Title
Isolation and molecular characterisation of the gene encoding eburicol 14α-demethylase (CYP51) from Penicillium italicum
Author
van Nistelrooy, J.G.M.
van den Brink, J.M.
van Kan, J.A.L.
van Gorcom, R.F.M.
de Waard, M.A.
Prins Maurits Laboratorium TNO
Publication year
1996
Abstract
The CYP51 gene encoding eburicol 14α-demethylase (P450(14DM)) was cloned from a genomic library of the filamentous fungal plant pathogen Penicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14α-demethylase from the yeast Candida tropicalis. The nucleotide sequence of a 1739-bp genomic fragment and the corresponding cDNA clone comprises an open reading frame (ORE) of 1545 bp, encoding a protein of 515 amino acids with a predicted molecular mass of 57.3 kDa. The ORF is interrupted by three introns of 60, 72 and 62 bp. The C-terminal part of the protein includes a characteristic haem-binding domain, HR2, common to all P450 genes. The deduced P. italicum P450(14DM) protein and the P450(14DM) proteins from Candida albicans, C. tropicalis and Saccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of the CYP51 family. Multiple copies of a genomic DNA fragment of P. italicum containing the cloned P450 gene were introduced into Aspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P450(14DM) activity, indicating that the cloned gene encodes a functional eburicol 14α-demethylase.
Subject
Biology
14α-demethylase
Cytochrome P450
Fungicides
Penicillium italicum
Sterol biosynthesis inhibitors
Cytochrome p450 isoenzyme
Enilconazole
Etaconazole
Fenarimol
Fungicide
Unclassified drug
Article
Controlled study
Gene isolation
Genetic analysis
Molecular cloning
Molecular genetics
Nonhuman
Penicillium
Priority journal
Sterol synthesis
Yeast
Amino Acid Sequence
Base Sequence
Blotting, Northern
Cloning, Molecular
Cytochrome P-450 Enzyme System
DNA Primers
Fungal Proteins
Fungicides, Industrial
Genes, Fungal
Lanosterol
Molecular Sequence Data
Oxidoreductases
Penicillium
Pyrimidines
Restriction Mapping
Sequence Analysis, DNA
Sequence Homology, Amino Acid
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http://resolver.tudelft.nl/uuid:510be056-261e-41ca-bf1c-de636f736526
DOI
https://doi.org/10.1007/s004380050126
TNO identifier
233310
ISSN
0026-8925
Source
Molecular and General Genetics, 250 (6), 725-733
Document type
article