Fig. 6
Definition of important cis-acting element(s) in the promoter region of glcwp1, Pglcwp1 using luciferase reporter. a Expression analysis of a series of luciferase reporters with various sizes of Pglcwp1. Pglcwp1s of various sizes (25–150 bp) were cloned to an empty vector, and the resulting plasmids were transfected to wild-type Giardia. Their luciferase activity at 15 h post-induction to encystation was compared. Three independent experiments were performed and analyzed from three experiments. Error bars represent the standard error of the mean. b Site-directed mutagenesis of putative GlMyb2-binding sites in the Pglcwp1-luc reporter. Five putative sequences suggested by the GlMyb2 consensus sequence were mutated in the Pglcwp1-luc reporter. Role of putative GlMyb2 binding sites in Pglcwp1 expression (c, d). c Comparison of luciferase activity of the promoter mutants MT1, MT2, and MT3 with that of their wild-type promoter of 75 bp. d Comparison of luciferase activity of the promoter mutants MT4 and MT5 with that of their wild-type promoter of 100 bp. Cells carrying these plasmids were incubated in an encystation for 6, 15, and 24 h and used for luciferase assays. Luciferase activity was measured in the presence of luciferin using a luminometer (TD-20210, Turner Designs). Specific bioluminescence was calculated by normalizing the relative light units (RLU) with protein concentration. Three independent replicates, each consisting of three technical replicates, were evaluated. Error bars represent the standard error of the mean