Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have identified the promoters for two inducible genes, in Streptomyces griseolus, that encode herbicide-metabolizing cytochromes P-450. They are in the class of promoters that have -35 and -10 sequences similar to those used in Escherichia coli by RNA polymerase E sigma 70. Transcription from either promoter was shown to be induced by sulfonylurea (chlorimuron ethyl) or phenobarbital. Mapping of mRNA showed that each cytochrome P450-encoding gene was transcribed on a separate multicistronic mRNA that encodes cytochrome P-450 (suaC or subC), ferredoxin (suaB or subB) and at least one other open reading frame. An inducible, site-specific DNA-binding activity was identified that bound to two similar 8-bp inverted repeat sequences within or near the sua promoter (suaP). A noninducible DNA-binding activity, distinct from that which bound to suaP, was found that bound to an 11-bp inverted repeat at the sub transcription start point.
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PMID:Phenobarbital and sulfonylurea-inducible operons encoding herbicide metabolizing cytochromes P-450 in Streptomyces griseolus. 155

It is apparent that the exposure of animals to xenobiotics has a profound effect upon the expression of cytochrome P450. In this manuscript, phenobarbital and 3MC have been selected from the many xenobiotics capable of eliciting this affect to demonstrate the magnitude of changes occurring at the level of the nucleic acids. We have seen that effects include increased levels of particular RNA polymerase isozymes; stabilization of ribosomes and increased synthesis of ribosomal RNA; increase in the amount of mature messenger RNA for individual cytochrome P450 components; and an increase in the amount of nuclear RNA precursors. How these effects come about is a matter for speculation. However, it is clear that the mechanisms by which this is accomplished differ substantially for both phenobarbital and 3MC. 3MC may induce cytochrome P450c through its interaction with a cytosolic "receptor" and subsequent activation of chromatin. The details must await further study.
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PMID:Induction of cytochrome P450 by xenobiotics. 643 66

Congenital adrenal hyperplasia (CAH) comprises a family of inherited human disorders caused by a defect in cortisol biosynthesis. We previously reported absent cholesterol side-chain cleavage enzyme cytochrome P450 (P450scc) expression in rabbits affected with CAH. Further molecular studies via Southern blotting analyses, using a full-length human P450scc cDNA probe and a cloned rabbit P450scc cDNA probe, demonstrated the absence of P450scc DNA fragments in CAH animals. Reverse transcriptase-based polymerase chain reactions revealed that P450scc mRNA was not detectable in the adrenals of CAH rabbits, confirming the previous findings of absent P450scc gene expression by Northern and Western blotting. Cloning and sequencing of a 1336-basepair fragment of rabbit P450scc cDNA (85% of the coding sequence) revealed an approximately 80% identical nucleotide sequence and a 76% identical amino acid sequence compared to the human P450scc cDNA. We conclude that a large deletion mutation in the P450scc gene is most likely responsible for the absent P450scc gene expression resulting in the lethal and feminizing form of CAH in the rabbit. Further investigation of adrenal and gonadal steroidogenic enzyme gene expression in this CAH animal model will provide a greater understanding of the molecular genetics of CAH, while wild-type P450scc gene transfer experiments using CAH adrenals in vitro or in vivo will ultimately characterize the molecular basis of CAH and provide a foundation for CAH gene therapy modality.
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PMID:Inherited congenital adrenal hyperplasia in the rabbit is caused by a deletion in the gene encoding cytochrome P450 cholesterol side-chain cleavage enzyme. 768 38

Previous work in our laboratory demonstrated that cytochrome P450 2B1 in rat liver was induced, but P450 2E1 was inhibited and inactivated, by diallyl sulfide (DAS), a compound derived from garlic. Such a selective effect on P450 enzymes is of considerable interest toward the understanding of dietary effects on xenobiotic metabolism. In the present study, the mechanism of P450 2B1 induction by DAS was investigated. Following a single dose of DAS (200 mg/kg body weight, ig), liver microsomal pentoxyresorufin dealkylase (PORd) activity, a representative activity of P450 2B1, was induced 3-, 16-, 26-, and 43-fold at 6, 12, 18, and 24 h after the treatment, respectively. A corresponding increase in the level of P450 2B1/2 protein was observed by immunoblot analysis. The level of P450 2B1/2 mRNA in rat liver also increased markedly, reaching a maximum at 12 h after the DAS treatment. Hybridization with the isozyme-specific oligonucleotide probes revealed that the mRNA levels of both P450s 2B1 and 2B2 were induced. In contrast, the level of P450 2E1 mRNA in the liver of DAS-treated rats was not changed. The results of nuclear run-on assay revealed that the transcriptional rate of P450 2B1/2 genes in the rat liver increased 13-fold at 6 h after DAS administration and returned to the control value at 24 h. The transcription of P450 2B1/2 genes was blocked completely by alpha-amanitin, an inhibitor of RNA polymerase II. These results clearly demonstrate that the induction of P450 2B1/2 in rat liver by DAS is mainly due to transcriptional activation. In the DAS-treated rats, P450 2B1/2 mRNA was also markedly induced in the stomach and duodenum. The maximal induction was found at 12 h after the treatment while the levels of P450 2B1/2 mRNA increased 66-fold in the duodenum and 23-fold in the stomach. DAS treatment, however, did not change the levels of P450 2B1/2 mRNA in the lung and nasal mucosa.
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PMID:Transcriptional activation of cytochrome P450 2B1/2 genes in rat liver by diallyl sulfide, a compound derived from garlic. 848 39

The heterologous expression and spectroscopic characterization of the [2Fe-2S] ferredoxin from the sexually transmitted human parasite Trichomonas vaginalis is described. Using oligonucleotide primers based on the deduced DNA sequence, the gene encoding the ferredoxin was amplified by polymerase chain reaction and cloned into a T7 RNA polymerase expression vector. Expression of the gene in Escherichia coli host HMS174(DE3) resulted in the high level production of the protein with the correctly assembled iron-sulfur cluster. The absorption, circular dichroism, resonance Raman, and EPR spectra of the recombinant protein revealed many differences from those of other [2Fe-2S] ferredoxins. The redox potential of the protein (-347 mV versus normal hydrogen electrode) was also determined. Whereas the amino acid sequence of T. vaginalis ferredoxin showed greatest homology to the [2Fe-2S] ferredoxins found in bacteria and vertebrate mitochondria which function in cytochrome P450 oxidation pathways, the spectroscopic properties showed substantial dissimilarity. Differences in the biophysical properties and function of T. vaginalis ferredoxin are proposed to result from the characteristic amino acid sequence of the parasite protein near the cysteine residues that ligate the valence-localized Fe(III) site of the reduced cluster.
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PMID:Expression and spectroscopic characterization of the hydrogenosomal [2Fe-2S] ferredoxin from the protozoan Trichomonas vaginalis. 866 34

To study the role of the cis-acting element(s) in controlling the expression of the cytochrome P450(BM-1) gene and its upstream regulatory gene, bm1P1, in Bacillus megaterium, various deletion derivatives were constructed. A 53-bp inverted repeat located midway between the P450(BM-1) gene and bm1P1 gene was found in vivo to negatively regulate the expression of both genes, the regulation of which may occur at the transcriptional level. The promoter of the P450(BM-1), gene was also identified and found to be similar to those recognized by the sigmaA RNA polymerase of Bacillus subtilis. Possible mechanisms by which the 53-bp inverted repeat regulates the gene expression are discussed.
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PMID:A 53-base-pair inverted repeat negatively regulates expression of the adjacent and divergently oriented cytochrome P450(BM-1) gene and its regulatory gene, bm1P1, in Bacillus megaterium. 898 10

The aim of the study was to investigate whether the adrenal renin-angiotensin system plays an independent role in the regulation of mineralocorticoid biosynthesis in the adrenal gland and to explore the mechanisms of this action. Twelve-week-old male Sprague-Dawley rats were studied: 22 rats were maintained on a regular diet; 27 and 22 rats received a low salt diet with and without treatment, respectively, with the angiotensin II (Ang II) AT1-subtype receptor antagonist losartan (10 mg/kg per day). A fraction of each group of rats underwent bilateral nephrectomy (n = 12, 15, and 10, respectively) and was killed 48 hours later. In an additional group of 24 (12 intact and 12 nephrectomized) rats, the effects of the Ang II AT2-subtype receptor antagonist PD123319 were investigated. In intact rats, plasma renin activity (PRA) and adrenal renin activity and expression were progressively raised by salt restriction and losartan, whereas aldosterone synthase mRNA and plasma aldosterone (PA) levels were increased by salt restriction and reduced by losartan. Forty-eight hours after nephrectomy, PRA fell to undetectable levels; in contrast, adrenal renin expression, assessed by semiquantitative reverse-transcriptase polymerase chain reaction (using GAPDH as a standard for gene expression), showed an 18-fold increase and was further increased after salt restriction and losartan (all P < .05). Also, adrenal renin activity was raised after nephrectomy and further increased after salt restriction (P < .05) and losartan. Cytochrome P450 aldosterone synthase expression in the adrenal cortex was stimulated by nephrectomy alone and by nephrectomy combined with low salt intake (P < .05), with consequent increases in PA concentrations. In losartan-treated salt-restricted nephrectomized rats, cytochrome P450 aldosterone synthase expression (P < .05 versus nephrectomy alone and nephrectomy plus salt restriction) and PA concentrations were diminished (P < .05) in spite of the observed increases of adrenal renin expression. The AT2-receptor antagonism did not significantly affect PRA, adrenal renin, and aldosterone biosynthesis and production in either intact or nephrectomized salt-restricted rats. These results demonstrate that the adrenal renin-angiotensin system plays an independent role in the regulation of mineralocorticoid biosynthesis in vivo. This action is mediated primarily via the Ang II AT1-subtype receptors.
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PMID:Role of tissue renin in the regulation of aldosterone biosynthesis in the adrenal cortex of nephrectomized rats. 935 60

S-Warfarin 7-hydroxylation, S-flurbiprofen 4'-hydroxylation, and diclofenac 4'-hydroxylation activities were determined in liver microsomes of 30 humans of which 19 were wild-type (Arg144.Ile359), 8 were heterozygous Cys (Cys144.Ile359), and 3 were heterozygous Leu (Arg144.Leu359) allelic variants of the cytochrome P450 2C9 (CYP2C9) gene. All of the human samples examined contained P450 protein(s) immunoreactive with anti-CYP2C9 antibodies in liver microsomes. Individuals with the Cys144 allele of CYP2C9 had similar, but slightly lower, activities for the oxidations of these substrates than those of wild-type CYP2C9. One of the three human samples heterozygous for the Leu359 allele had very low Vmax and high Km values for the oxidation of three substrates examined, while the other two individuals gave kinetic parameters comparable to those seen in the wild-type and Cys144 CYP2C9. Reverse transcriptase-polymerase chain reaction analysis, however, showed that all of the three human samples with the heterozygous Leu359 variant were found to express both Ile359 and Leu359 variants at relatively similar extents in liver RNA of three humans. These results suggest that the Cys144 variant of CYP2C9 catalyzes the CYP2C9 substrates at rates comparative to, but slightly lower than, those of wild-type CYP2C9, while the Leu359-allelic variant has slower rates for the oxidation of these drug substrates. Activities for the oxidation of these CYP2C9 substrates in humans with heterozygous Leu359 allele is likely to be dependent on the levels of expression of each of the wild- and Leu-variants in the livers. However, one of the humans with a heterozygous Leu allele was found to have very low activities towards the oxidation of CYP2C9 substrates. The basis of this defect in catalytic functions towards CYP2C9 substrates is unknown.
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PMID:Comparative studies on the catalytic roles of cytochrome P450 2C9 and its Cys- and Leu-variants in the oxidation of warfarin, flurbiprofen, and diclofenac by human liver microsomes. 969 79

Five murine cytochrome P450 (CYP) 2C cDNAs were cloned and characterized, including four new members of this subfamily: CYP2C37, CYP2C38, CYP2C39, and CYP2C40. The cDNAs ranged from 1716 to 1812 bp in length and encoded polypeptides of 490 amino acid residues except for CYP2C40, which contained an additional glutamic acid residue at the carboxyl terminus. The amino acid identity of the murine CYP2Cs ranged from 69 to 92%, while the overall amino acid identity was 60%; however, within the six putative substrate recognition sites the identity was only 25 to 41%, suggesting possible differences in substrate specificity and product profiles. The CYP2C cDNAs were expressed in Escherichia coli following modification of the N-terminus. All five recombinant CYP2Cs metabolized arachidonic acid, but with different metabolic profiles and catalytic rates. Based on coelution with authentic standards on reverse-phase HPLC, themajor metabolites were tentatively identified asfollows: CYP2C29 and CYP2C39 produced 14, 15-cis-epoxyeicosatrienoic acid (EET); CYP2C37 produced 12-hydroxyeicosatetraenoic acid (HETE); CYP2C38 produced 11,12-EET; and CYP2C40 produced an unidentified metabolite that coeluted with 16-,17-, and 18-HETEs. The turnover numbers for CYP2C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40 were 0.34, 1.12, 5.15, 0.51, and 0.15 nmol/nmol/min, respectively. Reverse transcriptase-polymerase chain reaction demonstrated the presence of CYP2C29 mRNA in liver as well as in extrahepatic tissues including brain, kidney, lung, heart, and intestine. CYP2C38 and CYP2C40 were found in liver, brain, kidney, and intestine, with trace amounts in lung and heart, while CYP2C37 and CYP2C39 appeared to be liver specific.
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PMID:Cloning and expression of murine CYP2Cs and their ability to metabolize arachidonic acid. 972 Nov 82

We have recently reported that beta-ionone induces cytochrome P450 (P450) 2B1 in rats. Effects of beta-ionone on the expression of other P450 isozymes and NADPH-P450 reductase were further investigated in Sprague Dawley rats. Administration of beta-ionone subcutaneously 72 and 48 h before sacrificing the animals not only significantly induced the liver microsomal activities of P450-associated enzymes and NADPH-P450 reductase, but also clearly increased in the level of P450 1A1/2, P450 2C, and NADPH-P450 reductase proteins. The induction of P450 1A1/2 and 2C by beta-ionone was much greater in male than in female as measured by western immunoblotting. Reverse transcriptase-polymerase chain reactions showed that, in addition to P450 2B1 and 2B2 mRNAs, P450 1A2, 2C6 and NADPH-P450 reductase mRNAs were increased when beta-ionone was administered. Our previous and present results indicated that beta-ionone may induce several P450s and NADPH-P450 reductase by the accumulation of their corresponding mRNAs.
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PMID:Effects of beta-ionone on the expression of cytochrome P450s and NADPH-cytochrome P450 reductase in Sprague Dawley rats. 974 58


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