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Nucleic Acids Research, 1985, Vol. 13, No. 20 7269-7288
© 1985

Articles

Autoregulation plus upstream positive and negative control regions associated with transcriptional activation of the mouse P1450 gene

Frank J. Gonzalez and Daniel W. Nebert1

Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health Bethesda, MD 20892, USA 1Laboratory of Developmental Pharmacology, National Institute of Child Health and Human Development, National Institutes of Health Bethesda, MD 20892, USA

Received July 29, 1985. Revised September 26, 1985. Accepted September 26, 1985.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to interact with a cytosolic receptor and, in turn, activate transcription of the mouse P1450 gene. Various lengths of DNA upstream of the P1450 gene were inserted into the pSV0-cat expression vector, with and without addition of the Harvey murine sarcoma virus (Ha-MSV) 72-bp repeat enhancer element. The constructs were cotransfected with pSV2-neo into mouse hepatoma wild-type cells and two variant cell lines. One variant is believed to result from a mutation in the P1450 structural gone and expresses high levels of P1450 mRNA constitutively; the other variant has a defect in nuclear translocation of the inducer-receptor complex. After selection in G418, the cells were treated with control medium, TCDD, cycloheximide, or TCDD plus cycloheximide and then assayed for chloramphenicol acetyltransferase (CAT) activity. The data are consistent with the presence of several functional regions within the upstream sequence: a promoter region, a region that is negatively autoregulated, possible repressorbinding and inducer-receptor complex-binding sites, and an upstream activation element that is required for transcriptional activation by TCDD. The Ha-MSV enhancer can substitute for this upstream activation element.


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