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An efficient method for gene disruption in Neurospora crassa

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Abstract

The frequency with which transforming DNA undergoes homologous recombination at a chromosomal site can be quite low in some fungal systems. In such cases, strategies for gene disruption or gene replacement must either select against ectopic integration events or provide easy screening to identify homologous site, double-crossover insertion events. A protocol is presented for efficient isolation of Neurospora crassa strains carrying a definitive null allele in a target gene. The protocol relies on the presence of a selectable marker flanking a disrupted plasmid-borne copy of the gene, and in the case presented led to a seven-fold enrichment for putative homologous site replacement events. In addition, a polymerase chain reaction assay is utilized for rapid identification of homologous recombinants among the remaining candidates. This protocol was used to identify 3 isolates, out of 129 primary transformants, which have a disruption in the Neurospora ccg-1 gene. The method should be applicable to a variety of fungal systems in which two selectable markers can be expressed, including those in which homologous recombination rates are too low to allow easy identification of homologous site insertions by the more traditional molecular method of Southern analysis. In addition to disrupting target genes for the purpose of generating null mutations, this method is useful for the targeting of reporter gene fusions to a native chromosomal site for the purpose of studying gene regulation.

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References

  1. Asch DK, Kinsey JA (1990) Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene. Mol Gen Genet 221:37–43

  2. Cambareri EB, Singer MJ, Selker EU (1991) Recurrence of repeatinduced point mutation (RIP) in Neurospora crassa. Genetics 127:699–710

  3. Case ME (1986) Genetical and molecular analyses of qa-2 transformants in Neurospora crassa. Genetics 113:569–587

  4. Connerton IF (1990) Premeiotic disruption of the Neurospora crassa malate synthase gene by native and divergent DNAs. Mol Gen Genet 223:319–323

  5. Cullen D, Leong SA, Wilson LJ, Henner DJ (1987) Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph. Gene 57:21–26

  6. Dhawale SS, Marzluf GA (1985) Transformation of Neurospora crassa with circular and linear DNA and analysis of the fate of the transforming DNA. Curr Genet 10:205–212

  7. Ebbole D, Sachs MS (1990) A rapid and simple method for isolation of Neurospora crassa homocaryons using microconidia. Fungal Genet Newsl 37:17–18

  8. Erlich HA, Gelfland D, Sninsky JJ (1991) Recent advances in the polymerase chain reaction. Science 252:1643–1651

  9. Fincham, JR (1989) Transformation in fungi. Microbiol Rev 53:148–170

  10. Fincham JR (1990) Generation of new functional mutant alleles by premeiotic disruption of the Neurospora crassa am gene. Curr Genet 18:441–445

  11. Frohman MA, Martin GR (1989) Cut, paste, and save: new approaches to altering specific genes in mice. Cell 56:145–147

  12. Grimm C, Kohli J, Murray J, Maundrell K (1988) Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol Gen Genet 215:81–86

  13. Gritz L, Davies J (1983) Plasmid-encoded hygromycin B resistance the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25:179–188

  14. Kim SY, Marzluf GA (1988) Transformation of Neurospora crassa with the trp-1gene and the effect of host strain upon the fate of the transforming DNA. Curr Genet 13:65–70

  15. Kim HS, Smithies O (1988) Recombinant fragment assay for gene targeting based on the polymerase chain reaction. Nucleic Acids Res 16:8887–8903

  16. Kirk KE, Morris NR (1991) The tubB alpha-tubulin gene is essential for sexual development in Aspergillus nidulans. Genes Dev 5:2014–2023

  17. Loros JJ, Denome SA, Dunlap JC (1989) Molecular cloning of genes under control of the circadian clock in Neurospora. Science 243:385–388

  18. Mansour SL, Thomas KR, Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336:348–352

  19. Marathe S, Connerton IF, Fincham JR (1990) Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product. Mol Cell Biol 10:2638–2644

  20. McClung CR, Phillips JD, Orbach MJ, Dunlap JC (1989) New cloning vectors using benomyl resistance as a dominant marker for selection in Neurospora crassa and other filamentous fungi. Exp Mycol 13:299–302

  21. Nickoloff JA, Reynolds RJ (1990) Transcription stimulates homologous recombination in mammalian cells. Mol Cell Biol 10:4837–4845

  22. Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene for β-tubulin from a benomyl-resistance mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461

  23. Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358

  24. Paietta JV, Marzluf GA (1985) Gene disruption by transformation in Neurospora crassa. Mol Cell Biol 5:1554–1559

  25. Porter GP, Brennwald P, Wise JA (1990) U1 small nuclear RNA from Schizosaccharomyces pombe has unique and conserved features and is encoded by an essential single-copy gene. Mol Cell Biol 10:2874–2881

  26. Rhounim L, Rossignol JL, Faugeron G (1992) Epimutation of repeated genes in Ascobolus immersus. EMBO J 11:4451–7

  27. Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–210

  28. Selker EU (1990) Premeiotic instability of repeated sequences in Neurospora crassa. Annu Rev Genet 24:579–613

  29. Selker EU, Garrett PW (1988) DNA sequence duplications trigger gene inactivation in Neurospora crassa. Proc Natl Acad Sci USA 85:6870–6874

  30. Selker EU, Cambareri E, Garrett P, Jensen B, Haack K, Foss E, Turpen C, Singer M, Kinsey J (1989) Use of RIP to inactivate genes of Neurospora crassa. Fungal Genet Newsl 36:76–77

  31. Staben C, Jensen B, Singer M, Pollock J, Schechtman M, Kinsey J, Selker EU (1989) Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation. Fungal Genet Newsl 36:79–81

  32. Vollmer SJ, Yanofsky C (1986) Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci USA 83:4869–4873

  33. Yamashiro CT, Yarden O, Yanofsky C (1992) A dominant selectable marker that is meiotically stable in Neurospora crassa: the amdS gene of Aspergillus nidulans. Mol Gen Genet 236:121–124

  34. Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci USA 81:1470–1474

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Author information

Correspondence to Jennifer J. Loros.

Additional information

Communicated by C. van den Hondel

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Aronson, B.D., Lindgren, K.M., Dunlap, J.C. et al. An efficient method for gene disruption in Neurospora crassa . Molec. Gen. Genet. 242, 490–494 (1994). https://doi.org/10.1007/BF00281802

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Key words

  • Gene disruption
  • Homologous recombination
  • Neurospora
  • ccg-1
  • Null allele