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Figure 1 Schematic diagram of a single-site crossover at the endogenous pppk-dhps gene with a transfection plasmid construct containing truncated pppk-dhps. The filled triangles represent introns. The arrows within the pppk-dhps gene represent positions where mutations have been introduced into the various allelic exchange constructs. Mutations have been found in codons 436, 437, 540, 581 and 613 (see Table I). The arrows labelled #1–#7 correspond to the position of the oligonucleotides used in this study. The diagram of the genomic DNA following integration assumes that crossing-over occurred upstream of the first mutation (position 436).
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 | Figure 2 Integration of truncated dhps alleles occurs at chromosome 8. Chromosomes of D10 and cloned parasites following transfection of D10 with the six dhps alleles were separated by PFGE and stained with ethidium bromide. Chromosomal DNA was then blotted to a nylon membrane and first probed with a 391 bp PCR-amplified dhps fragment then, after removal of the signal, reprobed with a 210 bp PCR-amplified T.gondii dhfr-ts probe.
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Figure 3 Restriction analysis of genomic DNA showing allelic replacement of the endogenous D10 dhps gene. DNA from D10 and the six integrated clones following allelic replacement were digested with EcoRI–ClaI (A) and AvaII (B) and the fragments separated electrophoretically before blotting to nylon membranes. Both blots were probed with the 391 bp dhps fragment represented by the solid bar in (C). (C) The structure of the integration events into the D10 dhps gene is shown, together with the position of the restriction enzyme sites EcoRI (R)–ClaI (C) and AvaII (Av) that were used for the Southern hybridization experiments shown in (A) and (B). The scale is shown at the bottom of the panel.  Av indicates the AvaII site which is generated when D10 is transfected with all constructs encoding G437 (see Table I).
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 | Figure 4 AvaII digestion of RT–PCR products derived from poly(A)+ RNA from the parasite clones containing the six integrated mutant dhps alleles. The left panel shows RT–PCR products from each clone digested with AvaII. The oligonucleotides used for amplification were located 364 bp 5' of the AvaII site in dhps [5'-TCCATTCCTCAT-GTGTATACAAC-3' (#7)] and within the hsp86 3' sequence (#2; see Materials and methods). The right panel is included as a control and shows that cDNA derived from the D10-ageaa integrated clone is free of contaminating genomic DNA (all samples were tested but only D10-ageaa is shown). D10 genomic DNA and D10-ageaa cDNA were amplified with oligonucleotides derived from a different gene involved in folate biosynthesis (pabAB). The oligonucleotides amplify a 545 bp fragment from genomic DNA and a fragment of 259 bp from cDNA because of the presence of two introns. The oligonucleotides used were 5'-AATTGAAATAGTGCCTAGGG-3' and 5'-AAAATATATATTCCACGTCAGTGT-3'.
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Figure 5 The PPPK-dhps enzyme is expressed in clones containing the integrated mutant dhps alleles. Proteins from synchronized trophozoites were separated by SDS–PAGE and blotted to nitrocellulose. The upper filter was probed with anti-dhps antibodies and the lower filter with antibodies to the P.falciparum hsp70 protein.
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