Mosaic-like transcription of var genes in single Plasmodiumfalciparum parasites�

Victor Fernandez a,*, Qijun Chen a, Annika Sundstrom a, Artur Scherf b, Per Hagblom a,Mats Wahlgren a

a Microbiology and Tumor Biology Center (MTC), Karolinska Institutet and Swedish Institute for Infectious Disease Control, Box 280, S-17177

Stockholm, Swedenb Unite de Biologie des Interactions Hote-Parasite, CNRS URA 1960, Institut Pasteur, 75724 Paris, France

Received 19 October 2001; received in revised form 2 February 2002; accepted 7 February 2002

Abstract

The var gene family of Plasmodium falciparum encodes the clonally variant adhesin PfEMP1 present on the surface of infected

erythrocytes. A poorly understood mechanism of allelic exclusion controls the expression of PfEMP1. Transcription of var genes is

developmentally and, most likely, epigenetically regulated. Here we have studied the transcriptional pattern of 28 members of this

multigene family in individual parasites, early in the intraerythrocytic cycle. The results show unique patterns (type and number) of

var transcripts in each individual PRBC, with 1�/15 mRNA species detected per cell at 2�/4 h post-invasion. When a panel of ten

single PRBC was analyzed, the var gene coding for the expressed PfEMP1 was transcribed in more cells than any other, although

transcripts from this gene did not give the strongest hybridization signal within each individual cell. Chromosomal mapping of

transcriptionally active var genes indicated that their distribution reflects that of var loci in the genome, including a pronounced

clustering in chromosome 4. These findings, taken together with existing data on var transcription at later developmental stages,

suggest that the mosaic-like transcription of multiple var genes detected at the ring stage and the steady transcription of the gene

encoding the expressed PfEMP1 are distinct although superimposed events, one of them random and the other taking place under

some form of imprinting. With its unique features, the expression of P. falciparum var genes may reveal new principles of gene

regulation. # 2002 Elsevier Science B.V. All rights reserved.

Keywords: Plasmodium falciparum ; Asexual stages; var -gene transcription; Single cell

1. Introduction

Malaria symptoms and pathology are caused by

Plasmodium falciparum during the intraerythrocytic

phase of its life cycle. Far from just concealing itself

inside the host cell, the parasite actively synthetizes

proteins that are transported to and inserted into the

erythrocyte membrane where they, besides performing

tasks thought to be vital for parasite survival, become

antigenic targets. One such protein is the adhesin

PfEMP1 which mediates the sequestration and accumu-

lation of infected erythrocytes (PRBC) in the brain, the

placenta and other organs by adhering to receptors on

the vascular lining (endothelial cytoadherence) and to

uninfected erythrocytes (rosetting). This allows the

parasite to avoid spleen-dependent clearance mechan-

isms [1,2].

PfEMP1 proteins, which are encoded by the highly

diverse var gene family [3,4], undergo antigenic varia-

tion as a means of evading the host immune response

[5�/7]. Switching in the expression of PfEMP1 variants

Abbreviations: DBL, duffy-binding-like; ORF, open reading frame;

PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1;

PFGE, pulsed-field gel electrophoresis; PRBC, Plasmodium

falciparum -infected red blood cell; RT-PCR, reverse transcriptase

polymerase chain reaction.�

Note: The sequences here reported are deposited in the DDBJ/

EMBL/GeneBankTM database under the accession numbers

AF003473, AF039275, AF039278, AF039282, AF039283, AJ007940,

AJ007941, AJ429501-21, U67959 and U67960.

* Corresponding author. Tel.: �46-8-4572553; fax: �46-8-310525.

E-mail address: victor.fernandez@mtc.ki.se (V. Fernandez).

Molecular & Biochemical Parasitology 121 (2002) 195�/203

www.parasitology-online.com

0166-6851/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 6 - 6 8 5 1 ( 0 2 ) 0 0 0 3 8 - 5

may occur at a rate as high as 10�2 per generation

among in vitro grown parasites [8], but through a yet

unknown mechanism of allelic exclusion individual

parasites synthesize and display only one (or a major)PfEMP1 type on the PRBC surface [9]. The var genes

have a two-exon structure and a total length of 8�/14 kb.

According to presently available estimates (prior to

completion of the P. falciparum genome annotation)

there are over 50 unique var genes per haploid parasite

genome [9�/11].

Analysis of var gene transcription in asexual batch

and individual parasites showed that the var mRNAcoding for the surface expressed PfEMP1 seemed to be

the only species in the mature trophozoite stage, while

low-level transcripts from several other var genes could

also be detected in single PRBC containing ring-stage

parasites [9,12,13], a phenomenon variously referred to

as ‘overflow’, ‘relaxed’, ‘leaky’ or ‘indiscriminate’ tran-

scription. The possible significance of this latter ob-

servation, in the context of var gene expressionregulation, is not clear. More recent studies have

revealed that multiple var transcripts can be detected

in monomorphic, phenotypically homogeneous bulk

mature trophozoites [14,15]. Additionally, another

group of transcripts, 1.8�/2.4 kb mRNAs mapping to

sections of the intron and the second exon of var , are

abundantly transcribed by erythrocytic stages [4]. It is

believed that these events concerning var mRNAdynamics in the PRBC do reflect basic features of the

mechanism(s) that govern transcription and allelic

exclusion in the var loci. We here extend the investiga-

tions on the transcription of var genes in asexual P.

falciparum by characterizing, immediately after erythro-

cytic invasion, the var mRNA contents in single infected

PRBC of a cloned parasite line of known adhesive

phenotype and defined surface expressed PfEMP1.

2. Materials and methods

2.1. Parasites

P. falciparum parasites were cultured as previously

described [16]. FCR3S1.2 parasites were obtained by

micromanipulation cloning from FCR3S1, which hadbeen previously cloned by limiting dilution from the

FCR3 strain originally isolated in The Gambia, West

Africa. FCR3S1.2 parasites were maintained in contin-

uous culture and periodically enriched for the rosetting

phenotype as described elsewhere [17].

2.2. Synchronization of cultures and parasite collection

Phenotypically monomorphic FCR3S1.2 parasites

were obtained by successive enrichment of rosetting

trophozoites on Ficoll�/Isopaque (Pharmacia) cushions

until the rosetting rate in the culture was steadily above

90% [18]. The parasites were then stage-homogenized by

three rounds of synchronization in 5% Sorbitol (Sigma)

over consecutive cycles [18]. For the experiments,parasites were collected from synchronized cultures 2 h

after the first intraerythroeytic ring forms were observed

during the re-invasion step following the last synchro-

nization round. Single PRBCs containing one ring were

aspirated in 5-mm micropipettes using a hydraulic MN-

188 micromanipulator (Narishige/Nikon) mounted on

an inverted microscope (Nikon), and immediately

frozen on dry ice [17,18].

2.3. Reverse transcription and semi-nested PCR

A schematic description of the steps followed to

analyze var gene transcription in single PRBC is shown

in Fig. 1. Preparation of cDNA and amplification ofDBL1 fragments was performed after modification of

methods previously described [9,12]. In short, cells in 15

Fig. 1. Analysis of var transcripts in individual intraerythrocytic P.

falciparum . The var genes of FCR3 represented in the array are: (1)

var1, (2) var 13, (3) var 70, (4) var 51, (5) var21, (6) var 50, (7) var C28,

(8) var 71, (9) var 49, (10) var 48, (11) var3, (12) var11, (13) var C22, (14)

var46, (15) var 6, (16) var 65, (17) var29, (18) var 12, (19) var C21, (20)

varC19, (21) var 9P, (22) var 52, (23) var 45, (24) var 10, (25) var CSA,

(26) var C6, (27) var 58, (28) var 44, (29) TM284S2var 1.

V. Fernandez et al. / Molecular & Biochemical Parasitology 121 (2002) 195�/203196

ml of a solution containing 13.3 mM Tris�/HCl pH 8.3,

6.7 mM MgCl2, 67 mM KC1, and 1.3 mM of each

dNTP were heat-lysed at 95 8C for 5 min and immedi-

ately cooled to 4 8C. Ten units DNase I (Stratagene)and 20 U RNase inhibitor (Perkin�/Elmer) were added

and DNA digested at 37 8C for 30 min. The DNase was

inactivated at 95 8C for 3 min and tubes were cooled to

4 8C. Fifty units MuLV reverse transcriptase (Perkin�/

Elmer), 50 pmol random hexamers (Perkin�/Elmer) and

20 U RNase inhibitor were added to make the reaction

volume to 20 ml and tubes were incubated at 37 8C for

30 min followed by 95 8C for 3 min. For the first PCRround, 80 ml of a master mix containing 5 U Taq DNA

polymerase (Perkin�/Elmer), 100 pmol each of primers

DBL1.1 (5?-GGW GCW TGY GCW CCW TWY MG-

3?) and DBL1.2 (5?-ARR TAY TGW GGW ACR TAR

TC-3?), and 4 ml each of Opti-Prime buffers #3 and #4

(Stratagene) were added to the reverse transcription

reaction to make 100 ml and final concentrations of 10

mM Tris�/HCl pH 8.3, 3.8 mM MgCl2, 50 mM KCl, 200mM of each dNTP and 1 mM of each primer. PCR was

performed starting with a hold step at 95 8C for 3 min

followed by 50 cycles of 93 8C for 30 s, 55 8C for 30 s

and 72 8C for 1 min, with a terminal extension step at

72 8C for 7 min. For the second (semi-nested) PCR

round, 5 ml of the first PCR were mixed with 95 ml of a

master mix containing 5 U Taq DNA polymerase, 100

pmol each of primers DBL1.3 (5?-GCA CGA AGTTTY GCA GA-3?) and DBL1.2, dNTPs and salts to

make final concentrations of 10 mM Tris�/HCl pH 8.3, 2

mM MgCl2, 50 mM KC1, 200 mM of each dNTP and 1

mM of each primer. PCR was started with a hold step at

93 8C for 3 min, then 50 cycles of 93 8C for 30 s, 52 8Cfor 30 s and 72 8C for 1 min, with a final extension hold

at 72 8C for 7 min.

2.4. Dot blot var-gene matrix and hybridization

DBLla sequence tags 450�/650 bp long were amplified

from FCR3S1.2 and FCR3 gDNA or cDNA using

primer pairs DBL1.1/DBL1.2 and varA5.2 (5?-GCC

TGY GCK CCR TWY AGR CG-3?)/varE3.2 (5?-ACA

TAA TCD AAA TWT GTR GGA AC-3?), respectively.

PCR products were cloned into pCR II (Invitrogen)

according to the manufacturer recommendations. Plas-mid DNA was isolated using an anion-exchange micro-

preparation kit (Invitrogen), inserts were sequenced

using the ABI PRISM BigDye terminators sequencing

kit (Applied Biosystems), and the sequences analyzed

using MAC VECTOR version 7.0 (Oxford Molecular Ltd.).

Plasmids containing unique DBL1a sequence inserts

were denatured by heating at 100 8C for 5 min and 0.5

mg blotted per dot onto a Hybond-N�/ nylon membrane(Amersham Pharmacia) using a Bio-Dot microfiltration

apparatus (Bio-Rad). RT-PCR products were labeled

with [a-32P]dCTP by random priming using Ready-To-

Go DNA labeling beads (Amersham Pharmacia) ac-

cording to the manufacturer recommendations. Filters

were prehybridized at 65 8C for 1 h in Rapid-hyb buffer

(Amersham Pharmacia) and denatured labeled probeadded and hybridized at 65 8C overnight. Filters were

washed twice in 2�/SSC/0.1%SDS, once in 1�/SSC/

0.1%SDS, once in 0.5�/SSC/0.1%SDS, once in 0.2�/

SSC/0.1%SDS and twice in 0.1�/SSC/0.1%SDS. The

washes times were always 15 min long and at a

temperature of 65 8C. Membranes were exposed to

storage phosphor plates which were scanned on a

phosphorimager 445 SI (Molecular Dynamics).

2.5. Pulsed-field eleclrophoresis and chromosomal

mapping of var genes

Agarose blocks of FCR3 and FCR3S1.2 parasites

were prepared as described previously [18,19]. Chromo-

somes were separated by pulsed-field gel electrophoresis

(PFGE) in 0.5 or 1% chromosomal grade agarose (Bio-

Rad) using 1�/TAE or 0.5�/TBE buffers at 14 8C. Theseparations were performed in a CHEF Mapper (Bio-

Rad). After depurination, denaturation and neutraliza-

tion, chromosome DNA was transferred to a Hybond-

N�/nylon membrane using a PosiBlot 30�/30 pressure

blotter (Stratagene) and cross-linked to the membrane

under UV light. The var DBL1a probes labeled by

random priming were hybridized to the membranes

under standard conditions and washed at high strin-gency (0.1�/SSC/0.1%SDS at 60 8C).

3. Results and discussion

3.1. Construction of a gene matrix for analysis of

transcriptional activation in var loci

A dot gene matrix was prepared for the screening of

var cDNAs in RT-PCR amplicons from single

FCR3S1.2 PRBC and bulk cultures. DBL1a sequence

tags 450�/650 bp long corresponding to 28 unique var-

gene variants present in the FCR3 genome were blotted

onto membranes and hybridization/washing stringency

conditions adjusted until single-gene discrimination was

achieved. All the sequences in the matrix were amplifiedfrom parasite gDNA or cDNA using primer pairs

DBL1.1/DBL1.2 [12] or varA5.2/varE3.2 [20], which

map to conserved boxes in DBL1a flanking approxi-

mately 2/3 of the total length of this domain including

the internal region with five highly polymorphic seg-

ments defined by the primers DBL1.3/DBL1.2 used for

the semi-nested PCR. Pairwise comparison of the 28

sequences showed that nucleotide identity ranged from45 to 84% and amino acid similarity from 44 to 83%.

The two closest sequences (var1 and var13) share several

large identical blocks. Detection of var1 and var13 in

V. Fernandez et al. / Molecular & Biochemical Parasitology 121 (2002) 195�/203 197

cDNAs generated in at least two independent reverse

transcription experiments indicated that these two

sequences belong to distinct var genes. In total, 34

different DBL1 sequences, assumed to correspond to anequal number of unique variants of this gene family,

have been identified to date in gDNA and cDNA from

parasites derived from the FCR3 strain ([4,9,12,13], and

the present study) (Fig. 2), and about 50 different genes

have been estimated to constitute the var gene pool of

this parasite [9]. Thus, the var array assembled here

includes 82% (28/34) of the known DBL1 sequences and

over 50% of the var genes of FCR3.

3.2. Different subsets of var genes are transcribed in

individual ring parasites

Previous RT-PCR studies showed that multiple var

genes are transcribed in bulk cultures of ring-stage P.

falciparum [9,12,14]. Also, multiple var transcripts have

been detected in parasite populations rigorously selected

for one adhesive phenotype [9,13,15]. Furthermore,transcripts of more than one var gene were found in

individual ring parasites [9]. To examine in detail the

extent and identity of var genes transcribed early during

the intra-erythrocytic cycle by parasites of a mono-

morphic, developmentally and phenotypically homoge-

neous population, we synchronized (3�/) and selected

for rosetting (3�/) a culture of the originally cloned

FCR3S1.2 until ]/95% of the mature parasites formedrosettes and, as assessed by morphological criteria,

stage-synchronicity was �/99%. From this culture,

individual PRBC containing a single ring were collected

by micro-manipulation 2�/4 h after the onset of ery-

throcyte re-invasion and the transcription products were

analyzed by RT-PCR and dot-blot hybridization (Table

1). Overall, 79% (22/28) of the var genes represented in

the filter array yielded positive hybridization signalswith the DBL1 probes amplified from ten different

single ring-stage cDNAs. We observed that the number

and type of detected var mRNAs was distinct in each

cell. The average number of distinct var transcripts

detected per cell in this panel of very young rings was

8.0, ranging from one single mRNA species (PRBC no.

9) to 15 transcripts (PRBC no. 4). Importantly, when 2�/

4 h post-invasion batch parasites from the same culturewere harvested, serially diluted, and RT-PCR products

obtained from the limiting dilution step, i.e. 5/1

parasite, the hybridization patterns on var arrays were

different in replicate experiments (not shown). The most

common mRNA appeared to be var1, for which

hybridization was positive in 9/10 PRBC (Fig. 3A).

The var1 gene encodes the PfEMP1 polypeptide trans-

ported to and expressed on the surface of the PRBCbearing mature stages of FCR3S1.2 [12]. This could

imply that multiple var loci are transcriptionally active

already at, or prior to, the time of invasion and that this

changeable transcription pattern transiently overlaps an

apparent steady production of the mRNA coding for

the surface expressed PfEMP1, which is also initiated

very early or before RBC invasion.

3.3. Levels of var transcripts shortly after RBC invasion

The most abundant and commonly detected var

mRNA at later time points during intraerythrocytic

growth is that coding for the PfEMP1 polypeptide

transported and expressed on the PRBC surface

[9,12,13,21,22]. In the present study of early ring-stage

parasites, the most common transcript appeared to be

var1. However, unambiguous strong hybridization to

other var genes was evident in a majority of the PRBC

analyzed, in contrast to the often weak var1 hybridiza-

tion signal (Table 1). In seven of the var1-positive PRBC

(no. 1�/6 and 8) the hybridization signal for other var

species was stronger or very much stronger, i.e. var70 in

PRBC number 1 and 2, var48 in PRBC number 3, var65

and var70 in PRBC number 4, var10 in PRBC number

5, var3 and var70 in PRBC number 6, and var27 in

PRBC number 8. These results thus seem to indicate

that var1 was not the dominant mRNA in a significant

number of early FCR3S1.2 rings. Examination of the

cumulative hybridization scores further suggests an

imbalance in favor of certain var transcripts, e.g.

var70, var65, var50, or var27, (Fig. 3B). Control

hybridizations with var70, var65, var50, and var27

probes prepared from plasmid inserts with the same

primers used for nested PCR from single PRBC cDNAs,

i.e. DBL1.3/DBL1.2, did not show cross-hybridization

with other var in the array or abnormally increased

signal intensity as compared with var1 (not shown). In

additional control experiments, a var1 probe weakly co-

hybridized with var13 at the high stringency conditions

used for hybridization of RT-PCR products. This

finding, not totally unexpected given the high degree

of homology in the DBL1 domain of these two genes,

indicates that var13 transcript frequency might be

overrated. Although overestimation of var1 transcrip-

tion cannot be totally ruled out, we consider it less likely

in a majority of the cells positive for transcripts of this

gene. Control hybridizations with var13 probes yielded

cross-hybridization signals in var1, which invariably

were at least one order of magnitude fainter. Hybridiza-

tion of filters with products amplified from genomic

DNA of a number of individual ring-infected PRBC did

not provide evidence of significant primer bias (Fig. 3C).

Taken together, the data suggest that while var1 is

steadily transcribed early in the intraerythrocytic life of

FCR3S1.2, products of transcriptional activation of

other var -gene loci make up the dominant var mRNA

species during the first hours post-invasion.

V. Fernandez et al. / Molecular & Biochemical Parasitology 121 (2002) 195�/203198

Fig. 2. Alignment of deduced amino acid sequences from the DBL1a domain in var genes of the FCR3 lineage. Thirty-one out of the 34 different known var genes of FCR3 parasites are included.

Sequence var-3 NIH has accession number L40609 [4]. The aligned sequences are defined by the primers DBL1.3 and DBL1.2, which are described in Section 2. Consensus amino acids are shaded in

dark gray (identity) or light gray (physicochemical similarity). Sequences were compared using the CLUSTALW algorithm.

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3.4. Chromosomal location of transcribed var genes

To determine the chromosomal location of cloned var

genes, parasite chromosomes were size-fractionated byPFGE and Southern analysis carried out using gene-

specific DBL1a probes. To date, we have mapped 17 out

of the 34 known var variants in FCR3, which are found

in all chromosomes except 1, 2, 6 and 14 (Fig. 4; see also

ref. [13]). In agreement with previous data indicating an

irregular distribution of var in the 14 chromosomes of

P. falciparum [10,23], and with early releases from the

Malaria Genome Project showing numerous ORF withthe features of var in the chromosome 4 of the 3D7

strain [24], we observed clustering of var genes in

chromosome 4 of FCR3. Over one-third (6/17) of the

var -specific probes, including that of var1, hybridized to

this chromosome. Five of the six var mapping to

chromosome 4, i.e. var1, var13, var48, var57 and

var70, were found to be transcribed. Overall, about

50% (allowing for overestimation of var13 hybridiza-tion) of the transcript detection events for var genes of

known chromosomal location involved loci mapping to

chromosome 4, suggesting that positional factors may

influence the likelihood of transcription.

Notably, probing of var70 yielded an equally intense

signal in chromosomes 4 and 9, even at the highest level

of stringency, suggesting the recognition of the entire

gene, or recombinant DBL1 fragments, in more thanone copy. Other expressed var , i.e. var3, var6, var10 and

var11, mapped to chromosomes no. 5, 7, 9 and 8,

respectively. Thus, transcriptionally active var genes are

not only found on different chromosomes but their

distribution seems to reflect that of var loci in the

genome, including a pronounced clustering in chromo-

some 4.

In conclusion, our analysis of var-gene transcripts inindividual PRBC collected from highly synchronized

and phenotypically homogeneous parasite populations

shows that distinct transcription events are taking place

within the first 4 h post-invasion or, possibly, already at

the merozoite stage. One of them is the transcription of

var1, encoding the surface expressed PfEMP1. Tran-

scripts of var1, which in very early ring stages are found

in the vast majority of the PRBC, were also present inmost of the later rings [9], and constituted the only var

mRNA detected in the mature trophozoites of

FCR3S1.2 [12]. Superimposed to the background of

var1 transcription, var transcripts varying noticeably in

type and number from cell to cell were detected short

after initiation of the erythrocytic cycle. This apparent

promiscuous activation involved distinct var subsets in

different parasites, resulting in ‘mosaic’-like patterns oftranscription overlapping the expression of var1.

The nature of the multiple mRNA species in ring

stages is not clear. Analysis with primers spanning

DBL1 sequences does not give information on whether

Table 1

Transcription of var genes in early (2�/4 h post-invasion) ring-stage P.

falciparum

PRBC number Hybridization

intensitya

var gene

1 5�4� var 70

3�2�1� var 1, var 13, var 50

2 5�4� var 70

3� var 46, var 50

2� var 27, var C22

1� var 1, var 11, var 13, var 49, var 57,

var 71, var C19 var C28

3 5�4� var 48

3� var 1, var 10

2�1� var 3, var 11, var 71

4 5� var 65, var 70

4� var 3, var 13, var 50

3� var 27, var 71, var C22

2� var 1, var 11, var 48, var 49, var 57,

var C19, var C28

1�5 5�

4� var 10

3�2� var 12, var 52

1� var 1, var 13, var 45, var C28

6 5�4� var 3, var 70

3�2� var 13, var 50, var 71

1� var 1, var 57, var C19, var C22

7 5�4�3�2� var 1, var 11, var 71

1� var 3, var 6, var 10, var 29, var 46

8 5� var 27

4�3�2� var 57, var 70, var 71

1� var 1, var 11 var 13, var 48, var 49,

var 50, var C22

9 5�4�3�2�1� var 1

10 5�4�3�2� var 49, var 71

1� var 45

Dot arrays of FCR3 var genes were probed with RT-PCR products

from single PRBC contaning one ring-stage parasite.a Hybridization was quantified by phosphoimagery and scored using

a relative scale from 0, no signal over background, to 5�, maximal

signal.

V. Fernandez et al. / Molecular & Biochemical Parasitology 121 (2002) 195�/203200

the products seen by RT-PCR represent full-length

spliced mature messages. On the basis of Northern

blot data, it has been suggested that only one var gene is

fully transcribed [14]. However, RT-PCR in bulk para-

sites with primers to the DBL1 and to the region across

the splice site demonstrates the transcription of multiple

full-length var genes in trophozoites of phenotypically

monomorphic populations [15]. Several models can be

contemplated to explain the gradual predominance of

var1 mRNA as the parasite grows. A higher stability of

var1 message relative to the rest of the var transcript

pool is an improbable contingency given that var -gene

expression seems to be regulated at transcription initia-

tion [13]. Bearing in mind that var1 mRNA is present in

the youngest rings, but as a minor product, a plausible

hypothesis is that a prolonged, steady transcription in

the ‘imprinted’ var locus takes place over a relatively

long interval during intraerythrocytic development, and

that the bulk of ‘relaxed’ transcripts, product of a

random short-lived activation of other var loci, rapidly

decays below the threshold of RT-PCR sensitivity.

Studies done in bulk cultures of the protozoan parasite

Trypanosoma brucei show that a progressive arrest of

RNA elongation occurs in all but one of the variant

surface glycoprotein telomeric expression sites [25]. A

possibility is that most or all var-gene promoters can

recruit the transcription machinery and compete for a

limited number of transcription factors. The observation

of randomly activated var subsets or mosaics is in line

with the latter scenario. This transitory transcription

may result or not in properly elongated messages.

Limitations in the sensitivity of Northern blot analysis

preclude excluding the possibility that the early tran-

scripts are full-length products present at low level in

RNA preparations from bulk parasites, despite being

dominant species in some individual PRBC.

The chromosomal mapping data is consistent with the

notion that all var genes, irrespective of their genomic

location or position within the chromosome, may be

capable of expression. Transcription of var genes can

occur in situ from both sub-telomeric and more internal

loci [13,20]. Recently, random telomere clustering invol-

ving 4�/7 chromosome ends in asexual and sexual stages

was observed [26]. This association of heterologous

chromosomes occurs among subtelomeric regions,

where recombination events are known to take place

at high rates. It is tempting to speculate that, besides

promoting the generation of diversity, events of chro-

Fig. 3. Frequency distribution of FCR3 var -gene mRNAs. (A) Filters were probed with RT-PCR products. Bars represent the percentage positive

detections of each var transcript in ten single ring-stage parasites. (B) Cumulative hybridization score for each var gene. Scale 0�/5� (see Table 1).

(C) Hybridization of filters with amplification products from five single PRBC, omitting DNase and RTase steps.

V. Fernandez et al. / Molecular & Biochemical Parasitology 121 (2002) 195�/203 201

mosome association and chromatin reconfiguration may

determine the set of active var loci in one cell cycle.

Control of var expression involves gene silencing by anepigenetic mechanism [13,27]. This silencing is estab-

lished during the S phase of the cell cycle and requires

cooperation between two control elements situated in

the 5?-flanking region and the intron, respectively [28].

From these findings it is implied that some change in

chromatin state might take place, for example through

reversible histone acetylation [29]. Histone acetylation/

deacetylation has been described in P. falciparum bloodstages [30]. It remains to be seen if the multiple

transcripts in ring stages reflect trailing events of the

cyclic reinstatement of silence in all var except one, or

whether they are the product of a transient overcoming

of a transcriptionally silent state already in place.

The available experimental evidence suggest that

regulation of var-gene transcription and allelic exclusion

of PfEMP1 in P. falciparum present unique features notfound in other parasite models of antigenic variation.

Research into this organism might uncover new princi-

ples of gene regulation.

Acknowledgements

This work was supported by grants from the Swedish

Research Council, the Swedish Society for Medical

Research and the European Union (grants No. IC18-

CT98-0362-DG12-CEOR, QLRT-2000-01302).

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Fig. 4. Chromosomal mapping of var genes of FCR3 parasites. Chromosomes were resolved and blots were probed as described in Section 2.

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