Analysis of regulatory sequences of the upstream element.

(A) The sequence of the upstream element of the bcl-xpromoter that has strong enhancerlike activity. Also depicted are the structures of several mutations created in that sequence that were analyzed for functional activity. Mutants 1 through 6 are indicated by brackets encompassing a sequence deleted from the normal sequence. Bases shown within a bracket indicate that those bases were substituted for the deleted sequence. The transcription factors indicated above the sequence were retrieved by the Transcription Element Search Software (www.cbil.upenn.edu/tess), based on consensus sequence motifs found by that program. The motifs of the factors shown are present in homologous sequences of the mouse and human bcl-x genes. GAGA factor has great importance for transcription in Drosophila, but no homolog of it has been identified in mammalian cells. (B) Results of transient transfection assays in HCD57 cells using constructs of the enhancer test vector, PGL3-Promoter Vector (Promega). This vector contains the SV40 promoter. The plasmids represented by the histogram columns contain, from left to right: vector alone; vector containing the SV40 enhancer; vector with bcl-x promoter sequence from −1804 through −1734 (upele70), forward orientation; vector withbcl-x promoter −1801 through −1213 (upele589), forward orientation; vector with upele589 in reverse orientation; vector with upele 589 placed downstream of luciferase gene. Other columns labeled as “del” represent experiments with upele70, mutated as indicated in (A), inserted upstream of the luciferase gene and in the normal orientation. Test plasmids were cotransfected with the control plasmid, pRL-TK, as an internal control for experimental variation. The activity of the unmodified PGL3-Promoter Vector was chosen as 1. Transfection experiments for each test plasmid were repeated 3 to 5 times. In all cases, the range of the determined values was equal to or less than 13% of the values shown.