Proc. Natl. Acad. Sci. USAVol. 90, pp. 12020-12024, December 1993Biochemistry

Cloning and characterization of subunit genes of ribonucleotidereductase, a cell-cycle-regulated enzyme, fromPlasmodium falciparumDEBOPAM CHAKRABARTI*tt, SHELDON M. SCHUSTERt§, AND RATNA CHAKRABARTI§*Department of Infectious Diseases, tInterdisciplinary Center for Biotechnology Research, and §Department of Biochemistry and Molecular Biology,University of Florida, Gainesville, FL 32611-0880

Communicated by George K. Davis, September 21, 1993

ABSTRACT Ribonucleotide reductase (EC 1.17.4.1;RNR), a cell-cycle-regulated enzyme, catalyzes the rate-limiting step in the de novo synthesis ofdeoxyribonucleotides bythe reduction of the corresponding ribonucleotides. The im-portant role of the RNR in DNA synthesis and cell divisionmakes this enzyme an excellent target for chemotherapy.However, nothing is known about this enzyme from the malariaparasite Plasmodium falciparum. We have isolated cDNAclones encoding both the large and small RNR subunits. Thesequences of full-length clones of the large and small RNRsubunits revealed an open reading frame encoding 806 and 349amino acids, respectively, and showed significant identity withother RNR sequences in the data base. RNA blot analysisshowed that the size of the large and small RNR subunittanscripts are 5.4 kb and 2.2 kb, respectively. Both the RNRsubunit transcripts fluctuate in level during the cell cycle,reaching a peak preceding maximal DNA synthesis activity. Anoligodeoxynucleotide phosphorothioate that is complementaryto sequences around the translational initiation codon of thesmall RNR subunit showed significant inhibition of growth, asmeasured by the inhibition in DNA synthesis.

Malaria is one ofthe most prevalent and devastating parasiticdiseases that afflict mankind. Deaths from malaria are be-tween 1 and 2 million each year, occurring most often inchildren (1). Malaria affects the lives of nearly half the worldpopulation, but the drugs available for the treatment of thisdisease have lost effectiveness because of the spread ofdrug-resistant strains, and the immediate possibility of auseful vaccine is uncertain. Therefore, there is an urgent needto understand the biology of the parasite in detail, so thatsusceptible metabolic targets can be identified. With this goalin mind, we have initiated a study to characterize proteinsthat are important for the progression of malaria parasitesthrough the cell cycle.One such protein is the enzyme ribonucleotide reductase

(EC 1.17.4.1; RNR). A continuous balanced supply ofdNTPsfor the replication and repair of DNA is maintained by thereduction of ribonucleotides by RNR. RNR plays a keyrate-limiting role in DNA synthesis by catalyzing the firststep in the dNTP production pathway (2). The RNR has beenintensively studied in many prokaryotes, eukaryotes, andviral systems (3, 4). However, nothing is currently knownabout this essential enzyme in Plasmodiumfalciparum. Theproperties of the RNR and its important role in DNA repli-cation and, hence, cell division make this enzyme an idealtarget for chemotherapy (5).RNR holoenzymes of the Escherichia coli type are iron-

containing heterodimeric enzymes of a232 configuration,where a represents the large subunit (R1) and a represents

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the small subunit (R2) (3, 4). In E. coli and eukaryotic cellsthe Rl protomers, binding to substrates and allosteric effec-tors, have a M, of 83,000-87,000 (6). The R2 protomers, witha Mr of 37,000-45,000, contain ferric iron centers and atyrosyl free radical essential for activity (7). The enzyme-substrate specificity and activity of RNR is regulated by avariety of nucleotides (2, 3).

In this paper, we report the cloning and sequencing of P.falciparum Rl and the large and small R2 protomer cDNAs.¶The cell-cycle regulation of the subunit genes in synchronousintraerythrocytic cultures was also studied. The treatment ofthe parasite culture with an antisense oligonucleotide phos-phorothioate directed against the R2 resulted in a significantgrowth inhibition; this suggests that the R2 is essential forcellular survival.

MATERIALS AND METHODSParasite Culture. P. falciparum strains HB3 and Dd2 were

maintained in an erythrocyte culture as described by Tragerand Jensen (7). If required, cultures were synchronized withtwo rounds of 5% D-sorbitol treatment (8).

Nucleic Acid Isolation and Blot Analyses. The genomicDNAand RNA samples were isolated from saponin-lysed P. fal-ciparum-infected erythrocytes by the SDS-proteinase Kmethod (9) and the acidic guanidinium-phenol/chloroformmethod (10), respectively. Southern and RNA Blots werehybridized with random primer 32P-labeled RNR probesaccording to standard methods (9). A phosphorimager (Mo-lecular Dynamics) was used to quantitate bands on a blot.

Library Construction and Screening. A AZap II directionalcDNA library constructed from P.falciparum strain Dd2 wasscreened by standard techniques (9) with 32P-labeled Rl andR2 probes. Positive clones were plaque purified, and coloniesfrom E. coli-harboring plasmid were obtained from AZap IIclones by in vivo excision with R408 helper phage.DNA Sequencing and Analysis. Double-stranded DNA se-

quencing was done on 5% Long-Ranger (AT Biochem, Mal-vein, PA) gels by the dideoxynucleotide chain-terminationmethod (11) using Sequenase (United States Biochemical).Sequence data were analyzed by Genetics Computer Group(Madison, WI) programs (12) on microVAX computers at theUniversity of Florida Interdisciplinary Center for Biotech-nology Research Biological Computing Facility.DNA Synthesis Measurement. The DNA synthesis was

measured in terms of [3H]hypoxanthine incorporation. Cul-tures (1 ml) were washed with and resuspended in serum- andhypoxanthine-free RPMI 1640 (GIBCO) medium and incu-bated with [3H]hypoxanthine (10 ,Ci per well; 1 ACi = 37

Abbreviations: RNR, ribonucleotide reductase; Rl, large subunit ofRNR; R2, small subunit of RNR.*To whom reprint requests should be addressed.'The sequences reported in this paper have been deposited in theGenBank data base (accession nos. U01322 and U01323).

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Biochemistry: Chakrabarti et al. Proc. Nati. Acad. Sci. USA 90 (1993) 12021

kBq) for 2-4 hr in 24-well cluster dishes. The radiolabeled clones for both Rl (Pf0367) and R2 (Pf0293) were identifiedgenomic DNA was isolated by SDS-proteinase K treatment on the basis of homology to RNR sequences from other(9) in the presence of carrier DNA, treated with RNase to species in the data base. These partial clones were used asremove any labeled RNA, and counted in a liquid scintillation probes to isolate several full-length cDNA clones. Twospectrometer. clones, pR155 (2.9 kb) and pR214 (1.4 kb), were selected for

further studies because they appeared to contain full codingRESULTS AND DISCUSSION sequences based on comparisons of the DNA sequences with

RNR sequences from other species at the National Center forIsolation and DNA Sequence Analysis of P. fakciparum Rl Biotechnology Information data base by BLAST (13) search.

and R2 cDNA Homologue Clones. By sequencing random The DNA sequence of the Rl (data not shown) contains twoclones from a P. falciparum cDNA library, in the University in-frame 5' ATGs. Although the extreme 5' ATG showedof Florida P. falciparum gene sequencing project, partial agreement with the Kozak consensus sequence for a eukary-

AHuman ......H....hV ikRdGrqErV mFD.... kItsRIqklc YG.....Lnmd fVdpaqItiuk 42

Vaccinia ......H....FV ikRnGykEnV mFD.... kItsRIrklc YG.....Lntd hIdpikIaisk 42Pfalciparum ..m..n...k........en.. .e...stsgr lsddgikrtp sgkpiqtMYV lnRkGeeEdI sFD.....qIlkRIqrls Y...... Lh.e lVdparVtqg 41

Yeast ......H.......... YV ikRdGrkEpV qFD.... kItsRItrls YG.... Ldpn rIdavkVtqr 42Ecoli ...................... ..mnqnLLV tkRdGstErI nLD.... kIhrvldwaa eG.... Lhn. .VsisqVelr 44

Herpes apldvcpgtp gpgadaggps avdphaptpe agaglaadpa varddaegls dprprlgtgt aypvpleltp enaeavarFl gdavnrepal mLEyfcrcar eetkRVpprt FGspprLted dfgllnyalv 380Co se sus ---u-- -M-- ------ ---- --FD--- RI----- - R- -V -FY--------- I---Y------- --130-3

Human viqglYsGVt TvEldtlaae taAtltTkh. PDYaiLAARi A.....vsnLhKeTkkv fsd.......... .vmEdLYny inphngkhsp mVakstldiV lankdrLnsa IiYDRDFsYn 144Vaccinia viqgiYnGVt TvEldtlaae iaAtctTqh. PDYaiLAARi A.....vsnLhKeTkkl fse.......... viEdLFny vnpkngkhsp iIssitxsdiV nkykdkLnsv IiYERDFsYn 144

Pfalciparum vingmYsGIk TcEldelaaq tcAymaTth. PDFsiL.AARi t.....tdnLhKnTsdd vae.......... .vaEaLYty kdvr.grpas lIskevydf I llhkdrLnke IdYtRDFnYd 142Yeast iisgvYsGVt TvEldnlaae tcAymtTvh. PDYatLAARi A..... isnLhKqTtkq fsk.......... .viEdLhdw inpatgkhap mIsdeiyniV metry.Lnsa IvYDRDFqYt 143Ecoli shiqfYdGIk TsDihetiik aaAdlisrda PDYqyLAARI A.....ifhLrKkaygq fep .. ......paLYdh v.vkmvemgk ydnihlledyt eeefkqMdtf IdhDRDmtFs 144

Herpes emqrlCldVp pvppnaympy ylreyvTrlv ngFkpLvsRs Arlyrilgvl vhLrirTrea sfeewlrske va,d,fg'lt'e'r lrehEaqLvi laqaldhydc lIhstphtlV erg... Lqsa lkYE.EFyLk 506Consensus----Y-GV- T-E------A---T--- PDY--LAAR- A-------L-K-T -- --------E-LY-- ------I----V-- --L--- I-YDRDF-Y- 260

Human YFGfKtLErs YLL. .kIngk VaERpQhMlm rVsvGi.. hkedidaai etYnlLSerw fthasPtLFN aGTnrpQLSS CFLlsmkdDS iEglYdTl.k qcalIsksaG GIGvaVscIR atGSyIaGtn 266Vaccinia YFGfKtLEks YLL. .kInnk IvERpQhHlm rVavGi.. hqwdidsai etYnlLSekw fthasPtLFN aGTsrhQMSS CFL1rnmidDS iEgIYdTl.k rcalIskmaG GIGlsIsnIR asGSyIsGtn 266

Pfalciparum YFGfKtLErs YLL. .rInnk IiERpQhLlm rVsiGi.. hiddidkal etYhlMSqky fthatPtLFN sGTprpQMSS CFL1smkaDS iEgIFeTl.k qcalIsktaG GIGvaVqdIR gqnsylrGtn 264Yeast YFGfKtLErs YLL..rlnge VaERpQhLvm rValGi.. hgsdiesvl ktYnlMSlry fthasPtLFN aGTplpQMSS CFLiaiskdDS iEgIYdTl.k ecamIlsktaG vls.hInnIR stGSyIaGtn 264Ecoli YaavKqLEgk YLvqnrVtge IyEsaQfLyi lVaaclfsny pretrlqyvk rfYdavStfk islptPiMsg vrTptrQFSS CvLie.cgDS lDsInaTs.s aivkyvsqra GIGinagrIR alGSpIrGge 272

Herpes rFGghyMEsv FqMytrIagf lacRatrgmr hIalGregsw wemfkf.. fFhrLydhq ivpstPaMLN lGTrnyytSS CYLvnpqatt nkatLraits nvsaIlarnG GIGlcVqafn dsGp....625Consensus YFG-K-LE-- YLL---I--- I-ER-Q-L-- -V--G--------- -Y--LS------P-LFN -GT---QMSS CFL----DS -E-IY-T-----I----G GIG--V--IR --GS-1-G-- 390

Human gnsnGlVPmsl rVYnntaryv dqggnkrpGa faiYlepWHi DIfefLdLkk n.tGkEeqRa RdLFfA1WIP DLFiuKRv.....etnqdWs LmcPnecPGL dEvW....GEE FEkLYasYEk qgrvrkv.Vk 385Vaccinia gisnGiIPml rVYnntaryi dqggnkrpGv maiYlepWHs DlmafLdLkk n.tGnEehRt RdLFiAlWIP DLFmKRv.....kddgeWs LmcPdecPGL dnvW. .. .GDE FErLYt1YEr erryksi.Ik 385

Pfalciparum gisnGlVPml rVFndtaryv dqgggkrkGs favYiepWHs DIfefLdLrk n.hGkEelRa RdLFyAvWVP DLFmKRv.....kenknWt LmcPnecPGL sEtW. .. .GEE FEkLYtkYEe ernmgkkt.Vl 383Yeast gtsnGlIPm.i rVFnntaryv trvvtrdlvl spfswshgmq isstkFdirk t.hGkEeiRa RdLFpAlWIP DLFmKRv.....qedgpWt LfsPsaaPGL dDvW. .. .GDE FEeLYtrYEr egrgk.t.Ik 382Ecoli afhtGcIPfy khFqtavksc sqggv.rgGa atlFypmWHl EVeslLvLkn n.rGvEgnRv RhMdygvqIn kLmsytRl.....lkgedit LfsPsdvPGL yDaFfadqEE FErLYtkYEk ddsirkqrVk 394

Herpes .gtasvmPal kVLdslvaah nkesarptGa .cvYiepWHt DVravLrMkg vlaGeEaqRc dniFsAlWmP DLFfKR1irh ldgeknvtWt LfdrdtsmsL aDf. .. .hGEE FEkLYqhlEv mgfgeqipI. 749Consensus ----G-IP-- V ----G--Y--WH- DI---L-L-- -G-E--R- R-LF-A-WIP DLF-KR --- ----W- L--P---PGL -D-W---GEE FE-LY--YE -------520

Human AqqLWyaIie sqteTGtPYm lYKDsCNrks n.qqnlgtIk cSNLCtEIVe ytsk.... .dEvAVCNL aSlaLnmYV. tseht.... .Y dFkkLaEvtk vvvrnLnkiI DinyYPVPeA clsNkrhRpi 501Vaccinia ArvvWkaIie sqieTGtPFi lYKDaCNkks n.qqnlgtIk cSNLCtEIIq yada.... .nEvAVCHL aSvaLnmFV. idgr ...F dFlkLkvvvk vivrnLnkiI DinyYPIPeA eisHkrhRpi 500

Pfalciparum AqdLWfaIlq sqieTGvPYm lYKDsCNaks n.qknlgtIk cSNLCcEIIe ytsp.... .dEvAVCNL aSiaLckFVd lekke.....F nFkkLyEitk iitrnLdkiI ErnyYPVkeA ktsHtrhRpi 500Yeast AgkLWyaIlq aqteTGtPFm vYKDaCNrkt n.qqnlgtIk sSNLCcEIVe yssp.... .dEtAVCNL aSiaLpaFVe vsedgktasY nFerLhEiak vithnLnrvI DrnyYPVPeA rnsNmkhRpi 503Ecoli AveLFslmmsq erasTGriYi qnvDhCHths pfdpaiapVr qSNLClEIal ptkplndvnd engEiAlCtL safnLgai .....n nLdeLeElai lavraLdall DyqdYPIPaA krgamgrRtl 513

Herpes .qeLaygIvr saatTGsPFv mFKDavNrhy iydtqgaaIa gSNLCtEIVh pask.... .rssgVCHL gSvnLarCVs rqt....F dFgrLrDavq acvlmvnimI DstlqPtPqc trgNdnlRsm 864Consensus A--LW--I-- --TG-P-- -YKD-CNH-I----- - -SNLC-EIV ------- --E-AVCHL -S--L--FV------F -F--L-E-----L---IDD---YPVP-A ---H---R-- 650

Human GIGVQGLAda FilMrypFES aeAqlLNKqI FEtiyygaLe aScDLAkeqG .pYetYegSp vskGILqYDm Wnv.tptd. 1..WDWkvL kekIakyGiR NOlLiApMPt astaQIlgnn EsiEPytsNi 624Vaccinia GIGVQGLAda FilLnypFDS leAqdiNKkI FEtiyygaLe aScELAekeG .pYdtYvgSy asnGILgYDi Wnv.vpsd. 1..WnWEpL kdkIrtyGLR HOiLvApLP1 hqhaQllgon EsvEPytsNi 623

Pfalciparum GIGVQGLAdt FmlLrypYES daAkeLNKrI FEtmyyaaLe mSvELAsihG .pYesYqgSp asqGILqFDm Wnakvdnk.. .yWDWDeL kakIrkhGLR NSlL1ApMPt astsQIlgnn EsfEPytsNi 624Yeast alGVQGLAdt YssutLrlpFES eeAgtLNKqI FEtiyhatLe aScELAqkeG .kYstFegSp askGILqFDm Wnak.pfg.. .mWDWEtL rkdIvkhGLR NSItmApMPt astsQIlgyn EcfEPvtsNms 626Ecoli GIGVinFAyy LandgkrYsd gsAnnLthkt FEaiqyylLk aSnELAkeqG .aCpwFnett yakGlLpiDt Ykkdldtian epihYDWEaL reslkthGLR NStLsAlMPs etssQIsnat ngiEPprgyv 641

Herpes GIGmQGLhta clkLgldLES aefqdLNKhl aEvmllsaMk tSnaLcvrga rpFnhFkrSm yraGrFhWEr Fpdarpryeg....EWEmL rqssuskhGLR NSqFvAlMPt aasaQIsdvs EgfaPlftNI 989Consensus GIGVQGLA-- F--L---FES --A--LNK-I FE-- --L- -S-ELA ---G --Y-- 0-- ---GIL--D- W --------WDWE-L ---I---GLR NS-L-A-MP----QI---- E--EP---HN- 780

Human YtrrvlsGEF qiVNPhLLkd LtErglwhee sknqiiacng siqsipei.. .Pddlkql YKTvWEis.Q KtvlkMaAER gaFIDQSqSL Nihiaepny. .gKltsiuhFy....gWkqG LKTOHYYlrT 741Vaccinia YtrrvisGEF qvVNPhLLrv LtErklwnde iknrimadgg sign.tnli ..Pedikrv YKTiWEip.Q KtiikMaADR gaFIDQSqSM Nihiadpsy. .sKltsmhFy....gWsIG LKTG84YYlrT 739

Pfaiciparum YyrrvlsGEF fvVNPhLLkd LfDrglwded mkqqliahng sioyisei.. .Pddlkel YKTvWEik.Q KniidMaADR giFIDQSqSL Niyiqkptf. .aKIssmhFy....gWekG LKTGaYYlrT 741Yeast YsrrvisGEF qvVNPyLLrd LvDIgiwdds mkoylitqng siqglpnv.. .Pqelkel YKTvWEis.Q KtiinMaADR aiYIDQShSL Hilfiqapsm. .gKitsmhFy....gWkkG LKTGMYYlrT 743Ecoli sikaskdGiL rqVvPdy.. ........ehlhda YeilWEmspgn dgylqLvgim qkFIDQSiSa Ntnydpsrfp sgKvpmqqLl kdiitaYkfG vKT.LYYqnT 734

Herpes FskvtrdGEt irpNtiLLke L.Er.tfsgkr Iievmlsda kqwsvaqaip ciePthpirr FKTaFD3yd.Q KllidLcADR apYVDhSqSM tiyvteka.. dgtlpastLv rlivhaYkrG LKTGMYYckv 1115Consensus Y-- --GEF --VNP-LL-- L-E ---------- -------P-- --YKT-WE---Q K----HM-ADR --FIDGS-SL H-------K----F-----W--G LKTOHYY--T 910

Human rpAanpiqFt ink........eklkd kekvskeeee kerntaamvc si. .enrdec imCgs 792Vaccinia kpAsapiqFt idk........dkik....... ...ppvc ....dseic tsCsg 771

Pfalciparum qaAtdaikFt vdthvaknav klknadgvqi trevsretis testvtqnvc plrrnndeqc lmCsg 806Yeast qaAsaaiqFt idp ...... 756Ecoli rdgaedaqdd ivps ......... .......iqddgces gaCki 761

Herpes rkAtnsgvFg gddnivcms ........... .......Cal 1137Consensus --A----F- -C-- 975

BHuman mlslrvplap itdpqqlqls plkg....ilsivdkentp palsgtrvla sktarrifqe ptepktkaaa pgvedepllr enprrfvifp ieYhdiwqmy kkaeasFWta EEvdlSkDiq hw.esLkpeE 123

Vaccinia................mepila pnpnrfvifp iqYydiwrnay kkaeasFWtv EEvdiSkDin dw.nkLtpdE 55Yeast mnpketpska didekdknne etrenv s lkekls ankyks hqhhi mkelnekrvlfp ikYheiwqay kraeasFWta EEidlSkDih dwnnrMnenE 130

Pfalciparum ...... ........madvi nisripifsk qerefsdlqk gkeinekiin kesdrftlyp iiYpdvwdfy kkaeasFWta EEidlSsDik df.ekLnenE 84Ecoli ...... .......... .....mayttfsqtk ndqlkepmfg o.pvnvarydq qkYdifekli ekqisfFWrp EEvdvSrDri dy.qaLpehE 69

Herpes.s.............dpavspa stdpidthas gagaapipvc ptpery.fyt sqCrdinhlr slsilnrWle tElvfvgDee d.vskLsegE 76Consensus --------------------------------------Y--------FW-- EE---S-D-----L---E 130

Human ryFishvLaF faAsDgIVnE NLverFsqev qItEarcFYg fqiamsEnIHS emYsl.ilId tyikDpkere flfnaIetmP cVkkKadWal rWigDkeaty gEr .......... .....224Vaccinia kyFikhvLaF faAsDgIVnE NLaerFctev qItEarcFYg fqmaiEnIHS emYsl. lId tyvkDsnekn ylfnaIetmP cVkkKadWaq kWihD.sagy gEr .......... .....155

Yeast rfFisrvLaF faAsDgIVnE HLvenFstev qIpEaksFYg fqimiEnIHS etYsl..lld tyikDpkese flfnaIhtiP eIgeKaeWal rWiqDadalf gEr .......... .....231Pfalciparum khFikhvLaF faAsDglVlE HLaskFirev qItEakkFYs fqiavEnIHS etYsI.1Ild nyikDekerl nlfhaIeniP aVknKalWaa kWinDtns.f aEr .......... .....184

Ecoli khifisnLkY qtllDsIqgr spnvaLlpii sIpEletWve twafsEtIHS rsYt. .hiIr nivnDps... vvfddIvtne qlqkraegis sYydEliemt sywhllgegt htvngktvtv slrelkkkly 194Herpes igFyrflFaF isAaDdiVtE NLgg.Lsglf eqkDilhYYv eqeciEvVHS rvYniiqlVl fhnnDqarra yvartInh.P aIrvKvdWle aRvrEcds.i pEk .......... .....176

Consensus --F----L-F --A-D-IV-E HL---F----I-E---FY----E-IHS --Y----I- -D------I---P ----K--W-- -W--D -----E--------- -----260

Human .vvafaavE GIfFsgSFAs IFwLkkrgIM pditfSneLI sRDEgLHcdf acImFkhlvh ....kpseerV re.....iiinAVriEq eFiteaiP.v kliGMNctlM ... kqYIeFv ADrLmleiGf 337Vaccinia .. iiafaavE GIfFsgSFAs IFwLkkrglM pGitfSneLI sRDEgLHcdf acImsFkhllh ....ppseetV rs.....iitdAVsiEq eFitaaiP.v kliGMNcemM ... ktYIeFv ADrLiselGf 268

Yeast ..livafasiE GVfFsgSFAs IFwLkkrgmM pGltfSneLI cRDEgLHtdf aciiFahikn ... kpdpaiV ek.....ivteAVeiEq rYfldalP.v allGHNadlM ... nqYVeFv ADrLlvafGn 344Pfalciparum .ivanacvE GIlFsgSFca IFwFkkqnkL hGltfSneLI sRDEgLHtdf ncliYslldn ... klpeqiI qn.....ivkeAVevEr sFicesiP.c dliGMNsrlM ... sqYIeFv ADrLleclGc 297

Ecoli lclmsvnalE aIrFyvSFAc sFaFaereiM eGnakiirLI aRDEaLHitg tqhmLnllrs gaddpemaeI aeeckqecyd ifvgAaqqEk dWadyifrdg smiGLNkdiL c....qYVeYi tnirisqavGl 321Herpes .filmiliE GVfFaaSFAa IayLrtnnlL rvtcqSndLI sRDEavHtta scyiYnnylg ghakpeaarV yr..... lf reAVdiEi gFirsgaP.t dssiLspgaL aaienYVrFs ADrLlgiihms 295

Consensus ------ GI-F--SFA- IF-L-H-- -GO---S--LI -RDE-LH-----F---------V------ --AV--E-OHH---P- MN--M-H---Y--F- AD-L----OG- 390

Human skvFrv.eNP fdf..meniS legkTNFFEk RVgeYqrmgV ins... .spten sftldadf 38)Vaccinia kkiYnv.tNP fdf..meniS legkTNFFEk RVgeYqkmgV m. .... sqedn hfsldvdf 319

Yeast kkyYkv.eNP fdf..meniS lagkTNFFEk RVsdYqkagV mskstkqeag aftfnedf 399Pfalciparum skiFhs.kNP fnw..mdliS lqgkTNFFEk RVadYqksgV maq... rkdq vfclntef 349

Ecoli dipFqtrsNP ipwintwIvS dnvqvapqEv eVssYlvgqI dsevdtddls nfql.... 375Herpes cplYsa.paP dasfplslmS tdkhTNFFEc RstsYagavV ndl ....... 337

Consensus --------NP ---------S ----TNFFE- RV--Y----V ---------- 448

FIG. 1. Comparison of RNR subunit-deduced amino acid sequences with RNR sequences in the National Center for BiotechnologyInformation data base using PILEUP and PRETTY programs of the Genetics Computer Group package. (A) Rl. (B) R2. The second in-framemethionine of the P. falciparum Rl is assumed to be the translational start site.

12022 Biochemistry: Chakrabarti et al.

otic initiation site (14), the sequence alignment with otherRNR sequences (Fig. 1A) indicated that the second ATG atamino acid residue 42 is most likely the translation start site.At the 3' end, the eukaryotic poly(A) signal AAUAAA is notseen, but an 18-base poly(A) tail could be detected. An openreading frame of 2418 bp extends from the second 5' AUG tothe termination codon UAA. The 3' untranslated region is 127bp long. Translation of the cDNA results in a predicted Rlpolypeptide of 806 amino acids.The RNR protomer sequences from human, vaccinia,

yeast, P. falciparum, E. coli, and herpes virus were alignedby using several Genetics Computer Group programs (Fig. 1A and B). The alignment for the Rl, shown in Fig. 1A, startedwith amino acid 251 of the herpes simplex virus. The herpessimplex virus Rl protomer is unusually large with a Mr of136,000 (15), and the nonhomologous NH2 terminal end is notincluded in the alignment. The overall identity of Plasmo-dium Rl with human Rl is =67%. A lesser degree of identitywas seen between Rl from different sources at the NH2terminal and COOH-terminal ends. The NH2 terminal regionof Rl has been implicated in the regulatory activity (16). Alow identity may indicate a difference in the allosteric regu-lation of the P. falciparum enzyme from its human host. TheCys-225 and Cys439 of the E. coli protein that are importantfor nucleotide binding (4) are conserved in P. falciparumRNR. The COOH-terminal cysteines that participate in theinteraction with thioredoxin and glutaredoxin are also con-served in P. falciparum, as is the Asp-57 of the mammalianenzyme that is involved in the dATP feedback inhibition.The R2 cDNA sequence contained an open reading frame

of 1047 bp, encoding a 349-amino acid protein. The consensuspolyadenylylation signal is also not detected in the R2 se-quence. The R2 3'-untranslated region is 231 bp, ending witha poly(A)18 tail. The Plasmodium R2 is -57% identical withthe human R2. As with the Rl protomer, the identities at theNH2 terminal and the COOH-terminal ends are lower. In fact,the NH2-terminal sequences of the R2 from different sourcesare quite divergent (Fig. 1B), indicating that these sequencesmay be dispensible for the enzyme activity. Several con-served residues that are reported to be important for thecatalytic activity of RNR in E. coli (17) are conserved inPlasmodium R2 also (Fig. 1B). They are as follows: Glu-115,His-118, Glu-204, Glu-238, His-241 for iron ligands; Tyr-122,Phe-208, Phe-212, Ile-234, Ser-211 for the tyrosyl radicalenvironment; Asp-237 and Trp-48 for the hydrogen-bondingnetwork; and Asp-58, Arg-236, Glu-350, and Tyr-356, whichare involved in the binding to the Ri. The amino acid numbersmentioned reflect the corresponding numbers in E. coli RNR.By using the MOTIF program, the PROSITE data base (a

dictionary of protein sites and patterns, University ofGeneva) was searched for putative motifs present in the RNRprotomer-translated amino acid sequences. Several proteinkinase C (positions 102, 192, 208, 220, 232, 280, and 420),tyrosine kinase (128, 147, 275, 322, 478, 528, and 529), and asingle cAMP (291) phosphorylation sites were detected in theRl-deduced amino acid sequence. The R2 showed the exis-tence of a single protein kinase C phosphorylation site atamino acid 38. It would be of interest to study whether or notthese phosphorylation sites are used in vivo. Because theRNR plays such an important role in DNA replication and,hence, in cellular proliferation, the phosphorylation sites onRNR subunits may be important for the regulation of prolif-erative responses.

Organization ofRNR Protomer Genes and Expression. TheSouthern blot analysis ofPlasmodium genomic DNA digestswith restriction enzymes, probed with full-length Ri (pR155)and R2 (pR214) clones, showed only a single band withenzymes that do not have sites in the coding sequences (Fig.2A). This banding pattern is consistent with the presence ofa single-copy gene for both RNR protomers. The results of

Akb 1 2 3 4 5 6 7 8 9 10

12 -

8-6-

4-

2-

1-

B

kb 1 2

7.4 -

5.4 -

2.2 -

FIG. 2. (A) Southern blot analysis of the restriction enzyme-digested P. falciparum strain Dd2 genomic DNA (3 pg) probed withRl (lanes 1-5) or R2 (lanes 6-10) full-length cDNA clones. Lanes: 1and 6, EcoRI; 2 and 7, EcoRV; 3 and 8, HindIII; 4 and 9, Spe I; Sand10, Xba I. For Rl, Xba I has no site, and for R2, EcoRV and Spe Ienzymes have no sites. (B) RNA blot analysis of total P. falciparumRNA from asynchronous culture probed with 32P-labeled pR155 (forR1) and pR214 (for R2).

hybridizational analysis of Southern blots of P. falciparumchromosomes showed positive signals corresponding tochromosome 14 (data not shown) when either probe wasused. Because the genes for both Ri and R2 are located onchromosome 14, it would be of interest to determine whetherthey are linked. Many glycolytic enzymes, such as glucosephosphate isomerase, aldolase, and 3-phosphoglycerate ki-nase, are also located on the stable chromosome 14 (18).RNA blot analysis revealed a 5.4-kb Ri transcript (Fig. 2B,

lane 1). This result suggests that the Ri mRNA has a verylong 5'-untranslated region. The combined length of the Ricoding region and the 3'-untranslated sequences is 2541 bp.The 2.9 kb of 5'-untranslated region is extremely long but isnot unusual for P. falciparum. For example, the transcriptsize of 3-phosphoglycerate kinase was found to be =1.7 timesthat of the coding sequences (18). Similar results were alsoobtained with tubulin genes (19). An indication of a 7.4-kbtranscript hybridizing with the Rl probe was also detected;this could be an unprocessed or an alternate Rl transcript.The transcript size of the R2 was measured to be 2.2 kb.During its progression through the cell cycle, P.falciparum

undergoes distinct morphological changes: (i) rings (the format the initiation of intraerythrocytic life cycle without anyhemozoin pigment), (ii) trophozoites (mononucleated cellscontaining hemozoin pigment), and (iii) schizonts (multinu-cleated cells). The complete developmental cycle takes=42-48 hr. To address the question of the periodicity of Riand R2 gene expression during intraerythrocytic growth ofthe malaria parasite, the synchronized cultures were pro-cessed every 6 hr to monitor parasitemia, DNA synthesis interms of [3H]hypoxanthine incorporation, and RNR subunitexpression, as determined by the RNA analysis followed byquantitation in a phosphorimager. The filter for RNA analysiswas also hybridized with a Plasmodium berghei small rRNAprobe, and the values obtained for a small rRNA band at eachtime point were used to normalize the level of Ri and R2transcripts. Fig. 3A shows that the DNA synthesis, asmeasured by [3H]hypoxanthine incorporation, was initiatedafter 12-hr postsynchronization and reached its peak at =24hr, at which time :85% of the parasites were mature tro-phozoites. Fig. 3B shows that the steady-state transcriptlevels of the Rl and R2 are coordinately regulated during the

Proc. Natl. Acad. Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 12023

A10

8

.6 Q U Ring8 U Early Troph

1Mature Troph4 13 Schizonta,L sDNA

. 2 Synthesis

0

6 12 18 24 30 36 42Time after Synchrony, hr

6 12 8 24 30 36 42Time after Synchrony, hr

FIG. 3. (A) Distribution ofrings, trophozoites (Troph), schizonts,and DNA synthesis activity in a synchronized P. falciparum in-traerythrocytic culture. Samples were processed every 6 hr aftersynchronization for slide preparation for microscopy and DNA-synthesis measurement. To analyze DNA synthesis, a 1-ml culturewas incubated with 10 jICi of [3H]hypoxanthine for 2 hr in a

hypoxanthine- and serum-free medium. (B) Cell-cycle-dependentexpression ofRl and R2. Total RNA was isolated every 6 hr from P.falciparum strain Dd2 erythrocyte cultures. Approximately 20 pg ofRNA was electrophoresed on a formaldehyde gel, analyzed on aNorthern blot, and quantitated in a phosphorimager. The results ofquantitation are represented as counts, as determined by volumeintegration (sum of all pixels corresponding to the band area), afternormalization with the values obtained for the small rRNA band foreach time point.

cell cycle, reaching peak expression at 18 hr. This coordinateinduction of expression of the RNR subunit genes is ex-pected, as both subunits are needed for a functional enzyme.The peak Rl and R2 mRNA levels were attained at 18 hr, justpreceding the peak in the rate of [3H]hypoxanthine incorpo-ration that occurred 24 hr after synchronization. Theseresults suggest that the abundance of RNR subunit tran-scripts in P. falciparum are regulated during the cell cycle,being expressed just before and during the S phase. Similarresults were observed with mammalian RNR, where both Riand R2 transcripts increase in parallel correlated with theDNA synthesis (20). However, the Ri polypeptide level inmurine cells is constant, and the expression of R2 polypep-tide is linked with the S phase, indicating a longer half-life ofRi protein (20). It would be ofinterest to study whether or notsimilar mechanisms are operative in the malaria parasite. Theinduction of expression of the Ri transcript in P. falciparumappears higher compared with that of R2 (Fig. 3B). Anotherinteresting observation was that the 7.4-kb Ri transcriptdetected by the RNA analysis (Fig. 2B) was not present in theS phase but could be detected in the ring stage (data notshown). The 7.4-kb transcript may be a precursor that is

processed to yield the mature transcript. Another possibilityis that the 7.4-kb band, as in Saccharomyces cerevisiae (21),corresponds to an alternative Rl transcript that is present forDNA repair. At this point, the significance of the existenceof this transcript hybridizing with Rl probe is unclear.

Anfimalarial Activity of an Oligodeoxynucleotide Phospho-rothioate Complementary to R2 Sequences. Antisense oligonu-cleotides by virtue oftheir ability to inhibit gene expression arepotential chemotherapeutic agents (22, 23). Recently, nu-clease-resistant phosphorothioate-modified oligonucleotidescomplementary to sequences around the translational initia-tion site of the P. falciparum dihydrofolate reductase-thymidylate synthetase gene showed significant antimalarialactivity (24). Only erythrocytes infected with P. falciparumare permeable to- labeled oligodeoxynucleotides (24). Theoligonucleotides most likely enter the parasite through therecently described parasitophorous ducts (25). This duct sys-tem allows an ideal system for the antisense oligonucleotidemediated malaria chemotherapy. Because the RNR is anessential enzyme that provides a continuous supply ofdNTPsfor DNA synthesis (2), a study was initiated to test the effectof oligodeoxynucleotide phosphorothioate that is complemen-tary to P. falciparum RNR sequences. The R2 transcriptsappeared to exist in limiting amounts (Fig. 3B); therefore, inthe initial study reported here, the translational initiationregion ofthe R2 sequences were chosen to synthesize a 20-merantisense oligonucleotide phosphorothioate. Asynchronous P.falciparum strain Dd2 culture (chloroquine resistant) wasincubated for 48 hr with the antisense oligonucleotide phos-phorothioate. The inhibition ofgrowth in terms of inhibition inthe DNA synthesis was measured by [3H]hypoxanthine in-corporation. It is evident from the results described in Table1 that the oligodeoxynucleotide phosphorothioate comple-mentary to the R2 sequences around the translational initiationcodon (DEC56) showed significant inhibition of growth at 1.0uM concentration. Similar results were obtained with syn-chronous cultures (data not shown). In contrast, the nonspe-cific oligonucleotide phosphorothioate was ineffective at anequimolar concentration. The IC50 for DEC56 oligonucleotideis between 0.5 and 1.0 ,uM. Comparable IC50 values were alsoobtained with specific antisense oligonucleotides directedagainst the dihydrofolate reductase-thymidylate synthetasegene of P. falciparum (24). However, different regions of thesubunit genes must be probed to obtain maximal growthinhibition. These results show that the R2 is essential forsurvival of the malaria parasite, and the RNR sequences, inaddition to the P. falciparum dihydrofolate reductase-

Table 1. Inhibition of P. falciparum growth by oligonucleotidephosphorothioate directed against R2

DNAsynthesis,* Inhibition,

Oligonucleotide cpm x 10-3 %None 54.6Nonspecific (DEC57) (1.0 ,M) 44.7 17Specific (DEC56) (0.5 ,uM) 37.3 31Specific (DEC56) (1.0 ,uM) 21.4 60

Oligonucleotides were as follows: DEC56, complementary tosequences around the initiation codon (-7 to +13) of the R2,5'-TAACATCAGCCATTCTATTC-3'; DEC57, nonspecific mis-matched sequence as described in ref. 24, 5'-GCCGGCGGC-CCGCGGCGCCGG-3'. DNA synthesis was measured in terms of[3H]hypoxanthine incorporation. After antisense oligonucleotidetreatment for 48 hr with a change of medium containing oligonucle-otide at 24 hr, cultures were washed and resuspended in serum- andhypoxanthine-free medium. To measure DNA synthesis, the [3H]hy-poxanthine incorporation was continued for 4 hr. Results representthe average of three separate experiments.*cpm x 10-3 per 6 x 107 parasites.

c0

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Biochemistry: Chakrabarti et al.

12024 Biochemistry: Chakrabarti et al.

thymidylate synthetase gene, could be exploited for potentialantisense oligonucleotide-mediated chemotherapy.

We thank Michael Weig for his excellent technical assistance andDavid Allred for comments on the manuscript. This work wassupported by grants from the Division of Sponsored Research,University of Florida.

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