RODENT MALARIA PARASITES :

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RODENT MALARIA PARASITES :

MOLECULAR KARYOTYPES CHARACTERIZE SPECIES, SUBSPECIES AND LINES

AHAMADA SAID.*, WERY M.*, HAMERS R**

Summary :

The molecular karyotypes of the African murine malaria parasites P. berghei (3 strains, 2 lines) P. yoelii (2 strains) P. chabaudi (3 strains, 1 line) and P. vinckei (4 strains) have been studied using orthogonal field alternation gel electrophoresis (OFAGE).

The genome of each species was resolved into 9 to 11 distinct chromosomal DNA bands molecules of varying intensities which seem to represent 14 chromosomes ranging in size from 6 0 0 kb to 3 5 0 0 kb. The position of certain chromosomes allowed the identification of a unique karyotype for each of the strains and lines under study. P. yoelii appears by criteria of chromosome size, chromosome numbers and localisation of DNA probes to differ considerably from the other three rodent malaria species. The chromosomal location of 5 DNA probes allowed the identification of corresponding chromosomes in rodent malaria parasites and the differentiation between species and strains. Assignment of the

"PMMSA" gene of P. c. chabaudi IP-PC ] enables the distinction of the four rodent malaria species. The molecular karyotype combi- ned to chromosomal assignment of DNA probes provides a useful tool for a more precise characterization by a genetic definition of malaria parasites.

KEY WORDS : Rodent plasmodium. species. strains. lines. clones. karyotype.

PFGE. DNA probes.

ABBREVIATIONS : PMMSA, precursor to the major merozoïte surface anti- gens ; OFAGE, orthogonal field alternation gradient electrophoresis ; PFGE, pulse field gradient electrophoresis.

Résumé : PLASMODIUMS DE RONGEURS : CARACTÉRISATION DES ESPECES, SOUS-ESPECES ET LIGNÉES PAR CARYOTYPAGE MOLÉCULAIRE.

Le caryotype moléculaire des plasmodiums de rongeurs P. berghei (3 souches, 2 lignées), P. yoelii (2 souches), P. chabaudi (3 souches,

1 lignée) et P. vinckei (4 souches) a été étudié par électrophorèse en gel à champs orthogonaux alternés (OFAGE). Le génome de chaque espèce a été résolu en 9 à 1 bandes supposées représenter 14 chromosomes dont la taille varie de 600 à 3 500 kilobases. Après migration, la position de certains chromosomes a permis l'identifica- tion d'un caryotype original pour chaque souche étudiée. P. yoelii apparaît très différent des trois autres espèces par la taille et le nombre des chromosomes ainsi que l'affectation des sondes d'ADN.

La localisation chromosomique des 5 sondes choisies a permis l'identification des chromosomes homologues dans les différentes espèces et souches. La localisation du gène "PMMSA" de P.c. cha- baudi IР-РС 1 permet la distinction des quatre espèces de plasmo- diums de rongeurs. Le caryotype moléculaire combiné à la localisation des sondes sur certains chromosomes fournit un outil pré- cieux pour une caractérisation plus fine des parasites du paludisme.

MOTS CLES : Plasmodiums de rongeurs. espèce. souche. lignée. clone. caryo- type. électrophorèse ά champs alternés. sondes ADN.

ABREVIATIONS : PMMSA, précurseur de l'antigène majeur de la surface des mérozoïtes ; OFAGE, électrophorèse en gel à champs orthogonaux alternés ; PFGE, électrophorèse à champs puisés.

INTRODUCTION

R

odent malaria parasites are useful laboratory m o d e l s for h u m a n malaria research. T h e deve- lopment o f the malaria parasite varies with the parasite s p e c i e s and strain within o n e given host. T h e c l a s s i f i c a t i o n o f s p e c i e s a n d s u b s p e c i e s o f Plasmodium has classically b e e n b a s e d o n m o r p h o l o - gical and physiological characters. B i o c h e m i c a l c h a - racters w e r e added during the last t w o d e c a d e s or so (Carter, 1 9 7 3 ; Walliker et al, 1 9 8 7 ) . Classical c y t o g e - n e t i c studies are difficult to carry out with malaria parasites b e c a u s e o f the c o m p l e x i t y o f the life cycle a n d o f the fact that c o n d e n s e d c h r o m o s o m e s h a v e not b e e n demonstrated at any stage.

Electron m i c r o s c o p y , h o w e v e r , has revealed the pre- s e n c e o f I t k i n e t o c h o r e s in P. falciparum w h i c h

* Laboratory for Protozoology, Institute of Tropical Medicine, 155 Nationalestraat, B-2000 Antwerpen.

** Vrije Universiteit Brussel. Paardenstraat, St. Genesius Rode.

Correspondence address : Ahamada Said, Institute of Tropical Medicine. 155 Nationalestraat. B-2000 Antwerpen. Belgique.

w e r e c o n s i d e r e d as the haploid c h r o m o s o m e s num- b e r (Prensier & Slomianny, 1 9 8 6 ) . T o date there have b e e n few DNA markers w h i c h would b e useful for a m o r e precise g e n e t i c definition o f lines used in malaria research projects. A n e w tool to study the g e n o - mic organisation has b e e n provided by the technique o f p u l s e d - f i e l d g r a d i e n t g e l ( P E G ) e l e c t r o p h o r e s i s (Schwartz et al, 1 9 8 3 ) w h i c h allows the separation o f DNA m o l e c u l e s w i t h i n t h e 3 0 to 3 . 0 0 0 k i l o b a s e s range. Using PFG m a n y studies have b e e n carried out o n the c h r o m o s o m e s n u m b e r o f p l a s m o d i a . T h e s e studies have s h o w n that the c h r o m o s o m e s are highly polymorphic in size and that the n u m b e r o f c h r o m o - s o m e s s e e m s to b e 14 ( K e m p et al, 1 9 8 5 ; Sharkey et al., 1 9 8 8 ; J a n s e et al., 1 9 8 9 ; P o n z i et al., 1 9 9 0 ) . Laboratories w h e r e several s p e c i e s or s u b s p e c i e s o f rodent malaria parasites are simultaneously in use for different research studies n e e d specific markers for detecting m i x e d infections. In the present study this t e c h n i q u e w a s a p p l i e d to t h e m o l e c u l a r k a r y o t y p e chracterization o f rodent malaria parasites. Five DNA m a r k e r s ( 2 c h r o m o s o m e s - s p e c i f i c p r o b e s , 2 cDNA c l o n e s and o n e g e n o m i c DNA marker) were located.

Parasite. 1994. /. 31-38

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MATERIALS AND METHODS

CHARACTERIZATION OF PARASITES

Strains, lines and c l o n e s in this work w e r e defined as f( )\\ s :

- A strain is a sample o f parasites c o l l e c t e d from a naturally infected host o n a single o c c a s i o n ;

- Lines are parasites w h i c h have u n d e r g o n e particular passage series in the laboratory ;

- A c l o n e ( o p p o s e d to p o p u l a t i o n ) is c o m p o s e d o f genetically identical organisms derived from a single cell by asexual division.

Before analyse, all strains and lines w e r e maintained for a while in white m i c e from cryopreserved stabi- lates ;

- P. berghei K 1 7 3 R line is resistant to c h l o r o q u i n e : - All parasites are populations e x c e p t 8 4 1 7 , 8 4 9 5 and IP-PCI which are c l o n e s .

D N A PREPARATION

Parasites w e r e prepared for electrophoresis as descri

b e d by K e m p et al. ( 1 9 8 5 ) . Parasitised red b l o o d cells w e r e c o l l e c t e d from mice and l e u c o c y t e s r e m o v e d by p a s s a g e through a C F 1 1 c e l l u l o s e p o w d e r c o l u m n . After saponin ( 0 . 0 2 % ) hemolysis the parasites w e r e w a s h e d twice in p h o s p h a t e buffered saline ( P B S ) at

1 0 . 0 0 0 tr/mn for 1 mn.

T h e pellet w a s r e s u s p e n d e d in P B S to an a p p r o x i m a t e c o n c e n t r a t i o n o f 3 . 1 08 p a r a s i t e s per ml and added to an equal v o l u m e o f 2 % low-melting point agarose at 3 7 ° C. T h e blocks were incubated at 4 2 ° С for 48h. in 0.5 M EDTA/0.01 M TRIS/1 % sarkosyl/2 mg m l- 1 proteinase K.

T h e b l o c k s w e r e then stored in the s a m e solution at 4° C.

PULSED-FIELD GRADIENT GEL F1ECTROPHORF.SIS

T h e apparatus is a h o m e - m a d e modification o f the O r t h o g o n a l F i e l d A l t e r n a t i o n G e l E l e c t r o p h o r e s i s

( O F A G E ) set u p a n d d e s c r i b e d b y S c h w a r t z et al.

( 1 9 8 3 ) . A third o f e a c h b l o c k was inserted into the slots o f a 1 % agarose gel in 0.5x T r i s / B o r a t e / F D T A ( T B E ) buffer and scaled into the slots with the s a m e a g a r o s e gel. T h e pulse times used w e r e 6 0 , 1 2 0 o r 200 s e c o n d s depending o n the DNA m o l e c u l e s size to b e separated. T h e gel electrophoresis was run at 270 V for 2 0 hours at 14° C.

After running, the gel was stained in 0 . 5 x T B E contai

ning e t h i d i u m b r o m i d e (1 m g / m l ) for 3 0 min and p h o t o g r a p h i é e ! u n d e r an UV t r a n s l u m i n a t o r with a polaroid film (type 55 or 5 7 ) .

D N A PROBES

P r o b e s 5 ( 5 K b ) and 7 ( 3 . 5 K b ) are g e n o m i c DNA c l o n e s from P. berghei Anka 8 4 1 7 w h i c h are specific for c h r o m o s o m e s 5 and 7 respectively.

Clones W and Y and X are cDNA c l o n e s from /'. cha- baudi I P - P C I a n d c o r r e s p o n d t o t h e f o l l o w i n g e x p r e s s e d proteins :

\Y is a cDNA c l o n e (n° 1 0 0 ) o f 2.4 k b c o r r e s p o n d i n g t o t h e c e n t r a l part o f t h e chabaudi P M M S A (Deleersnijder et ai. 1 9 9 0 ) ;

Y is a cDNA c l o n e (n° 1 1 0 ) o f 1.1 kb c o r r e s p o n d i n g to a schizont-specific protein ;

X is a g e n o m i c DNA c l o n e ( X 2 A ) o f 2.3 k b corres

p o n d i n g to a part o f a p h o s p h o p r o t e i n a s s o c i a t e d with the erythrocyte cell m e m b r a n e (Deleersnijder et al., 1 9 9 2 ) .

SOUTHERN TRANSFER AND HYBRIDIZATION

A g a r o s e g e l s w e r e s o u t h e r n - b l o t t e d to H y b o n d - N m e m b r a n e s ( A m e r s h a m ) . DNA p r o b e s w e r e radiola

belled by m e a n s o f multiprime DNA labelling system ( A m e r s h a m ) . H y b r i d i z a t i o n w a s p e r f o r m e d u n d e r standard conditions at 6 5 ° C. After hybridization the blots w e r e w a s h e d at low stringency in 6 x SSC for 10 min at r o o m t e m p e r a t u r e f o l l o w e d b y an a n o t h e r wash in 2x S S c / 0 . 1 % SDS at 6 5 ° С for 3 0 min.

Autoradiography was performed using an intensifying s c r e e n with Fuji RX X-ray film.

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RESULTS

P

FG g e l e l e c t r o p h o r e s i s w a s applied o n three strains o f P. berghei, t w o strains o f P.yoelii.

three strains o f P. chcibaudi, three strains o f P. riuckei. t w o lines o f P. berghei and o n e line o f P. chabaudi. D i f f e r e n t p u l s e t i m e s w e r e u s e d t o achieve the separation o f small or large c h r o m o s o m e s (results not s h o w n ) . T h e karyotype o f the P. berghei c l o n e Anka 8 4 1 7 w a s c h o s e n as reference for chro- m o s o m e s n u m b e r b e c a u s e it can u n d e r g o the full life cycle and therefore is unlikely to lack essential g e n e - tic information (Ahamada Said et al., 1 9 8 6 ) .

The results show in P. berghei and P. chabaudi up to eleven well distinguishable c h r o m o s o m e bands and ten in P. vinckei. Apparently this restricted resolution is s o far t h e b e s t a p p r o a c h t o t h e b a s i c n u m b e r o f 14 c h r o m o s o m e s (Janse st al., 1 9 8 9 : Sharkey et al., 1988 ; S h e p p a r d et al.. 1 9 8 9 ) . In o u r e x p e r i m e n t s , c h r o m o s o m e s 9 to 12 appear as a non resolved entity.

On the other hand, the two strains o f P. yoelii differ considerably and show only 6 or 7 intense c h r o m o s o - mal bands.

MOLECULAR KARYOTYPE

P. berghei

T h e n u m b e r o f c h r o m o s o m e s within e a c h isolate is very similar but their s i z e m a y b e different. S o m e c h r o m o s o m e s comigrate in the gel : C h r o m o s o m e s 1- 2 in t h e K 1 7 3 strain, K 1 7 3 R line and SP11 strain ; c h r o m o s o m e s 2-3 in t h e SP11 strain ; c h r o m o s o m e s 5-6 in t h e K 1 7 3 R line ; c h r o m o s o m e s 5-6-7 in t h e 8 4 9 5 line : c h r o m o s o m e s 9-10-11 in all strains.

The position o f certain c h r o m o s o m e s allows an easy identification o f each strain and line : chromosomes 1.

2. 3 and 4 o f K173 strain are smaller than in the other strains and lines ; c h r o m o s o m e 7 o f the K173 and SP11 strains is smaller than the homologous chromosomes o f the Anka strain ; c h r o m o s o m e 7 o f Anka 8417 is larger than the h o m o l o g o u s c h r o m o s o m e in the Anka line 8495 ; c h r o m o s o m e 9 o f the Anka strain is larger than the homologous c h r o m o s o m e in the other strains and m i g r a t e s t o g e t h e r with t h e n o n r e s o l v e d c h r o m o - somes : in clone 8417 the straining intensity o f chromo- s o m e 7 with ethidium bromide decreased significantly after numerous mechanical passages as shown in fig.

2A (passage n° 6 3 ) .

l-ïg. I.

Karyotypes of different populations of rodent malaria parasites separated by pulsed field gel electrophoresis.

OFAGE was performed in a standard home made electrophoresis chamber at 270V.

with linean increasing pulse times from 60 to 200s. Run time was 20 hours at 14° C.

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Fig. 2.

A. K a r y o t y p e s o f different p o p u l a t i o n s o f r o d e n t m a l a r i a p a r a s i t e s s e p a r a t e d b y P F G . O F A G E w a s p e r f o r m e d in a s t a n d a r d h o m e m a d e c h a m b e r at 2 7 0 V w i t h l i n e a r y i n c r e a s i n g f o r w a r d p u l s e t i m e s f r o m 6 0 t o 1 0 0 s , run t i m e w a s 2 0 h o u r s at 1 4 ° C.

B . E t h i d i u m b r o m i d e s t a i n i n g o f O F A G E s e p a r a t e d c h r o m o s o m e s a n d S o u t h e r n b l o t o f t h e s a m e g e l h y b r i d i z e d to p r o b e W .

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Fig. 3.

Ethidium bromide staining of OFAGE

separated chromosome and Southern blot of the same gel hybridized to probe 7 and probe Y.

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Tableau I. - Karyograms of rodent malaria parasites examined in this study.

P. berghei Anka 8417 was taken as reference karyotype and the chromosomes were numbered consecutively from smallest to largest. Mapping of cDNA clones (W - Y)

and genomic DNA clones ( 7 - 5 and X) to chromosomes of parasites under study.

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MOLECULAR KARYOTYPES CHARACTERIZE RODENT MALARIA PARASITES P. yoelii

Most o f t h e c h r o m o s o m e s o f t w o P. yoelii strains comigrate and e a c h strain s h o w s an u n i q u e pattern w h i c h a l l o w to d i f f e r e n t i a t e t h e m from t h e o t h e r strains and lines u n d e r study.

P. chabaudi

E a c h o f the four P. chabaudi e x a m i n e d exhibited a different c h r o m o s o m e migration pattern. A n u m b e r o f c h r o m o s o m e s comigrate as was previously o b s e r v e d in P.c. adami and P.c. chabaudi C B ( S h e p p a r d et al, 1 9 8 9 ) .

P. vinckei

T h e karyotypes o f the three P. vinckei strains are dif

ferent. T h e position o f c h r o m o s o m e s 5, 6 and 7 per

mit to discriminate them. C h r o m o s o m e s 1-2, 3-4 and 10-11-12 comigrate.

ASSIGNMENT OF D N A SEQUENCES TO SPECIFIC CHROMOSOMES

T h r e e DNA p r o b e s from P. chabaudi w e r e m a p p e d to c h r o m o s o m e s b y SoLithern hybridization in P F G blot : p r o b e X r e c o g n i s e s comigrating c h r o m o s o m e b a n d s ( 1 0 - 1 1 a n d 1 2 ) in all t h e i s o l a t e s , t h e t w o others p r o b e s ( Τ and W ) hybridize to single c h r o m o s o m e b a n d i n d i c a t i n g that t h e y c o u l d b e u s e d as c h r o m o s o m e s s p e c i f i c m a r k e r s for r o d e n t m a l a r i a p a r a s i t e s . In P. yoelii t h e p r o b e W r e c o g n i z e s t h e s a m e comigrating c h r o m o s o m e s as p r o b e Y ( 1 0 - 1 1 - 1 2 ) . T w o specific m a r k e r s r e s p e c t i v e l y for c h r o m o s o m e s 5 and 7 isolated from a g e n o m i c library o f P.

berghei Anka (Ponzi et al, 1 9 9 0 ) w e r e u s e d to iden

tify c h r o m o s o m e s sharing h o m o l o g o u s g e n e t i c infor

mation in the different isolates and lines used in this study. E a c h p r o b e hybridized to single c h r o m o s o m e b a n d o f different m o l e c u l a r w e i g h t . T h e results o f c h r o m o s o m e specific hybridization o f all the p r o b e s are summarized in table I. T h e three p r o b e s c o d i n g for e x p r e s s e d proteins localise c h r o m o s o m e s o f simi

lar size in P. berghei, P. chabaudi and P. vinckei.

DISCUSSION

^ I ^he m o l e c u l a r karyotype o f the four s p e c i e s o f rodent malaria parasites have b e e n characteri- JL zed by P F G . It must b e b o r n in mind that for total p o p u l a t i o n s t h e k a r y o t y p e is clearly different after cyclical transmission and unstable after m e c h a n i cal p a s s a g e (Ahamada Said, 1 9 8 7 ) . T h e s e observations suggest that total populations are c o m p o s e d o f a mix

ture o f parasites with different karyotypes. P. yoelii a p p e a r s to differ c o n s i d e r a b l y from the three o t h e r rodent malaria s p e c i e s b y k a i y o t y p e and g e n e locali

sation.

NUMBER AND POSITIONS OF THE BANDS

C o n s i s t e n t with r e p o r t e d studies o f r o d e n t malaria k a r y o t y p e ( S h e p p a r d et al, 1 9 8 9 ; S h a r k e y et al, 1988 ; Langsley et al, 1987 and J a n s e et al, 1 9 8 9 ) ; the p r e s e n t analysis s h o w s an important k a r y o t y p e p o l y m o r p h i s m within a n d b e t w e e n s p e c i e s , strains and lines. E a c h c l o n e d parasite exhibits an u n i q u e karyotype pattern w h i c h remains stable after m e c h a n i c a l o r c y c l i c a l p a s s a g e s ( A h a m a d a S a i d , 1 9 8 7 ) . However, continuous m e c h a n i c a l passages s e e m e d to e x e r t a s e l e c t i v e p r e s s u r e in f a v o u r o f p a r t i c u l a r karyotype mutants. Such conditions take p l a c e only in laboratory and are thus easy to control. A decrea

sing intensity o f c h r o m o s o m e 7 and the loss o f g a m e - tocyte infectivity o c c u r e d simultaneously after 58 and 63 m e c h a n i c a l passages o f the c l o n e 8 4 1 7 . H o w e v e r there is n o direct correlation b e t w e e n karyotype and g a m e t o c y t e infectivity. T h i s d e c r e a s i n g intensity o f c h r o m o s o m e 7 can b e e x p l a i n e d by a variation in the n u m b e r o f c o p i e s o f a s u b t e l o m e r i c repetitive e l e ment ( 2 . 3 K b ) w h i c h s e e m e d to b e involved in the c h r o m o s o m e size polymorphism (Ponzi et al, 1 9 9 0 ) . Molecular karyotype t e c h n i q u e s w e r e able to diffe

rentiate t h r e e P. berghei isolates s h o w i n g identical i s o e n z y m e or morphological patterns.

THE USE OF PROBES

Strains and lines o f the four rodent malaria s p e c i e s w e r e identified for the first time b y c h r o m o s o m e spe

cific and DNA p r o b e s recognizing h o m o l o g o u s chro

m o s o m e s or c h r o m o s o m e s sharing the s a m e genetic information. T h e three P. chabaudi p r o b e s and the two P. berghei c h r o m o s o m e specific p r o b e s all hybri

dized to the four rodent plasmodia species.

P r o b e s 5 and 7 s h o w e d m a r k e d c h r o m o s o m a l loca

tion differences in all species, lines and strains indica

ting that they can b e used as specific markers for all t h e r o d e n t m a l a r i a p a r a s i t e s . T h e p r o b e W w h i c h represents the PMMSA is localised o n c h r o m o s o m e s o f slightly different size in the four species. Moreover, within e a c h species, very little size variation is obser

v e d and p r o b e W is therefore a useful species diffe

rentiator k e y after isoenzymes.

T h e other g e n e s ( X , Y ) , corresponding to e x p r e s s e d proteins, are also located in P. chabaudi, P. berghei and P. vinckei o n c h r o m o s o m e s showing relative size conservation. This c o u l d b e the effect o f c l o s e phylo- g e n e t i c relation. P. yoelii s e e m s to differ from t h e o t h e r three rodent malaria s p e c i e s b y c h r o m o s o m e size, lack o f small c h r o m o s o m e s and localisation o f p r o b e s . B a s e d u p o n the analysis o f the small subunit ribosomal RNA s e q u e n c e s , P. berghei s e e m e d to b e phylogenetically c l o s e r to P. vinckei than to P. yoelii

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three a n d s h o u l d n o t b e g r o u p e d with P. berghei as is usually d o n e . A m o l e c u l a r tree m a d e with c h r o m o s o - m a l a s s i g n m e n t o f D N A p r o b e s a l s o s h o w e d that P. berghei is c l o s e r t o P. vinckei t h a n t o P. yoelii (results n o t s h o w n ) .

Molecular karyotype c o m b i n e d to assignment o f DNA p r o b e s (15, 7, W a n d Y ) p r o v i d e a n u n a m b i g u o u s characterization a n d a m o r e p r e c i s e g e n e t i c definition o f parasites lines a n d isolates u s e d in i m m u n o l o g i c a l and g e n e t i c research. T h e s e markers c a n b e u s e d for d e t e c t i o n o f accidentally cross c o n t a m i n a t i o n arising b y m a n i p u l a t i n g different s p e c i e s o r s u b s p e c i e s o f rodent malaria involved in different areas o f research.

T h e p r o b e s c a n also b e u s e d as g e n e t i c markers in crossing e x p e r i m e n t s a n d as valuable tools at t a x o n o - mie level.

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Accepté le 22 novembre 1993

3 8 Mémoire

ACKNOWLEDGEMENTS

Ж А / ƒ e t h a n k D r M. Ponzi (Istituto Superiore di

\ Ж / Sanita, R o m e ) for providing the p r o b e s 5

• • a n d 7 a n d D r W . D e L e e r s n i j d e r (Vrije Universiteit, B r u s s e l s ) for providing the p r o b e s W , X and Y .

This w o r k w a s supported by EEC contract TSD-M-052.