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Versatile EGFP reporter plasmids for cellular localization of recombinant gene products in filamentous fungi

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Abstract.

The recent development of variants of the green fluorescent protein (GFP) with altered codon composition facilitated the efficient expression of this reporter protein in a number of fungal species. In this report, we describe the construction and application of a series of plasmids, which support the expression of an enhanced gfp (egfp) gene in filamentous fungi and assist the study of diverse developmental processes. Included were a promoterless egfp vector for monitoring the expression of cloned promoters/enhancers in fungal cells and vectors for creating translation fusions to the N-terminus of EGFP. The vectors were further modified by introducing a variant hygromycin B phosphotransferase (hph) gene, lacking the commonly found NcoI site. Instead, this site, which contained an ATG start codon, was placed in front of the egfp gene and thus was made suitable for the cloning of translational fusions. The applicability of these vectors is demonstrated by analyzing transcription regulation and protein localization and secretion in two ascomycetes, Acremonium chrysogenum and Sordaria macrospora. In the latter, the heterologous egfp gene is stably inherited during meiotic divisions, as can easily be seen from fluorescent ascospores.

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Fig. 1A–C.
Fig. 2A, B. Fig. 3A, B. Fig. 4A, B. Fig. 5A, B.
Fig. 6.

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Acknowledgements.

The authors wish to thank Silke Giessmann, Ingeborg Godehardt, Swenja Ellßel and Gisela Isowitz for their excellent technical assistance, Gabriele Frenßen-Schenkel for help in the artwork and Drs. Theiß and Mannherz for help and support with confocal laser microscopy. We thank Biochemie AG (Kundl) and RheinBiotech (Hilden) for support throughout this work. U.K. thanks Dr. Inglis for providing plasmid pEGFP/gpd/tel. This work was funded by a grant from the Landesförderung NRW (PTJ-FKZ 001-9910-0v08) and a grant from the Deutsche Forschungsgemeinschaft (SFB 480) to S.P. and U.K.

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Authors and Affiliations

  1. Department for General and Molecular Botany, Ruhr-University Bochum, 44780, Bochum, Germany

    Stefanie Pöggeler, Sandra Masloff, Birgit Hoff, Severine Mayrhofer & Ulrich Kück

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  1. Stefanie Pöggeler
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  2. Sandra Masloff
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  3. Birgit Hoff
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  4. Severine Mayrhofer
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  5. Ulrich Kück
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Correspondence to Ulrich Kück.

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Communicated by S. Hohmann

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Pöggeler, S., Masloff, S., Hoff, B. et al. Versatile EGFP reporter plasmids for cellular localization of recombinant gene products in filamentous fungi. Curr Genet 43, 54–61 (2003). https://doi.org/10.1007/s00294-003-0370-y

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  • DOI: https://doi.org/10.1007/s00294-003-0370-y

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