MOLECULAR AND CELLULAR BIOLOGY, Oct. 1991, p. 5244-52500270-7306/91/105244-07$02.00/0Copyright C) 1991, American Society for Microbiology

Vol. 11, No. 10

Amplification of the Multidrug Resistance Gene pfmdrl inPlasmodium falciparum Has Arisen as Multiple

Independent EventsTONY TRIGLIA,* SIMON J. FOOTE, DAVID J. KEMP, AND ALAN F. COWMAN

The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia

Received 20 May 1991/Accepted 25 July 1991

The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genesthat results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) inPlasmodium fakciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P.falciparum have amplified levels of the pfmdrl gene. To investigate the nature and origin ofpfmdrl amplicons,we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificialchromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel methodemploying inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We showthat the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flankthe breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandemamplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shownin 26 independent CQR isolates, 16 of which contain amplified copies of pfmdrl, that amplification of thepfmdrl gene in P. falciparum has arisen as multiple independent events. These results suggest that this regionof the genome is under strong selective pressure.

The multidrug resistance (mdr) genes that can encoderesistance to a broad range of cytotoxic drugs in mammaliantumor cells (9) have counterparts involved in diverse biolog-ical phenomena. mdr-like genes have been implicated incystic fibrosis (27), amino acid (13) and protein (21) transportin bacteria, pigment transport in Drosophila melanogaster(35), and transport of a mating pheromone in Saccharomycescerevisiae (18). The mdr genes encode an ATP-bindingglycoprotein (P-glycoprotein), which has been implicated inthe multidrug-resistant (MDR) phenotype in mammaliantumor cells and is thought to function as an energy-depen-dent pump that exports the cytotoxic drugs out of the cell,thus lowering the intracellular concentration to sublethallevels. Overexpression of this protein usually involves am-plification of the mdr gene.The development of resistance in the malaria parasite

Plasmodium falciparum to clinically useful antimalarialdrugs, in particular chloroquine, has become a major prob-lem (24). Chloroquine resistance (CQR) can be reversed bymany of the same drugs that modulate the MDR phenotypein mammalian tumor cells, suggesting that it may be medi-ated by an analogous mechanism (22). Chloroquine is accu-mulated less by resistant than by sensitive parasites (15), andefflux of chloroquine from resistant parasites has beenshown to be increased 40- to 50-fold compared with that inchloroquine-sensitive (CQS) parasites (17). The similaritybetween the phenotypes of CQR and MDR prompted asuccessful search for mdr gene homologs in P. falciparum,and two homologs, designated pfmdrl and pfmdr2, wereidentified (12, 43). The pfmdrl gene encoded a P-glycopro-tein homolog consisting of two homologous halves that eachcontain six hydrophobic transmembrane regions and a nu-cleotide-binding fold (12). The pfmdrl gene was amplified insome but by no means all CQR isolates of P. falciparum.

* Corresponding author.

Five copies of the pfmdrl gene were located on amplicons ofabout 110 kb in clone B8, derived from ItG2, and of 20 kb orless in Indochina 1 (IC1). Such amplification has generatedextensive size polymorphism of chromosome 5. Amplifica-tion ofpfmdrl has also been observed in an in-vitro-selectedmefloquine-resistant isolate W2mef (43).

Identification of amino acid differences in the pfmdrl genethat were strongly linked to CQR strengthened the hypoth-esis that the protein product of the pfmdrl gene is involvedin the CQR phenotype (11). The product of the pfmdrl genewas found by immunoelectron microscopy to be located inthe membrane of the food vacuole, the most likely site ofaction for chloroquine (6). An apparent contradiction, how-ever, is that a genetic cross between CQR and CQS clones ofP. falciparum showed no linkage of the pfmdr genes with theCQR phenotype (41).

In mammalian tumor cells, amplification of mdr genesalways occurs when cell lines are selected for high levels ofresistance but is not required for low levels of drug resis-tance (9). However, the events occurring at the breakpointsof these amplifications in MDR have not been examined.Amplification of selectable genes such as ampC (8) and thelac operon (37) in Escherichia coli have indicated that theamplified units are arranged in tandem arrays and that breaksoften occur at short repetitive stretches of DNA. In ampli-fications of the dihydrofolate reductase (dhfr) and CADgenes in mammalian cells, amplicons are arranged in tandemor as inverted palindromic repeats (34). The breakpointsanalyzed in a number of these systems indicate that AT-richelements are often involved in mammalian DNA amplifica-tions (19, 34).

In order to investigate the structure of the pfmdrl ampli-con, we have isolated segments of P. falciparum DNAcloned in the yeast artificial chromosome (YAC) system (2).We report here the structure of the breakpoints of the 110-kbamplicon in the CQR clone B8 (1) and show that an amplifiedunit with these breakpoints is present only in B8 among a

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AMPLIFICATION OF THE pfmdrl GENE IN P. FALCIPARUM 5245

total of 28 CQR isolates examined. The sequences adjacentto the breakpoints suggest a mechanism for the tandemamplification. We conclude that amplification of the pfmdrlgene in CQR isolates of P. falciparum has arisen in multipleindependent events.

MATERIALS AND METHODS

Parasite DNA. Parasites maintained in asynchronous cul-tures were used for preparation of DNA in solution or asagarose blocks (5). The parasites were grown in RPMI-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-5.8% NaHCO3 with 10% human serum as previously de-scribed (38).YAC DNA. DNA from individual yeast cultures was

pooled to facilitate screening by using a polymerase chainreaction (PCR)-based method (14). Agarose blocks contain-ing yeast DNA were prepared by a standard method (30).The B8 YAC library has been described previously (39).

Cloning, sequencing, and hybridization. The 252L probe inFig. 1 was obtained by inverted PCR (IPCR) (40) across thecloned left boundary of the YAC 252 clone to obtain theflanking P. falciparum sequence. YAC 252 DNA, digestedwith RsaI and ligated at a low DNA concentration, wasamplified by using primer 38 (position 5247 to 5230 on theantisense strand, CGCAAGAGGCCCGGCAGT) and primer39 (position 5418 to 5438 on the sense strand, GGCGACCACACCCGTCCTGT), which are derived from the pBR322sequence contained within the YAC4 vector (2). A fragmentof approximately 360 bp was obtained from the left end ofYAC 252. Fragments obtained by IPCR which were to besequenced were 5' phosphorylated and then subcloned intothe SmaI site of the M13mp8 vector. Sequencing wasperformed by using the chain termination method of Sangeret al. (29). DNA was separated electrophoretically in normalagarose gels or by pulsed-field gradient electrophoresis in acontour-clamped homogeneous electric field apparatus (4)and blotted to Hybond N (Amersham) and hybridized with32P-labeled probes.

Nucleotide sequence accession number. The nucleotidesequence shown in Fig. 4 has been assigned the EMBLaccession number X56851.

RESULTS

It has been shown previously that in the CQR B8 clone ofP. falciparum one breakpoint for amplification of the pfmdrlgene is just 3' to the coding region of the gene (12). In themap shown in Fig. 1A, the regions on either side of this 3'breakpoint are identified as regions 1 and 2. At the other endof the amplicon (defined as the 5' end with respect to thepfmdrl gene), the corresponding sequences on either side ofthe breakpoint are identified as regions 3 and 4 (Fig. 1A).From the structure expected for a tandem repeat of such anamplicon, each internal breakpoint should contain region 1adjacent to region 3 (Fig. 1C). Figure 1A also shows thelocation of the probes P2, P1, and 510 from pfmdrl and its 3'flanking region (12) used here. The precise location of theseprobes is given in the legend to Fig. 1.

Isolation of YACs that include the pfmdrl gene. In order toobtain a cloned DNA fragment including the internal break-point of B8, a YAC library (39) made from B8 was screenedfor the pfmdrl gene by the PCR. YACs 252, 255, and 387were positive for the pfmdrl gene, and the two largest clones(YACs 252 and 255), each of about 100 kb, were analyzedfurther. The YACs 252 and 255 contain the pfmdrl gene,

A 5' br.pt in B8413 Bg3' br.pt in B8

3D7

B

C

D

300 z P2 T''252L510

10kb

pfmdr 14 3 1 2

Bg P

3D7

110kb

5 br.pt Internal Intemal Intemal Intemal 3' br.ptbr.pt br.pt br.pt br.pt

4 3 1 3 1 3 1 3 1 3 1 2

BgBg Bg p Bg P Bg P

252 255

110kb

58 61

413 1

GGG(A)3CAATA

110kb

B8

ii 1 11 11 11 B8I 3 1)3

\ /

CAT(A) CAATA30

113 l1Z

CAT(A) TATAT30

FIG. 1. Location of probes and YACs within the B8 amplicons.(A) A portion of chromosome S surrounding the unamplified pfmdrlgene of the 3D7 isolate. Note that in panel A, the lengths of theprobes are not to scale. (B) The 3D7 region surrounding the pfmdrlgene drawn to the same scale as the B8 amplicons in panels C and D.(C) The locations and approximate sizes of YACs 252 and 255. Themaps shows sites for the enzymes BglII (Bg) and PstI (P). (D) TheB8 amplicons with the DNA sequence across each breakpointshown. The location of the primers 58 and 61 used to amplify theDNA across the internal breakpoint of B8 are shown. In panel A, P2is a probe from the coding region of pfmdrl. The derivation of 252Lfrom YAC 252 is described in Materials and Methods. The 510 probeextends from an XbaI site approximately 350 bp 5' to the SspI siteat position 6869 (Fig. 4). The P1 probe originates at the BamHI siteat position 7062 (Fig. 4) and extends in the 3' direction for approx-imately 1.5 kb. The 300 probe was PCR amplified from 3D7 DNA byusing primers 59 and 60 (Fig. 4). The numbers 1, 2, 3, and 4 refer tothe regions ofDNA surrounding each breakpoint with respect to the3D7 sequence as described in the text.

since the probe P2 from the coding region hybridized toDNA from 3D7 (a parasite with a single copy of the pfmdrlgene), more intensely to DNA from B8 (since it has fivecopies of pfmdrl), and to DNA from both of these YACs(Fig. 2A). Figure 2B shows an overexposure of the sameSouthern blot hybridized with probe P1, from region 2. P1fails to hybridize to DNA from YAC 252 but hybridizes toYAC 255, B8, and 3D7 DNAs. Since the pfmdrl gene islocated near the middle of both of these YACs (data notshown), YAC 252 must be located within the amplifiedregion, whereas YAC 255 spans region 2 encompassing theP1 probe (Fig. 1C).

VOL. 11, 1991

5246 TRIGLIA ET AL.

YAC 88YA

3D7

252 255

kb

25

15

3.6

B X B X BX B X B X B X BX B XFIG. 2. YAC 252 spans the internal breakpoint in B8. DNAs

from YAC 252, B8, YAC 255, and 3D7 were digested with BamHI(B) or XbaI (X), fractionated electrophoretically, blotted, andprobed with the P2 (A) or P1 (B) probe shown in Fig. 1. Both probeshybridized to YAC 255, but only P2 hybridized to 252.

The internal breakpoint in B8 occurs at a string of Aresidues. In order to more accurately position the site of thebreakpoint in B8, DNA from YAC 252, YAC 255, and B8was digested with various enzymes, blotted, and probedwith the 510 probe, which is from region 1, 5' to thebreakpoint. YACs 252 and 255 give different patterns offragments, but B8 shows both patterns (Fig. 3). Since the

Y252 Y255 B8

A S Sau A S Sau A S Sau

bp

- 9400

1600

- 1050

680

FIG. 3. The internal breakpoint in B8 is located 3' to the 510probe. DNAs from YAC 252, YAC 255, and B8 were digested withAseI(A), Scal (S), or Sau3A1 (Sau), fractionated electrophoreti-cally, and blotted. They were then probed with the 510 probe (Fig.1), which is located just 3' to pfmdrl. Since the AseI and Scalfragments differ in size between YAC 252 and YAC 255, the 3'breakpoint must occur within the region spanned by these frag-ments, which is only -800 bp in the case of the AseI fragment.

amplified fragments in B8 correspond to the fragments inYAC 252, this confirms that YAC 252 spans the internalbreakpoint of B8 (Fig. 1C) and localizes the breakpointbetween the 510 and P1 probes.To determine the sequence of the internal breakpoint in

B8, we used the IPCR technique (39) to jump in the 3'direction across the breakpoint within YAC 252 from region1 to region 3. DNA from YAC 252 was digested with SspI,ligated at a low DNA concentration to enhance the formationof circles, and then amplified with two oligonucleotides (56and 58, Fig. 4) by the IPCR. A fragment of 577 bp wasobtained, sequenced, and compared with the same sequencein 3D7 DNA. The sequence of region 1 in YAC 252 wasidentical to that of 3D7 up to a string of 30 A residues butdiffered after this string. The sequence of region 3 thereforecommenced after the string of A residues (Fig. 4, position137 to 496).The 5' breakpoint in B8. The 360 bp of sequence from

region 3 obtained as above was used to jump by the IPCRacross the 5' breakpoint in 3D7 DNA to region 4 (Fig. 1B).This assumed that the amplicons in B8 were arranged headto tail (Fig. 1D), as suggested from preliminary evidence(12). Thus, oligonucleotides 61 and 62 (shown in Fig. 4) wereused to amplify a sequence 5' to the breakpoint by using theIPCR and SspI-cut 3D7 DNA. A fragment of 224 bp,corresponding to region 4, was obtained, and part of itssequence is shown in Fig. 4 (position 1 to 136). Once again,the breakpoint in B8 occurs at a string of 30 A residues.The sequence surrounding the internal breakpoints and

the 5' and 3' breakpoints in B8 is shown in Fig. 4. Thesequences of the four internal breakpoints are assumed to beidentical. An important aspect of the B8 amplicon is thatonly the 3' end of each amplicon contains the string of 30 Aresidues.The amplicons in B8 are arranged in a head-to-tail tandem

array. Preliminary evidence already indicated that the am-plicons in B8 were arranged in a tandem array (12). Thisassumption has been confirmed here in two ways. First,primers 58 and 59 (Fig. 4) across the B8 internal breakpointwere used in PCR amplification reactions with each primerseparately or both together. Only the combination of primers58 and 59 gave the expected fragment (data not shown),whereas either primer alone would be expected to generate afragment if head-to-head or tail-to-tail amplicons existed inB8 DNA. Second, only single fragments in both PstI andPstI-BglII doubly digested B8 DNA, probed with eitherprobe 300 or 252L (Fig. 5), were detected. Since these siteseach occur only once within each amplicon, this resultconfirmed that all amplicons were arranged in head-to-tailtandem arrays.Only isolate ItG2 has the B8 internal breakpoint. By using

primers spanning the B8 internal breakpoint (58 and 61, Fig.4), 30 isolates or clones thereof were analyzed by the PCRfor the presence of a 258-bp fragment. Of the 28 CQRisolates, two were previously known not to have an ampli-fied pfmdrl gene (Vi and Kl), while four others werepreviously known to be amplified (W2mef, IC1, FCR3, andItG2). The results indicated that only three isolates, ItG2,FCR3, and FCB, gave the expected 258-bp fragment (datanot shown). Although FCR3 and FCB were supposedly ofAfrican and ItG2 of South American origin, the possibility ofprevious cross-contamination of these widely used isolatesat some stage was suspected. Probing HindIII-digested DNAfrom a number of isolates showed that FCR3, ItG2, and FCBwere derived from the same parent, since they gave the samepattern of bands with a repetitive probe (Fig. 6). Despite

A BYAC YAC252 BS 255 3D7

MOL. CELL. BIOL.

AMPLIFICATION OF THE pfmdrl GENE IN P. FALCIPARUM

120

Sspl5' breakpoint in B8

4 1 360

AATATATAACAATATATAACAAAGAACAAAAGAATTAAATAATAAAAGCATAAAAGAATGGATTAAAATGTACCTATATATATATATATATATATATATAGAA

61

360

62

240

59

GGAAGGATTAAATATT........ 110 kb pfrndrl segment...... AATATTCTTTTAAGCATTCACAACTATAAACCATATGTTCACGAAATGCATTACAACTAASspl Sspl 6

58 56

CAGTAGCTCCTATTTTTTCGAGTGATTTTATGGTTCTTAAATGAGATAAATGTGCCGTACTATGAAAAGCCATATTTTCAAGCATTACGCTCATACCTTGTTCATTTACTTTGTCGTTTA

3' breakpoint in B8

BamHl

TTTCTTCGTATCTGGATCCGCACTTTACATATAAACCTGCCACA AAAAA MTAAAATATATAACATGTATGTAAAATTTTCATAAAAAAAAAAAAAAAAATATATAT

480

6928

7048

7168

ATACTTACATATATACATATATATATACATAAATATATACATACATATATACATACATATATACATACATATATATATATATATATATATATATATATAT .............................7268

FIG. 4. Sequence surrounding the 5' and 3' breakpoints of B8 in 3D7. (A) The sequence in wild-type CQS 3D7 DNA is shown. Breakageand amplification does not actually occur in 3D7, but the position of these breaks in B8 are indicated on the 3D7 sequence. The SspI sitesand oligonucleotides (56, 58, 61, and 62) used in the IPCR experiments are shown together with the locations of primers 59 and 60. Thelocations of the primers 58 and 61 across the internal breakpoint are also shown.

this, FCR3 lacks part of exon 1 of the RESA gene (Fig. 6), acharacteristic genetic marker (3, 24). The conclusion that theisolates FCR3, ItG2, and FCB that are commonly used inmalaria laboratories actually are descendants of the samelaboratory isolate has been independently reached by others(28). Hence, we conclude that of 26 independent CQR

isolates examined, only ItG2 (the parent of B8) had the B8breakpoint.

Amplification status of CQR isolates lacking the B8 break-point. The amplification status of the pfmdrl gene in 21 of the

A B C D

A

P P/Bg

kb

110

40

B

P P/Bg rep20

kb

- 110

70

RESA * e*

kb

- 9.4

- 4.4

- 2.0

- 1.3

- 2.0

-1.3

FIG. 5. The pfmdrl amplicons in B8 are in a tandem array. B8DNA prepared in agarose plugs was digested with PstI (P) or bothPstI and BglIH (P/Bg), fractionated electrophoretically in a contour-clamped homogeneous electric field apparatus with a pulse time of 8s for 16 h at 180 V, and probed with the 300 (A) or 252L (B) probeas shown in Fig. 1.

FIG. 6. Isolates FCB, FCR3, and B8 are derived from the sameparent. DNAs from FCB (A), FCR3 (B), B8 (C), or FC27 (D) weredigested with Hindlll, fractionated electrophoretically, blotted, andprobed with rep20 (23) or a RESA fragment (3). FC27 is an isolatefrom Papua New Guinea and is unrelated to the other isolates.

VOL. 11, 1991 5247

5248 TRIGLIA ET AL.

TABLE 1. Amplification status of CQR isolates lackingthe B8 breakpoint"

Isolate pfmdrl Origin and datecopy numberb of isolation'

313d 0.6 S. America, 1984IEC54/84 0.7 S. America, 1984W2d 0.7 S.E. AsiaViet Smith 0.9 S.E. Asia241 1.0 S.E. Asia, 19891935 1.3 PNG, 1987119 1.4 S.E. Asia, 19891904 1.8 PNG, 19871775 2.2 PNG, 19871776 2.3 PNG, 1987BT3 2.6 S.E. Asia, 1989125 2.6 S.E. Asia, 1989Nigeria 14 2.7 Africa, 19891933 3.1 PNG, 19871934 3.2 PNG, 1987120 3.9 S.E. Asia, 1989122 4.9 S.E. Asia, 1989135 5.1 S.E. Asia, 1989121 6.1 S.E. Asia, 1989252 6.5 S.E. Asia, 1989123 9.3 S.E. Asia, 1989B8d 5.0 S. America

" DNA from each isolate was digested to completion with BamHI andEcoRI, blotted to Hybond N, and probed with a fragment from the single-copydihydrofolate reductase gene (dhfr) and with a pfmdrl gene fragment. Thedhfr and pfmdrl fragments hybridized to 5.0- and 1.6-kb bands, respectively.The results were analyzed by using a Molecular Dynamics phosphorimager(Sunnyvale, Calif.) and ImageQuant software. For each isolate, the intensityof the pfmdrl and dhfr bands was compared and adjusted relative to a pfmdrlcopy number of 5.0 for the B8 isolate (12).

b Each value is the average of two independent experiments.' Specific information on these isolates can be found in reference 11. PNG,

Papua, New Guinea.d Cloned parasite line.

previously discussed CQR isolates was determined, and theresults are presented in Table 1. Thirteen of the CQRisolates, mainly from Southeast Asia, had a twofold orgreater amplification of the pfmdrl gene compared with acontrol gene (DHFR-TS) (7).

DISCUSSIONThe finding that no other isolates with the same amplicon

as B8 were represented in the 24 independent CQR isolatesexamined (FCR3, FCB, and B8 are together called oneindependent isolate) makes it probable that amplification ofthis 110-kb pfmdrl-bearing segment does not occur at a hotspot for recombination. Sixteen of the 26 CQR isolatesexamined have an amplified pfmdrl gene, yet none of thesehave the same amplicon as B8. Amplification of pfmdrlindependently at different breakpoints is consistent with

other amplifications studied both in E. coli and in mamma-lian cells (33, 34). Only one example of an apparent ampli-fication hot spot, occurring near the adenylate deaminaselocus of Chinese hamster fibroblasts, has been reported (16).The data presented here suggests that amplification of the

pfmdrl gene in P. falciparum has occurred as a number ofindependent events. The finding that three supposedly dis-tinct isolates of P. falciparum (FCR3, FCB, and ItG2) werein fact all derived from the same parent explains the anomalyof how FCR3 could be CQR and yet isolated from Africabefore CQR isolates were described on that continent (11).This argues for subsequent cross contamination ofFCR3 andFCB and ItG2. A number of recent results from otherlaboratories have provided independent evidence that theFCR3, FCB, and ItG2 isolates used around the world arederived from the same parental isolate, ItG2. The erythro-cyte-binding antigen (EBA 175) sequence from both FCR3and ItG2 was found to be identical at two positions butdifferent to all other isolates examined (31) and FCR3 andItG2 have the same merozoite surface antigen (MSA2)repeating unit (32, 36).Both breakpoints in the ItG2 pfmdrl-bearing amplicon

occur 3' to a string of 30 A residues. The unequal sisterchromatid exchange model which relies on initial misalign-ment between sister chromatids because of pairing in a smallregion of homology (33) can be used to explain the pfmdrlamplification, since the string of A residues provides thehomology prior to misalignment (Fig. 7). Reciprocal recom-bination in this model leads to an initial gene duplication,which can then be further amplified in later generations.Since this is the first example of the sequence at thebreakpoints of an intrachromosomal amplification in P.falciparum, it is not yet known whether the string of A's willbe involved in other such amplifications. The 82% ATcontent of P. falciparum DNA means that there are frequentruns of A's in the genome. There have been a number ofexamples of subtelomeric chromosome breakage in P. falci-parum which have been followed by telomere healing.However, the sequences at these breaks have not immedi-ately followed a string of A or T residues (3, 26), although inone case a homopolymeric string was implicated in inversionof exon 1 of the RESA gene (3). In amplifications of the lacIgene in E. coli, the breakpoints have usually involved shortrepeated sequences of less than 15 bp (42), while in mam-malian DNA, amplifications of AT-rich elements are fre-quently involved (34).

Finally, the pfmdrl-bearing amplicons reported here arearranged in a head-to-tail tandem array. This is the simplestform of amplified repeating units found in mammalian cellsand has been found in amplified rRNA genes in Drosophilacells (10) and in amplified dhfr genes in hamster cells (20),though most mammalian amplifications contain invertedrepeating units. Simpler organisms usually have tandemly

-T 30 pfmdrl 30T30 T30

MISALIGNMENT

_ A30 1 A30mT30 LMdrT30

30 pfmdrl 30- T30 T30

-MA30 ~ A30p.*TMT30 pfmdrl T30

A3-- A30___A30pmdr

T pfmdrlIAT- A

DUPLICATION

DEA30EITM30 ~

DELETION

FIG. 7. Unequal sister chromatid exchange model for amplification of the genome surrounding the pfmdrl gene. The figure shows thesimplest form of the model, resulting in one chromatid with a tandem duplication and a second chromatid with a deletion, which wouldpresumably be lethal. It is not known whether the events occurred in mitosis or meiosis.

MOL. CELL. BIOL.

AMPLIFICATION OF THE pfmdrl GENE IN P. FALCIPARUM 5249

arrayed amplicons. For example, amplifications of the ampCgene (8) are all arranged in tandem arrays. The demonstra-tion of independent amplification events suggests that thisarea of the genome is under strong selective pressure.

ACKNOWLEDGMENTS

We thank Dennis Kyle and Sodsri Thaithong for providing someof the parasite isolates used in this study. We thank HeatherSaunders for typing the manuscript.

This work was supported by the National Health and MedicalResearch Council of Australia and the John D. and Catherine T.MacArthur Foundation. AFC is supported by a Wellcome Austra-lian Senior Research Fellowship.

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