Figure 3

Single nucleotide polymorphisms and differential polyA+-choices are reflected in SAGE tag datasets. [A] Screening for reciprocal frequency patterns of nearest neighbor SAGE tags identifies potential strain-specific polymorphisms within the SAGE library datasets. Out of more than 400 tags containing SNPs, a selected set of five SNP-containing SAGE tags that represent all known biallelic patterns are shown [31] (I, III versus II; I, II versus III; II, III versus I). SAGE tag frequencies from all nine libraries are listed; VEG post-sporozoite SAGE libraries; Sp = sporozoite, D4 = Day-4 post-sporozoite infection, D6 = Day-6, D7 = Day-7, D15 = Day-15, pH = alkaline shifted parasites. [B] RFLP analysis was performed to verify strain-specific polymorphisms in two selected SAGE tags. Primers were designed from sequence flanking each SAGE-tag position within Toxoplasma genomic sequence. PCR fragments were amplified from genomic DNA from each of the three genotypic strains, purified, and digested with the appropriate restriction enzyme. Molecular mass standards are displayed. The sequence for Type II SAGE tag CATGCTCCGCTACA is found on chromosome V-1,674,592 and contains a mutation in the Aci I binding site. The Type II SAGE tag CATGACATCGAGGA is located on chromosome X-2,748,371 and within the coding region for the Toxoplasma homolog of SUL1 (AAF76883.1). A single nucleotide change in Type I and III SAGE tag occurs within the binding site for restriction endonuclease Mnl I. Note that in each case shown the sequence variation is biallelic; I, III versus II. [C] SAGE tag clusters indicate strain-specific differences in polyA+-addition. A 3'RACE reaction was performed on Type I (RH), Type II (ME49B7) and III strain (VEG) total RNA using a GRA7-specific primer in conjunction with a modified oligo-d(T) primer. The distance of the Nla-III site (anchor site for each SAGE tag) from the GRA7-specific primer was correlated to the 3'-RACE products and the 3'-RACE products were verified by cloning and sequencing. The corresponding tag frequencies for the top two 3'RACE products are indicated below the band in each strain. Four tags with similar frequency were obtained from the Type II library that correlate with Type II 3'-RACE products; 3'-RACE 951 bp product = tag with 842 frequency, 600 bp = 609 tags, 520 bp = 136 tags, 409 bp = 401 tags. The 951 bp and 600 bp 3'-RACE products were dominant in RH and VEG strains.