high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

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 recom...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 103; no. 27; pp. 10352 - 10357
Main Authors Colot, H.V, Park, G, Turner, G.E, Ringelberg, C, Crew, C.M, Litvinkova, L, Weiss, R.L, Borkovich, K.A, Dunlap, J.C
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 05.07.2006
National Acad Sciences
Subjects
Ascospores
Biological Sciences
Cloning
DNA Primers - genetics
DNA repair
functional genomics
Fungal Proteins - genetics
Fungal Proteins - metabolism
Fungi
Gene Deletion
Gene Expression Regulation, Fungal
Genes
genetic recombination
Genetics
genomics
Hyphae
knockout mutants
Mutagenesis, Insertional - genetics
Mutagenesis, Insertional - methods
Mutation
Mutation - genetics
Neurospora
Neurospora - genetics
Neurospora - growth & development
Neurospora - metabolism
Neurospora crassa
Phenotype
Phenotypes
Polymerase chain reaction
recombination cloning
Sexual development
Transcription factors
Transcription Factors - deficiency
Transcription Factors - genetics
Transcription Factors - metabolism
Yeasts
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Summary: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.
Bibliography:http://www.pnas.org/contents-by-date.0.shtml
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Author contributions: H.V.C., G.P., G.E.T., K.A.B., and J.C.D. designed research; H.V.C., G.P., G.E.T., C.R., C.M.C., and L.L. performed research; H.V.C., G.P., G.E.T., C.R., C.M.C., R.L.W., K.A.B., and J.C.D. contributed new reagents/analytic tools; H.V.C., G.P., G.E.T., and K.A.B. analyzed data; and H.V.C., G.P., G.E.T., K.A.B., and J.C.D. wrote the paper.
Present address: School of Psychology, Georgia Institute of Technology, 654 Cherry Street, Atlanta, GA 30332-0170.
Edited by David D. Perkins, Stanford University, Stanford, CA, and approved March 29, 2006
H.V.C. and G.P. contributed equally to this work.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0601456103