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The genomic region upstream from exon F (exon IV) of the human aldolase A gene has been studied for its ability to direct the transcription of a reporter gene in vivo. Transfection experiments in human hepatoma cells (Hep 3B) followed by CAT assay, and S 1mapping analysis, demonstrated that: (i) this region is able to drive CAT gene transcription; (ii) all the transcriptional control elements of this promoter are downstream from nucleotide −384 of the longer ubiquitous RNA start site and the sequences between −384 and −262 play a crucial role in transcriptional efficiency; (iii) initiation starting points for two mRNAs exist 61 bp apart. Gel retardation and footprinting assays demonstrated the presence of DNA-protein complexes mainly in the region between −384 and −262 and such ubiquitous binding factors as Sp1 and AP-1.