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Non‐Ap‐1 tumor promoter 12‐ O ‐tetradecanoylphorbol‐13‐acetate‐responsive sequences in the human ornithine decarboxylase gene
Author(s) -
Kim YongJoo,
Pan Huichin,
Verma Ajit K.
Publication year - 1994
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/mc.2940100308
Subject(s) - biology , microbiology and biotechnology , chloramphenicol acetyltransferase , tata box , reporter gene , 5' flanking region , gene , exon , caat box , 12 o tetradecanoylphorbol 13 acetate , promoter , intron , ornithine decarboxylase , luciferase , primer extension , fusion gene , enhancer , gene expression , transfection , genetics , messenger rna , biochemistry , enzyme , phorbol ester , phosphorylation , protein kinase c
To define the mechanisms of 12‐ O ‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced transcription of the ornithine decarboxylase (ODC) gene, we isolated a genomic clone (hODC41 B) of ODC from a human leukocyte genomic DNA library. The restriction endonuclease map, in comparison with the previously published sequences of the human ODC gene, indicated that hODC41B contained a 15.7‐kb sequence that extended from the sixth exon to about 10 kb upstream of the ODC gene. A 2.5‐kb genomic fragment containing the 5′ flanking region and the first exon was subcloned and sequenced. Sequence analysis revealed multiple putative promoter/enhancer elements (a TATA box, a CAAT box, 17 GC boxes, and a cAMP‐responsive element) but no consensus AP‐1 sequences (TGAGTCA) in the 2.5‐kb 5 flanking region. However, three AP‐1 sequences were located in introns 3, 5, and 11. We constructed a series of chimeric genes containing part of the first exon and increasingly longer 5′ flanking sequences of the ODC gene fused to either bacterial chloramphenicol acetyltransferase (CAT) or luciferase reporter genes. TPA inducibility was determined by transient transfection and measurement of CAT or luciferase expression in HeLa cells. The induction of CAT activity by TPA decreased with decreasing lengths of the 5′ flanking sequences up to nt‐82. The TPA induction from the construct‐72 ODC CAT was threefold to sevenfold, and the TPA inducibility of the same fragment was about ninefold to 30‐fold with the luciferase reporter gene. Further deletion analysis revealed TPA‐responsive sequences in ODC nt‐42 to +54. Gel mobility shift assays using α‐ 32 P‐end labeled ODC nt‐42 to +60 revealed that nt‐42 to +60 specifically bound HeLa cell nuclear proteins. HeLa cell nuclear protein binding to ODC nt‐42 to +60 could not be completely competed by AP‐1‐, AP‐2‐, AP‐3‐, or SP1‐responsive sequences. © 1994 Wiley‐Liss, Inc.