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A reporter system for analysis of regulatable promoter functions in the basidiomycete fungus Phanerochaete chrysosporium
Author(s) -
Birch P.R.J.,
Sims P.F.G.,
Broda P.
Publication year - 1998
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.1998.853468.x
Subject(s) - phanerochaete , biology , promoter , chrysosporium , microbiology and biotechnology , gene , reporter gene , untranslated region , dna , gene expression , genetics , messenger rna , enzyme , biochemistry
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 phl R 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’ phl R colonies. Reverse transcriptase‐polymerase chain reaction analysis of gene expression revealed that the cbhI.1 5′ UTR/ phl R 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.