Figure 8

Live imaging of FcB1 parasites transfected with a construct allowing expression of PfA-M1-GFP chimera confirms its marginal association with the FV. A. Schematic representation of the integration of the PfA-M1-GFP plasmid at the PfA-M1 locus in the P. falciparum genome. The genomic environment of the PfA-M1 gene (MAL13P1.56) in wild type parasites (wt) is draw to scale with indication of the two flanking Hind III sites (H). Integration of the PfA-M1-GFP plasmid creates a fused gene and a truncated gene in transfected parasites (51). The red bar corresponds to the 1024-bp fragment encoding the C-terminal part of PfA-M1 that was cloned in the plasmid and subsequently used as probe on Southern blots. B. Nuclear DNA from wild type (wt) or transfected parasites (51) was digested by using Hind III, separated on 0.7% agarose gels in 1×TBE, transferred to nylon and hybridized with the [³²P]-labelled 1024-bp fragment as previously described [6]. The observed pattern indicates, as expected, a wild-type Hind III fragment (Wt, ~8-kb) that is replaced, in transfected parasites (51), by a 5'-fused gene (~6.8-kb), a 3'-truncated gene (~6.2-kb) and a plasmid (~5-kb) Hind III bands. Note that this pattern is similar to that obtained by Dalal and Klemba [26]. C. Soluble proteins from transfected parasites (51) were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-GFP (lane 1) or anti-MAP1 [6](lane 3) antibodies as described in the Methods procedures section, revealing the recombinant PfA-M1-GFP protein, at ~150-kDa and some free GFP. Mw markers in kDa are in lane 2. Mw markers are as described on Figure 4. D to I: live imaging of transfected parasites showing: D, E. ring stages. F, G, trophozoite stages; H. young schizont. I. segmenting schizont. First column: GFP (green) and nuclei (Hoechst 33342 staining, 4 μg.ml^-1); second column: phase contrast; third column: overlay. Scale bar, 5 μm.