Functional characterization of the 5' proximal flanking fragment of Plasmodium falciparum GBP130 gene.

OBJECTIVE: To identify the putative regulation elements with strength- and stage-specificity in 5' proximal flanking sequence of P. falciparum GBP130 gene. METHODS: Plasmids containing different deletions of upstream of the GBP130 promoter were constructed. For strength-specific analysis, pGBPCAT delta 2, pGBP delta 2/400 and pGBP delta 2/800 were electroporated into ring-stage P. f. respectively, and the expression level of CAT in each plasmid was detected by liquid scintillation counts(LSC). For stage-specific analysis, transfectants with pGBP delta 2/400 and pGBP delta 2/800 were harvested at 5 hours post-transfection(h), 15 h and 46 h respectively, and the CAT expression levels were detected. RESULTS: In strength-specific analysis, the expression level of CAT in pGBP delta 2/800 and pGBPCAT delta 2 was similar, down-regulated significantly in pGBP delta 2/400. The CAT level showed significant difference between pGBP delta 2/400 and the control. In stage-specific analysis, the CAT level of pGBP delta 2/400 was higher than that of pGBP delta 2/800 at the time point of 5 h, and lower at 15 h and 46 h. CONCLUSION: This strength-specific promoter activity was due to the difference of 5'UTR length: the longer the 5'UTR the higher the promoter strength, and two poly (dA:dT) tracts in the proximal sequence could enhance the promoter activity. The length of 5'UTR regulated the promoter activity in a stage-specific manner. The shorter 5'UTR was functional at ring stage, while the longer one prompted transcription at trophozoite and schizont stage. The functional role of poly (dA:dT) tracts in stage-specific regulation of GBP130 remains unclear.