The green fluorescent protein gene functions as a reporter of gene expression in Phanerochaete chrysosporium

The enhanced green fluorescent protein (GFP) gene (egfp0 was used as a repo rter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter i n Phanerochaete chrysosporium. Four different constructs were prepared. pUG GM3' and pUGiGM3' contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3' and pUMiGM3' contain the P. chrysosp orium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3' untranslated region. In pUGGM3' and pUMGM3', the promoters were fused directly with egfp, whereas in pUGiG M3' and pUMiGM3', following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were in serted at the 5' end of the egfp gene. All constructs were ligated into a p lasmid containing the ural gene of Schizophyllum commune as a selectable ma rker and were used to transform a Ural1 auxotrophic strain of P. chrysospor ium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activi ty. The transformants containing a construct with an intron 5' of the egfp gene (pUGiGM3' and pUMiGM3') exhibited maximal fluorescence under the appro priate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that t he insertion of a 5' intron resulted in more egfp RNA than was found in tra nsformants carrying an intronless egfp. These results suggest that the pres ence of a 5' intron affects the expression of the egfp gene in P. chrysospo rium. The expression of GFP in the transformants carrying pUMiGM3' paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, s uggesting that this construct sill be useful in studying cis-acting element s in the mnp1 gene promoter.