Figure 6

Role of URE1 in the transcriptional activity of EhrabB. (A) Schematic representation of the EhrabB promoter region analyzed and its putative cis-acting consensus sequences (boxes). (B) Left, schematic representation of the relevant features of the plasmids used for promoter activity assays. EhrabB, 5'-flanking fragments of EhrabB, containing -428 (pRab428), -415 (pRab415) (without the URE1-like sequence), and -369 (pRab369) nt from the first transcription initiation site.Right, CAT activities obtained from trophozoites transfected with constructions showed at left. Activities are relative to that obtained with 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) EMSA using the ³²P-labeled sequences URE1 from EhrabB (URE1 EhrabB) or from hgl5 (URE hgl5) gene promoters as probes and 30 μg of trophozoite nuclear extracts (NE). Lanes 1–5, EMSA using the URE1 sequence from EhrabB as a probe. Lane 1, free probe. Lane 2, no competitor. Lane 3, specific competitor (SC) (150-fold excess of the same cold fragment). Lane 4, URE1 from the hgl5 gene promoter used as competitor (URE1^C) (150-fold excess). Lane 5, unspecific competitor (UC) (150-fold excess of a mingled oligonucleotide with similar composition). Lanes 6–8, EMSA using the URE1 sequence from hgl5 as a probe. Lane 6, no competitor. Lane 7, unspecific competitor (UC) (150-fold excess of a mingled oligonucleotide with similar composition). Lane 8, free probe. Arrowheads indicate the two DNA-protein complexes formed by probe-nuclear extracts interaction.