 bp fragm

nce of a strong signal in the 1277 bp fragment of A1s in agreement with results of Williamson et al.[1] whoetected a bubble pattern in this region in two-dimensiels. They suggested the presence of anori in the 1026 bphaI–ScaI region of each sector of the IR which is co


ered by the 1277 bp fragment of A1820 in our experiments(Fig. 1).

3.3. Competitive PCR analysis confirms differential oriactivity within two IR regions

To confirm whetherori sequences throughout the IR weredifferentially utilized during initiation of plDNA replicationwe adopted the method of competitive PCR that has beenused for mappingori in mammalian cells[27,32,33]as wellas the determination of abundance of sequences withinori re-gions in nascent DNA preparations[28,29]. A fixed amountof DNA sample enriched in nascent DNA (i.e., low molecularweight DNA emanating fromori) is coamplified with increas-ing amounts of a quantified reference template (competitor)so that the two templates compete for the same primer set andamplify at the same rate. The ratio between the final ampli-fication products of the two species is a precise reflection ofthe ratio between the initial amounts of the two templates andis used to evaluate the amount of the unknown nascent DNAtemplate. This method of quantification of nascent DNA hasshown a high level of sensitivity and fidelity for sequencesthat are believed to be at or nearori [28,34]. The isolation ofnascent DNA in the size range of 0.3–1.5 kb ensures maxi-mal elimination of broken genomic parental DNA and largen allyr nts(

IRo(T hint 988s ignalsi n-

F d IV ins 1820 a

control regions III and IV lie within plDNA segments A1919and A1880, both of which have been previously reported tolackori activity [2] (Table 1andFig. 4). A fixed amount ofnascent DNA isolated from parasites at the late trophozoite-early schizont stage was added to the PCR mix withincreasing amounts of the corresponding competitor DNA.The ratio of the competitor and template reaction products(C/T) was calculated after densitometric analysis of the ethid-ium bromide-stained gels and was plotted against the numberof competitor molecules added to each reaction (Fig. 5). Thenumber of competitor molecules whenC/T= 1 was calcu-lated from the plot equation. This value corresponds to theprecise number of molecules of the target template (nascentDNA) added to the PCRs. Competitive PCR analysis carriedout for regions I and II revealed that region II was representedby ∼3 times higher number of molecules than region I(6× 104 molecules representing region II and 1.9× 104

molecules representing region I) (Fig. 5A and B). Nonascent DNA template amplification product was obtainedfor control non-ori regions (III and IV) although the primersamplified corresponding fragments from total parasite DNApreparations (Fig. 5C) indicating that DNA sequences fromthese regions were not represented in the nascent DNAsample prepared by us. The difference in number of nascentDNA molecules representing regions I and II suggests thata ncesa arem

larg lyu hat ab deo Amo in

ascent DNA fragments including sheared DNA (typicanging from 25 to 50 kb) as well as Okazaki fragme25–300 nt) from replication forks.

Competitor DNA fragments were constructed for twori regions (I and II) as well as two non-ori control regionsIII and IV) of P. falciparumplDNA (Table 1andFig. 4).he twoori regions selected for the experiment lie wit

he 1277 bp and 973 bp fragments of the A1820 and A1egments, respectively. These regions had given high sn 5′ end-labelling experiments described above. The noori

ig. 4. Location ofori regions I and II and non-ori control regions III anhown with relevant restriction enzyme sites as well as positions of A

P. falciparumplDNA (diagram not to scale). The IRA sector of plDNA isnd A1988.

lthough replication initiation sites around both sequere utilized during plDNA replication those near region IIore active.Although the mechanism by which unit-length, circu

enomes ofP. falciparumplDNA are generated is not fulnderstood there is substantial evidence to indicate tidirectional ori/D-loop mechanism is the primary mof plDNA replication with a minor population of plDNolecules following a rolling circle mode[1,2,18]. Previ-us identification of two replication initiation sites with


Fig. 5. Competitive PCR reveals greater abundance of sequences derived from region II in plDNA nascent DNA preparations. Determination of the numberofmolecules representing regions I and II are shown in panels A and B, respectively. A fixed amount (1�l) of nascent DNA template was added to competitive PCRsfor the two regions. PCR products were resolved on an 8% polyacrylamide gel and stained with ethidium bromide. The intensity of the bands correspondingto the template target (T) and competitor (C) was determined by densitometric analysis. The ratio between the two PCR products for each reaction (C/T) wasplotted against the number of competitor molecules added to the reaction. A linear correlation between theC/T ratio and the quantity of competitor added to thereaction was observed. Correlation coefficients (R2) are reported for each plot. The number of target template molecules, that equal the number of competitormolecules whenC/T= 1, was calculated from the equation of the line fitting the experimental points. Panel C shows results of PCR amplification of controlnon-ori regions III and IV andori region I from a nascent DNA preparation (lanes 3, 5, and 7) and total genomic DNA (lanes 2, 4, and 6).

each IR sector of plDNA had led us to suggest parallels withOenotherachloroplast replication where two D-loops in eachIR have been identified[2]. However, evidence for the pres-ence of multiple initiation sites within each IR sector now im-plies that the IR serves as a replication initiation zone wheremore than twoori in each sector may be utilized at the sametime. Moreover, differential levels of activation of replicationori observed by us indicates that allori may not be utilizedfor replication of a plDNA molecule and that there may bea certain degree of redundancy inori requirement. The iden-tification and characterization of origin-binding proteins inthe apicoplast would be the next step towards understandingthe mechanism ofori activation and initiation of replicationof P. falciparumplDNA.

Acknowledgements

We thank JP Srivastava for technical assistance. DS, AKand EVSRR are recipients of research fellowships from theCouncil for Scientific and Industrial Research. This work wassupported by a CSIR Young Scientist Grant to SH and theCSIR Network Project SMM0003.

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multiple replication origins within the inverted repeat region of the plasmodium falciparum...

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