RESUMO
Transient transformation of Toxoplasma using the CAT (chloramphenicol acetyl transferase) reporter gene has been used to map promoter elements of four genes encoding dense granule proteins (GRA1, GRA2, GRA5 and GRA6). Intense CAT activities (GRA1 > GRA5 > GRA2 > GRA6) are detected for constructs containing 379bp, 276bp, 209bp and 265bp upstream of the transcription start site of the GRA1, GRA2, GRA5 and GRA6 genes, respectively. Deletion analysis shows that optimal promoter activity of each gene is contained in the proximal region of the transcription start site: -129 to -47 for GRA1, -87 to -37 for GRA2, -156 to -30 for GRA5 and -146 to -27 for GRA6. Quantitative CAT assay and mutation analysis show that repeated motifs (A/TGAGACG) found in either orientation with respect to transcription are critical elements of these defined promoter regions. We have found such sequence elements in the upstream region of other Toxoplasma genes such as Tub1 and within the stretch of 27bp repeats of the SAG1 promoter.
