Fluorescent and bimolecular-fluorescent protein tagging of genes at their native loci in Neurospora crassa using specialized double-joint PCR plasmids

Fungal Genet Biol. 2011 Sep;48(9):866-73. doi: 10.1016/j.fgb.2011.05.002. Epub 2011 May 31.

Abstract

The double-joint polymerase chain reaction (DJ-PCR) is a technique that can be used to construct vectors for targeted genome integration without laborious subcloning steps. Here we report the availability of plasmids that facilitate DJ-PCR-based construction of Neurospora crassa tagging vectors. These plasmids allow the creation of green or red fluorescent protein (GFP or RFP) tagging vectors for protein localization studies, as well as split-yellow fluorescent protein (YFP) tagging vectors for bimolecular fluorescence complementation (BiFC) analyses. We have demonstrated the utility of each plasmid with the tagging of known meiotic silencing proteins. Microscopic analysis of the tagged strains indicates that SMS-2 and QIP form macromolecular complexes in the perinuclear region during meiosis.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Fungal Proteins / genetics*
  • Fungal Proteins / metabolism
  • Gene Targeting / methods*
  • Luminescent Proteins / genetics*
  • Luminescent Proteins / metabolism
  • Neurospora crassa / genetics*
  • Neurospora crassa / metabolism
  • Plasmids / genetics*
  • Plasmids / metabolism
  • Polymerase Chain Reaction / methods*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Fungal Proteins
  • Luminescent Proteins
  • Recombinant Fusion Proteins