POLYMORPHISM IN A PLASMODIUM FALCIPARUM MEROZOITE SURFACE PROTEIN GENE IN SRI LANKA DEMONSTRATED BY SOUTHERN HYBRIDISATION

The gene for a 45kDa merozoite surface protein of Plasmodrum falc~parum (GYMSSA) was amplified by the polymerase chain reaction in fifteen blood samples collected from patients in Dambulla, Galewela, Kurunegala and Polgahawela hospitals The amplified DNA was hybndised in Southern blots to repet~tive reaon-based oligonucleotide probes specific for the two major allelic forms of GYMSSA identified in culture-adapted laboratory llnes ofP falc~parum The FC27 allelic form of GYMSSA was detected in thirteen blood samples and the 3D7 allelic form in the remaining two samples


INTRODUCTION
The 185-200 kDa precursor to the major merozoite surface antigen (termed PMMSA, MSA-1, MSP-1) and the 45kDa glycosylated and myristilated smaller surface antigen (termed GYMSSA, MSA-2 or MSP-2) are candidates for malaria vaccine development since antibodies against the molecules inhibit parasite growth and reinvasion in in vitro cu1tures.lTwo major allelic forms of GYMSSA, differing chiefly in a centrally located repetitive sequence, have been identified in in vitro cultured lines and in patient isolates.These are represented by sequences in the K I and 3D7 cultured P. falciparum isolate^.^,^Determining the nature and extent of allelic variation in vaccine candidate molecules is important for vaccine development and may also provide strainspecific markers for epidemiological studies.We report here on the use of repeatregion specific oligonucleotides to examine allele distribution in Southern blots of polymerase chain reaction (PCR) amplified GYMSSA DNA, in fifteen isolates obtained from patients in Dambulla, Galewela, Kurunegala and Polgahawela hospitals located in the central and north-western provinces.

METHODS AND MATERIALS
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Isolation of DNA from infected blood: Fifteen 1 ml samples of microscopically confirmed P, falciparum-infected blood (parasitaemia 0.06-0.90%)were collected in acid citrate dextrose from the Dambulla (3 samples), Galewela (2 samples), * correiPonding author Kurunegala (8 samples) and Polgahawela (2 samples) hospitals.After removal of plasma, the cells were treated with 7.5ml of 6M guanidine hydrochloride in 0.1M sodium acetate (pH 5.5) and stored at -80°C until use.Five hundred pl of lysed blood cells were centrifuged at 100,000xgfor 20min a t 4OC.The supernatant was transferred to microfuge tubes containing 20pl glassmilk (Prep-a-gene, Biorad, USA) and the DNA purified and collected according to the manufacturer's instructions.PCR amplification: This was carried out essentially as described previ~usly.~
Allelespecific oligonucleotide probes: Two oligonucleotide probes described previously were used3: The K 1 repeat-specific probe R9213 (ATCACAAACTACTACTC) and th,e 3D7 repeat-specific probe R9214 (GGTGGTAGTGCTGGTGGTAG)..The oligonucleotides were synthesised a t the La Trobe University, Department of Biochemistry, Australia.10 pmoles of the probes were 5' end labelled with 50pCi of [a-32P] ATP (specific activity 3000 Cilmmole, Amersham, UK) using T4 polynucleotide kinase according to standard procedure^.^Southern hybridisation: Eight pl of PCR-2 amplified product separated on 1.2% agarose gels were denatured with 0.5N NaOH and then transferred to nitrocellulose membranes by standard procedure^.^The membranes were treated with pre-hybridisation solution containing 6 x SSC (standard saline citrate pH7.0), 5 x Denhardts solution (0.5g ficoll, 0.5g polyvinyl pyrollidone and 0.5g bovine serum albumin), 0.5% sodium lauryl sulphate and 50 pglml salmon sperm DNA, for l h at 37OC.Hybridisation was carried out for 16 h a t 37OC by adding 10 pmoles of the [32P] labelled probe to the pre-hybridisation solution.After hybridisation, the membrane was washed twice at 37OC in GxSSC, 0.1% sodium lauryl sulphate and autoradiographed for 16 h.

RESULTS
The PCR amplified GYMSSA DNA gave a single major band of approximately 500bp on agarose gels stained with ethidiwn bromide.In Southern blots of the amplified DNA, R9213 reacted with thirteen of the fifteen samples (Fig. 1).The two R9213 non-reactive samples termed D3 (Dambulla) and P2 (Polgahawela), reacted with probe R9Y4 (Fig. 2).

DISCUSSION
The results show that GMMSSA in P. falciparum isolated from patients in Sri Lanka is represented by at least the two major allelic families (K1 and 3D7) that were previously detected in cultured lines.Since this work was initiated, similar GYMSSA alleles were reported in P, falciparum isolates from patients in C ~l o m b i a , ~ Irian Jaya7 and Papua New G ~i n e a .~ The complete sequence of the GYMSSA gene in two isolates, D2 (Dambulla) and P2 (Polgahawela), have been determined confirming the presence of the two types of allelic families in Sri Lar~ka.~We did not investigate, in our samples, the occurrence of sub-families within the two major allelic types and the limited recombination between alleles that have been r e p ~r t e d .~ The presence of possible mixed infections in some of the blood samples was also not excluded.A larger number of blood samples need to be examined in order to determine the relative prevalences of the K1 and 3D7 allotypes of GYMSSA in Sri Lanka.However our results suggest that a malaria vaccine for use in Sri Lanka should preferably be based on N and C terminal sequences of GYMSSA that are conserved between alleles and that probes specific for the repetitive region of GYMSSA are useful in differentiating P. falciparum strains in Sri Lanka.
DNA: A pair of primers from the N and C terminal conserved regions of GYMSSA in the K l laboratory isolate of P. falciparum were used for PCR amplifi~ation.~The primers for PCR were synthesised with the M13 forward and reverse sequencing primer sequences located at their 5' ends for possible additional use of the primers in sequencing (M13F = TGTAAAACGACGGCCAG; M13R = CAGGAAACAGCTATGACC).The N-terminal primer, termed VM 59111, corresponded to nucleotides 100-120 in K1 and had the.sequence M13F -GCTTATAATATGAGTATAAGG.4The Cterminal primer, termed VM 59112, corresponded to nucleotides 638-657 in K l and had the sequence M13R -CATATGTCCATGTTGTCCTG.4The two primers were kindly provided by Vicky Marshall of the Walter and Eliza Hall Institute for Medical Research, Melbourne.