Abstract
Two separate enhancers were identified both upstream and downstream of the promoter of the human epidermal growth factor receptor gene. Transcriptional enhancer activity was found to be associated with a region 1172 and 852 base pairs upstream of the most upstream RNA start site, and within a 530-base pair fragment located in the first intron about 2000 base pairs downstream of the most upstream RNA start site. These two separate enhancers stimulated promoter activity cooperatively in HeLa cells synthesizing the epidermal growth factor receptor. The enhancer downstream of the promoter functioned only in the presence of the other upstream enhancer. Several areas of sequences homology with viral and cellular enhancers were noted in both the upstream and downstream enhancers. The region essential for the downstream enhancer activity has 10 nuclear factor-binding sites.
Highlights
Two separate enhancers were identified bothupstream and downstream of the promoter of the human epidermal growth factor receptor gene
To delimitate the upstream enhancer, the fragments generated by Sau3A or PstI digestion of the EcoRI-Hind111 1.1-kb fragment were inserted using BamHI linker into the BamHI site of pERCAT1, and theplasmids shown in Fig. 5A were generated
For delimitation of the downstream enhancer, the fragments generated by SstI or PstI digestion of the PuuII2.2-kb fragment were inserted using BamHI linker into the BamHI site of pERCAT2, and theplasmids shown in Fig. 5 8 were generated
Summary
The EcoRI-HindIII 1.1-kb (nucleotides -2200 to -1109) fragment containing the upstream enhancer was inserted into the BamHI site of pERCATl using BamHI linker in both orientationsto generate pERCATlUE(C) andpERCATlUE(R). To delimitate the upstream enhancer, the fragments generated by Sau3A or PstI digestion of the EcoRI-Hind111 1.1-kb fragment (nucleotides -2200 to -1109) were inserted using BamHI linker into the BamHI site of pERCAT1, and theplasmids shown in Fig. 5A were generated. For delimitation of the downstream enhancer, the fragments generated by SstI or PstI digestion of the PuuII2.2-kb fragment were inserted using BamHI linker into the BamHI site of pERCAT2, and theplasmids shown in Fig. 5 8 were generated. The gel retardation assay and DNase I footprint assay were performed as described by Singh et al [29] and Briggs et al [30]
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