Multiple cis- and trans-acting elements involved in regulation of the neu gene.
A 2.4-kb rat neu genomic DNA fragment that hybridized to the 5'-most coding sequence of the rat neu cDNA was cloned. S1 nuclease mapping identified multiple transcriptional initiation sites. DNA sequence analysis revealed that this fragment contained 64 bp of the first intron, 81 bp of the first exon, and the upstream noncoding sequence of the neu gene. The sequence immediately upstream of the translation start site was G + C rich (greater than 75%) and contained a consensus CCAAT sequence despite the absence of a TATA box. An Sp1-binding site was found, in addition to various sequence motifs common to the promoters of the human neu gene (erbB2), the epidermal growth factor receptor gene, and the simian virus 40 enhancer. A 2.2-kb EcoRI-Narl fragment containing sequences upstream from the 3'-most transcriptional start site was fused to the bacterial chloramphenicol acetyltransferase reporter gene and shown to promote transcription efficiently. A series of promoter deletion constructs was made, and results from transfection and subsequent chloramphenicol acetyltransferase assays suggested the presence of multiple cis-acting elements that contributed either positively or negatively to the transcription activity. Cotransfection competition experiments using subcloned cis-acting elements confirmed the existence of trans-acting factors interacting with these DNA fragments. In addition, a gel retardation assay was performed to demonstrate the physical binding of nuclear factors to certain fragments. The results complemented those of the deletion studies and led us to conclude that transcriptional regulation of the neu proto-oncogene involves at least one negative and three positive trans-acting factors interacting with different cis-acting elements along the neu gene promoter.
Suen
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Department of Tumor Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Hung
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MC
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009857:Oncogenes; D000090063:Proto-Oncogene Mas; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase