The Ah locus: Correlation of intranuclear appearance of inducer-receptor complex with induction of cytochrome P1-450 mRNA

Tukey, Robert H.; Hannah, Rita R.; Negishi, Masahiko; Nebert, Daniel W.; Eisen, Howard J.

doi:10.1016/0092-8674(82)90427-5

Cited by 175 publications

(45 citation statements)

References 24 publications

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“…In mouse hepatoma cells, the prototypical compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) activates transcription of the cytochrome P1-450 gene (3,4,16). Studies of receptor-defective cells reveal that this response requires the formation of a TCDD-receptor complex and an interaction between the inducer-receptor complex and the nucleus (3, 4).…”

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In situ protein-DNA interactions at a dioxin-responsive enhancer associated with the cytochrome P1-450 gene.

Durrin¹,

Whitlock²

1987

Mol. Cell. Biol.

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We used an in situ exonuclease III protection technique (C. Wu, Nature [London] 309:229, 1984) to analyze protein-DNA interactions at a dioxin-responsive enhancer. Our results imply that the 2,3,7,8-tetrachlorodibenzo-p-dioxin-receptor complex interacts with the dioxin-responsive enhancer to activate transcription of the cytochrome Pl-450 gene.Halogenated aromatic hydrocarbons are environmental contaminants that produce diverse biological effects (11,12). In mouse hepatoma cells, the prototypical compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) activates transcription of the cytochrome P1-450 gene (3,4,16). Studies of receptor-defective cells reveal that this response requires the formation of a TCDD-receptor complex and an interaction between the inducer-receptor complex and the nucleus (3, 4). A cis-acting dioxin-responsive element (DRE) located upstream of the cytochrome P1-450 gene mediates the action of the TCDD-receptor complex (2, 5, 7). The DRE contains two TCDD-responsive domains, which have properties typical of transcriptional enhancers and require TCDD-receptor complexes for their function (5-7). Here, we use an exonuclease protection technique (18,19) to show that both the DRE and the TCDD-receptor complex participate in the formation of a stable nucleoprotein complex within the cell nucleus. These results imply that the TCDDreceptor complex interacts with the DRE in vivo. Figure 1 shows the relevant restriction sites in the DNA upstream of the mouse cytochrome P1-450 gene. The hatched boxes indicate the position of the two DREs (8). To examine this region for resistance to exonuclease III (Exo III), nuclei were prepared by Dounce homogenization and centrifugation (1) and were digested with BamHI (Bethesda Research Laboratories, Inc.) (300 U/ml, 30 min, 30°C) to nick the DNA downstream of the proximal DRE. Next, chromatin (at a DNA concentration of about 1 mg/ml) was digested for 30 min with various concentrations of Exo III (BRL) as previously described (9). The BamHI-Exo IIIdigested DNA was purified by phenol-chloroform extraction, digested with Si nuclease (BRL) (4,000 U/ml, 15 min, 30°C) as previously described (18), and then digested to completion with HindIII (BRL). The repurified and ethanolprecipitated DNA was fractionated by agarose gel electrophoresis, transferred to a nylon membrane (15), and hybridized to a nick-translated (13) probe complementary to the 5' end of the HindIll-HindIII fragment. The 32P-labeled bands were detected by autoradiography, with Kodak XAR-5 film and an intensifying screen.The results in Fig. 2 reveal that BamHI digested the cytochrome P1-450 regulatory region in nuclei from TCDDinduced cells (Fig. 2B, lane b) to a greater extent than in nuclei from uninduced cells (Fig. 2A, Fig. 2A, lane b). However, digestion with Exo III (Fig. 2A, lanes c to g) failed to generate a smaller HindlIlExo III subband(s). In contrast, Exo III digestion of nuclei from TCDD-induced cells generated a 0.67-kilobase HindlIlExo III subband (Fig. 2B, lanes c to g)

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confidence: 99%

In situ protein-DNA interactions at a dioxin-responsive enhancer associated with the cytochrome P1-450 gene.

Durrin¹,

Whitlock²

1987

Mol. Cell. Biol.

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“…Phenobarbital-induced mouse liver mRNA does not hybridize on Northern blots (13) or by Rot analysis (14) to mouse P1-450 cDNA, nor does 3-methylcholanthrene-induced rat liver mRNA hybridize measurably to rat P-450b (15,16), rat P-450e (17), or mouse P-450b (18) cDNA clones. The 3-methylcholanthrene-inducible P-450 gene family is regulated by the cytosolic Ah receptor (19).…”

Section: Introductionmentioning

confidence: 99%

Mouse cytochrome P₃-450: complete cDNA and amino acid sequence

1984

Self Cite

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A full-length cDNA clone (1,894 homology, and nucleotides 1068 to 1260, 69Z homology, with portions of rat P-450b exons 2 and 7, respectively. P3-450 shows 62% homology in the socalled "highly conserved region" of 39 nucleotides in the rat P-450b and P-450e and the mouse P-450b. These results indicate that P3-450, P-450b and P-450e arose from a common ancestral gene. Cysteinyl peptide-coding regions were examined: P3-450 nucleotides 1405 to 1464 exhibit 61% homology, and nucleotides 502 to 552 exhibit 37% homology, when compared with their corresponding regions in the rat P-450b gene. These data support the likelihood that cysteine 456 is the thiolate ligand to the heme iron in the P3-450 enzyme active-site. INTRODUCTIONA large portion of drug-metabolizing enzyme activity arises from the membrane-bound multicomponent cytochrome P-450 system (1, 2). Endogenous substrates of P-450 include steroids, fatty acids, biogenic amines, prostaglandins and leukotrienes. Foreign substrates include almost all drugs, chemical carcinogens, and other environmental pollutants numbering in the tens of thousands; hence, the recommended nomenclature for P-450-mediated catalytic activity is "multisubstrate monooxygenase" (3).

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“…Elucidation of the structure and organization of genes encoding certain xenobiotic-inducible forms of cytochrome P-450 has brought to light several interesting features including the existence of multiple genes (10)(11)(12) and unusually large mRNA molecules (13-15) as well as possible mechanisms involved in genetic regulation (11,12,(16)(17)(18)(19). However, little is known concerning the structure and organization of the genes and.…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.

John¹,

Ashley²,

MacDonald³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

109

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Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts -425 and ':950 base pairs long, respectively, which are specific for bovine cholesterol sidechain cleavage cytochrome P-450 (P-450CC), have been identified by using two differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNAs as hybridization probes, an RNA species was identified that had the properties expected of mRNA specific for P-450, with respect to tissue specificity, corticotropin (ACTH)-mediated regulation of synthesis, and size of the protein product synthesized in vitro. In RNA samples obtained from bovine adrenal cortex, from bovine corpus luteum, and from cultured bovine adrenocortical cells, it was found that P450s is encoded by mRNA species -2000 bases long, a mujority of which are polyadenylylated. P-450s, mRNA was not detected in RNA samples prepared from bovine heart, liver, and kidney. Treatment of cultured bovine adrenocortical cells with ACTH resulted in the appearance of elevated levels of P-450, mRNA within 8 hr. Thus, ACTH promotes the enhancement of P-450scc gene transcription or acts to stabilize the transcripts. When pBSCC-2 cDNA was used to probe high molecular weight bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450O may be encoded by a single gene.Cytochromes P-450 are a diverse group of heme-containing mixed-function oxidases that are involved in the biotransformation of a wide variety of exogenous and endogenous substrates. The best-studied examples of those forms that metabolize endogenous substrates are the group of cytochromes P-450 required for steroid hormone biosynthesis. In steroidogenic tissues such as adrenal cortex, testis, ovary, and placenta, the initial and rate-limiting step in the pathway leading from cholesterol to steroid hormones is the cleavage of the side chain of cholesterol to yield pregnenolone (1). This reaction, known as cholesterol side-chain cleavage, is catalyzed by a specific form of cytochrome P-450 (P-450,cc) that is localized in the inner mitochondrial membrane (2,3). This protein is synthesized on cytoplasmic ribosomes as a higher molecular weight precursor (Mr 54,500) that is proteolytically processed to the mature form (Mr 49,000) upon insertion into the mitochondrion (4,5). By utilizing bovine adrenocortical cells in primary monolayer culture, it has been demonstrated in this laboratory that the synthesis of P-450,"c can be modulated by corticotropin (ACTH) and that this action of ACTH is mediated by cyclic AMP (6,7). A similar pattern of regulation of P-450Scc synthesis has been observed upon stimulation of bovine ovarian granulosa cells with follicle-stimulating hormone (8) and rat testicular Leydig cells with human chorionic gonadotropin (9). Thus, it appears that the levels of P-4Sscc are maintained in vivo in response to trophic hormone stimulation and that cyclic AMP plays a key role in the regulation of P-45scc synth...

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The Ah locus: Correlation of intranuclear appearance of inducer-receptor complex with induction of cytochrome P1-450 mRNA

Cited by 175 publications

References 24 publications

In situ protein-DNA interactions at a dioxin-responsive enhancer associated with the cytochrome P1-450 gene.

In situ protein-DNA interactions at a dioxin-responsive enhancer associated with the cytochrome P1-450 gene.

Mouse cytochrome P₃-450: complete cDNA and amino acid sequence

Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.

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The Ah locus: Correlation of intranuclear appearance of inducer-receptor complex with induction of cytochrome P1-450 mRNA

Cited by 175 publications

References 24 publications

In situ protein-DNA interactions at a dioxin-responsive enhancer associated with the cytochrome P1-450 gene.

In situ protein-DNA interactions at a dioxin-responsive enhancer associated with the cytochrome P1-450 gene.

Mouse cytochrome P3-450: complete cDNA and amino acid sequence

Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.

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Mouse cytochrome P₃-450: complete cDNA and amino acid sequence