Fig. 6.

Strategy for modification of the MSP1 secondary processing site in P. falciparum by single cross-over homologous recombination.A. Integration construct (pMSP1chimWT or pMSP1chimSUB2to1) used for targeted homologous recombination. The plasmid contains 1036 bp of targeting sequence (dark grey) derived from the 3D7 msp1 gene, fused in frame to synthetic recodonized sequence (sMSP1-19+) encoding the C-terminal 119 residues of the Wellcome-type MSP1, including its GPI anchor sequence. The position of the PfSUB2 cleavage (the secondary processing site) is arrowed. In pMSP1chimSUB2to1 this site is modified to encode a PfSUB1-sensitive sequence, but otherwise the two constructs are identical. Because the recodonized sequence shares low identity with the authentic msp1 sequence, cross-over (dotted lines) is expected to occur only within the targeting sequence. Pbdt 3′ untranslated region (UTR), UTR of the Plasmodium berghei dihydrofolate reductase gene to ensure correct transcription termination and polyadenylation of the modified gene. hdhfr-ts, human dihydrofolate reductase–thymidylate synthase cassette, providing resistance to the antifolate drug WR99210.B. The 3D7 P. falciparum genomic msp1 locus.C. Expected result of integration, which displaces the 3′ end of the msp1 gene downstream, and replaces it with a chimera of the native sequence and the recodonized Wellcome-type sequence. As the integration plasmid contains only a partial msp1 open reading frame, not preceded by a promoter, the only copy to be transcribed is the modified chimeric msp1 gene directly downstream of the endogenous promoter. The positions of epitopes recognized by mAbs X509 and 111.4 are indicated. Restriction sites used for Southern blot analysis (see Fig. 8), the predicted sizes of restriction products, and the position of hybridization of the probe used for Southern analysis are shown.