Molecular and Biochemical Parasitology, 47 ( 1991) 197 206 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100194X

197

MOLBIO 01563

7H8/6, a multicopy DNA probe for distinguishing isolates of

Plasmodiumfalciparum T e m d u a n g L i m p a i b o o n l*, Martin W. Shirley 2.* D a v i d J. K e m p 2 and Allan Saul ~ ~Queensland Institute of Medical Research, Bramston Terrace, Herston, Queensland, Australia; and 2Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia (Received 10 December 1990; accepted 8 March 1991 )

A method using DNA fingerprinting to distinguish Plasmodiumfalciparum isolates has been developed using a clone, 7H8/6, isolated by screening a genomic expression library of the Malayan Camp strain with a monoclonal antibody. Unlike other P.falciparum DNA probes that contain internally repetitive sequences, this probe contains a single full-length open reading frame and lacks any repetitive sequences. Chromosome mapping located the hybridizing sequences to most chromosomes. 7H8/6 sequences are located at sites near the subtelomeric regions, although they are further from the telomeres than the subtelomeric repetitive sequences rep20. The probe gave multiple polymorphic bands on Southern blots of genomic DNA allowing all unrelated isolates tested to be readily distinguished. Hybridization patterns of parent and progeny from the genetic cross of two P. falciparum clones showed that most bands were inherited independently and no new bands were generated during the cross. This probe is useful for distinguishing isolates of P.falciparum and also for analyzing relationships between closely related isolates. Key words: Plasmodiumfalciparum; DNA fingerprinting; Polymorphic probe; Chromosome mapping

Introduction

Plasmodium falciparum shows extensive variation between isolates. Methods used to study isolate variation include isoenzyme electrophoresis [ 1,2], two dimensional gel electrophoresis of parasite proteins [3], drug resistance [4] and the reaction of parasite antigens with panels of monoclonal antibodies (mAbs) [5,6]. A few studies of isolate characterization by hybridization have been reCorrespondence address: T. Limpaiboon, c/o A. Saul, Queensland Institute of Medical Research, Bramston Terrace, Herston, Qld., Australia 4006. Present addresses: *Dept. Clinical Chemistry, Faculty of Associated Medical Science, Khon Kaen University, Khon Kaen, 40002, Thailand; and "*Houghton Laboratory Institute for Animal Health, Houghton, Huntingdon, Cambs., PE 17 2DA, U.K. Abbreviations: PFGE, pulsed-field gradient electrophoresis; CHEF, contour-clamped homogeneous electric field; RESA, ring-infected erythrocyte surface antigen; GARP, glutamic acid rich-protein; Tel, telomere.

ported [7-9]. In most of these studies, the DNA probes used were derived from the subtelomeric repeat of P. falciparum [9,10]. The region of the chromosomes carrying this sequence is not stable, with deletions occurring during long-term culture as well as in nature, sometimes resulting in loss of functional genes ofP.falciparum [ 11-13]. Extensive rearrangements have been observed in the progeny of a genetic cross [ 14]. Recently, we isolated a clone, 7H8/6 [15] (GenBank Accession No. M63817), from a genomic expression library using the mAb 7H8 [ 16]. While this clone had a single large open reading frame containing no internally repetitive elements, with a codon usage characteristic of P.falciparum coding regions, it gave a complex Southern blotting pattern. In this paper, we describe the use of a probe based on this clone for distinguishing P. falciparum isolates and cloned lines. Investigation of its chromosomal arrangement revealed that it is located further from the telomere than rep20. In the analysis of the

198

progeny of a cross between HB3 and 3D7 [14] no evidence of rearrangement was found suggesting that this probe may be useful for following changes in field populations of parasites. Materials and Methods

Parasites. P. falciparum isolates used in this study were the D10 clone derived from isolate FCQ-27/PNG, Malayan Camp, Palo Alto, AE7, T9/94, FCQ-2/PNG, FCQ-49/PNG, clone HB3 derived from isolate H1 and clone 3D7 derived from isolate NF54. Cloned progeny X5, XI0, X 11, XP8, XP9, XP12 and XP13 were derived from a cross of HB3 and 3D7 [ 14]. Cultures of asexual erythrocytic forms of P.falciparum were grown in flasks using a modification of the method of Trager and Jensen (1976) [17]. The cultures were maintained in human erythrocytes, type O, at 5% hematocrit in RPMI-1640 medium containing 10% human serum, in an atmosphere of 5% COz, 5% 02 and 90% N2. Parasite DNA extraction. Parasites were freed from host erythrocytes by treating with 0.15% saponin, pelleted and washed in PBS. The pellet was resuspended and left overnight at 37°C in lysis buffer containing 0.01 M Tris-C1/0.01 M EDTA/ 0.01 M NaCI/2% SDS, pH 8.0, with 100/ag ml t of proteinase K. The DNA was extracted with phenol/ chloroform and treated with DNase-free RNase ( 100/ag ml -~) at 37°C for 2 h. The DNA was then reextracted with phenol/chloroform and ethanol precipitated. DNA was dissolved in 10 mM Tris-C1/l mM EDTA, pH 8.0 (TE), and dialysed against TE buffer with several changes at room temperature. Southern blot analysis. Analyses were performed on DNA cut with restriction enzymes [ 18], and on chromosomal DNA separated by pulsed field gradient electrophoresis. DNA (2/ag) was digested with the appropriate restriction enzymes and separated on 0.8% (w/v) or 0.5% (w/v) agarose gels. The electrophoresed DNA was transferred to nylon membranes (Hybond-N, Amersham, U.K.) [ 18]. Hybridization was carried out in a buffer containing 0.9 M NaCI/0.09 M trisodium citrate (6 x SSC)/5 x Denhardt's solution]2 mM phosphate buffer, pH 6.5/0.1% SDS/5 mM EDTA/sonicated

salmon sperm DNA (100/ag ml ~) and the insert from clone 7H8/6 amplified and 3'-P-labeled by the polymerase chain reaction [19,20] (50 ng 7H8/6 insert/20 ng m 13 forward and reverse primers/25 /aM dATP/25/aM dGTP/25/aM dTTP/100/aCi [oc32p]dCTP/2.5 units Taq polymerase/10 cycles of 1 min each at 94°C, 55°C and 72°C). Washing was performed in 2 x SSC, l x SSC and 0.1 x SSC, respectively, in the presence of 0.1% SDS at 65°C. The blots were analyzed by autoradiography.

Pulsed field gradient electrophoresis of chromosomes. Agarose blocks of parasites were prepared and the embedded parasites lysed as described [21 ]. Pulsed field gradient gel electrophoresis (PFGE) was performed in a CHEF-DRII apparatus (BioRad Laboratories, Richmond, CA, U.S.A.) [22]. Electrophoresis was performed on a 1% agarose gel with pulse time of 150-270 s (ramping) at 100 V for 24 h, then with 270 s at 100 V for 20 h and followed by 999 s at 60 V for 52 h. Chromosome mapping. Two-dimensional PFGE was performed as described by Corcoran et al. [ 11 ]. Purified chromosome l, 2 and 9 were separated as described previously [11,23]. Chromosomes were digested with restriction enzymes and digested fragments were usually fractionated by PFGE with 90 s pulses at 160 V for 16-24 h. The AccI digested fragments of chromosome 1 and 2 were separated by PFGE with 3-s pulses at 160 V for 8 h. Analysis of genetic cross. Bands present in the HB3 and 3D7 parents were numbered consecutively from the largest band. The bands observed in each progeny were then numbered corresponding to the parental band numbers. Exhaustive pairwise comparisons were then made of the bands present in each of the progeny and the parents to find those bands which independently segregated. Results

Hybridization patterns in unrelated isolates. Unrelated isolates of P.falciparum were studied by comparison of polymorphic patterns on Southern blots. The hybridization pattern from six different isolates and cloned lines, D10, Malayan Camp, AE7, T9/94, FCQ-2/PNG and FCQ-49/PNG, di-

199

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Fig. 1. Hybridization of probes to malarial genomic DNA. DNA from D10 ( 1), Malayan Camp (2), AE7 (3), T9/94 (4), FCQ-2/PNG (5) and FCQ-49/PNG (6) clones and isolates of P.falciparum were digested with Accl, HinfI and Alul and hybridized to 7H8/6 (A). Hybridization patterns ofAccI-restricted DNA from Malayan Camp and Palo Alto probed with 7H8/6 and with rep20 (B). Electrophoresis was performed on 0.8% agarose gel. Blot was hybridized at 55°C and washed at 65°C.

gested with restriction enzymes (AccI, HinfI and AluI) and probed with 7H8/6 is shown in Fig. 1A. There was no hybridization of 7H8/6 to P. vivax or human DNA digested with Hinfl. The hybridization patterns of the different P.falciparum isolates are readily distinguished. At least 20 different fragments were detected on Southern blots by this DNA probe. Since AccI gave a convenient distribution of bands which readily distinguished different isolates it has been used routinely. From 40 lines examined so far (data not shown), all unrelated lines were readily distinguishable with the exception of Malayan Camp and Palo Alto (Fig. 1B). These differ in only 2 bands. The same blot probed with rep20, showed that Malayan Camp and Palo Alto gave related banding patterns but many bands differed (Fig. 1B).

Hybridization patterns in related clones.

The relationship between the banding patterns of related clones is shown in Fig. 2. The blot of AccI-restricted DNA fragments from parents and progeny of the cross separated on 0.5% agarose was probed with 7H8/6 (Fig. 2A). The band of approximately 400 bp separated on 0.8% agarose hybridized to 7H8/6 is shown in Fig. 2B. All of the bands in the parent clones were found in at least one of the progeny clones and no new bands were produced during the cross-fertilization. An exhaustive pairwise analysis of the presence or absence of each parental band in the pattern of each cloned offspring showed that of 38 distinguishable bands in the two parents, 23 bands showed independent segregation. The remaining 15 bands fell into six apparently linked groups. However as only seven clones were available for analysis, many of these apparent linkage groups could have arisen by chance and so most, if

200

23.1

hybridization varied considerably. The exceptions were chromosome 5 of 3D7 and X5 and chromosome 12 and 13 of D10 which lacked detectable 7H8/6 sequences. Although we cannot rule out the possibility that there is a very divergent 7H8/6 copy on these chromosomes, this result suggests that 7H8/6 is not a necessary component of each chromosome such as a centromere.

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B Fig. 2. Hybridization patterns of AccI restricted DNA from cloned parents and progeny of a genetic cross probed with 7H8/6. Electrophoresis on 0.5% (A) and the approx. 400 bp band on 0.8% agarose gel (B).

not all of these bands show independent segregation in a genetic cross.

Genomic

distribution

of

7H8/6

sequences.

Chromosomes from P. falciparum clones D10, HB3, 3D7, X5 and XP8 were separated by PFGE, blotted and hybridized with a 7H8/6 probe. Most chromosomes hybridized (Fig. 3) but the extent of

Localization of 7H8/6 sequences on P. falciparum chromosomes 1,2 and 9. Chromosome 1. Our mapping studies demonstrated that there are copies of 7H8/6 located near the subtelomeric regions at both ends of chromosome 1 in the D 10 and E 12 clones of FCQ-27/PNG. As can be seen in Fig. 4, 7H8/6 hybridized to more than one fragment in ApaI and BglI digests of chromosome 1 from these clones. In order to identify these fragments, the 7H8/6 probe was stripped from the filter and probes for RESA, GARP (data not shown), Tel and rep20 were successively hybridized to the filter. The maps derived from this data and from data in Corcoran et al. [11] are shown in Fig. 5. In D10 the ApaI fragments bearing both telomeres of chromosome 1 comigrate at about 150 kb. E 12 has 2 telomeric ApaI fragments, one of about 150 kb and the other of about 12-15 kb. In both D 10 and E 12 the RESA gene is located on the 150-kb telomeric ApaI fragment at the left end of chromosome 1 (Fig. 5 and ref. 11 ). 7H8/6 hybridized to this telomeric ApaI fragment in both D10 and E12, and also to a larger fragment in E 12. The latter fragment hybridized to rep20 (Fig. 5) and so both 7H8/6 sequences on chromosome 1 are located toward the telomeres (Fig. 6). These locations were confirmed in BglI digests. D 10 has 2 telomeric BglI fragments, the smaller of which is shared by E 12 (Fig. 4). However, the larger telomeric BglI fragment of E 12 is shorter than that of D 10. RESA is located on the much larger internal BglI fragment. 7H8/6 is located on the smaller telomeric fragment in both D 10 and E 12, as well as the larger telomeric fragment of D 10. However it is not present on this second telomeric fragment of E 12. Instead it is present on a fragment that is slightly larger and is located internally to the right telomere as it does not hybridize to rep20 (Figs. 4 and 5). Further, in both D10 and E12 the large non-telom-

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Fig. 3. Assignmentof 7H8/6 sequences to P.falciparum chromosome by PFGE. Chromosomes from clones D 10 (D), HB3 (H), 3D7 (3), XP8 (8) and X5 (5) were fractionated by PFGE and are visible on the ethidium bromide-stained gel (left). Chromosomes were hybridized with the 7H8/6 probe (right). Yeast chromosomes (Y) strain YNN295 were used as markers: sizes are shown in kb.

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C

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Fig. 4. Mapping 7H8/6 sites on chromosome 1. Chromosome 1 from D10 (D) and El2 (E) was purified by PFGE and digested with Apal or B,elI.The fragments were fractionated by PFGE at 160 V for 16 h with 90-s pulses, blotted and hybridized with a 7H8/6 probe (A). After autoradiography the probe was stripped and the filter was rehybridized with a Tel probe (B), a RESA probe (C) and then a rep20 probe (D), Sizes are indicated at the left in kb. eric BglI f r a g m e n t is also labeled by 7H8/6. W e cannot tell w h e t h e r it is a third copy or whetherBglI has cut w i t h i n a single 7 H 8 / 6 c o p y at the left or right end. F r o m this data a n d p r e v i o u s m a p p i n g studies we

c o n c l u d e that 7 H 8 / 6 s e q u e n c e s are located at 2 w i d e l y separated sites o n c h r o m o s o m e 1 o f D 1 0 and E l 2 as s h o w n in Fig. 5. In studies on other isolates, we f o u n d that c h r o m o s o m e 1 from F C R 3 o n l y bore 7H8/6 s e q u e n c e s at the right e n d (data not

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