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1.
Curr Genet ; 37(1): 65-73, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10672447

ABSTRACT

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.


Subject(s)
DNA, Complementary/genetics , Genes, Fungal/genetics , NADPH-Ferrihemoprotein Reductase/genetics , Phanerochaete/enzymology , Phanerochaete/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular , Cytochrome P-450 Enzyme System/metabolism , DNA, Complementary/analysis , Flavin Mononucleotide/metabolism , Flavin-Adenine Dinucleotide/metabolism , Gene Library , Humans , Introns/genetics , Molecular Sequence Data , NADP/metabolism , NADPH-Ferrihemoprotein Reductase/chemistry , NADPH-Ferrihemoprotein Reductase/metabolism , Phylogeny , Response Elements/genetics , Sequence Alignment , TATA Box/genetics
2.
Biochem Biophys Res Commun ; 268(2): 345-53, 2000 Feb 16.
Article in English | MEDLINE | ID: mdl-10679206

ABSTRACT

The filamentous fungus Cunninghamella utilizes cytochrome P450 system(s) in the metabolism of a broad range of polyaromatic and aliphatic pollutants and a variety of drugs, but prior attempts at isolation of P450 system components of this fungus have been generally unsuccessful. We report upon the cytochrome P450 oxidoreductase (CPR) gene from two widely studied species, C. elegans and C. echinulata. The C. elegans CPR gene was obtained by screening a genomic library using as probe a PCR amplicon obtained with degenerate primers based on known CPRs. The 2420 bp coding region contained two apparent introns (149 bp and 138 bp). Northern blot analysis showed that the CPR gene is transcriptionally expressed in C. elegans and appears to be inducible by an alkane substrate, n-tetradecane. Phylogenetic comparison of the deduced C. elegans CPR (710 aa) suggested that it is more closely related to animal CPRs (41-42%) than to yeast (38-41%) and plant (35-36%) forms. A 2074 bp sequence containing most of the CPR gene homolog from C. echinulata was also isolated.


Subject(s)
Cunninghamella/genetics , NADPH-Ferrihemoprotein Reductase/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cunninghamella/enzymology , DNA, Fungal/analysis , Introns , Molecular Sequence Data , NADPH-Ferrihemoprotein Reductase/classification , NADPH-Ferrihemoprotein Reductase/metabolism , Phylogeny , Polymerase Chain Reaction , Protein Structure, Tertiary , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Transcription, Genetic
3.
Mutat Res ; 430(1): 109-19, 1999 Nov 29.
Article in English | MEDLINE | ID: mdl-10592322

ABSTRACT

This work tested the hypothesis that the content of spontaneous micronuclei in lymphocytes in an apparently healthy normal human subject, who exhibited an unusually high micronucleus frequency, was non-random. Several DNA probes were used in fluorescent in-situ hybridization (FISH), beginning with a probe generated from the subject's micronuclei. Micronuclei obtained from peripheral blood lymphocytes by microdissection were subjected to random amplification of polymorphic DNA (RAPD-PCR), and a unique PCR product was then used to isolate a cosmid clone from a human genomic library. This clone hybridized to chromosome 2. Subsequently, commercial probes were included in FISH analyses of micronuclei from the subject and age- and sex-matched controls. No significant differences were found between subject and controls in the percentages of micronuclei hybridizing with a centromere probe for the X chromosome or a painting probe for chromosome 3. However, the subject had a very highly significant increase (p<0.0001) in chromosome 2 in micronuclei over a level that might be expected to be present by chance. Characterization of micronuclei may be a promising tool in studies of mechanisms of inherited or induced chromosome instability. The strength of the strategy employed in this study is that, by characterizing the chromosomes present in micronuclei, this work has advanced from an observation of chromosomal instability to a foundation for study of the mechanism underlying the observation.


Subject(s)
Chromosomes, Human, Pair 2/genetics , Micronuclei, Chromosome-Defective/genetics , Adult , Aged , Cells, Cultured , Chromosome Mapping , Chromosomes, Human, Pair 3/genetics , Cosmids/genetics , DNA Probes/genetics , Female , Humans , In Situ Hybridization, Fluorescence , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , X Chromosome/genetics
4.
Gene ; 226(2): 139-46, 1999 Jan 21.
Article in English | MEDLINE | ID: mdl-9931473

ABSTRACT

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.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Genes, Fungal , Mixed Function Oxygenases/genetics , Saccharomycetales/enzymology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cytochrome P-450 CYP4A , DNA, Fungal , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Sequence Homology, Nucleic Acid
5.
Appl Environ Microbiol ; 61(9): 3221-6, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7574631

ABSTRACT

Two mycobacterial strains previously isolated from fossil-fuel-contaminated environments and shown to degrade four- and/or five-ring polycyclic aromatic hydrocarbons were further characterized. The two strains, PYR-I and RJGII-135, had similar growth characteristics, colony morphologies, and scotochromogenic pigmentations. DNA amplification fingerprints obtained with total genomic DNA indicated some strain similarities but with several distinctly different bands. Moreover, phylogenetic analysis based upon essentially full-length 16S rRNA gene sequences separates the two strains as distinct species within the fast-growing group of mycobacteria. Although both strains are thermosensitive, strain PYR-I has the bulged U between positions 184 and 193 characteristic of thermotolerant mycobacteria. Both strains are of potential use for reintroduction into and bioremediation of polycyclic aromatic hydrocarbon-contaminated soils.


Subject(s)
Mycobacterium/genetics , Mycobacterium/metabolism , Phylogeny , Polycyclic Aromatic Hydrocarbons/metabolism , Base Sequence , Biodegradation, Environmental , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Molecular Sequence Data , Mycobacterium/growth & development , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , Soil Pollutants/metabolism , Species Specificity
6.
Plant J ; 7(5): 761-70, 1995 May.
Article in English | MEDLINE | ID: mdl-7773307

ABSTRACT

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.


Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/biosynthesis , Fungal Proteins/biosynthesis , Nicotiana/genetics , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/biosynthesis , Plants, Toxic , Recombinant Fusion Proteins/biosynthesis , Saccharomyces cerevisiae/genetics , Sterols/biosynthesis , Triazoles/pharmacology , Cholestadienols/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Drug Resistance , Fungal Proteins/genetics , Herbicides/pharmacology , Itraconazole/pharmacology , Ketoconazole/pharmacology , Oxidoreductases/genetics , Oxidoreductases/metabolism , Plants, Genetically Modified , Saccharomyces cerevisiae/enzymology , Silanes/pharmacology , Sterol 14-Demethylase
7.
J Ind Microbiol ; 14(1): 10-6, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7766062

ABSTRACT

The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity.


Subject(s)
DNA Fingerprinting/methods , Pseudomonas/isolation & purification , Industrial Microbiology , Species Specificity
10.
Lipids ; 28(11): 963-7, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8277826

ABSTRACT

The identification of the precise structural features of yeast sterol molecules required for the essential "sparking" function has been a controversial area of research. Recent cloning and gene disruption studies in Saccharomyces cerevisiae have shown that C-24 methylation (ERG6), C-5 desaturation (ERG3) and delta 8-delta 7 isomerization (ERG2) are not required, while C-14 demethylation (ERG11) and C-14 reduction (ERG24) are each required for aerobic viability. Earlier observations had indicated that C-14 demethylase deficient strains could be restored to aerobic growth by suppressor mutations that caused a deficiency in C-5 desaturase. These strains were reported to synthesize some ergosterol, indicating that they contained leaky mutations in both ERG11 and ERG3, thereby making it impossible to determine whether the removal of the C-14 methyl group was required for aerobic viability. The availability of the ERG11 and ERG3 genes has been used in this study to construct strains that contain null mutants in both ERG11 and ERG3. Results show that these double disruption strains are viable and that spontaneously arising suppressors of the ERG11 disruption are erg3 mutants. The erg11 mutants of S. cerevisiae are compared to similar mutants of Candida albicans that are viable in the absence of the erg3 lesion.


Subject(s)
Candida albicans/genetics , Cytochrome P-450 Enzyme System/genetics , Oxidoreductases/genetics , Saccharomyces cerevisiae/genetics , Sterols/biosynthesis , Aerobiosis , Candida albicans/growth & development , Cell Division , Cycloheximide/pharmacology , Genes, Fungal/genetics , Genes, Suppressor , Mutation , Saccharomyces cerevisiae/growth & development , Sterol 14-Demethylase , Transformation, Genetic
11.
J Bacteriol ; 175(18): 6062-6, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8376354

ABSTRACT

Eubacterial consensus oligonucleotide primers were used to amplify by polymerase chain reaction the nearly full-length 16S rRNA gene of isolate C7, a gram-negative rod capable of aerobic degradation of azo dyes. The DNA product was cloned and sequenced. Phylogenetic analysis based upon this DNA sequence places C7 within the alpha subdivision of proteobacteria, most closely related to Caulobacter subvibrioides. The phospholipid fatty acid pattern resembles that of caulobacters, with monounsaturated 16- and 18-carbon fatty acids predominating. C7 is unusual in having a monounsaturated branched fatty acid in the phospholipids and exclusively 2-hydroxy fatty acids in the lipid-extracted residue. This organism is of potential use in bioreactors operated for azo dye degradation.


Subject(s)
Azo Compounds/metabolism , Gram-Negative Bacteria/classification , Phylogeny , RNA, Ribosomal, 16S/genetics , Aerobiosis , Base Sequence , Biodegradation, Environmental , Cloning, Molecular , DNA, Bacterial , Gram-Negative Bacteria/genetics , Gram-Negative Bacteria/metabolism , Molecular Sequence Data , Polymerase Chain Reaction
12.
J Biol Chem ; 267(3): 2046-56, 1992 Jan 25.
Article in English | MEDLINE | ID: mdl-1730736

ABSTRACT

The major cytochrome P450 in the yeast Saccharomyces cerevisiae, lanosterol 14 alpha-demethylase (ERG11), catalyzes an essential reaction in the biosynthesis of ergosterol, the predominant sterol of yeast. Protein levels of this cytochrome P450 are known to be affected by carbon source, oxygen, and heme, as well as the growth state of the culture. We have determined that ERG11 message levels increase during growth on glucose, in the presence of heme, and during oxygen limiting growth conditions and, unexpectedly, during anaerobic growth. To determine the cis-acting regions responsible for regulation of expression of the ERG11 promoter under optimal conditions of fermentative growth, deletion analysis was performed using the Escherichia coli lacZ as a reporter gene. Two upstream activating sequences, UAS1 and UAS2, and an upstream repressor element, URS1, plus a second possible or cryptic repressor element, URS2, were identified in the ERG11 promoter. The HAP1 protein product apparently participates in activation from UAS1 but not from UAS2. Sequences resembling ERG11 UAS2 were identified in seven additional oxygen-regulated genes. Repression of ERG11 expression was dependent upon the ROX1 repressor and additional repressor(s) designated as Old (overexpression of lanosterol demethylase). These data indicate that ERG11 is a member of the hypoxic gene family which includes ANB1, COX5b, CYC7, and HEM13. Furthermore, NADPH-cytochrome P450 reductase (CPR1), another component in this P450 system, appears to be coordinately regulated with ERG11.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Genes, Fungal , Oxidoreductases/genetics , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Saccharomyces cerevisiae/genetics , Base Sequence , Chromosome Deletion , Cloning, Molecular , Cytochrome P-450 Enzyme System/isolation & purification , Cytochrome P-450 Enzyme System/metabolism , Escherichia coli/genetics , Genotype , Molecular Sequence Data , Oxidoreductases/isolation & purification , Oxidoreductases/metabolism , Plasmids , RNA, Fungal/genetics , RNA, Fungal/isolation & purification , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Restriction Mapping , Saccharomyces cerevisiae/enzymology , Sequence Homology, Nucleic Acid , Sterol 14-Demethylase , beta-Galactosidase/isolation & purification , beta-Galactosidase/metabolism
13.
J Steroid Biochem Mol Biol ; 43(8): 1107-16, 1992 Dec.
Article in English | MEDLINE | ID: mdl-22217856

ABSTRACT

Eukaryotes characteristically express a cytochrome P450-catalyzed sterol 14α-methyl demethylase as an essential step in the production of membrane sterols. Lanosterol 14α-demethylase of Saccharomyces cerevisiae is the best characterized representative of these enzymes among fungi and provides a model system for the molecular genetic analysis of the reaction. The gene for this P450 and the gene for the S. cerevisiae NADPH-cytochrome P450 reductase have been examined by mutational inactivation and for their regulation of expression. Our results have contributed to a better understanding of sterol biosynthesis in relation to mechanisms of resistance to fungicidal demethylase inhibitors, and promote the rationale for using S. cerevisiae in the further characterization of structure function relationships among sterol 14α-demethylases.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Enzymologic , Lanosterol/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sterol 14-Demethylase/metabolism , 14-alpha Demethylase Inhibitors/pharmacology , Antifungal Agents/pharmacology , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/genetics , Drug Resistance, Fungal , Gene Expression Regulation, Fungal , Mutation , NADPH-Ferrihemoprotein Reductase/genetics , NADPH-Ferrihemoprotein Reductase/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sterol 14-Demethylase/chemistry , Sterol 14-Demethylase/genetics
14.
Yeast ; 7(6): 627-30, 1991.
Article in English | MEDLINE | ID: mdl-1767591

ABSTRACT

DNA sequencing and analysis of genomic DNA using the polymerase chain reaction were used to demonstrate that SOD1 and ERG11 are adjacent genes in Saccharomyces cerevisiae S288c and to establish the correct intergenic sequence of this segment on chromosome VIII.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , DNA, Fungal/chemistry , Oxidoreductases/genetics , Saccharomyces cerevisiae/genetics , Superoxide Dismutase/genetics , Base Sequence , DNA, Fungal/analysis , Electrophoresis, Agar Gel , Molecular Sequence Data , Polymerase Chain Reaction , Restriction Mapping , Saccharomyces cerevisiae/enzymology , Sterol 14-Demethylase
15.
J Biol Chem ; 265(27): 16428-36, 1990 Sep 25.
Article in English | MEDLINE | ID: mdl-2118906

ABSTRACT

The gene coding for the Candida tropicalis NADPH-cytochrome P-450 oxidoreductase (CPR, NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was isolated by immunoscreening of a C. tropicalis lambda gt11 expression library and colony hybridization of a C. tropicalis genomic library. The C. tropicalis CPR gene produces a 2.35-kilobase mRNA transcript, levels of which were shown to be increased 16-fold in cells grown on tetradecane relative to cells grown on glucose as the sole carbon source. A 3-kilobase DNA fragment was sequenced, including 554 and 397 base pairs of 5'- and 3'-noncoding sequence, respectively. A single open reading frame of 2040 base pairs was identified and predicts a 76,683-Da polypeptide of 680 amino acid residues. The deduced C. tropicalis CPR amino acid sequence was compared with each of the CPR sequences reported from other organisms and invariant residues were identified. Multiple pairwise alignments of divergent members of protein families, previously recognized for their sequence similarities in their respective binding domains for FMN, FAD, and NADPH, have allowed identification of a subset of these invariant residues. From these analyses we infer the importance of 25 of the 680 amino acid residues.


Subject(s)
Alkanes/pharmacology , Candida/genetics , Genes, Fungal , NADPH-Ferrihemoprotein Reductase/genetics , Amino Acid Sequence , Animals , Base Sequence , Candida/drug effects , Candida/enzymology , Enzyme Induction , Gene Library , Genes, Fungal/drug effects , Molecular Sequence Data , NADPH-Ferrihemoprotein Reductase/biosynthesis , NADPH-Ferrihemoprotein Reductase/isolation & purification , RNA, Messenger/genetics , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Restriction Mapping , Sequence Homology, Nucleic Acid , Software
16.
Biochem Biophys Res Commun ; 160(3): 1257-66, 1989 May 15.
Article in English | MEDLINE | ID: mdl-2543395

ABSTRACT

Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14 alpha-demethylase. Resistance is restored through complementation by the plasmid-borne wild type gene from either S. cerevisiae or Candida tropicalis. Neither Southern hybridization nor Western immunoblot techniques provided evidence for a second NADPH-cytochrome P450 reductase gene, suggesting that an alternate pathway may provide for the functions of this reductase in S. cerevisiae.


Subject(s)
Cytochrome P-450 Enzyme System , Genes, Fungal , Ketoconazole/pharmacology , NADPH-Ferrihemoprotein Reductase/genetics , Saccharomyces cerevisiae/genetics , Blotting, Western , Candida/genetics , DNA Restriction Enzymes , DNA, Fungal/isolation & purification , Drug Resistance, Microbial/genetics , Leucine/physiology , Nucleic Acid Hybridization , Oxidoreductases/antagonists & inhibitors , Phenotype , Plasmids , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/growth & development , Sterol 14-Demethylase , Transformation, Genetic
17.
Gene ; 76(1): 121-36, 1989 Mar 15.
Article in English | MEDLINE | ID: mdl-2663647

ABSTRACT

The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures are discussed for their possible role in the inducibility of this gene. Expression of the P450alk gene was achieved in Saccharomyces cerevisiae using the yeast alcohol dehydrogenase expression system after removal of the P450alk gene flanking regions. The resultant expressed protein had a molecular mass slightly greater than that of P450alk from C. tropicalis. This alteration did not prevent the function and the localization of P450alk expressed in S. cerevisiae, as this organism showed an acquired microsome-bound activity for the terminal hydroxylation of lauric acid. The deduced P450alk amino acid sequence was compared with members of the nine known P450 gene families. These comparisons indicated that P450alk had a low relationship with these members and was therefore the first member (A1) of a new P450 gene family (LII).


Subject(s)
Candida/genetics , Cytochrome P-450 Enzyme System/genetics , Gene Expression Regulation , Genes, Fungal , Genes , Multigene Family , Algorithms , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Codon , Electronic Data Processing , Genetic Vectors , Molecular Sequence Data , Promoter Regions, Genetic , Restriction Mapping , Saccharomyces cerevisiae/genetics
18.
DNA ; 7(9): 617-26, 1988 Nov.
Article in English | MEDLINE | ID: mdl-3068024

ABSTRACT

We report the nucleotide sequence of the gene and flanking DNA for the cytochrome P450 lanosterol 14 alpha-demethylase (14DM) from the yeast Candida tropicalis ATCC750. An open reading frame (ORF) of 528 codons encoding a 60.9-kD protein is identified. This ORF includes a characteristic heme-binding domain, HR2, common to all P450 proteins. This protein and the 14DM from Saccharomyces cerevisiae share 66.5% identical and 23.1% conservatively replaced amino acids in a 516-amino-acid alignment, and thus are orthologous forms of the P450LIA1 gene. Conversely, C. tropicalis 14DM shares relatively little sequence similarity with P450alk, the predominant P450 protein present when this organism is grown on n-alkanes. Sequence information of these three yeast P450s will be useful for structure-function analyses in the future.


Subject(s)
Candida/genetics , Cytochrome P-450 Enzyme System/genetics , DNA, Fungal/ultrastructure , Genes, Fungal , Oxidoreductases/genetics , Base Sequence , Codon , Saccharomyces cerevisiae/genetics , Sterol 14-Demethylase
19.
Proc Natl Acad Sci U S A ; 85(19): 7221-5, 1988 Oct.
Article in English | MEDLINE | ID: mdl-3050990

ABSTRACT

Proteins from eight eukaryotic families in the cytochrome P-450 superfamily share one region of sequence similarity. This region begins 275-310 amino acids from the amino terminus of each P-450, continues for approximately 170 residues, and ends 35-50 amino acids before the carboxyl terminus. The region can be divided into four domains of sequence similarity, each possessing its own pattern of invariant, conserved, and variable amino acids. The four domains are 56, 20, 59, and 28 residues long and are connected by three shorter segments of limited sequence similarity. The number of residues in these short segments varies with the P-450 protein but ranges from 0 to 20 residues. Consensus sequences based on these similarities can be used to determine whether the sequence of an unidentified peptide resembles that expected for a P-450. Sequence similarities between proteins sometimes reflect constraints imposed by the requirements of a common function. The fourth domain of the P-450s, for example, contains an invariant cysteine that provides the axial thiolate ligand to the heme iron. Other relationships between the four domains and P-450 function can be examined by in vitro mutagenic procedures that alter the conserved amino acids or modify the distance between domains.


Subject(s)
Cytochrome P-450 Enzyme System/analysis , Isoenzymes/analysis , Adrenal Cortex/enzymology , Amino Acid Sequence , Animals , Cattle , Liver/enzymology , Molecular Sequence Data , Pseudomonas/enzymology , Rats , Saccharomyces cerevisiae/enzymology , Structure-Activity Relationship
20.
DNA ; 6(6): 529-37, 1987 Dec.
Article in English | MEDLINE | ID: mdl-3322742

ABSTRACT

We have sequenced the structural gene and flanking regions for lanosterol 14 alpha-demethylase (14DM) from Saccharomyces cerevisiae. An open reading frame of 530 codons encodes a 60.7-kDa protein. When this gene is disrupted by integrative transformation, the resulting strain requires ergosterol and, as expected, grows only in the absence of oxygen. The deduced amino acid sequence of 14DM includes a hydrophobic segment near the amino terminus which may be a transmembrane domain. The deduced sequence has been compared with those of eight other eukaryotic P450s, each from a different family within the P450 superfamily. These comparisons indicate that this yeast gene is the first member of a new P450 family, P450LI. The P450, designated P450LIA1, is more closely related to mammalian P450s than to the bacterial P450cam. In fact, both the yeast P450 and several mammalian P450s have equivalent alignment scores when each is compared with the bovine P450scc. Matrix comparisons of the amino acid sequence of this P450 with those of mammalian P450s reveal three conserved regions. The DNA region 5' to the structural 14DM gene includes poly(dA:dT) sequences and a repeating hexamer sequence.


Subject(s)
Cytochrome P-450 Enzyme System , Genes, Fungal , Oxidoreductases/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Molecular Sequence Data , Saccharomyces cerevisiae/enzymology , Sterol 14-Demethylase , Transcription, Genetic
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