title: A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.
abstract: The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.
authors.affiliation: *Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755.
authors.forename: Hildur V
authors.initials: HV
authors.lastname: Colot
authors.affiliation: Department of Plant Pathology, University of California, Riverside, CA 92521; and.
authors.forename: Gyungsoon
authors.initials: G
authors.lastname: Park
authors.affiliation: Department of Chemistry and Biochemistry, 405 Hilgard Avenue, University of California, Los Angeles, CA 90095.
authors.forename: Gloria E
authors.initials: GE
authors.lastname: Turner
authors.affiliation: *Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755.
authors.forename: Carol
authors.initials: C
authors.lastname: Ringelberg
authors.affiliation: Department of Plant Pathology, University of California, Riverside, CA 92521; and.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Crew
authors.affiliation: Department of Plant Pathology, University of California, Riverside, CA 92521; and.
authors.forename: Liubov
authors.initials: L
authors.lastname: Litvinkova
authors.affiliation: Department of Chemistry and Biochemistry, 405 Hilgard Avenue, University of California, Los Angeles, CA 90095.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Weiss
authors.affiliation: Department of Plant Pathology, University of California, Riverside, CA 92521; and.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Borkovich
authors.affiliation: *Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755; jay.c.dunlap@dartmouth.edu.
authors.forename: Jay C
authors.initials: JC
authors.lastname: Dunlap
mesh_terms: D017931:DNA Primers / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D025301:Hyphae; D016254:Mutagenesis, Insertional / Q000235:genetics / Q000379:methods*; D009154:Mutation / Q000235:genetics; D009491:Neurospora / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D010641:Phenotype; D014157:Transcription Factors / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D005656:Fungal Proteins; D014157:Transcription Factors