Control of ferredoxin and Gal/GalNAc lectin gene expression in Entamoeba histolytica by a cis-acting DNA sequence

Abstract

The ferredoxin (fdx) and lectin (hgl5) promoters of Entamoeba histolytica contain the DNA sequence motif TATTCTATT (URE3). Previously we showed that mutation of the URE3 motif in the hgl5 lectin promoter results in an increase in promoter reporter activity. Mutation of this motif in the fdx promoter led to a 40-to-50% decrease in fdx promoter activity as measured by reporter gene activity and abundance of mRNA. E. histolytica nuclear proteins exhibited sequence-specific binding to the URE3 motif in electrophoretic mobility shift assays. These results support the regulation of both ferredoxin and lectin promoters by a nuclear protein(s) which recognizes the URE3 motif.

FIG. 1

Mutation of URE3 motif decreases fdx promoter activity. (A) Luciferase reporter gene activity in amebae transfected with wild-type or URE3-mutated fdx promoters in growth-arrested and exponentially growing trophozoites (x̄ ± standard error, n = 3 where n is the number of independent experiments, each of which was done in triplicate). (B) luc and neo mRNA in exponentially growing amebae transfected with wild-type or URE3-mutated fdx promoters. (The image was generated with the PhosphorImager [Molecular Dynamics model 425] in conjunction with the Adobe Photoshop 3 software program.)

FIG. 2

Electrophoretic mobility shift analysis of the URE3 motif in fdx and hgl5. Electrophoretic mobility shift assays were performed with Klenow-radiolabeled fdx URE3 (A) and hgl5 URE3 (B) double-stranded DNA. Increasing concentrations (5×, 10×, and 20× [A] or 5×, 25×, and 50× [B]) of unlabeled fdx URE3, hgl5 URE3, hgl5 MUT, or an unrelated oligonucleotide (Olig-1) were added as competitors to the binding reactions as indicated. See Table 1 for sequences of the oligonucleotides. (The image was generated with the PhosphorImager [Molecular Dynamics model 425] in conjunction with the Adobe Photoshop 3 software program.)