title: Fluorescent and bimolecular-fluorescent protein tagging of genes at their native loci in Neurospora crassa using specialized double-joint PCR plasmids.
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.
authors.affiliation: Division of Biological Sciences, University of Missouri, Columbia, MO 65211, United States.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Hammond
authors.forename: Hua
authors.initials: H
authors.lastname: Xiao
authors.forename: David G
authors.initials: DG
authors.lastname: Rehard
authors.forename: Erin C
authors.initials: EC
authors.lastname: Boone
authors.forename: Tony D
authors.initials: TD
authors.lastname: Perdue
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Pukkila
authors.forename: Patrick K T
authors.initials: PK
authors.lastname: Shiu
mesh_terms: D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D009492:Neurospora crassa / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins