Figure 3

Candidate motifs identified upstream of the micronemal protein-encoding genes, upstream location, site-directed mutagenesis and results of reporter assays. Motifs MICA and MICB display an additive effect in the regulation of the gene encoding microneme 8. (a) Sequence logos represent the consensus sequence for each candidate motif. The y-axis represents information content at each position. (b) Occurrences and positions of the motifs in the promoter region relative to the translational start site of each gene. The gene names are abbreviated as shown in Table 1. The underlined gene name indicates the representative promoter used in reporter assays. Motif MICA, found in both E. tenella and T. gondii, is denoted by a circle and motif MICB, exclusive to T. gondii, is denoted by a square. (c) The WT motifs and their mutagenized (MUT) versions in the representative promoter are represented. (d) The graphs depict luciferase activity as ratios of firefly:renilla activity in relative luciferase units (RLU) from the different constructs containing either WT or mutagenized versions of MICA, MICB, or both motifs. All luciferase readings are relative to an internal control (α-tubulin-renilla). Error bars represent standard error calculated across the means of three independent electroporations. p-values describe the probability that the difference in expression between the WT and mutagenized promoters may be due to chance.