The life cycles of the conidiating species of

The life cycles of the conidiating species of

The phenobarbitone-responsive minimal promoter has been shown to lie between nt -179 and nt + 1 in the 5' (upstream) region of the CYP2BI/B2 gene in rat liver, on the basis of the drug responsiveness of the sequence linked to human growth hormone gene as reporter and targeted to liver as an asialoglycoprotein-DNA complex in vivo. Competition analyses of the nudear protein-DNA complexes formed in gel shift assays with the positive (nt -69 to -98) and negative (nt -126 to -160) cis elements (PE and NE, respectively) identified within this region earlier indicate that the same protein may be binding to both the elements. The protein species purified on PE and NE affinity columns appear to be identical based on SDS/PAGE analysis, where it migrates as a protein of 26-28 kDa. Traces of a high molecularweight protein (94-100 kDa) are also seen in the preparation obtained after one round of affinity chromatography. The purified protein stimulates transcription of a minine construct containing the 179 nt on the 5' side of the CYP2BI /B2 gene linked to the I exon in a cell-free system from liver nuclei. The purified protein can give rise to all the three complexes (I, II, and III) with the PE, just as the crude nuclear extract, under appropriate conditions. Manipulations in vitro indicate that the NE has a significantly higher affnity for the dephosphorylated form than for the phosphorylated form of the protein. The PE binds both forms. Phenobarbitone treatment of the animal leads to a significant increase in the phosphorylation of the 26-to 28-kDa and 94-kDa proteins in nuclear labeling experiments followed by isolation on a PE affinity column. We propose that the protein binding predominantly to the NE in the dephosphorylated state characterizes the basal level of transcription of the CYP2BI /B2 gene. Phenobarbitone treatment leads to phosphorylation of the protein, shifting the equilibrium toward binding to the PE. This can promote interaction with an upstream enhancer through other proteins such as the 94-kDa protein and leads to a significant activation of transcription.Many members of the cytochrome P-450 (CYP) gene superfamily are inducible. However, knowledge regarding the mechanisms of induction is restricted to a few members, although transcriptional activation is a common feature (1, 2). The mechanism of transcriptional activation of CYPlAl gene, induced by the prototype chemical 3-methylcholanthrene, has been studied in detail. It involves interaction of the ligand with Ah receptor, translocation of the receptor to the nucleus, and interaction with upstream cis elements (3, 4). However, the CYP2BI/B2 gene, one of the first mammalian cytochrome P-450 genes to be identified and cloned, has presented certain unique features of regulation (5). Although phenobarbitone (PB) induces this gene (Bi and B2 are 97% homologous and hence treated as a unit at this stage) essentially through transcriptional activation, the mechanistic details are not clear. It has not yet been possible to identify a receptor for PB. ...

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Heme deficiency precipitated by CoCl 2 administration to rats leads to a striking decrease in the inducibility of CYP2B1/B2 mRNA levels and its transcription by phenobarbitone (PB), besides decreasing the basal levels. Exogenous hemin administration counteracts the effects of CoCl 2 administration. The binding of nuclear proteins to labeled positive cis-acting element (؊69 to ؊98 nucleotides) in the near 5-upstream region of the gene is inhibited by CoCl 2 administration to saline or PB-treated rats, as assessed in gel shift assays. Administration of exogenous hemin to the animal or addition in vitro to the extracts is able to overcome the effects of CoCl 2 treatment. The protein mediating this effect has been purified from CoCl 2 administered nuclear extracts by heparin-agarose, positive element oligonucleotide affinity, and heme affinity column chromatography. This 65-kDa protein manifests very little binding to the positive element, but in the presence of certain other nuclear proteins, shows a strong heme-responsive binding. The purified protein binds heme. It is also able to stimulate transcription of a minigene construct of the CYP2B1/B2 gene containing ؊179 nucleotides of the 5-upstream region and the I exon in a cell-free system, manifesting heme response. It is concluded that the 65-kDa protein mediates the constitutive requirement of heme for the transcription of CYP2B1/B2 gene.The transcriptional regulation of the cytochrome P-450 (CYP) supergene family is of considerable interest. The CYP1A1 gene of rat liver, induced by the prototype drug 3-methylcholanthrene, has been studied in detail. It involves interaction of the ligand, 3-methylcholanthrene, with the Ah receptor, translocation of the receptor to the nucleus, and interaction with specific upstream elements (1, 2). The details regarding the mechanism of transcriptional activation of the CYP2B1/B2 gene (2B1 and B2 are 97% homologous and hence treated as a unit) by the prototype drug, phenobarbitone (PB), 1 are beginning to emerge. Studies in this laboratory have led to the identification of a positive cis-acting element (Ϫ69 to Ϫ98 nt) and a negative cis-acting element (Ϫ126 to Ϫ160 nt) in the near 5Ј-upstream region of the CYP2B1/B2 gene (3-5). The positive element includes the 17-base pair PB-responsive consensus element, referred to as Barbie Box, identified first by Fulco and co-workers (6 -8) in Bacillus megaterium, rat, mice, and other organisms of barbiturate inducible cytochrome P-450 and other proteins. There have also been other PB-responsive sequences identified in the CYP2B1/B2 gene of the rat. Shephard et al. (9) have identified two sequences, located between Ϫ183 to Ϫ199 nt and Ϫ31 to Ϫ72 nt, to be PB-responsive, although these do not include the "Barbie" elements. In addition, Trottier et al. (10) have located a sequence between Ϫ2155 and Ϫ2318 nt to be PB-responsive and Ramsden et al. (11) have identified an upstream enhancer as far as Ϫ20 kilobases. Studies in this laboratory have led to the development of a model which propose...