One of the most commonly known genes involved in chronic diffuse liver diseases pathogenesis are genes that encodes the synthesis of glutathione-S-transferase (GST), known as the second phase enzyme detoxification system that protects against endogenous oxidative stress and exogenous toxins, through catalisation of glutathione sulfuric groups conjugation and decontamination of lipid and deoxyribonucleic acid oxidation products. The group of GST enzymes consists of cytosolic, mitochondrial and microsomal fractions. Recently, eight classes of soluble cytoplasmic isoforms of GST enzymes are widely known: α-, ζ-, θ-, κ-, μ-, π-, σ-, and ω-. The GSTs gene family in the Human Gene Nomenclature Committee, online database recorded over 20 functional genes. The level of GSTs expression is considered to be a crucial factor in determining the sensitivity of cells to a broad spectrum of toxins. Nevertheless, human GSTs genes have multiple and frequent polymorphisms that include the complete absence of the GSTM1 or the GSTT1 gene. Current review supports the position that genetic polymorphism of GST genes is involved in the pathogenesis of various liver diseases, particularly non-alcoholic fatty liver disease, hepatitis and liver cirrhosis of different etiology and hepatocellular carcinoma. Certain GST allelic variants were proven to be associated with susceptibility to hepatological pathology, and correlations with the natural course of the diseases were subsequently postulated.

Colletotrichum lini is used as an industrial stain for the dihydroxylation of steroid compound dehydroepiandrosterone (DHEA) to biosynthesize 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA), a key intermediate of the most popular oral contraceptive "Yasmin". This work aimed to enhance 7α,15α-diOH-DHEA production in C. lini CGMCC 6051 through ethanol induction. With 0.6% (v/v) ethanol induction and 10 g/L DHEA concentration, the 7α,15α-diOH-DHEA molar yield reached 58.8%, which was increased by 67.5% than that of the control. iTRAQ-based quantitative proteomic analysis was applied to explore the probable molecular mechanism of C. lini response to ethanol induction. A total of 50 differential expressed proteins was affected by ethanol induction, and could be related to multiple metabolic pathways. Most of differently expressed proteins were functionally mapped into pathways of transport, steroids metabolism, or redox reaction. Other proteins for energy, transcription and translation, and carbohydrate metabolism might have important roles in the cellular response to ethanol induction. In addition, the levels of cytochrome P450 and NAD(P)H-cytochrome P450 reductase were remarkably higher under ethanol induction, and their functions on DHEA dihydroxylation were first proposed in C. lini. Our results provide critical clues in revealing the dihydroxylation mechanism and are important for efficient microbiological hydroxylation of steroidal compounds in the future. BIOLOGICAL SIGNIFICANCE: iTRAQ strategy was first used to compare the proteomes of ethanol induction during the dihydroxylation reaction by Colletotrichum lini CGMCC 6051. The changes in protein provided a comprehensive overview of DHEA dihydroxylation in C. lini, including the proteins for steroids metabolism, redox reaction, transport, transcription and translation, energy and carbohydrate metabolism. Cytochrome P450, NADPH-cytochrome P450 reductase, and NADH-cytochrome b5 reductase were highlighted due to their outstanding contribution to DHEA dihydroxylation. The results help us understand the molecular mechanism underlying ethanol induction in C. lini and would guide strain engineering to further improve dihydroxylation efficiency.

P450(BM3) (CYP102A1), a fatty acid hydroxylase from Bacillus megaterium, has been extensively studied over a period of almost forty years. The enzyme has been redesigned to catalyse the oxidation of non-natural substrates as diverse as pharmaceuticals, terpenes and gaseous alkanes using a variety of engineering strategies. Crystal structures have provided a basis for several of the catalytic effects brought about by mutagenesis, while changes to reduction potentials, inter-domain electron transfer rates and catalytic parameters have yielded functional insights. Areas of active research interest include drug metabolite production, the development of process-scale techniques, unravelling general mechanistic aspects of P450 chemistry, methane oxidation, and improving selectivity control to allow the synthesis of fine chemicals. This review draws together the disparate research themes and places them in a historical context with the aim of creating a resource that can be used as a gateway to the field.

Many Drosophila cytochrome P450 or Cyp genes are induced by caffeine and phenobarbital (PB). To understand the induction mechanism, we created Drosophila S2 cell lines stably transformed with different luciferase reporter plasmids carrying upstream DNAs of Cyp6a8 allele of the resistant 91-R strain, and the 1.1-kb upstream DNAs of Cyp6g1 of the 91-R and the susceptible 91-C strains. Following 24 h treatment with dichlorodiphenyltrichloroethane (DDT), caffeine or PB, luciferase activity of all cell lines was determined. Results showed that the 0.1-kb DNA of Cyp6a8 and the upstream DNAs of Cyp6g1 from both strains are not induced by these chemicals in S2 cells. However, the 0.2-, 0.5- and 0.8-kb DNAs of Cyp6a8 showed 13-24-, 4-5- and 2.2-2.7-fold induction with caffeine, PB and DDT, respectively. These DNAs also showed a 2-3-fold synergistic effect of caffeine and PB but not of caffeine and DDT. The results suggest that the cis-regulatory elements for all three chemicals are located within the -11/-199 DNA of Cyp6a8. Furthermore, caffeine and PB inductions appear to be mediated via different cis-elements, whereas caffeine and DDT induction may involve common regulatory elements. These stably transformed cell lines should help understand the mechanism of resistance-associated Cyp gene overexpression in Drosophila.

Methods for specific immobilization, isolation and labeling of proteins are central to the elucidation of cellular functions. Based on bacterial repressor proteins, which bind to specific target sequences in response to small molecules (macrolide and tetracycline antibiotics) or environmental parameters (temperature), we have developed a set of protein tags (RepTAGs), which enable reversible immobilization of the protein of interest on a solid support for the isolation and quantification as well as for the specific labeling of target proteins with fluorescent dyes for tracking them within a complex protein mixture. Similarly, live mammalian cells were specifically labeled with a fluorescent operator sequence bound to RepTAGs, which were directed towards the cell surface for easy discrimination between transfected and untransfected cell populations. Based on the drug-responsive RepTAG-DNA interactions, it was also possible to quantify or discover antibiotics in environmental samples or compound libraries by means of rapid, sensitive detection methods involving fluorescence polarization and bioluminescence. We believe that the universally applicable RepTAGs will become essential for the analysis and manipulation of proteins in the most diverse areas of protein chemistry and cell biology.

Cytochrome P450 of the 2B subfamily is easily induced by many xenobiotics. In spite of intensive investigations, the molecular mechanisms of regulation of the CYP2B genes are not clear. The nuclear receptor CAR is shown to play a crucial role in the activation of CYP2B genes by xenobiotics, but many problems of CAR activation in different animal species and humans remain unsolved. This review focuses on signaling pathways involved in the control of CYP2B gene expression in mammals.

Available data indicate that there are significant differences in individual susceptibility to lung cancer within the human population. It is believed to be underlie by inherited genetic predispositions related to the genetic polymorphism of several enzymes involved in the detoxification and xenobiotic metabolism. In this review, we collect and discuss the evidence reported up to date on the association between lung cancer and genetic polymorphism of cytochromes P450, N-acetyltransferase, glutathione S-transferases, microsomal epoxide hydrolase, NAD(P)H:quinone oxidoreductase, myeloperoxidase and glutathione peroxidase. All these genes might appear to be candidates for lung cancer susceptibility genes, nevertheless, the present state of the art still offers only a limited explanation of the link between such polymorphisms and increased risk of lung cancer.

We have developed a general profile for the proteins of the TetR family of repressors. The stretch that best defines the profile of this family is made up of 47 amino acid residues that correspond to the helix-turn-helix DNA binding motif and adjacent regions in the three-dimensional structures of TetR, QacR, CprB, and EthR, four family members for which the function and three-dimensional structure are known. We have detected a set of 2,353 nonredundant proteins belonging to this family by screening genome and protein databases with the TetR profile. Proteins of the TetR family have been found in 115 genera of gram-positive, alpha-, beta-, and gamma-proteobacteria, cyanobacteria, and archaea. The set of genes they regulate is known for 85 out of the 2,353 members of the family. These proteins are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity. The regulatory network in which the family member is involved can be simple, as in TetR (i.e., TetR bound to the target operator represses tetA transcription and is released in the presence of tetracycline), or more complex, involving a series of regulatory cascades in which either the expression of the TetR family member is modulated by another regulator or the TetR family member triggers a cell response to react to environmental insults. Based on what has been learned from the cocrystals of TetR and QacR with their target operators and from their three-dimensional structures in the absence and in the presence of ligands, and based on multialignment analyses of the conserved stretch of 47 amino acids in the 2,353 TetR family members, two groups of residues have been identified. One group includes highly conserved positions involved in the proper orientation of the helix-turn-helix motif and hence seems to play a structural role. The other set of less conserved residues are involved in establishing contacts with the phosphate backbone and target bases in the operator. Information related to the TetR family of regulators has been updated in a database that can be accessed at www.bactregulators.org.

The presence of four nucleotide changes and a three base-pair deletion in the GABA A alpha6-subunit promoter is described in Sardinian alcohol non-preferring rats, selectively bred for their ethanol aversion. These mutations are associated with the R100Q alpha6 intragenic mutation that was previously characterized in the same animals. The possibility that these mutated nucleotides alter the ethanol-induced upregulation of the alpha6 gene was investigated by measuring cerebellar alpha6 mRNA levels after a chronic ethanol liquid diet in sNP rat. Real-time quantitative PCR showed an increased alpha6 gene expression after ethanol ingestion in normal and mutated rats. However, lower amounts of alpha6 mRNA levels were detected both in control and in ethanol-treated sNP rats carrying the five promoter and the intragenic mutations in a homozygous state. Using the electromobility shift assay, specific DNA binding sites were found in cerebellar extracts of the alpha6 regions comprising the five mutations. These results suggest that one or more of the mutated binding sites that were found in the 5' flanking alpha6 region may be a consensus sequence for regulatory factors which are responsible for both basal and ethanol-induced alpha6 gene expression.

Induction of drug metabolism was described more than 40 years ago. Progress in understanding the molecular mechanism of induction of drug-metabolizing enzymes was made recently when the important roles of the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), two members of the nuclear receptor superfamily of transcription factors, were discovered to act as sensors for lipophilic xenobiotics, including drugs. CAR and PXR bind as heterodimeric complexes with the retinoid X receptor to response elements in the regulatory regions of the induced genes. PXR is directly activated by xenobiotic ligands, whereas CAR is involved in a more complex and less well understood mechanism of signal transduction triggered by drugs. Most recently, analysis of these xenobiotic-sensing nuclear receptors and their nonmammalian precursors such as the chicken xenobiotic receptor suggests an important role of PXR and CAR also in endogenous pathways, such as cholesterol and bile acid biosynthesis and metabolism. In this review, recent findings regarding xenosensors and their target genes are summarized and are put into an evolutionary perspective in regard to how a living organism has derived a system that is able to deal with potentially toxic compounds it has not encountered before.

Microsomal glutathione transferase (MGST1) and prostaglandin E synthase (PGES) are both members of the MAPEG (Membrane Associated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily. In humans, their organ distribution is quite distinct with the former being widely and constitutively expressed whereas PGES is largely inducible. In order to study the basal expression of these genes, we characterized the promoter regions and identified the elements and the transcription factors required using in vitro assays, including reporter analysis of deletion and mutant clones and EMSA. The results indicate that Sp1 is the protein mediating the basal transcription of MGST1. It appears that both the Sp1 and Sp3 proteins are important for the basal expression of PGES. In addition, mutational analysis of two Barbie-box elements in the PGES promoter showed that these were not involved in the down-regulation of PGES by phenobarbital (PB). These results provide the first description of the basal regulation of these genes in humans.

In Drosophila, the insecticide resistant 91-R strain is an overproducer and susceptible 91-C and ry(506) strains are the underproducers of CYP6A8 mRNA encoded by a cytochrome P450 gene, Cyp6a8. Low expression of Cyp6a8 in the underproducer strains is due to a downregulatory effect of a putative repressor locus, which is thought to be mutant in the overproducer strain. In the present investigation, organization of Cyp6a8 and promoter activity of its upstream DNA were analyzed. Cyp6a8 has two introns of which intron II is similar to the introns of other insect CYP genes with respect to its length and position. Intron I is only 36 bp long and lacks consensus splice sites. It is also in-frame with the CYP6A8 open reading frame. Therefore, inefficient splicing of intron I may produce two isoforms of CYP6A8. Analysis of Cyp6a8 upstream DNA of the overproducer 91-R strain showed that DNA sequences between -199 and -761 bp are required for the highest constitutive and barbital-induced expression of Cyp6a8. This region has six barbie boxes and binding sites for various transcription factors. Promoter activity of the -11/-761 DNA of the overproducer 91-R strain was found to be 4-fold lower in the genome of underproducer ry(506) strain, which is wild type for the putative repressor gene, than in the genome of F1 hybrids of 91-R and ry(506) strains. These results suggest that -11/-761 Cyp6a8 DNA of the 91-R strain can respond to the active repressor present in the hybrid genome and further support our previous findings that overexpression of Cyp6a8 is a result of mutation of a repressor gene rather than mutation of the cis-regulatory sequences.

The positive element (PE) (-69 to -98 bp) within the 5'-proximal region of the CYP2B1/B2 gene (+1 to -179 bp) of rat liver is essential for phenobarbitone (PB) response and gives a single major complex with the rat liver cytosol in gel shift analysis. This complex corresponds to complex I (top) of the three complexes given by the nuclear extracts. PB treatment of rats leads to a decrease in complex I formation with the cytosol and PE and an increase in the same with the nuclear extract in gel shift analysis. Both the changes are counteracted by simultaneous okadaic acid administration. The nuclear protein giving rise to complex I has been isolated and has an M(r) of 26 kDa. The cytosolic counterpart consists of two species, 26 and 28 kDa, as revealed by Southwestern blot analysis using labeled PE. It is concluded that PB treatment leads to the translocation accompanied by processing of the cytosolic protein species into the nucleus that requires protein dephosphorylation. It is suggested that PB may exert a global regulation on the transcription of many genes by modulating the phosphorylation status of different protein factors involved in transcriptional regulation.

Primary structural information including the complete nucleotide sequence of the first insect aldehyde oxidase (AO) was obtained from the common house mosquito Culex quinquefasciatus (Say) through cloning and sequencing of both genomic DNA and cDNA. The deduced amino-acid sequence encodes a 150-kDa protein of 1266 amino-acid residues, which is consistent with the expected monomeric subunit size of AO. The Culex AO sequence contains a molybdopterin cofactor binding domain and two iron-sulfur centres. A comparison of the partial sequences of AO from insecticide resistant and susceptible strains of C. quinquefasciatus shows two distinct alleles of this enzyme, one of which is amplified in the insecticide resistant strain on a 30-kb DNA amplicon alongside two resistance-associated esterases. The amplified AO gene results in elevated AO activity in all life stages, but activity is highest in 3rd instar larvae. The elevated enzyme can be seen as a separate band on polyacrylamide gel electrophoresis. The role of AO in xenobiotic oxidation in mammals and the partial inhibition of elevated AO activity by a range of insecticides in Culex, suggest that this AO may play a role in insecticide resistance.

Phenobarbital (PB) response elements are composed of various nuclear receptor (NR)-binding sites. A 51-bp distal element PB-responsive enhancer module (PBREM) conserved in the PB-inducible CYP2B genes contains two NR-binding direct repeat (DR)-4 motifs. Responding to PB exposure in liver, the NR constitutive active receptor (CAR) translocates to the nucleus, forms a dimer with the retinoid X receptor (RXR), and activates PBREM via binding to DR-4 motifs. For CYP3A genes, a common NR site [DR-3 or everted repeat (ER)-6] is present in proximal promoter regions. In addition, the distal element called the xenobiotic responsive module (XREM) is found in human CYP3A4 genes, which contain both DR-3 and ER-6 motifs. Pregnane X receptor (PXR) could bind to all of these sites and, upon PB induction, a PXR:RXR heterodimer could transactivate XREM. These response elements and NRs are functionally versatile, and capable of responding to distinct but overlapping groups of xenochemicals.

The P450 monooxygenases of insects are important in the metabolism of numerous endogenous and exogenous compounds. However, identity of the P450 isoform(s) involved in these reactions is rarely known. A critical first step in the identification of important P450s is the cloning and sequencing of their genes. Toward this goal we report the genomic sequence of a new cytochrome P450, termed CYP6D3, from the house fly, Musca domestica. CYP6D3 is part of a P450 gene cluster located on chromosome 1 and is located upstream of a related gene, CYP6D1. The similar genetic structures of CYP6D3 and CYP6D1 (5 exons and 4 introns of similar length) suggest one of these genes may have been the result of a duplication event. The CYP6D3 deduced amino acid sequence indicates a protein with 518 amino acids and a molecular weight of 59.3 kDa. The CYP6D3 protein is most similar to house fly CYP6D1 (78%) and Cyp6D2 (56%) from Drosophila melanogaster. The deduced amino acid sequences of CYP6D3 and CYP6D1 are identical at the Helix I and heme binding regions.

Hlx is a homeobox transcription factor gene required for normal intestinal and hepatic growth in development. We previously found high sequence identity and 17 conserved consensus cis-regulatory/transcription factor binding elements in the mouse and human Hlx 5' regions. A 594 bp sequence in the Hlx 5' region possessing the same activity in driving luciferase expression as larger Hlx 5' sequences had three segments each necessary but not sufficient for luciferase expression in NIH 3T3 cells (which express Hlx). Nine of the conserved putative regulatory elements are positioned within these segments, including two CCAAT boxes on opposite strands within a conserved 44 bp inverted repeat sequence. To test the hypothesis that these elements are required for promoter activity, we compared the reporter expression activity of segments containing mutations of these elements with activity of the parent Hlx promoter sequence. We found that mutation of either CCAAT box or a conserved AP-2 site resulted in a significant decrease in promoter activity. Restoration of the inverted repeat with complementary mutations of both CCAAT boxes did not restore activity. Further, mutation of other portions of the inverted repeat did not affect promoter activity. Mutation of other elements had no effect on promoter activity.

The gene of a fatty-acid hydroxylase of the fungus Fusarium oxysporum (P450foxy) was cloned and expressed in yeast. The putative primary structure revealed the close relationship of P450foxy to the bacterial cytochrome P450BM3, a fused protein of cytochrome P450 and its reductase from Bacillus megaterium. The amino acid sequence identities of the P450 and P450 reductase domains of P450foxy were highest (40.6 and 35.3%, respectively) to the corresponding domains of P450BM3. Recombinant P450foxy expressed in yeast was catalytically and spectrally indistinguishable from the native protein, except most of the recombinant P450foxy was recovered in the soluble fraction of the yeast cells, in marked contrast to native P450foxy, which was exclusively recovered in the membrane fraction of the fungal cells. This difference implies that a post (or co)-translational mechanism functions in the fungal cells to target and bind the protein to the membrane. These results provide conclusive evidence that P450foxy is the eukaryotic counterpart of bacterial P450BM3, which evokes interest in the evolutionary aspects concerning the P450 superfamily along with its reducing systems. P450foxy was classified in the new family, CYP505.

Extrahepatic tissue distribution of the mRNAs for the four human CYP2Cs (2C8, 2C9, 2C18, and 2C19) was examined in kidney, testes, adrenal gland, prostate, brain, uterus, mammary gland, ovary, lung, and duodenum. CYP2C mRNAs were detected by RT-PCR using specific primers for each individual CYP2C. CYP2C8 mRNA was detected in the kidney, adrenal gland, brain, uterus, mammary gland, ovary, and duodenum. CYP2C9 mRNA was detected in the kidney, testes, adrenal gland, prostate, ovary, and duodenum. CYP2C18 mRNA was found only in the brain, uterus, mammary gland, kidney, and duodenum and CYP2C19 mRNA was found only in the duodenum. Immunoblot analysis of small intestinal microsomes detected both 2C9 and 2C19 proteins. In addition, genomic clones for CYP2C8 were sequenced, and long-distance PCR was performed to determine the complete gene structure. CYP2C8 spanned a 31 kb region. Comparative analysis of the 2.4 kb upstream region of CYP2C8 with CYP2C9 revealed two previously unidentified transcription factors sites, C/EBP and HPF-1, and the latter might be involved in hepatic expression. Although CYP2C8 has been shown to be phenobarbital inducible, neither a barbiturate-responsive regulatory sequence (a Barbie box) nor a phenobarbital-responsive enhancer module (PBREM) was found within the upstream region analyzed.

Cytochromes P-450 (P-450) are members of a multigene superfamily of hemoproteins consisting the microsomal monooxygenase system with NADPH P-450 reductase (reductase) and/or reducing equivalents. Expression of many P-450 isoforms in hepatocytes is shown to be regulated at the level of transcription through interaction between cis-acting elements in the genes and DNA-binding (transacting) factors. Some isoforms of the CYP1A, 2B, 2E, and 3A subfamilies are regulated at the posttranscriptional level. For the topology of P-450 and reductase molecules in ER membrane of hepatocytes, models from stopped flow analysis and electron spin resonance are proposed. The densities of total P-450 and reductase molecules are revealed to be high enough to support the cluster model, suggesting that about ten P-450 molecules form an aggregate and surround one reductase molecule, and therefore the two enzymes form large micelles. ER proliferation after PB administration, which had been correlated with increase in P-450 level, is shown to be probably independent of the increase in P-450 level. There are considerable discrepancies among results reported on sublobular expression of various P-450 isoforms. Causes of the discrepancies are likely to be differences in experimental conditions of histochemical detection carried out and/or in species, strain, and/or sex.

We have cloned two complementary DNAs (cDNAs), RL-Mtx-1 and RL-Mtx-2, corresponding to the bile acid- sensitive methotrexate carrier from rat liver by direct full-length rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) using degenerated primers that were deduced from published sequences of tumor cell methotrexate transporters. When expressed in Xenopus laevis oocytes and cosM6 cells, both clones mediate methotrexate and bumetanide transport. RL-Mtx-1 consists of 2,445 bp with an open reading frame of 1,536 bp. The corresponding protein with 512 amino acids has a molecular weight of 58 kd. RL-Mtx-2 (2,654 bp) differs by an additional insert of 203 bp. This insert is located in frame at position 1,196 of the RL-Mtx-1 and contains the typical splice junction sites at the 5' and 3' end, indicating that the RL-Mtx-2 messenger RNA (mRNA) is generated by alternative splicing. The insert contains a stop codon that shortens the RL-Mtx-2 protein to 330 amino acids (38 kd). Both cDNAs contain the binding site sequence for the dioxin/nuclear translocator responsive element (Ah/Arnt-receptor) in conjunction with a barbiturate recognition sequence (Barbie box). Preliminary results show that the Barbie box acts as a negative regulatory element. The two liver cDNA clones show homologies to the published sequences of folate and the reduced folate carriers, but no homology is found to the transport systems for organic anions like the Ntcp1, oatp1, OAT-K1, and OAT1. Expression of the mRNA for the methotrexate carrier is found in liver, kidney, heart, brain, spleen, lung, and skeletal muscle, but not in the testis as revealed by Northern blot analysis. The highest abundance of the mRNA is found in the kidney.

The phenobarbital-inducible rat cytochrome P450 (CYP) 2B1 and 2B2 proteins are encoded by homologous genes whose promoters contain a mammalian-apparent long terminal repeat retrotransposon (MaLR). An NF-kappaB-like site within the MaLR forms multiple protein-DNA complexes with rat liver and HeLa cell nuclear extracts. Using antibody supershift assays, we have identified these complexes as NF-kappaB and RPB-Jkappa/CBF1. Competition assays using a series of single site mutant oligonucleotides reveal that the recognition sites for these two factors overlap. We also show that the CYP2B1/2 NF-kappaB element, but not the Igkappa NF-kappaB element, can repress transcription in vitro when positioned upstream of the heterologous adenovirus major late core promoter. In addition, RBP-Jkappa over-expressed in COS-7 cells repressed expression in vivo from an SV40-luciferase reporter construct that contained the CYP2B1/2 NF-kappaB element. Finally, we observe similar levels of NF-kappaB and RBP-Jkappa binding activities in nuclear extracts prepared from control and phenobarbital-induced rat livers. The results suggest that RBP-Jkappa/CBF1 binds an atypical NF-kappaB site in the CYP2B1/2 promoters and may help to maintain a low level of expression in the absence of inducer.

The phenobarbitone (PB) responsiveness of the 5'-proximal region of the CYP2B1/B2 gene was examined in detail with plasmid DNA constructs containing G-free cassette as reporter, using in vivo targeting of the same DNA constructs into rat liver as galactosylated-polylysine complexes. The contribution of the proximal region (-1 to -179 bp) and the positive element (-69 to -98 bp) identified earlier in this laboratory to PB responsiveness was assessed. The results obtained on PB treatment of rats subjected to receptor-mediated gene delivery to liver were conclusive and dramatic, with the control (saline-treated) rats manifesting very little expression of the reporter, reflecting the in vivo picture of CYP2B1/B2 gene expression. The positive element conferred PB responsiveness to homologous and heterologous promoters. Deletion of the positive element led to elimination of PB response. The entire -179 bp region was significantly more effective in responding to PB treatment than the region up to -98 bp, both containing one copy of the positive element. Thus, the positive element and its flanking sequences in the 5'-proximal region are involved in conferring PB responsiveness to the CYP2B1/B2 gene.

5-Aminolevulinate synthase (ALA-S) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of the heme biosynthesis. There are two ALA-S isozymes encoded by distinct genes. One gene encodes an isozyme that is expressed exclusively in erythroid cells, and the other gene encodes a housekeeping isozyme that is apparently expressed in all tissues. In this report we examine the mechanisms by which phenobarbital and cAMP regulate housekeeping ALA-S expression. We have determined that cAMP and phenobarbital effects are additive and the combined action is necessary to observe the cAMP effect on ALA-S mRNA in rat hepatocytes. The role of the cAMP-dependent protein kinase (PKA) has been examined. A synergism effect on ALA-S mRNA induction is observed in rat hepatocytes treated with pairs of selective analogs by each PKA cAMP binding sites. A 870-bp fragment of ALA-S 5'-flanking region is able to provide cAMP and phenobarbital stimulation to chloramphenicol O-acetyltranferase fusion vectors in transiently transfected HepG2 cells. ALA-S promoter activity is induced by cotransfection with an expression vector containing the catalytic subunit of PKA. Furthermore, cotransfection with a dominant negative mutant of the PKA regulatory subunit impairs the cAMP analog-mediated increase, but the phenobarbital-mediated induction is not modified. Our data suggest that the transcription factor cAMP-response element binding protein (CREB) is probably involved in PKA induction of ALA-S gene expression. Finally, heme addition greatly decreases the basal and phenobarbital or cAMP analog-mediated induction of ALA-S promoter activity. The present work provides evidence that cAMP, through PKA-mediated CREB phosphorylation, and phenobarbital induce ALA-S expression at the transcriptional level, while heme represses it.

Availability of complete bacterial genomes opens the way to the comparative approach to the recognition of transcription regulatory sites. Assumption of regulon conservation in conjunction with profile analysis provides two lines of independent evidence making it possible to make highly specific predictions. Recently this approach was used to analyze several regulons in eubacteria and archaebacteria. The present review covers recent advances in the comparative analysis of transcriptional regulation in prokaryotes and phylogenetic fingerprinting techniques in eukaryotes, and describes the emerging patterns of the evolution of regulatory systems.

Specific cytochrome P450 enzymes show tissue-specific induction, and different regulatory units for expression of these enzymes have been identified. The regulation of the phenobarbital (PB)-inducible P450 genes has been relatively well characterized in terms of PB induction, but less so with regard to tissue-specific expression. CYP2B2 is not expressed in the rat lung, whereas cytochrome P450 2B1 (CYP2B1) is a dominating enzyme in the same tissue. The constitutive expression of CYP2B1 and CYP2B2 in liver is low, but inducible by PB, whereas the pulmonary expression of CYP2B1 is not induced by PB. This indicates utilization of different regulating mechanisms in the two organs. A gene construct consisting of the structural gene for LacZ coupled to a 1.3-kb 5' fragment of the rat CYP2B1 gene was used to generate transgenic mice in order to further elucidate the mechanism behind tissue-specific expression and PB induction of the CYP2B1 gene. Using reverse transcriptase-polymerase chain reaction on total RNA extracted from lung and liver tissue, a lung-specific transcription of the transgene was observed. Transcription of the construct was also observed in livers from PB-treated transgenic animals. By histochemical staining of lung sections with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), we demonstrated expression at the protein level in bronchiolar cells. In conclusion, our results revealed that the region extending to -1. 3 kb in the 5' flanking region of the CYP2B1 gene included sequences that could partly account for the lung-specific transcription of CYP2B1 and the hepatic induction of CYP2B1 transcription by PB.

A cryptic plasmid (pSR1) isolated from Selenomonas ruminantium HD4 was previously cloned into the HindIII site of pBR322 and a restriction map was constructed using HindIII, ClaI, BamHI, and PvuII (S. A. Martin and R. G. Dean, Appl. Environ. Microbiol. 55(12), 3035-3038, 1989). Analysis of the nucleotide sequence of pSR1 revealed two major open reading frames (ORFs) located in the minus strand at different frames. Analysis of ORF-1 revealed that it has 325 amino acids with a predicted MW of 36,588, and ORF-2 has 379 amino acids with a predicted MW of 42,651. The ORF-1 amino acids showed 30 to 32% sequence homology to the hypothetical protein YtqA in Bacillus subtilis and another hypothetical protein in the thermophilic bacterium Aquifex aeolicus. ORF-2 showed limited homology (23%) to the hypothetical protein ICFG in the photosynthetic cyanobacteria Synechocystis PCC6803.

CYP6D1 is a cytochrome P450 found in the house fly, Musca domestica. Expression is greater in pyrethroid-resistant vs. -susceptible strains and can be induced by phenobarbital in adult susceptible flies. CYP6D1 is expressed only in adult flies. To gain information about possible regulatory elements involved in CYP6D1 expression, and to confirm the gene sequence that was previously determined by polymerase chain reaction (PCR), we screened a house-fly library prepared with genomic DNA from the pyrethroid-resistant LPR strain. A CYP6D1v1 clone was isolated and sequenced. This clone contained 887 nucleotides 5' to the open reading frame and a previously unknown 2.4-kb intron. Using polymerase chain reaction with primers based on the CYP6D1v1 allele, the sequences 5' to the ORF were obtained from five pyrethroid susceptible strains. The transcription initiation site (TIS) was identified at the same position in LPR and two susceptible strains (86 nucleotides upstream from the translation start site). A comparison of the 5' flanking sequences revealed a high degree of similarity for most regions, although differences in the sequences were identified. The possible roles of these sequence differences in regulation of CYP6D1 expression are discussed.

The halotolerant alkane-assimilating yeast Debaryomyces hansenii was examined for P450 alkane hydroxylase genes known to be required for alkane assimilation in Candida. Four distinct P450alk gene segments and an allelic segment were isolated using PCR based on degenerate primers derived from the CYP52 family of alkane-inducible P450 genes. A screen of a genomic library (15-20kb inserts) constructed for this study, using a probe based on the PCR-isolated segments, yielded seven clones. This has led to the isolation and sequence of two full-length genes DH-ALK1 and DH-ALK2. These genes, each with an ORF of 1557 bp (519 aa), contained no apparent introns and showed 64% nucleotide sequence homology (61% based on the deduced amino acid sequences). The deduced proteins had predicted molecular weights of 59,254Da (DH-ALK1) and 59,614Da (DH-ALK2) and have been designated CYP52A12 and CYP52A13 by the P450 Nomenclature Committee. Phylogenetic analysis based on Neighbor Joining Tree showed that DH-ALK1 and DH-ALK2 constitute new genes located on two distinct branches and are most related to the gene CYP52A3 (60% deduced aa homology) and are least related to the gene CYP52C2 (41% deduced aa homology), both of C. maltosa. The isolated genes will provide tools to better understand the diversity of the P450alk family in eukaryotic microorganisms adapted to varied environmental conditions.

The levels of one or more cytochrome P450 (CYP) enzymes and the respective mRNAs are found to be higher in insecticide-resistant insects than in susceptible insects. To understand better how insects regulate the levels of CYPs, we examined the expression of the Cyp6a2 gene in various strains of Drosophila melanogaster. We also took a transgenic approach to understand the molecular mechanisms that are involved in strain variation of Cyp6a2 expression. RNA blot analysis showed that the constitutive expression of Cyp6a2 varies from strain to strain; the level of CYP6A2 mRNA is barely detectable in the underproducer ry506 strain, whereas it is very high in the overproducer 91-R and MHIII-D23 strains. The long terminal repeat (LTR) of mobile element 17.6 that is found in the 3' untranslated region (UTR) of the Cyp6a2 gene of some strains does not appear to have any role on the steady-state CYP6A2 mRNA level. We also found that the Cyp6a2 gene is inducible by barbital in 91-R, ry506 as well as 91-C, which carries an LTR insertion. To examine the genetic background of the underproducer ry506 strain with respect to Cyp6a2 expression, we transformed the ry506 strain with the Cyp6a2 allele of the overproducer 91-R strain (Cyp6a2-91 R) and measured the constitutive and barbital-induced expression of the Cyp6a2-91 R transgene in the transformed flies. The Cyp6a2-91 R transgene carrying 129 bp of DNA upstream of the ATG codon did not show any constitutive or barbital-induced expression in the ry506 host genome. However, transgenes with 1331 and 985 bp upstream DNA showed similar levels of constitutive expression that were higher than that of the endogenous Cyp6a2 gene of the ry506 host strain, but lower than the expression of the same gene in the 91-R strain. Both these transgenes, with 1331 and 985 bp upstream DNA, also showed induction with 0.1 M barbital. DNA sequence analysis revealed that in both 91-R and ry506, the upstream DNA between +1 and -985 bp contains a distal and a proximal group of three potential barbie boxes, i.e. cis-elements that are thought to be involved in barbiturate-mediated induction of CYP genes. Except for four bases located near the distal cluster of barbie boxes and two other bases, the base sequence of the upstream DNA is identical in ry506 and 91-R strains. These results suggest that the underproducer ry506 strain has the trans-regulatory factors to support constitutive and induced expression of the Cyp6a2-91 R allele carrying DNA between -129 and -1331 bp regions. Possible reasons for low constitutive expression of the endogenous Cyp6a2 gene and moderate level of expression of the Cyp6a2-91 R allele in the ry506 genetic background are discussed.

To study the effect of induction we gave six male volunteers 10 mg nortriptyline three times a day for 4 weeks and 0.2 gm pentobarbital on days 8 to 21. Plasma and urinary levels of nortriptyline and metabolites were measured. The rate and extent of induction of the enzyme(s) were estimated by a model with use of nortriptyline concentrations. There was a marked decrease of nortriptyline levels after 2 days of pentobarbital treatment. Total clearance of nortriptyline increased more than twofold (range, 1.6-fold to 4.1-fold). Apparent metabolic clearance by 10-hydroxylation increased markedly. The decrease in nortriptyline levels was more rapid than the increase after pentobarbital cessation, fitting with the theory of the model. The induction of nortriptyline metabolism is probably mainly the result of an increase in a non-CYP 2D6 P450 isozyme, possibly CYP 3A4 or a CYP 2C form. More knowledge of induction characteristics of drugs should lead to better predictions of decreased effects and appearance of adverse effects. The kinetic model used for analysis of our data could then be useful.

The rat can be protected against aflatoxin B1 (AFB1) hepatocarcinogenesis by being fed on a diet containing the synthetic antioxidant ethoxyquin. Evidence suggests that chemoprotection against AFB1 is due to increased detoxification of the mycotoxin by one or more inducible drug-metabolising enzymes. The glutathione S-transferase (GST) isoenzymes in rat liver that contribute to ethoxyquin-induced chemoprotection against AFB1 have been identified by protein purification. This approach resulted in the isolation of several heterodimeric class alpha GST, all of which contained the A5 subunit and possessed at least 50-fold greater activity towards AFB1-8,9-epoxide than previously studied transferases. Molecular cloning and heterologous expression of rat GSTA5-5 has led to the demonstration that it exhibits substantially greater activity for AFB1-8,9-epoxide than other rat transferases. The A5 homodimer can also catalyse the conjugation of glutathione with other epoxides, such as trans-stilbene oxide and 1,2-epoxy-3-(4'-nitrophenoxy)propane, and possesses high catalytic activity for the reactive aldehyde 4-hydroxynonenal. Western blotting has shown that the A5 subunit is not only induced by ethoxyquin but that it is also induced by other cancer chemopreventive agents, such as butylated hydroxyanisole, oltipraz, benzyl isothiocyanate, indole-3-carbinol and coumarin. In addition to GSTA5, we have identified a novel aflatoxin-aldehyde reductase (AFAR) that is similarly induced by ethoxyquin. However, immunoblotting has shown that GSTA5 and AFAR are not always co-ordinately regulated by chemoprotectors. In order to gain a better understanding of the mechanisms responsible for the induction of GSTA5 protein, the GSTA5 gene has been cloned. It was isolated on two overlapping bacteriophage lambda clones and found to be approximately 12 kb in length. The transcriptional start site of GSTA5 has been identified 228 bp upstream from the ATG translational initiation codon. Computer-assisted analysis of the upstream sequence has indicated the presence of a putative antioxidant responsive element (located between -421 and -429 bp) which may be responsible for the induction of GSTA5 by chemopreventive agents.

Gene expression of the canalicular conjugate transporter mrp2 is inducible by treatment with the DNA-damaging agents 2-acetylaminofluorene (50 and 100 microM), and cisplatin (20 microM) in primary rat hepatocytes as well as in the rat hepatoma cell line H4IIE. Furthermore, phenobarbital (1 and 2 mM) induces mrp2 gene expression, probably explaining the increase in bile-salt-independent bile flow caused by phenobarbital, while the cholestatic drug ethinyl estradiol (10(-6) M) leads to an increase in mrp2 mRNA but decreases Mrp2 protein level probably via a posttranscriptional mechanism. The 5'-flanking region of the rat mrp2 gene was sequenced and cloned into a luciferase reporter vector. Transient transfection assays with reporter vectors containing unidirectionally deleted 5'-flanking regions using H4IIE cells indicate that two different sequences of 17 and 37 bases comprising a Y-Box and a GC-Box are required for mrp2 gene basal expression. Sequences mediating 2-AAF induction are located within a region 250 bases upstream of the translation start site while the inducing effect of phenobarbital seems to be mediated by another domain located further upstream.

The Bm1P1 protein was previously proposed to act as a positive transcription factor involved in barbiturate-mediated induction of cytochrome P450BM-1 in Bacillus megaterium. We now report that the bm1P1 gene encodes a protein of 217 amino acids, rather than the 98 amino acids as reported previously. In vitro gel shift assays indicate that the Bm1P1 protein did not interact with probes comprising the regulatory regions of the P450BM-1 gene. Moreover, disruption of the bm1P1 gene did not markedly affect barbiturate induction of P450BM-1 expression. A multicopy plasmid harboring only the P450BM-1 promoter region could increase expression of the chromosome-encoded P450BM-1. The level of expression is comparable with that shown by a multicopy plasmid harboring the P450BM-1 promoter region along with the bm1P1 gene. These results strongly suggest that the Bm1P1 protein is unlikely to act as a positive regulator for barbiturate induction of P450BM-1 expression. Finally, deletion of the Barbie box did not markedly diminish the effect of pentobarbital on expression of a reporter gene transcriptionally fused to the P450BM-1 promoter. This suggests that the Barbie box is unlikely to be a key element in barbiturate-mediated induction of P450BM-1.

Hepatic cytochrome P450s play a critical role in the metabolism of hydrophobic xenobiotics. One of the major unsolved problems in xenobiotic metabolism is the molecular mechanism whereby phenobarbital induces hepatic enzymes, particularly CYP2B1 and CYP2B2 in rat liver. By using primary rat hepatocytes for transfection analyses, we previously identified in the CYP2B2 5'-flank a 163-base pair Sau3AI fragment that confers phenobarbital inducibility on a cat reporter gene and that has the properties of a transcriptional enhancer. Transfection experiments with sub-regions of the Sau3AI fragment now indicate that a central core together with an upstream or downstream accessory element within the fragment can confer phenobarbital responsiveness. One such accessory element, AF1, was identified and localized. DNase I footprinting analysis revealed the presence of a footprint overlapping this AF1 element. It also identified three other major protected regions, two of which are putative recognition sites for known transcription factors. Site-directed mutagenesis indicated that a putative glucocorticoid response element as well as a nuclear factor 1 site and an associated nuclear receptor hexamer half-site are essential for conferring maximal phenobarbital inducibility. Taken together, the results indicate that phenobarbital induction of CYP2B2 requires interactions among multiple regulatory proteins and cis-acting elements constituting a phenobarbital response unit.

Hepatic microsomal cytochrome P450 (equivalent to rat P4502B1 isozymic form, a CYPIIB gene product) can be induced by pentobarbital (PB) in the adults of the semiaquatic frog, Rana pipiens (as in other terrestrial vertebrates), but not in adults of the aquatic frog Xenopus laevis or in bluegill sunfish (Lepomis macrochirus). The activity of PB-induced P450 (2B1) towards aldrin and pentoxyresorufin increases respectively by about 2- and 10-fold. This enzyme is not inducible during larval and postlarval stages of R. pipiens. However, cytochrome P4501A1 (CYPIA1 gene product) is inducible by beta-naphthoflavone in all these species. Both CYPIA and CYPIIB genes are expressed, as determined by the catalysis of their protein products, during larval, postlarval, and adult stages of R. pipiens. The concentration of P450 increases slightly during the postlarval stages until the adult stage, ready to migrate to land, is reached. This increase seems to be mostly due to 2B1-type cytochrome P450 as judged by a large increase in aldrin epoxidase but not of 7-ethoxyresorufin O-deethylase activity. It is hypothesized that the evolution of true terrestrialness, and not the evolution of air-breathing lungs alone, is required for the transcriptional activation of CYPIIB gene by PB.

1. We review here the molecular mechanisms underlying the xenobiotic induction of genes encoding cytochrome P450 (CYP) enzymes in the liver and other tissues. We will focus on four major families of CYP genes. 2. Members of the CYP1 gene family are induced by polycyclic aromatic hydrocarbons and this process is mediated by the basic helix-loop-helix proteins: the Ah receptor and its heterodimeric partner Arnt. Considerable progress has been made in elucidating the molecular details of this induction process. 3. CYP4 genes are activated by peroxisomal proliferators, a group of structurally diverse chemicals that also induce peroxisome proliferation. The transcriptional response is dependent on the peroxisome proliferator-activated receptor and its partner RXR, both members of the nuclear receptor superfamily; their role in the induction process has been well characterized at the molecular level. 4. In contrast, the mechanism of gene induction of CYP2 genes by phenobarbital and other structurally diverse inducers is not well understood and a specific phenobarbital-responsive receptor has not been identified. 5. Induction of the CYP3 gene family by the glucocorticoid dexamethasone appears to involve the glucocorticoid receptor, but this receptor is not apparently required for induction by metapyrone and a complete molecular understanding of the induction processes is lacking at present.

Chicken CYP2H1 promoter constructs express strongly in chick embryo hepatocytes at a level comparable with that of Rous sarcoma viral promoter. We have identified the transcription factors responsible for the active CYP2H1 promoter. Binding sites for transcription factors were located within the first 160 bp of promoter sequence using promoter deletion experiments and DNase I footprint analysis. Sequence analysis revealed characteristic sites for the liver-enriched transcription factors of the HNF-1, HNF-3, and C/EBP families and for the ubiquitous factor, USF. Protein binding to these sites was established by gel mobility shift assays. Mutagenesis and transient transfection experiments demonstrated that these sites, in combination, were responsible for the strong promoter activity with a substantial contribution from HNF-1 and HNF-3. The promoter was also active in mammalian HepG2 and COS-1 cell lines where expression was dependent on the identified transcription factor binding sites but promoter activity in the HeLa cells was low. Transactivation experiments revealed that promoter expression could be activated through the appropriate binding sites by exogenously expressed rat HNF-1alpha or HNF-1beta, rat HNF-3alpha or HNF-3beta and chicken C/EBP alpha. Transcriptional synergism between HNF-1 and C/EBP was observed in these transactivation experiments. A Barbie box-like sequence overlapped the USF element but was not functional. The results demonstrate that liver-enriched transcription factors and USF direct strong expression of the CYP2H1 promoter in transiently transfected cells. By comparison, in vivo expression of this gene in uninduced chick embryo hepatocytes is low but markedly increased by phenobarbital. Drug induction may therefore substantially reflect derepression of this inherently active promoter.

The cytochrome P450 gene Cyp6a2 from Drosophila melanogaster is located on the right arm of chromosome 2 at position 43A1-2 and comprises two exons separated by a 69-bp intron. Phenobarbital treatment of flies leads to a rapid increase in the level of CYP6A2 mRNA and to an increased production of the CYP6A2 protein. DNA from the Cyp6a2 promoter region was functional when linked to a luciferase reporter gene and transfected into D. melanogaster Schneider cells. Moreover, a dose-dependent induction of luciferase activity by phenobarbital indicated that elements necessary for phenobarbital induction are located within 428 bp of the translation start site. Heterologous expression of the CYP6A2 protein in lepidopteran cells infected with a Cyp6a2-recombinant baculovirus was observed by Western blotting of cell lysates and by spectral characterization of the reduced-CO complex of the P450. The CYP6A2 protein produced in this system metabolized aldrin and heptachlor to their epoxides and metabolized the insecticide diazinon by desulfuration to diazoxon and by oxidative ester cleavage to 2-isopropyl-4-methyl-6-hydroxypyrimidine. Metabolism in lysates of cells infected with recombinant baculovirus was greatly enhanced by the addition of purified housefly NADPH cytochrome P450 reductase and cytochrome b5. These results show that CYP6A2 catalyzes the metabolism of organophosphorus insecticides and they implicate Cyp6a2 overexpression in metabolic resistance. The Cyp6a2 gene appears to be a suitable model for a genetic analysis of the phenobarbital induction process.

Bacillus megaterium contains a soluble cytochrome P450 termed BM-3, which is highly inducible by barbiturates, peroxisome proliferators, and nonsteroidal antiinflammatory drugs. In rats and mice, the chronic administration of peroxisome proliferators induces a sustained oxidative stress in hepatic tissue and may be responsible for the nongenotoxic carcinogenesis observed with prolonged treatment. Here it is shown that ibuprofen induces a variety of enzymes associated with the oxidative stress response in Bacillus, including catalase, glucose-6-phosphate-dehydrogenase, and aldehyde reductase in a dose-related manner. Furthermore, evidence is presented to show that the expression of cytochrome P450 in Bacillus is associated with a marked depletion in cellular glutathione levels and that it renders these cells considerably more sensitive to oxidant insult. Finally, this work reports that a variety of structurally diverse antioxidants such as ascorbic acid, reduced glutathione, alpha-tocopherol acetate and the artificial antioxidant, butylated hydroxyanisole, all dramatically attenuate the expression of the cytochrome P450BM-3 gene and its repressor, Bm3R1, following ibuprofen treatment. These observations provide the first evidence that the expression of cytochrome P450 genes can lead to increased oxidant sensitivity but can be strongly modulated by dietary and artificial antioxidants, as well as antioxidant enzymes. The important implications of this phenomenon are also discussed.

Neuronal nitric oxide synthase (nNOS) is known to share some structural and functional similarities with the cytochrome P450 mixed function oxidase system. Unlike P450, it does not require a second enzyme, reductase, to transfer electrons. This characteristic is similar to P450(BM-3) of Bacillus megaterium. P450(BM-3) and certain mammalian subfamilies of P450, such as P4502B, are known to be induced by phenobarbital (PB), and these P450s share a consensus sequence called the Barbie box. Because of the similarities nNOS shares with P450(BM-3) and other mammalian P450s, we have examined whether nNOS also responds to PB treatment. We have used semi-quantitative PCR, Western blot analysis, a functional assay, and immunohistochemistry in order to answer this question. These data show a threefold increase in nNOS mRNA expression, more modest nNOS protein and activity induction, and no discernible changes in localization of nNOS within the cerebellum following PB treatment.

Heme deficiency precipitated by CoCl2 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 CoCl2 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 CoCl2 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 CoCl2 treatment. The protein mediating this effect has been purified from CoCl2 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.

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.

The level of expression of the Cyp6a2 gene is much higher in the DDT-resistant 91-R strain than in the susceptible 91-C strain of Drosophila melanogaster (Waters et al. (1992b) Proc. Natl. Acad. Sci. USA, 89, 4855-4859). To understand the role of Cyp6a2 and related genes in insecticide resistance, we have isolated and characterized two new Cyp6 genes from the 91-R strain. The polypeptides encoded by these two genes, Cyp6a8 and Cyp6a9, show 77 and 75% amino acid sequence similarity, and 60 and 55% identity with Cyp6a2 of D. melanogaster, respectively. In the genome, Cyp6a8 and Cyp6a9 genes are closely clustered within 4 kb and map at region 51C of the second chromosome. In between them another Cyp gene is present which is more related to Cyp6a9 than to Cyp6a8. The Cyp6a8 gene which is transcriptionally highly active in 91-R, moderately active in ry506 and silent in the 91-C strain hybridizes with 2.0- and 1.8-kb RNAs. Two different-sized RNAs, 2.1 and 1.8 kb, also hybridize with the Cyp6a9 and/or Cyp6a9-related genes. While the level of 2.1-kb RNA is similar in all three strains, the level of 1.8-kb RNA is highest in the 91-R strain and barely detectable in 91-C strain. Transgenic experiments showed that a 8.3-kb BamHI fragment contains the cis-regulatory elements for the expression of both Cyp6a8 and Cyp6a9-related genes. Barbital induces all these genes in all three strains and increases the levels of the two Cyp6a8 transcripts and the 1.8-kb RNA produced by the Cyp6a9 and/or Cyp6a9-related genes. Expression of the Cyp6a8 gene is down-regulated in the hybrids of 91-R and 91-C strains despite the fact that the hybrids carry one copy of the highly active allele of the Cyp6a8 gene of the 91-R strain. Based on these results we propose that the Cyp6a8 gene in 91-C strain may be turned off by an active repressor which might be inhibited by barbital treatment. In the 91-R strain, the putative repressor may be defective, allowing high level of constitutive expression of the Cyp6a8 gene.