Fig. 1. Generation of P.berghei parasites lacking either or both P25 or P28 proteins and analysis of expression of these proteins in ko parasites. (A) Schematic diagram of the p25-p28 locus and the replacement vectors used to disrupt p25 (25Sko), p28 (28Sko) and both genes (28/25Dko). All vectors contain the TgDHFR/TS selection cassette (grey arrow) flanked by p25/p28 sequences. Correct integration of the vectors results in the disruption of the genes as shown. In the wt locus, the ORFs are separated by a 1.4 kb intergenic region. The transcription start sites (bent arrows) of both genes are indicated in addition to the direction of transcription (arrow from Margos et al., 1998). In the disrupted loci, the sizes of appropriate restriction fragments used for Southern analysis (see B) are shown. For Southern analysis of the correct integration events, three probes (hatched lines) were used: probe A, recognizing the ORF of p28; probe B, hybridizing to the TgDHFR/TS gene; and probe C, hybridizing to the 3′UTR of the PbDHFR/TS gene, which flanks the TgDHFR/TS gene in the selection cassette. H, HindIII; C, ClaI; P, PstI; B, BclI; Bs, BsmI; S, SmaI; H2, HincII; E, EcoRI; N, NdeI. (B) Southern analysis of digested genomic DNA of wt and ko parasites demonstrates correct integration of the replacement vectors in the different ko parasites. The size of the restriction fragments of the disrupted genes and the probes that recognize these fragments are shown in (A). Probe C recognizes both the integrated vector and the endogenous DHFR/TS of P.berghei (the 14 kb H3/NdeI fragment and the 1.4 kb BclI fragment). (C) RNA blot analysis of ko and wt parasites. RNA was isolated from purified gametocytes and hybridized with probes against the ORFs of p28 (probe A) and p25 (probe D). After disruption of p28, no transcription of p28 was detected. After disruption of the p25 gene, aberrant sized p25 mRNA species are produced that are slightly larger and smaller than the mRNA (1.4 kb) of the undisrupted gene. (D) Protein blot analysis of expression of P25 and P28 in mature ookinetes. Parasites were reacted with polyclonal anti-ookinete immune serum (lanes 2–5), which recognizes both P25 and P28, and with a Mab (13.1) (lane 1) against P28. Lane 5 is overexposed to demonstrate the complete absence of both proteins in the Dko parasites. (E) Expression of P25 and P28 in mature ookinetes, as shown by immune fluorescence analysis of acetone-fixed parasites. Parasites were labelled with Mab 13.1 to antigen P28 (M28), a monospecific anti-Pbs25 polyclonal antiserum (M25) (Rodriguez et al., 2000) and a polyclonal anti-ookinete immune serum (P25/28).