Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of living cells from the filamentous fungus Acremonium chrysogenum

Curr Genet. 2005 Feb;47(2):132-8. doi: 10.1007/s00294-004-0546-0. Epub 2004 Nov 30.

Abstract

Using bimolecular fluorescence complementation assays, we were able to demonstrate protein-protein interaction of the transcription factors AcFKH1 and CPCR1 in living cells from the filamentous fungus Acremonium chrysogenum. This was accomplished by splitting the gene for the enhanced yellow fluorescent protein (EYFP) into two parts encoding the N- and C-terminus. Both fragments were fused to different gene derivatives of the fungal transcription factors. The recombinant plasmids were used to generate transgenic fungal strains for subsequent confocal laser microscopy. Only when the full-length transcription factors were fused to EYFP fragments yellow fluorescence was observed due to the bimolecular complementation of both chimeric proteins. The nuclear localization of the protein-protein interaction was verified by staining fungal cells with the nucleic acid dye TOTO-3.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acremonium / metabolism*
  • Acremonium / ultrastructure
  • Base Sequence
  • Cell Nucleus / metabolism*
  • DNA Primers
  • Microscopy, Fluorescence
  • Plasmids
  • Protein Binding
  • Subcellular Fractions / metabolism
  • Transcription Factors / metabolism*

Substances

  • DNA Primers
  • Transcription Factors