Molecular and Biochemical Parasitology, 13 (1984) 333-342

Elsevier

MBP 00491

333

BIOSYNTHESIS OF THE TARGET ANTIGENS OF ANTIBODIES BLOCKING

TRANSMISSION OF PLASMODIUM FALCIPAR UM

NIRBHAY K U M A R and R I C H A R D CARTER

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of

Health, Bethesda, MD 20205, U.S.A.

(Received 30 April 1984; accepted 26 June 1984)

We have studied the biosynthesis of three proteins of M r 260 000, 59 000 and 53 000 previously identified

on the surface of extracellular gametes of Plasmodiumfalciparum as the targets of monoclonal antibodies

which block infectivity of P. falciparum to mosquitoes. In cultures of P. falciparum pulse labeled with

[35S]methionine we have found that these proteins are synthesized by gametocytes from an early stage in

their maturat ion but are not synthesized by asexual blood stage parasites. The target proteins synthesized

by the gametocytes become expressed on the surface of the extracellular gametes but the gametes themselves

no longer synthesize these proteins. The 59 000 and 53 000 M proteins do not result from processing from

the 260 000 M protein. The 59 000 and 53 000 M protein, but not the 260 000 M proteins, were

glycosylated by either glucosamine or mannose.

Key words: Plasmodium falciparurn; Gametocytes; Gametes; Biosynthesis; Glycosylation;

Transmission-blocking immunity

INTRODUCTION

Transmission of malaria from the vertebrate host to the mosquito vector is mediat- ed by the intraerythrocytic gametocytes. After ingestion by the mosquito the gameto- cytes emerge from their host red blood cells (RBC) and undergo gametogenesis and fertilization in the lumen of the mosquito midgut. The zygotes continue their develop- ment to establish the infection in the mosquito tissues. In several species (chickens, rodents, monkeys), immunization of the vertebrate host with extracellular gametes of the malaria parasite suppressed infectivity of subsequently induced blood infections to the mosquitoes [1-3]. Recently in our laboratory, target antigens of monoclonal antibodies (MAb) blocking infectivity of the human malaria parasite Plasmodium

Abbreviations: MAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec-

trophoresis; B~B, bromophenol blue; NMS, normal mouse serum; PBS, phosphate-buffered saline; RBC,

red blood cell(s); NETT, 150 mM NaCI, 5 mM EDTA, 50 mM Tris, 0.5% Triton X-100

0166-6851/84/$03.00 © 1984 Elsevier Science Publishers B.V.

334

falciparum to mosquitoes have been identified [4]. A set of three proteins of apparent M r of 260 000, 59 000 and 53 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were immunoprecipitated by these MAbs from extracts of surface radio-iodinated gametes of P. falciparum. A similar set of three gamete surface proteins was also found to be immunoprecipitated by MAbs blocking infectiv- ity of the chicken malaria parasite, P. gallinaceum, to mosquitoes [5]. The present study was undertaken to characterize the biosynthesis of these antigens in P. falcipa- rum.

MATERIALS AND METHODS

Reagents. [35S]Methionine (approx. 1200 Ci mmol-l), o-[6:H]glucosamine (30,3 Ci mmol -~) were obtained from Amersham, Arlington Heights, IL and D-[2- 3H(N)]mannose (24.3 Ci mmo1-1) was from New England Nuclear, Boston, MA. Protein A-Sepharose was purchased from Pharmacia Fine Chemicals, Piscataway, NJ.

Monoclonal antibodies. Details of production of various MAbs (IA3-B8, IIC5-B 10 and IID2-A10), identification of their target antigens on the surface of P. falciparum gametes and their effect on the infectivity of parasites to mosquitoes have been presented [4].

Parasite cultures. P. falciparum line 7G8, a clone of Brazilian isolate IMTM22 was cultured in human erythrocytes [6]. New cultures were set up by expansion of previously growing cultures with fresh RBC and medium. Under these conditions predominantly asexual parasites are present up to the 5th or 6th day after expansion with fresh RBC. During the next 2-3 days a wave of gametocytes appear in culture. These, thereafter, undergo maturation as a more or less synchronous cohort. The stages (I to V) of maturation of P. falciparum gametocytes have been described [7]. Cultures contained predominantly stage I gametocytes 7-9 days after expansion with fresh RBC, stage II after 9-12 days, stage III after 11-14 days, stage IV after 13-16days and stage V, which were less numerous than the earlier stages, reached maximum densities after about 16 days. The majority of parasitized cells in culture after 16 days were gametocytes.

Purification of extracellular gametes. Cultures of P. falciparum containing morpholo- gically mature gametocytes (I 7-18 days after expansion with fresh RBC) were centri- fuged at 1 000 )< g for 7 min and the cells resuspended in a solution that stimulates gametogenesis (7 mM Tris, 117 mM NaC1, 8 mM glucose, 30 mM NaHCO3 and 10% normal human serum at a final pH of 8.1). After 30 min incubation at room tempera- ture, the gametes were purified using a discontinuous Percoll (Pharmacia Fine Chemi- cals) gradient [4]. The extracellular gametes separated in a layer at the 30/42% interface.

335

Biosynthetic labeling of cuhured parasites. Cultured parasites (10 ml culture volume at approximately 3% hematocrit) or purified gametes were washed three times in medium RPMI-1640 (without methionine) and incubated in methionine-free RPMI-1640 containing 0.5 mCi [35S]methionine for 2-3 h at 37°C. After incubation, cells were washed by centrifugation (approx. 500 × g for 1 min) three times with RPMI-1640 containing 10% normal human serum or plasma. All the operations were carried out at 37°C using prewarmed glassware, pipettes and medium etc.

Preparation of soluble extract and immunoprecipitation. Biosynthetically labeled para- sites after washing with medium to remove excess of extracellular [35S]methionine were lysed with 1% Triton X-100 in 10 mM Tris and 150 mM NaCI, pH 7.4, for 45-60 min at 0°C. The following protease inhibitors were added during extraction of the cells; 2 mM phenylmethylsulfonyl fluoride (Sigma Chemical Company, St. Louis, MO), 0.2 mM 1-L-tosylamide-2-phenylmethylchloromethyl ketone (Sigma), 0.2 mM p-tosyl-L-lysinechloromethyl ketone (Sigma), 10 mM EDTA, 0.25 lag m1-1 leupeptin (Boehringer-Mannheim, Indianapolis, IN) and 10 lag m1-1 pepstatin (Sigma). Cell lysates were centrifuged at 40 000 X g for 45 min at 0-4°C (Sorvall rotor SS-34). Supernatants thus obtained were adsorbed with Protein A-Sepharose beads (100 lal packed volume) for 60 min at 4°C and used for immunoprecipitation.

For immunoprecipitation, typically 500 000 trichloroacetic acid-insoluble counts (cpm) were incubated with 15-20 lal of various MAbs (50% ammonium sulfate precipitate of ascites dialyzed against phosphate-buffered saline (PBS); 5 mg ml -t) in a final volume of 100-150 lal of 50 mM Tris, 150 mM NaC1, 5 mM EDTA and 0.5% Triton X-100, pH 7.4 (NETT) at 0-4°C for 2-3 h. Labeled proteins bound to antibodies were adsorbed to Protein A-Sepharose beads (150 lal of a 25% suspension) by rocking at room temperature for 60 min. Beads were washed twice with NETT containing 0.5 M NaC1, twice with NETT and twice with NET (NETT buffer without

Triton X-100).

SDS-PAGE andfluorography. Washed beads were extracted in 60 lal of sample buffer (62.5 mM Tris, 10% glycerol, 0.005% bromophenol blue and 5% SDS pH 6.8) by boiling for 3-5 min. Samples were analyzed by polyacrylamide (5-15% gradient) gel electrophoresis in the presence of SDS [8]. Gels were briefly stained with Coommassie Blue and destained. Low and high molecular weight protein markers (Biorad, Rich- mond, CA) were used to calibrate the gels. For detection of radiolabeled protein bands, gels were soaked in 'Autofluor ' (National Diagnostics, Somerville, N J) for 1-2 h before drying. Dried gels were then exposed to Kodak X-omat XAR5 films at -80°C.

RESULTS

Gametocytes from cultures of P.falciparum line 7G8, expanded 12 days previously

336

with fresh h u m a n RBC, were incubated in the presence of [35S]methionine. The

a m o u n t of label incorporated into proteins precipitated by the yE a isotype MAb,

IA3-B8, increased in a l inear fashion dur ing 2 h of incubat ion . MA b IA3-B8 precipitat-

ed biosynthet ical ly labeled proteins of M r 260 000, 59 000 and 53 000 (Fig. 1, lane b,

panels 2-6) which corresponded to proteins precipitated by this M A b from extracellu-

lar gametes of P. falciparum surface labeled with ~25I (Fig. 1, panel 7). A n u m b e r of

other labeled proteins were immunoprec ip i ta ted non-specifically from the [35S]me-

thionine labeled material. A m o n g these is a doublet of M r abou t 66 000 which was

precipitated from the labeled extracts by normal mouse serum (Fig. l, lane a, panels

2-6).

To determine whether the high and low molecular weight proteins specifically

precipitated by IA3-B8 exist in a precursor-product relat ionship, gametocytes of

M r x l O -3

Top - -

200 -

1 1 6 - - 97.4 - - 66.2 - -

4 5 - -

B ~ B - -

1 2 3 4 5 6 7

i ~ i i ..4_

O - -

a b a b a b a b a b a b a b

Fig. 1. SDS-PAGE/fluorography of samples biosynthetically labeled with [35S]methionine. Gametocytes from culture 12 days after expansion with fresh RBC were washed with serum and methionine-free RPMI-1640 and incubated at 37°C in the presence of [35S]methionine (0.5 mCi) in the same medium. 1.0 ml aliquots were removed at indicated time intervals, cells were washed and extracted in 1% Triton X-100 as described under Materials and Methods. Immunoprecipitations were done with NMS (lane a) or MAb IA3-B8 (lane b) using equal volumes of cells removed and Triton X-100 extracted after 15 min (panel 1), 30 min (panel 2), 60 min (panel 3), 120 min (panel 4), 150 min (panel 5) and 180 min (panel 6). Panel 7 shows the immunoprecipitation from the extracts of ~2~I-surface labeled extracellular gametes as in [4]. Samples were analyzed using 5-15% (acrylamide gradient) SDS-PAGE under reducing conditions. BioRad high molecular weight standards were used to calibrate the gels. Arrows indicate positions of [3~S]methionine labeled proteins corresponding to those precipitated by IA3-B8 from surface radioiodinated gametes.

337

P. falciparum, 7G8, were incubated with [35S]methionine for 30 min or 60 min at 37°C in

serum and methionine free RPMI- 1640 medium. The cells were then washed at 37°C in medium containing non-radioactive 2 mM L-methionine and 10% human serum followed by incubation in this medium at 37°C. At various intervals samples were removed for extraction and immunoprecipitation with IIC5-B10, a 72a MAb pre- viously shown to precipitate the same proteins from 7G8 as does IA3-B8. Immediately after the 30 min pulse all the three proteins, 260 000 and the 59 000 and 53 000 proteins (seen as a broad band) were labeled (Fig. 2, lane b). After increasing periods of chase (Fig. 2, lanes c to e) or a 60 min pulse and chase (Fig. 2, lanes f to i) the relative intensity

of label in the 260 000 as well as 59 000 and 53 000 proteins increased. These results do not indicate that the 260 000 protein is a precursor of the 59 000 and 53 000 proteins.

The stage specificity of synthesis of these proteins was studied using cultures at

Mrx lO -3 TOP --

200 --

116.2 - - 97 .4

6 6 . 2 - -

4 5 . 0

31.0

21.5 14.4

i

m

B ~ B - -

a b c d e f g h i Pulse (rnin.) 30 30 30 30 30 60 60 60 60

Chase (rnin.) 30 0 30 60 120 0 30 60 120

4 1 1 1 - - "

i ~ i i ~

Fig. 2. Pulse-chase labeling ofPlasmodiumfalciparum gametocytes with [35S]methionine and immunopreci-

pitation with MAb IIC5-B10. Gametocytes, from cultures expanded 13 days previously with fresh RBC,

were labeled at 37°C with [~SS]methionine for either 30 min (lanes a to e) or 60 min (lanes f to i). After

labeling, the cultures were washed and incubated at 37°C in the medium containing 10% normal human

serum and 2 mM non-radioactive L-methionine for different time periods. Immediately following the

initial labeling period and after each chase period (as indicated) the cells were extracted with Triton X-100

and immunoprecipitated with MAb IIC5-B10 (lanes b to i) or NMS (lane a). Immune precipitates were

analysed using a 5-15% acrylamide SDS-PAGE.

338

d i f f e r e n t t i m e s (2, 6, 9, 12 a n d 15 d a y s ) a f t e r e x p a n s i o n w i t h f r e s h R B C . C u l t u r e s o f

t he se ages were i n c u b a t e d f o r 2 h w i t h [35S]me th ion ine . I m m e d i a t e l y a f t e r l a b e l i n g , t he

cel ls we re w a s h e d a n d e x t r a c t e d in T r i t o n X-100 . E x t r a c t s c o n t a i n i n g e q u a l n u m b e r s

o f t r i c h l o r o a c e t i c a c i d - p r e c i p i t a b l e c o u n t s we re i m m u n o p r e c i p i t a t e d w i t h )'2a M A b s

M r x l O -3 1 2 3 4

TO - . . . . . . . . .

200 - -

1 1 6 - - 97 .4 - -

66.2 - -

4 5 - -

3 1 - -

2 1 . 5 ~ B~B ~ -

a b c d

5

a b c d a b c d a b c d a b c d

(A)

100 I i i i 80 I n ~ 60

40 (B)

I I I o - _ n n • _ _ _ u u _ _ _ n n x I>1 × ~ x ~ x I>1 x

4-

< I~ < ~ < I~ ~ < I ~ < I~ Fig. 3. (A) SDS-PAGE analysis of [3SS]methionine labeled polypeptides synthesized by parasites of different stages in culture. Cultures of Plasmodiurafalciparum after expansion in fresh RBC for 2 days (panel 1), 6 days (panel 2), 9 days (panel 3), 12 days (panel 4) and 15 days (panel 5) were labeled with [35S]methionine for 2 h and extracted in Triton X-100 as described under Materials and Methods. Extracts equivalent to 500 000 trichloroacetic acid-insoluble cpm were immunoprecipitated with NMS (lane a), MAbs IA3-B8 (lane b), IICS-BI0 (lane c) and IID2-A10 (lane d). Reduced samples were analyzed on a 5-15% S DS-PAGE. Molecular weight markers are indicated and positions of target antigens are marked by arrows. The gel in panel 2 was overexposed to detect presence, if any, of biosynthetically labeled target antigens. (B) Relative proportions of different stages of Plasmodium falciparum in cultures used for biosynthetic labeling in (A). The blood films were made on the day of labeling, fixed with methanol and stained with Giemsa stain. Bars represent each stage (asexual or gametocytes I, II, III or IV and V) as percent of total number of parasites. Because of difficulties in distinguishing stage I gametocytes from trophozoites the proportions of asexual parasites and stage I gametocytes in panels 3 and 4 could vary.

339

IA3-BS , I I C S - B I 0 a n d I I D - A 1 0 ( M A b o f s imi la r p rope r t i e s to IA3-B8) (Fig . 3).

P ro t e in s p r e v i o u s l y iden t i f i ed as non-spec i f i ca l ly p r e c i p i t a t e d by M A b s o r n o r m a l

m o u s e s e rum were p re sen t at the ear ly s tages o f cu l tu re (up to 6 days pos t e x p a n s i o n

wi th f resh R B C ) (Fig . 3A, pane l s 1 and 2) at a t ime w h e n few o r no g a m e t o c y t e s were

de tec t ib le in the cul tures . H o w e v e r as s o o n as y o u n g g a m e t o c y t e s c o u l d be f o u n d in

s ign i f ican t n u m b e r s ( f r o m day 9 o n w a r d s ) the 260 000, 59 000 a n d 53 000 p ro te ins were

l abe led a n d i m m u n o p r e c i p i t a t e d by all the three a n t i - g a m e t e M A b s (Fig. 3A, pane l s

3-5) . T h e in tens i ty o f label in these p ro t e in s inc reased re la t ive to tha t in the non - spec i -

f ical ly p r ec ip i t a t ed p ro t e in s as the g a m e t o c y t e s inc reased in m a t u r i t y (Fig. 3A).

M o r p h o l o g i c a l d a t a on the cu l tu res used fo r l abe l ing is s h o w n in Fig . 3B.

O n the o t h e r hand , w h e n ex t r ace l l u l a r game te s pur i f i ed f r o m m a t u r e g a m e t o c y t e s

M r x l O - 3

T O P - -

2 0 0 - -

1 1 6 . 2 - -

9 7 . 4 - -

6 6 . 2 - -

4 5 . 0 - -

3 1 . 0

2 1 . 5 - - 1 4 . 4 - -

~i!!~i!!!ii i ! ¸ !!

B O B - - '

a b c d

Fig. 4. SDS-PAGE of labeled proteins immunoprecipitated from the surface of intact extracellular gametes purified from biosynthetically labeled mature gametocytes. Gametocytes of Plasmodium falciparum (18 days after expansion with fresh RBC) were labeled with [3SSlmethionine for 2 h at 37oc in methionine-free RPMI and then incubated for an additional 24 h under the normal culture conditions. Exflagellation was induced and gametes purified on a Percoll gradient as in Materials and Methods. Intact gametes were then incubated for 30 min with MAbs or NMS at room temperature for immunoprecipitation of labeled proteins from the surface. Cells were then washed three times with medium RPMI-1640 (Immufuge 1 min X 300 X g) and extracted in 1% Triton X-100 for 20 min at 0-4°C. Cell lysates were centrifuged for 15 min at 25 lb/inch 2 in an Airfuge and supernatants incubated with 50 ILl (packed volume) of Protein A-Sepharose beads for 50 rain at room temperature. The beads were washed and immune complexes extracted in SDS-buffer for SDS-PAGE as described in Materials and Methods. Non-reduced samples were analyzed by SDS-PAGE (5-15% acrylamide gradient). Lane a, NMS, lane b, MAb IA3-B8; lane c, MAb IICS-B10;and lane d, MAb IID2-A 10. Positions of standard markers are shown. Arrows indicated the target antigens of MAbs.

340

were labeled with [35S]methionine, there was no detectible synthesis of any of these three proteins (data not shown).

These results suggested, therefore, that the three P. falciparum gamete surface proteins of M r 260 000, 59 000 and 53 000 are synthesized only in the gametocytes during their maturation in the blood but not in the extracellular gametes and zygotes themselves. To confirm that the proteins synthesized in the gametocytes are in fact expressed on the surface of the extracellular gametes the following experiment was conducted.

Gametocytes from 18 day-old cultures were biosynthetically labeled for 2 h with [35S]methionine as before. Thereafter the gametocytes were cultured for a further 24 h and then stimulated to undergo gametogenesis. The purified gametes and zygotes were reacted intact with MAbs by incubation for 30 rain at room temperature for immunoprecipitation of labeled proteins from the surface. Following reaction with antibody on the surface, the cells were washed three times to remove unreacted antibody, extracted with Triton X-100 and the extracts reacted with Protein A-Sepha- rose beads followed by SDS-PAGE. As shown in Fig. 4, all three MAbs (IA3-B8, IIC5-B 10 and I ID2-A 10) immunoprecipitated the three target proteins. These results demonstrate that these proteins synthesized in the gametocytes became expressed on the surface of the extracellular gametes.

Mr xlO -3 1 2 3

Top- -

2 0 0 - -

116 97.4, 6 6 . 2 - -

i: 1

5 m ........

B~B-- a b a b c a b c

Fig. 5. SDS-PAGE analysis of Plasmodium falciparum polypeptides labeled with (1) [35S]methionine, (2)

[3H]glucosamine or (3) [~H]mannose. Parasites in culture expanded 12 days previously with fresh RBC were

given a continuous pulse of [3SS]methionine or radioactive sugars for 24 h at 37°C as described under

Materials and Methods. Triton X-100 extracts were immunoprecipitated using NMS (lane a), MAb IA3-B8

(lane b) or MAb IIC5-BI0 (lane c). Arrows indicate the position of target antigens.

341

To determine whether any of the three gamete surface antigens recognized by these

MAbs are glycosylated, parasites in cultures 13 days after expansion with fresh RBC

were incubated in the presence of [3H]glucosamine or [3H]mannose. The cells were extracted with Triton X-100 and immunoprecipitated with MAbs IA3-B8 or IIC5-

BI0. As shown in Fig. 5 the M r 59 000 and 53 000 proteins (seen as a single broad band) were labeled by both glucosamine and mannose. The M r 260 000 protein was not

labeled with either sugar.

DISCUSSION

We have previously reported that three proteins of apparent M r 260 000, 59 000 and 53 000 present on the surface of gametes of P.falciparum are the targets of monoclonal antibodies which block infectivity of this parasite to An. freeborni mosquitoes [4]. These target proteins were identified by immunoprecipitation with the transmission blocking MAbs from extracts of the extracellular female gametes and zygotes of P. falciparum surface labeled with 1251 [4]. In the present report we have studied the

biosynthetic origin of these proteins in P. falciparum grown in culture. Cultures were grown under conditions for production and maturat ion ofgameto-

cytes. The cultures were pulse labeled with [35S]methionine and extracted and immu- noprecipitated with MAbs at different times during the formation and maturation of the gametocytes. In cultures in which gametocyte formation had not yet begun there was no detectible synthesis of the 260 000, 59 000 and 53 000 M r proteins. Shortly after young gametocytes appeared in the cultures synthesis of these proteins could be detected and increased as the proportion and maturity of the gametocytes increased. These proteins synthesized in mature gametocytes were shown to be subsequently expressed on the surface of the extracellular gametes following stimulation ofgameto- genesis. However during or shortly after gametogenesis, biosynthesis of the 260 000,

59 000 and 53 000 M r proteins could no longer be demonstrated. We conclude, therefore, that these gamete surface proteins are synthesized in gametocytes but not in asexual blood stages of P. falciparum and subsequently expressed on the gamete surface. Synthesis of these proteins ceases, however, at or shortly after gametogenesis.

The biosynthetic and biochemical relationships between the 260 000, 59 000 and 53 000 M r gamete surface proteins has also been investigated. In pulse chase studies no evidence was obtained to indicate that the low molecular weight proteins were processed products derived from the M r 260 000 protein. Moreover only the 59 000 and 53 000 M r proteins were glycosylated with both mannose and glucosamine whereas the 260 000 M r protein was not. This also suggests that the high and low molecular weight proteins are synthesized independently of each other.

ACKNOWLEDGEMENTS

We thank Dr. L.H. Miller for support and helpful discussion during this work, Mrs.

342

R o s a n n e H e a r n fo r t echn ica l s u p p o r t and Mrs. W i l m a D a v i s a n d Mrs . B r e n d a M a r t i n

fo r typing. These s tudies r ece ived a pa r t i a l f inanc ia l s u p p o r t o f the U N D P / W o r l d

B a n k / W H O special p r o g r a m m e for Resea rch and T r a i n i n g in T r o p i c a l Diseases and

S e n i o r f e l l owsh ip f r o m the B u r r o u g h s W e l l c o m e F o u n d a t i o n to N i r b h a y K u m a r .

REFERENCES

1 Carter, R., Gwadz, R.W. and McAuliffe, F.M. (1979) Plasmodium gallinaceum: Transmission blocking immunity in chickens. I. Comparative immunogenicity of gametocyte and gamete contain- ing preparations. Exp. Parasitol. 47, 185-193.

2 Mendis, K.N. and Targett, G.A.T. (1979) Immunization against gametes and asexual erythrocytic stages of rodent malaria parasite. Nature 277, 389-391.

3 Gwadz, R.W. and Green, I. (1978) Malaria immunization in rhesus monkeys: A vaccine effective against both sexual and asexual stages of Plasmodium knowlesi. J. Exp. Med. 148, 1311-1323.

4 Rener, J., Graves, P.M., Carter, R., Williams, J.L. and Burkot, T.R. (1983) Target antigens of transmission-blocking immunity on gametes ofPlasmodiumfalciparum. J. Exp. Med. 158,976-981.

5 Kaushal, D.C., Carter, R., Rener, J., Grotendorst, C.A., Miller, L.H. and Howard, R.J. (1983) Monoclonal antibodies against surface determinants on gametes of Plasmodium gallinaceum block transmission of malaria parasites to mosquitoes. J. Immunol. 131, 2557-2562.

6 Ifediba, T. and Vanderberg, J.P. (1981) Complete in vitro maturation of Plasmodium falciparum gametocytes. Nature 296, 364-366.

7 Carter, R. and Miller, L.H. (1979) Evidence for environmental modulation ofgametocytogenesis in Plasmodiumfalciparum in continuous culture. Bull. WHO 57, (Suppl. 1) 37-52.

8 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage- T4. Nature 227, 680-685.