FIG. 9.

The AAGGAATA elements in the pfs25 promoter recruit a mosquito stage-specific DNA-binding protein that activates transcription. (A) Oligonucleotide TFB25 (AATTCATAAGGAATATAG and its complement AATTCTATATTCCTTATG) was incubated with a nuclear extract derived from P. gallinaceum (g) or P. falciparum (Pfg) gametes or from an asynchronous P. falciparum blood-stage culture that contained both asexual parasites and gametocytes (Pfb). Competitions included either a 100-fold molar excess of the unlabeled oligonucleotide TFB25 or a 100-fold molar excess of an oligonucleotide containing a mutant version of the putative binding site (MUT; AATTCATAAGGCCGCTAG and its complement AATTCTAGCGGCCTTATG). (B) Control on the activity of the P. falciparum blood-stage nuclear extract. Radiolabeled oligonucleotide KAHRP (30) was incubated with a nuclear extract of the blood stages of P. falciparum parasites. The unlabeled oligonucleotide was added as a competitor DNA as indicated. (C) Radiolabeled probe C was incubated with a nuclear extract derived from P. gallinaceum gametes. A batch of nuclear extract different from that used in the experiments depicted in panel A was used, which resulted in the formation of an additional nonspecific complex, indicated by an asterisk. Binding reactions were supplemented with specific competitor DNAs as indicated. (D) Transfection of P. gallinaceum mosquito-stage parasites with plasmid pPFS25LUC and a version of the plasmid in which the two AAGGAATA elements were mutated to AAGGCCGC (pPFS25-LUC-MUT). Plasmids were cotransfected with pCAT-L16.1ΔSX, and luciferase activities were normalized to CAT activities. Values are from three transfections; error bars indicate standard deviations.