ABSTRACT
The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3′ and pUGiGM3′ contain the P. chrysosporium gpd promoter fused upstream of the egfpcoding region, and pUMGM3′ and pUMiGM3′ contain the P. chrysosporium mnp1 promoter fused upstream of theegfp 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 withegfp, whereas in pUGiGM3′ 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 inserted at the 5′ end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5′ of theegfp gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5′ intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5′ intron affects the expression of theegfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3′ paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studyingcis-acting elements in the mnp1 gene promoter.
Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells
