In addition, production of the MnP protein is dependent on, and transcription of the mnp gene is activated by, the presence of Mn(II) in the culture medium ( 7, 8, 15, 19). The production of the idiophasic proteins MnP and LiP is activated by the depletion of nutrient nitrogen in culture ( 13, 19, 33, 40, 49). MnP is an H 2O 2-dependent, heme-containing glycoprotein with an M r of ∼46,000 which oxidizes Mn(II) to Mn(III) ( 6, 21, 38, 40, 47, 48, 50) the latter acts as a diffusible mediator in the oxidation of lignin model compounds ( 6, 16, 50, 51). Under ligninolytic conditions, this fungus secretes two families of peroxidases, lignin peroxidase (LiP) and manganese peroxidase (MnP), along with an H 2O 2-generating system, which are the major components of its extracellular lignin-degrading system ( 16, 19, 21, 28, 30, 51). The white rot basidiomycete Phanerochaete chrysosporium has been the focus of numerous studies of the degradation of the plant cell wall polymer lignin ( 14, 19, 30) and various aromatic pollutants ( 10, 22, 42, 43). 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 studying cis-acting elements in the mnp1 gene promoter. These results suggest that the presence of a 5′ intron affects the expression of the egfp gene in P. 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. The transformants containing constructs with no introns exhibited minimal or no fluorescence. The transformants containing a construct with an intron 5′ of the egfp gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. 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. In pUGGM3′ and pUMGM3′, the promoters were fused directly with egfp, 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. In all constructs, the egfp gene was followed by the mnp1 gene 3′ untranslated region. chrysosporium mnp1 promoter fused upstream of the egfp gene. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3′ and pUMiGM3′ contain the P. 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.
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