A reporter system for analysis of regulatable promoter functions in the basidiomycete fungus Phanerochaete chrysosporium.
To establish a system for functional analysis of regulatable gene promoters involved in lignocellulose degradation by Phanerochaete chrysosporium, a DNA construct was made in which a minimal promoter region, 410 bp of the 5' untranslated region (UTR) of the cbhI.1 gene of P. chrysosporium, was fused to the phlR sequence of Streptoallotiechus hindustanus. This construct was used to transform P. chrysosporium to phleomycin resistance. Southern blot analysis revealed that the incoming DNA was maintained extrachromosomally in the transformants. The donor DNA was methylated by the fungus; inhibition of such methylation allowed transformants to be distinguished from 'false-positive' phlR colonies. Reverse transcriptase-polymerase chain reaction analysis of gene expression revealed that the cbhI.1 5' UTR/phlR sequence construct was transcribed, but that an intron within the cbhI.1 promoter was not excised from transcripts of the transforming DNA construct. Moreover, catabolite repression of cbhI.1 expression is relaxed in the construct. These observations suggest that normal function of the cbhI.1 promoter requires more than 410 bp.
Birch
P R
PR
Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, UK. pbirch@scri.sari.ac.uk
Sims
P F
PF
Broda
P
P
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D015825:Chromosomes, Fungal; D019175:DNA Methylation; D004271:DNA, Fungal; D004352:Drug Resistance, Microbial; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D020075:Phanerochaete / Q000235:genetics*; D010692:Phleomycins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic
D000900:Anti-Bacterial Agents; D004271:DNA, Fungal; D010692:Phleomycins; D001374:Azacitidine