Fig. 3. Expression and localisation of GRASP2 (A) Schematic representation of the transfection vector pARL-GRASP1^*/2-GFP. The complete genomic grasp sequence (exon 1: red; alternative start ATG and hydrophobic stretch: orange; exon 2: grey) was cloned into the pARL transfection vector in frame with GFP (green). A mutation was introduced into the 5' splice site (^*). The human Dhfr selection cassette is displayed in black. Relative positions of oligonucleotides used in RT-PCR are presented as bars. (B) Transcriptional analysis using appropriate oligonucleotides and either cDNA or genomic DNA (gDNA) derived from pARL-GRASP1^*/2-GFP-expressing parasites. RT-PCR reveals a single PCR product using cDNA (lane 1) but does not amplify a product on gDNA (lane 2). The absence of any contamination of the cDNA preparation with gDNA was confirmed using erd2-specific oligonucleotides (lane 4-5). (C) Disruption of the functional 5' splice site (GRASP1^*/2-GFP) leads to translation of a grasp transgene encoding GRASP2-GFP. Expression of the GFP fusion protein was confirmed with either anti-GRASP or anti-GFP specific antibodies. (D) In the double transgenic parasite line GRASP2-GFP/GRASP1-HA simultaneous expression of the fusion proteins was confirmed with either GFP- or HA-specific antibodies. (E) Localisation of GRASP2-GFP in unfixed parasites. Free merozoites display GFP fluorescence in one tightly defined compartment (a). A duplication of this compartment takes place in ring-stage parasites prior to nuclear division (b). As the parasite matures from trophozoite (c) to schizont (d) the organelle further multiplies until one compartment can be equally distributed among the forming progeny. (F) Colocalisation of the two GRASP proteins in the double transgenic parasite line. Immunofluorescence assay of GRASP2-GFP (green) with HA-specific antibodies representing GRASP1 (red). The merged image shows colocalisation of the two compartments. All images show the nucleus in blue (DAPI).