c-Ha-ras gene bidirectional promoter expressed in vitro: location and regulation.
Increased transcriptional activity of the c-Ha-ras gene product is correlated with induction of several important human tumor types. For this reason, we have investigated the nature of the c-Ha-ras promoter and the factors that regulate its expression. Using S1 and primer extension analysis of c-Ha-ras RNA from EJ cells, we have identified 18 initiation sites within an upstream exon (exon -1) whose 3' end (the donor splice site [D]) is located 1,105 base pairs (bp) upstream of the ATG codon. The furthest-upstream initiation site is located -191 bp relative to D, and the furthest downstream is located -16 bp relative to D. Transient expression assays, in which a series of mutants spanning this region were ligated to a promoterless chloramphenicol acetyltransferase vector, functionally confirmed the position and extent of this promoter. Mutational analysis further located a 47-bp element located between -243 and -196 relative to D that up-regulated transcriptional activity of the promoter region by 20- to 40-fold. This region contained both a GC box known to bind SP1 and a CCAAT box. Insertion of a simian virus 40 enhancer 5' to the promoter up-regulated transcription from each initiation site by approximately 10- to 20-fold. We have also localized, both by chloramphenicol acetyltransferase assay and by S1 analysis, a strong promoter operating in the direction opposite that of the gene and originating immediately 5' to the 47-bp regulatory region. The reverse promoter was found to have nine initiation sites between -248 and -278 relative to D.
Lowndes
N F
NF
Department of Genetics, College of Physicians and Surgeons of Columbia University, New York, New York 10032.
Paul
J
J
Wu
J
J
Allan
M
M
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005091:Exons; D005786:Gene Expression Regulation; D011905:Genes, ras; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism
D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase