The white rot fungus Phanerochaete chrysosporium metabolizes a range of xenobiotics via P450 mono-oxygenation, particularly under peroxidase-suppressing culture conditions. Here we report the cloning and analysis of the gene from this fungus for the cytochrome P450 oxidoreductase (CPR) and its differentially terminated cDNAs. Using a PCR-based approach with degenerate primers, a 285-bp genomic fragment was isolated from the two widely studied strains BKM-F 1767 and ME 446, and was identified as a CPR gene segment based on sequence comparison with the database. A clone containing the full-length CPR gene was isolated from a BKM-F 1767 genomic library using the PCR-generated segment as a probe, and the 3937-bp insert was sequenced by gene walking. Based on the detection of conserved CPR motifs, a coding region of 2381 bp was identified with a 991-bp segment 5' to the putative ATG start codon. Two cDNAs with differentially terminated transcripts were isolated and sequenced. Comparison of the gene and the cDNA sequences confirmed the presence of three introns (62 bp, 50 bp, and 58 bp). Sequence identity and a phylogenetic comparison of the deduced protein (736 aa) with other CPRs in the database suggested that P. chrysosporium CPR is the largest CPR known and is more closely related to animal (36-38%) and yeast (37-38%) CPRs than to plant CPRs (33-35%). The availability of this gene will facilitate further studies on understanding the potent xenobiotic mono-oxygenation systems in this model white rot fungus.

Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T-DNA in which the gene CYP51A1 encoding lanosterol-14-demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments. These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol-14-demethylase (OBT14DM) and LAN14DM) but were resistant to gamma-ketotriazole (gamma-kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. However, these transgenic calli were killed by mixtures of gamma-kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective. Further analysis of the transgenic calli grown in the presence of gamma-kt showed that their delta 5-sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substate obtusifoliol. In contrast, control calli when treated with gamma-kt, displayed a sterol content strongly enriched in 14 alpha-methyl sterols and depressed in physiological delta 5-sterols. When the transgenic calli were cultured in mixtures of gamma-kt and LAN14DM inhibitors sterol compositions enriched in 14 alpha-methyl sterols were obtained, reflecting a strong inhibition of both 'endogenous' OBT14DM and 'exogenous' LAN14DM. Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14-demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.

Environmentally recalcitrant compounds including 2,3,7,8-tetrachlorodibenzo-p-dioxin and hexachlorobenzene are metabolized in mammalian liver via react

Metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone, MTP) is used as an inhibitor of cytochrome P-450 enzymes, particularly those induced by phenobarbital (PB). We examined the effects of MTP on the microsomal dependent mutagenesis of a newly isolated promutagen, 3-(2-chloroethoxy)-1, 2-dichloropropene (CP), three S-chloroallyl thiocarbamate herbicides, and four model promutagens aflatoxin B1 (AFB), 2-acetylaminofluorene (2AAF), 2-aminoanthracene (2AA) and benzo[a]pyrene (BP). Samonella tester strains TA98, TA100 and TA1535 and liver microsomal preparations (S9) from rats induced with PB or Aroclor 1254 (PCB) were employed. For statistical analysis, mutagenesis data were transformed and subjected to two-way analysis of variance. Metyrapone alone was not mutagenic in the absence or presence of S9. In a dose-dependent manner, MTP inhibited mutagenesis of AFB for strains TA98 and TA100 and enhanced mutagenesis of 2AAF, 2AA and BP for these strains. 3-(2-Chloroethoxy) -1,2-dichloropropene and the herbicides diallate, triallate and sulfallate are all chloroallyl ethers. They are similar in their mutagenesis for Salmonella with respect to strain specificity, mutagenic potency, and requirement for activation by specifically-induced microsomes. Metyrapone inhibited the mutagenesis of CP, triallate and sulfallate for strain TA100 in the presence of either PB- or PCB-induced S9, and had no apparent effect on diallate mutagenesis; the same results were obtained for TA1535 with PCB-induced S9. On this basis, the mutagenic activation of diallate appears to be different from that of the other chloroallyl ethers tested. Our results indicate that MTP can inhibit as well as enhance microsomal dependent mutagenesis for Salmonella. We conclude that MTP may be a useful tool in characterizing pathways for promutagen activation.