Characterization of the Human N-ras Promoter Region
Overexpression of ras protooncogenes has been implicated in cancer development. We therefore initiated a study of the human N-ras promoter to determine the regions that control N-ras expression and their potential for interaction with DNA-binding proteins. N-ras CAT constructs were stably integrated into K562 cells by electric field-mediated gene transfer. A significant proportion of promoter activity was found to lie within a 438bp fragment comprising an untranslated exon (exon -1) with adjacent 5′ sequence and a small CpG island. A 107bp fragment at the 5′ end of exon -1 was essential for promoter activity, while a 44bp deletion from within this region decreased promoter activity by two thirds. Unlike the human H-ras promoter, the human N-ras promoter did not exhibit bidirectional activity. DNase footprinting of the 438bp fragment revealed seven protected regions, many of which contain sequences homologous to known DNA binding protein sites (MLTF/myc, CREB/ATF, AP-1, AP-2, myb, and E4TF1). Using purified MLTF and appropriate competitors in gel shift and DNase footprinting assays, we demonstrated binding of MLTF to the MLTF consensus sequence within exon -1.
KeywordsK562 Cell Negative Regulatory Element Untranslated Exon Major Late Promoter Human Fibrosarcoma Cell Line HT1080
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