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Regulation of expression of the rat CYP2B1 and CYP2B2 genes

Wong, Siew Cheng; (1999) Regulation of expression of the rat CYP2B1 and CYP2B2 genes. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

CYP2B1 and CYP2B2 proteins are highly induced in rat liver by phenobarbital (PB). CYP2B1 promoter sequences from -179 to -347 bp and -348 to -451 bp were observed in gel shift assays to bind liver nuclear protein that was either more abundant or activated from PB-treated than untreated rats. The DNA-binding activity of the protein bound to the sequence between -348 and -451 bp was enhanced when liver nuclear extracts from both untreated and PB-treated rats were treated with ATP prior to gel shift assays. While pre-treatment with either calf intestinal alkaline phosphatase (CIP) or 2-aminopurine (2-AP), a general protein kinase inhibitor inhibited complex formation. Thus, phosphorylation of this protein increases its binding to DNA and dephosphorylation inhibits binding. When primary hepatocytes or whole animals were treated with 2-aminopurine, it could totally suppress both basal and PB-induced CYP2B mRNAs expression in vitro but only partially suppress PB-induction of CYP2B mRNAs in vivo. A PB-responsive element (PBRE) has been identified in the distal region of the CYP2B2 promoter. The homologous region in the CYP2B1 promoter between -2142 and -2301 bp was cloned into a reporter gene construct and was shown to confer PB-responsiveness to the luciferase gene when transfected either into primary rat hepatocytes or directly into rat liver tissues. A higher fold induction was observed with in vivo DNA transfection. The PBRE sequence could activate gene expression better when the -348 to -451 bp sequence was included in the promoter region of the reporter construct. The CYP2B2 promoter, between -183 and -199 bp, also bound more liver nuclear protein from PB-treated rats in gel shift assay. An octamer consensus oligonucleotide competed for protein binding to this region. An antibody which recognised the DNA-binding domain of Oct-1 and Oct-2 inhibited complex formation and an Oct-1 specific antibody supershifted the protein-DNA complex.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Regulation of expression of the rat CYP2B1 and CYP2B2 genes
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Liver nuclear proteins
URI: https://discovery.ucl.ac.uk/id/eprint/10102360
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