Fig. 1. PfEMP3Δ3 transfection construct and integration events. (A) The plasmid construct, pHC1-EMP3Δ3, contains an insert of the 3′ repeat region of PfEMP3 exon 2 cloned downstream of the calmodulin promoter region in pHC1. The open arrowed box shows the pyrimethamine selection cassette for the generation of stable transfectants.The cross-hatched and grey boxes indicate the calmodulin 5′ and hsp86 3′ terminator sequences, respectively. A schematic representation of the gene encoding PfEMP3 is shown (Gardner et al., 1998). The gene has a two exon structure designated by I and II. The first exon (cross-hatched) encodes a putative signal sequence followed by a region that is non-repetitive (stippled) and two highly repetitive regions consisting of two sets of repeat regions (vertical hatching and solid filled box). The two repeat regions are followed by a non-repetitive unique region (empty box). R and H represent EcoRI and HincII restriction sites used in restriction site mapping. The cross indicates the region where gene disruption has taken place. (B) PFGE showing chromosomes from parental 3D7k⁺ and the transfected cloned lines EMPΔ1, EMPΔ2 and EMPΔ3. Identical filters were hybridized with pGem and KAHRP. (C) EcoRI genomic digests and Southern analysis of 3D7k⁺, EMPΔ1, EMPΔ2 and EMPΔ3 probed with PfEMP3 showing disruption of the endogenous PfEMP3 gene. (D) Map of the integration event for truncation mutants EMPΔ3, EMPΔ2 and EMPΔ1. Numbers refer to the length of designated restriction fragments in kb. All integrants have in common the >9.0 kb endogenous EcoRI band and the 4.2 kb plasmid band. Numbers separated by a backslash represent sizes of restriction fragments for EMPΔ2 and EMPΔ1 versus EMPΔ3, respectively. Square brackets show the plasmid repeat unit arising from multiple copies of the plasmid integrating into the PfEMP3 gene. The number of plasmid copies integrated per clone is shown outside the brackets for EMPΔ3, EMPΔ2 and EMPΔ1, respectively. The PfEMP3 coding region is designated as shown in (A).