Proc. Natl. Acad. Sci. USAVol. 90, pp. 8292-82%, September 1993Microbiology

Genes necessary for expression of a virulence determinant and fortransmission of Plasmodium falciparum are located ona 0.3-megabase region of chromosome 9

(malaria/adhesion/gametocytogenesis/antigenic variation/subtelomeric deletions)

K. P. DAY*tt, F. KARAMALIS*, J. THOMPSON*, D. A. BARNES*, C. PETERSON§, H. BROWN*, G. V. BROWN*,AND D. J. KEMP**The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3050, Australia; and *The Medical Service, San Francisco General Hospital andDepartment of Medicine, University of California at San Francisco, CA 94710

Communicated by Robert M. May, June 1, 1993

ABSTRACT Virulence of the human malaria parasitePlasmodium falciparum is believed to relate to adhesion ofparasitized erythrocytes to postcapillary venular endotheium(asexual cytoadherence). Transmission of malaria to the mos-quito vector involves a switch from asexual to sexual develop-ment (gametocytogenesis). Continuous in vitro culture of P.fakiparum frequently results in irreversible loss of asexualcytoadherence and gametocytogenesis. Field isolates andcloned lines differing in expression of these phenotypes werekaryotyped by pulse-field gel electrophoresis. This analysisshowed that expression of both phenotypes mapped to a 0.3-Mbsubtelomeric deletion of chromosome 9. This deletion fre-quently occurs during adaptation of parasite isolates to in vitroculture. Parasites with this deletion did not express the variantsurface agglutination phenotype and the putative asexual cy-toadherence ligand designated P.fakiparum erythrocyte mem-brane protein 1, which has recently been shown to undergoantigenic variation. The syntenic relationship between asexualcytoadherence and gametocytogenesis suggests that expressionof these phenotypes is genetically linked. One explanation forthis linkage is that both developmental pathways share acommon cytoadherence mechanism. This proposed biologicaland genetic linkage between a virulence factor (asexual cytoad-herence) and transmissibility (gametocytogenesis) would helpexplain why a high degree of virulence has evolved and beenmaintained in falciparum malaria.

Based on the conventional wisdom that parasites evolve to beharmless to their hosts (1), the high degree of host mortalityinduced by Plasmodium falciparum compared with otherhuman malarias, has been interpreted as a consequence oftherecent phylogenetic origin of the parasite (2). Theoreticalconsiderations of evolution of virulence indicate, however,that virulence can be maintained by natural selection, de-pending on whether host morbidity and mortality influencetransmissibility (the number of new hosts infected from oneinfectious host) (3-5). Recent selection experiments (6) haveconfirmed this modem view ofparasite virulence and indicatethat a more empirical approach to understanding evolution ofvirulence of P. falciparum may be achieved by defining therelationship between virulence and transmissibility for thisparasite. As in many bacterial systems, virulence (i.e., theability to induce disease) of P. falciparum relates to theexpression of a parasite adhesin. Parasites that have lost thisadhesive property (asexual cytoadherence) produce low-density, avirulent infections in experimental animal models(7). Polymorphisms in the asexual cytoadherence phenotypehave been suggested as the cause ofthe spectrum ofvirulence

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associated with mild and severe malarial disease (8, 9). In thispaper we explore the genetic relationship between asexualcytoadherence and the ability to initiate sexual development(gametocytogenesis), which is necessary for transmission tothe mosquito vector.

In vitro culture ofP.falciparum has been reported to resultin irreversible loss of expression of both asexual cytoadher-ence (10) and gametocytogenesis (11). Subtelomeric dele-tions that occur during continuous in vitro culture of P.falciparum account for the irreversible loss of a number offunctional genes (12-17). For example, subtelomeric dele-tions ofchromosome 2 result in loss of expression ofthe geneencoding the knob-associated histidine-rich protein (13, 16,17). Parasites with knob-associated histidine-rich proteindeletions do not produce surface membrane deformationscalled "knobs," which are possible points of cytoadherencebetween the parasitized erythrocyte and vascular endothelialcells during sequestration. Knobless parasites are generallyunable to cytoadhere in vivo and in vitro, although someknobless cloned lines with knob-associated histidine-richprotein deletions can retain cytoadherence (18, 19) andcontinue to express the P. falciparum erythrocyte membraneprotein 1 (PfEMP1) (19), a putative parasite cytoadherenceligand. Consequently loci other than those involved in knob-associated histidine-rich protein deletions are required forexpression of asexual cytoadherence and Pf EMP1. Theproperty of gametocytogenesis is also readily lost during invitro culture of P. falciparum, but to date no subtelomericdeletions have been linked to expression of this phenotype(11). We present here karyotype analysis aimed to identifyregions of the P. falciparum genome responsible for expres-sion of asexual cytoadherence and gametocytogenesis.

MATERIALS AND METHODSParasite Isolates and Clones. Isolates ofP. falciparum were

obtained in Madang, Papua New Guinea, as detailed (20).Clones 3D7 and HB3 were provided by D. Walliker (14).Clones C10 and B9 were obtained by limit dilution, asdescribed (21).

Karyotype Analysis. Isolates were cultured in vitro (20) andharvested when cultures contained 8-10% healthy trophozo-ites; chromosomes were prepared as described (21). Chro-mosomes were separated by pulse-field gel electrophoresis

Abbreviations: Pf EMP1, P. falciparum erythrocyte membraneprotein 1; IRBC, infected red blood cells; PFGE, pulse-field gelelectrophoresis; GC, gametocyte(s); TSP, thrombospondin; C32MC,C32 melanoma cells.tPresent address: Department ofBiology, Imperial College, London,SW7 2BB, United Kingdom.§To whom reprint requests should be sent at the present address.

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Proc. Natl. Acad. Sci. USA 90 (1993) 8293

(PFGE) for 44 hr at 120 V with a pulse time of 270 s and for20 hr at 70 V using 999-s pulse time.Phenotype Analysis. At the time of preparation of chromo-

somes for PFGE, isolates and clones were examined forability of asexual stages to cytoadhere to C32 melanoma cells(C32MC), as described (19). Results were expressed as thenumber of infected red blood cells (IRBC) bound per 100C32MC. In addition, isolates and clones were examined forgametocyte (GC) production using standard GC culture con-ditions as follows. Cultures were initiated at 5% hematocritand 0.5% ring-stage parasitemia. Cells were cultured for GCin 0,Rh+ human erythrocytes and RPMI 1640/Hepes (20)/hypoxanthine at 50 Ag/ml/10% human serum in an atmo-sphere of 5% C02/1% 02/94% N2. Gametocytogenesis re-sults are expressed as the number of GC produced per 100IRBC in a Giemsa-stained blood fllm taken 20 days afterestablishing GC cultures. Asexual cytoadherence was alsoassessed by binding of trophozoite-infected cells to purifiedreceptors CD36, thrombospondin (TSP), and fibronectinimmobilized on plastic as described (19). Results were ex-pressed as the number of IRBC bound per mm2 of plasticcoated with CD36 (1 ,g/ml), TSP (50,ug/ml), and fibronectin(1 ,ug/ml). Agglutination reactions were done and scored, asdescribed (20), by using 1:10 dilutions of hyperimmune serumfrom adult Papua New Guineans who were life-long residentsof a malaria endemic area. A normal human serum fromMelbourne was used as a negative control. Rabbit antiserumwas raised against trophozoite-infected red blood cells ofisolate 1776 by using a described immunization method (19).

Selection for Asexual Cytoadherence. The lines HB3, 1934,and C10 were selected for cytoadherent cells by bindingtrophozoite-infected cells to C32MC and a subsequent cul-ture of bound cells, as described (22). Four consecutiveselections were done for line HB3, resulting in selected linesdesignated HB3-Sell, -2, -3, and -4. Two consecutive selec-tions were done for isolate 1934, resulting in the line desig-nated 1934-Sel2. Three independent selections of line C10failed to result in isolation of any cytoadherent cells. Chro-mosomes of the original and selected lines were prepared andelectrophoresed as described above. The phenotype of thesecell lines was determined as above.

Surface Radioiodination and Immunoprecipitation. Tropho-zoite-infected erythrocytes were surface radioiodinated byusing the lactoperoxidase technique (23) incubated withtrypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone(TPCK) (Worthington; 10 pLg/ml in phosphate-buffered sa-line) or with trypsin plus an excess of soybean trypsininhibitor. Immunoprecipitations were done as described (19).

RESULTSIndependent clones and isolates of P. falciparum often un-dergo subtelomeric deletions of chromosome 9 that havesimilar breakpoints (21). To determine whether loss of ex-pression of asexual cytoadherence and/or gametocytogene-sis related to size changes in chromosome 9 we describedkaryotype changes in field isolates from Papua New Guinea(20) during the first 4-6 weeks of adaptation to in vitroculture. Fig. 1A shows PFGE analysis of chromosome 9 inearly passages of these field isolates (recent isolates) and thelong-term-cultured and cloned lines 3D7, C10, and B9. Sizeheterogeneity in chromosome 9 was observed within five ofthe recent isolates and one of the clones examined (Fig. 1A).Recent isolates 1934, 1904, 1775, 1933, 1935, and clone 3D7showed two populations of parasites differing in the size ofchromosome 9. PFGE analysis of chromosome 2 (Fig. 1B)showed size heterogeneity of chromosome 2 among therecent isolates and the clones examined but no differenceswithin any one recent isolate. Inability to detect mixedpopulations of parasites with respect to size of chromosome

Rt 19 (D - to CO (IF)to Dcm cm 4m P r- m

'of- Vs

A -r, v- v-, r____ _ _C) cn 1 ,

B

C

Chromosome 9

Chromosome 2

AA Bg A Bs Bg Bg IDBs,Bg A ABg ABs BgBg A Bsg.Bg A Bs BgBg

-.

A Bs,Bg_~ ZJ t

- 1.0

1776

C1o

Telomere 7H8/6 liii HARP

Rep2O 0 RhopH3 > MSA1

FIG. 1. PFGE of recent Papua New Guinean isolates of P.falciparum designated 1934, 1904, 1916, 1917, 1775, 1703, 1933, and1935 and cloned lines of P.falciparum designated 3D7, C10, and B9.(A) Hybridization with the chromosome 9-specific probe, the geneencoding merozoite surface antigen (MSA-1) (21), showed sizevariation (1.8 and 1.5 Mb) in chromosome 9 within recent isolatesduring adaptation to in vitro culture and in the cloned line 3D7. (B)Hybridization with the chromosome 2-specific probe, the genecoding for MSA-2 (16), showed no variation in size of chromosome2 within any of the recent isolates. The cloned line 3D7 showed twosizes of chromosome 2. (C) Maps of chromosome 9 from parentisolate 1776 and the clone derived from this isolate designated C10.The maps from ref. 21 have been revised. The presence of additionalBgl or Bssh sites near the telomeres cannot be excluded. Apa I (A),Bgl (Bg), and Bssh2 (Bs) are indicated together with the markersshown. The presence of the 7H8/6 sequences is inferred from studieson other isolates. Cytoadherence and gametocytogenesis results foreach clone are given as follows: isolate 1934-40 IRBC/100 C32MC,9 GC/100 IRBC; isolate 1904-2 IRBC/100 C32MC; 0 GC/100IRBC; isolate 1916-384 IRBC/100 C32MC, 46 GC/100 IRBC;isolate 1917-328 IRBC/100 C32MC, 38 GC/100 IRBC; isolate1775-346 IRBC/100 C32MC, 56 GC/100 IRBC; isolate 1703-660IRBC/100 C32MC, 44 GC/100 IRBC; isolate 1933-243 IRBC/100C32MC, 26 GC/100 IRBC; isolate 1935-247 IRBC/100 C32MC, 37GC/100 IRBC; isolate 1776-460 IRBC/100 C32MC, 64 GC/100IRBC; clone 3D7-302 IRBC/100 C32MC, 32 GC/100 IRBC; cloneB9-44 IRBC/100 C32MC, 0 GC/100 IRBC; clone C10-0 IRBC/100 C32MC, 0 GC/100 IRBC. Rep2O, RhopH3, HARP, MSA-1, and7H8/6 are as in ref. 21.

2 of the recent isolates indicated that either no detectable(>100 kb) subtelomeric deletions had occurred in chromo-some 2 or parasites with such deletions were not selectedduring adaptation to culture. In contrast, the subtelomericdeletions seen in chromosome 9 occurred frequently amongrecent isolates, and parasites with this deletion were rapidlyselected during adaptation to culture.Recent isolates were screened for expression of gameto-

cytogenesis and asexual cytoadherence phenotypes at thetime of preparation of chromosomes for PFGE analysis (Fig.1). The presence of the large form of chromosome 9 in anisolate always correlated with a high number of cytoadherentcells-i.e., >240 IRBC per 100 C32MC and >26% conversion

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

to GC. Recent isolates 1934 and 1904, which had predomi-nantly the small form of chromosome 9, had few cytoadher-ent cells (<40 IRBC per 100 C32MC) and low (<10 GC per100 IRBC) or zero GC production. The association betweensize of chromosome 9 and expression of asexual cytoadher-ence and gametocytogenesis was also examined in threecloned lines ofP.falciparum-i.e., 3D7, C10, and B9 (Fig. 1).Many cytoadherent cells and GC were seen in clone 3D7 (302IRBC per 100 C32MC; 32 GC per 100 IRBC), which hadlarge-sized chromosome 9. The cloned lines B9 and C10,derived from the parental isolate designated 1776, had smallforms of chromosome 9 and few (44 IRBC per 100 C32MC)or no cytoadherent cells and zero GC production.

Previously (21) we showed in a limited number of isolatesthat the small form of chromosome 9 was generated from thelarge form by a subtelomeric deletion in the right end ofchromosome 9 as well as a much smaller deletion in the leftend, as shown in Fig. 1C. To verify that similar deletionsresulted in the size changes found during adaptation of fieldisolates to in vitro culture, two of these recent isolates withthe small form of chromosome 9 were mapped. Recentisolates 1904 and 1934, as well as clones B9 and C10, revealeda change in the size ofthe MSA-1 (21) bearing Apa I fragment,but none ofthose examined (B9, C10, and 1904) showed a sizechange in the RhopH3 (21)-bearing Apa I fragment (Fig. 1C).Similar mapping data were obtained for later passages ofrecent isolates 1776, 1916, 1917, and 1703. After long-termculture these isolates had predominantly the small form ofchromosome 9 compared with earlier passages shown in Fig.1. Hence, the major deleted region ofchromosome 9 in recentisolates must be located at the right end of this chromosome.To demonstrate that the subtelomeric deletion in the right

end of chromosome 9 was linked to expression of asexualcytoadherence and gametocytogenesis we repeatedly se-lected for cytoadherent cells by binding to C32MC. Aftereach selection the population of binding cells was expanded,karyotyped by PFGE, and phenotyped. Passages of the lineHB3 and the isolate 1934 that had predominantly small formsofchromosome 9 and were low in expression of both asexualcytoadherence (<20 IRBC per 100 C32MC) and gametocy-togenesis (<10GC per 100 IRBC) were used for this selectionexperiment. In addition, the clone of parent isolate 1776designated C10, which did not cytoadhere or produce game-tocytes and had a small form of chromosome 9 was alsoselected.Cytoadherent cells were successfully selected from line

HB3 and isolate 1934, indicating that these passages origi-nally contained mixed populations of binding and nonbindingcells. For line HB3, selection of cytoadherent cells greatlyenriched for populations of cells with a large form of chro-

N X Rt4)7i 7a 0i0 (a 0A

co lm m m co r:I I I _

04 N

0)0)0-

T- CYO CO

-1.5

Chromosome 9

FIG. 2. PFGE analysis of chromosome 9 of the original andselected lines of HB3 and 1934 as well as isolate 1776 and clone C10.Chromosome 9 is identified with the chromosome 9-specific probe-i.e., the gene for merozoite surface antigen (MSA-1) (21). Bands 1.8and 1.5 indicate chromosome size in Mb.

mosome 9 (Fig. 2). Line HB3 and the lines selected from it allshowed the same polymorphism of the Apa I site boxed inFig. 1 and so are clearly derived from the same parentalclone. Hence, we can be confident that these selected lineswere not simply contaminated with another cell line duringselection. PFGE analysis of the chromosomes of HB3 beforeand after selection showed no differences in size of all the 14chromosomes except for a larger size of chromosome 9 afterselection. Comparison of the 1934 lines before and afterselection by PFGE revealed that chromosome 9 was larger insize after selection (Fig. 2). The only other karyotypic changein isolate 1934 seen after selection was in chromosome 3,which was smaller in size, presumably because of a subtelo-meric deletion (data not shown). As the size of chromosome3 in line HB3 remained unchanged after selection, the ob-served change in size ofchromosome 3 in isolate 1934 was notconsidered to relate to loss of asexual cytoadherence. Wewere unable to select any binding cells from clone C10 afterthree attempts, indicating that this cloned line contained onlynonbinding cells.To determine whether known features of the asexual

cytoadherence phenotype ofP.falciparum were linked to theobserved deletion on chromosome 9 the cytoadherence phe-notypes of asexual stages of the original and selected lines ofHB3, as well as isolate 1776 and clone C10, were character-ized in detail (Table 1). The leukocyte differentiation antigenCD36 (23, 24) and TSP (25) but not fibronectin have beenshown to be receptors for cytoadherence of asexual stages ofP.falciparum. Line HB3-sel4 and isolate 1776 bound to CD36and TSP but not to fibronectin (Table 1). In contrast, theunselected HB3 line and clone C10 did not bind to any ofthese purified receptors.

Expression of an isolate-specific agglutinogen on the sur-face of trophozoite-intected cells has been shown to beassociated with expression of the asexual cytoadherencephenotype (20, 26). Line HB3-sel4 and isolate 1776 expressedcell-surface agglutinogens, as detected by the isolate-specificpatterns of agglutination with hyperimmune sera and a rabbitantiserum to trophozoite-infected cells of isolate 1776 (Table

Table 1. Effect of selection over C32MC on cytoadherencephenotype, agglutination, and gametocyte production

Cell line

Characteristic HB3 HB3-sel4 1776 CloSize of chromosome 9 Small Large Large SmallCytoadherenceC32MC 11 510 460 0CD36 0 1490 2230 0TSP 0 270 3360 0FN 0 0 0 0

Agglutination scoreHS 1 - 3+ - -HS 2 - - - -

HS 3 - 3+ 2+ -HS4 - - 3+ -

HS 5 - 4+ 1+ -HS 6 - - - -HS 7 - - 2+ -HS 8 - 1+ - -HS9 - 3+ 3+ -HS 10 - - - -Rabbit 1776 - - 4+ -

NHS - - - -GC productionGC/100 IRBC 5 51 64 0FN, fibronectin; TSP, thrombospondin; NHS, normal human

serum.

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Proc. Natl. Acad. Sci. USA 90 (1993) 8295

1). Clone C10 and the unselected HB3 line were not agglu-tinated by these sera.

Expression of both the cytoadherence phenotype and anisolate-specific agglutinogen has been associated with thepresence of a trypsin-sensitive high-molecular-weight anti-gen on the surface of trophozoite-infected cells (27). Thismolecule designated Pf EMP1, which appears to undergoantigenic variation (28, 29) and can be radioiodinated andimmunoprecipitated by hyperimmune sera, was found on thesurface of trophozoite-infected cells of parasites bearing thelarge form of chromosome 9 (HB3-sel4 and 1776) but was notpresent on those with the deleted form of chromosome 9(HB3 and C10) (Fig. 3).The original and selected lines were also examined for GC

production (Table 1). Line HB3-sel4 showed 51% conversionof infected cells to GC after 20 days in culture under condi-

A T+I T -T E T+ T -T E M

200

HB3..

HB3-Sel4

B TI T -T E T+ T -T E

c

1 93

67

- 43

M

200

- 93

-l. - 67

M SE Abl Ab2 NHS SE Abl Ab2 NHS

HB3-Sel4 HB3

200

93

67

43

FIG. 3. PAGE analysis of surface-radioiodinated trophophozo-ite-infected cells of P. falciparum lines differing in size of chromo-some 9. (A) PAGE analysis of Triton X-100-insoluble pellets ofsurface-radioiodinated HB3 (small size chromosome 9) and HB3-sel4cells (large size chromosome 9) incubated with trypsin at 10 pg/mlplus excess soybean trypsin inhibitor (T+I), incubated with trypsin(T) at 10 ig/ml and no treatment (-T) as well as the TritonX-100-soluble extract (E) of untreated cells. Molecular weight mark-ers (x 10-3) are shown in lane M, and arrows indicate the high-molecular-weight radioiodinated bands. (B) PAGE analysis of TritonX-100-insoluble pellets of isolate 1776 (large chromosome 9) and itsclone C10 (small chromosome 9) treated as described for A. (C)Immunoprecipitation analysis of surface-radioiodinated proteins ofHB3 and HB3-sel4 cells treated with hyperimmune sera (Abl andAb2) from adult Papua New Guineans resident in a malaria endemicarea and with normal human serum (NHS). Molecular weight mark-ers (x 10-3) are shown in lane M, and the SDS extract of the TritonX-100-insoluble pellet is shown in lane SE.

tions known to induce gametocytogenesis, whereas unse-lected HB3 line produced very few GC under identicalculture conditions.

DISCUSSIONKaryotyping data and the above selection experiment havedemonstrated that a 0.3-Mb subtelomeric deletion in the rightend of chromosome 9 resulted in (i) loss of gametocytogen-esis, (ii) loss of ability of mature asexual blood stages tocytoadhere to C32MC via mechanisms dependent on CD36and TSP, and (iii) loss of expression of an isolate-specificagglutinogen and the putative cytoadherence ligand PfEMP1. This deletion may encode structural genes involved inexpression of both asexual cytoadherence and gametocyto-genesis. Alternatively this region of chromosome 9 mayencode products such as transcription factors regulatingexpression of both these phenotypes.P. falciparum has recently been shown to undergo clonal

antigenic variation of the surface agglutinogen phenotype invitro (28-30), and different variants of PfEMP1 have beenassociated with this antigenic switching (28, 29). We haveshown that expression of Pf EMP1 is associated with a0.3-Mb region ofchromosome 9, indicating that this region ofchromosome 9 may be involved in the molecular mechanismresponsible for antigenic variation. The observation thatsimilar breakpoints were involved in the deletions seen inchromosome 9 of independent clones and isolates of P.falciparum raises the question of whether the breakpointreflects site-specific recombination akin to that seen in du-plicative transposition for the variant surface glycoproteinsoftrypanosomes (31). The region is small enough to be clonedin yeast artificial chromosomes and studied in detail.Loss of expression of Pf EMP1 in cells with the deleted

form of chromosome 9, and concomitant loss of expressionof both cytoadherence and agglutination phenotypes, pre-sents the strongest evidence to date that this molecule is boththe parasite cytoadherence ligand and the variant surfaceantigen of P. falciparum. How the role of Pf EMP1 incytoadherence can be reconciled with experiments showingthat parasite cytoadherence is inhibited by synthetic peptidesbased on motifs present in the human red cell protein, band3 (32), requires further analysis of the deleted region ofchromosome 9 in a transfection system.Karyotype analysis of recent isolates showed that the

observed deletion in the right end of chromosome 9 occurredfrequently during adaptation of P. falciparum to in vitroculture. This result explains why it has proven difficult tomaintain cytoadherence and gametocytogenesis phenotypesduring long-term culture. The frequency with which thisdeletion was observed in newly adapted isolates suggests thatit is the most common genetic mechanism whereby isolateslose expression of the above phenotypes, although it is notthe only mechanism whereby isolates can lose expression ofgametocytogenesis (P. Alano, D. Read, and K.P.D., unpub-lished observations).Having mapped expression of asexual cytoadherence and

gametocytogenesis to the same chromosome, we must askwhether this syntenic relationship is relevant to the biologyof P. falciparum or merely a coincidence. Cytoadherence isa critical process for maturation of both the asexual bloodstages and the sexual stages (GC) in the infected human host.Differences in the site specificity of sequestration of theselife-cycle stages have led to speculation that the mechanismsof cytoadherence are distinct in the two developmentalpathways (27). Location of genes involved in expression ofasexual cytoadherence and gametocytogenesis to a 0.3-Mbregion of chromosome 9 suggests that expression of bothphenotypes may be linked. A biological explanation for thisproposed linkage may be that this region of chromosome 9 is

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

involved in regulation of gametocytogenesis. If both devel-opmental pathways share a common cytoadherence mecha-nism, it would be logical to propose that control ofexpressionof cytoadherence would be linked to control of expression ofgametocytogenesis. This hypothesis, if correct, describes abiological and genetic linkage ofa virulence determinant (i.e.,asexual cytoadherence) to transmissibility (gametocytogen-esis including GC cytoadherence). Consideration of such abiological linkage in the context of modem theories ofevolution (3-6) would indicate that asexual cytoadherence, avirulence factor, has evolved and been maintained by naturalselection infalciparum malaria because of its relationship totransmissibility.

We thank Andrew Boyd and David Wilkinson for purified CD36and TSP and Kaye Wycherley for culture of melanoma cells.Financial support was provided by the Australian National Healthand Medical Research Council and the John D. and Catherine T.MacArthur Foundation and Saramane Pty Ltd. Saramane is a jointventure between the Walter and Eliza Hall Institute of MedicalResearch, The Queensland Institute of Medical Research, TheCommonwealth Serum Laboratories, Biotechnology Australia PtyLtd., and the Australian Industry Development Corporation. Sara-mane receives financial support from the Australian Government.

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82% bficrobiology: Day et al.