Figure 3

Promoter activity of different fragments from the 5'-flanking region of the EhrabB gene. (A) Schematic representation of the EhrabB promoter region and its putative cis-acting consensus sequences (boxes). Arrows indicate the transcription initiation sites. Arrow with sense to left show the transcription initiation site and transcription sense of the Ehcp112 gene. (B) left, schematic representation of the relevant features of the plasmids used for promoter activity assays. EhrabB, 5'-flanking fragments of EhrabB, containing different 5'-ends (from -683 to -39 upstream of the first initiation transcription site) and 96 nt downstream from the first initiation transcription site (+96). CAT, chloramphenicol acetyl transferase reporter gene. 3'ACT, 3'-flanking region of the actin gene. Act, 480 bp of the 5'-flanking region of actin gene. P, PstI. H, HindIII. B, BamHI. X, XhoI. E, EcoRI. K, KpnI. Arrows indicate the transcription initiation sites. Right, CAT activities obtained from trophozoites transfected with constructions at left. Activities are relative to that obtained with the actin promoter gene, used as positive control. Each bar corresponds to the average of CAT activities ± S.D. representative of three independent experiments performed by duplicate. (C) Strand-specific RT-PCR of cat reporter gene. cDNA of trophozoites transfected with pRab683 (lanes 1–3) or with pRab428 (lanes 4–6) was synthesized using sense (lanes 2, 5) or antisense (lanes 3, 6) primers of the cat gene; then, PCR assays were performed and analyzed in agarose gels. Lanes 1 and 4, PCRs using cDNA synthesized with an oligo(dT) primer.