SPECIES-SPECIFIC POLYMORPHISM IN THE PROMOTER OF THE APOLIPOPROTEIN-A-I GENE - RESTORATION OF HUMAN TRANSCRIPTIONAL EFFICIENCY BY SUBSTITUTION AT POSITIONS -189, -144 AND -48 BP

Previous studies indicate that species-specific differences in apolipo protein A-I (ape A-I) expression could be largely explained by cis-act ing factors located within or near the 5' flanking region (-231 to +22 3 bp, where +1 is the start site of transcription). In the present stu dies, we have localized 7 sites within the (-231 to -15 bp) region of the African green monkey apo A-I gene that differ from the human apo A -I gene 5' flanking region. To identify which of the 7 polymorphic sit es were essential for the species-specific differences in apo A-I gene expression, mutated promoter constructs were transfected into HepG2 c ells and reporter gene expression was measured. Each of the 7 sites wi thin a defined 5' flanking region of the human gene was individually m utated to the African green nucleotide sequence found at that position . Three of the sites (-189, -144 and -48) were found to raise the huma n apo A-I promoter activity to approx. 60-65% of the African green pro moter. While double mutations (-144/-48 bp and -189/-144 bp), restored the human apo A-I promoter activity to 100% of that found with the Af rican green monkey promoter. Additional studies revealed similar DNA: protein interactions with DNA probes from either human or African gree n monkey and HepG2 cell nuclear extract. In conclusion, these studies demonstrate that double and triple nucleotide substitutions within the human apo A-I promoter are sufficient to restore gene expression in H epG2 cells to levels seen with the African green monkey promoter. Thes e data suggest that sites -189, -144 and -48 bp are involved in signif icantly altering the binding affinity of a nuclear factor determining the species-specific level of apo A-I gene transcription.