Microarray analysis of strain-specific differences at the toxofilin locus. (A) Microarray data from the 14 toxofilin probes from the bradyzoite microarray derived from a type II strain. Parasite cDNA was labeled with Cy5 (red) and cohybridized with a Cy3-labeled common reference. Data are shown from triplicate arrays for the type II and type III parents, and single arrays were done for each of the 18 F₁ progeny. LOD scores as determined by R/QTL software are shown for each probe. (B) Structure of the toxofilin locus. The putative promoter and predicted N-terminal region of the protein are more conserved between type II and type III than the predicted C terminus and putative 3′ UTR. The locations of the 5′ ends of the cDNAs on the microarray are shown below the locus, sorted by the start position on the genome. The percentage of identity between the sequenced portion of the probe and the type III toxofilin sequence, along with the LOD score for each probe, is also shown, demonstrating the inverse correlation between the percentage of identity and the LOD score.

Analysis of strain-specific differences in 46.m01601 promoter activity. (A) Structure of the 46.m01601 locus, showing the upstream 1,004 bp used as the putative promoter and the 17 polymorphisms that distinguish the type II and type III sequences. Transcription starts at about position −441 relative to the start codon, based on alignment of EST sequences to the locus. The truncated version of the promoter used in luciferase assays is also shown. SP, signal peptide sequence. (B) Effects of truncation of the type II and type III promoters, engineering of chimeric promoter constructs, and effect of mutation of multiple SNPs in the type III promoter on luciferase production. All data were normalized to the level with the type II promoter. Data are shown for the type II and type III wild-type promoters, the type II and type III truncated promoters (type II and type III, −480 to −1), the chimeric promoter constructs [e.g., type II (−1004 to −481)/type III (−480 to −1)], and the type III promoter mutated to the type II sequence at either SNP1, SNPs 2, 3, and 4, or SNPs 6 and 7 [e.g., type III (−1004 to −1; type II SNP 1)]. Asterisks indicate significant differences from the results with the full type III promoter by one-way analysis of variance and Tukey's multiple-comparison posttest. **, P < 0.01; ***, P < 0.001. (C) Versions of the 46.m01601 promoter were constructed where SNP5 was mutated in the type II promoter to the type III nucleotide [type II (−1004 to −1; type III at SNP5)] or in the type III promoter to the type II nucleotide [type III (−1004 to −1; type II at SNP5) and fused to firefly luciferase. Luciferase production for each mutated construct was compared to that for its wild-type counterpart, and data are represented as ratios of the firefly luciferase signal to the Renilla luciferase signal. Means with the same letter are not significantly different (P > 0.05). SEM, standard error of the mean.

Genomic locations of two cis-mapping transcripts corresponding to gene models 57.m03117 and 57.m00004. (A) Genome browser view (www.toxodb.org) encompassing bp 999000 to 1012000 on chromosome IX. The positions of the three cDNA probes with hybridization intensities that mapped to this locus are shown (probes 1 to 3). (B to D) Log₂ ratios of probes 1 to 3, respectively, in the parental lines used in this study, showing the higher hybridization of type III strains to probe 1 and the higher hybridization intensity of type II strains with probe 3.