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Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi

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Abstract

Random insertional mutagenesis is an efficient tool for studying molecular mechanisms of many genetically determined processes. An improved variant of this method is REMI (Restriction Enzyme Mediated Integration) mutagenesis. In this method, the insertion cassette is introduced into the recipient cell together with restriction endonuclease. As a result, the REMI cassette insertion occurs in sites recognized by the restriction enzyme. The use of restriction endonucleases enhances transformation rate and provides cassette insertion in virtually any locus. A mutation is tagged by the insertion cassette, which can be identified by isolating the REMI cassette together with the flanking genomic DNA regions. The review describes general requirements to REMI. The mechanisms of REMI mutagenesis are surveyed with special reference to yeast Saccharomyces cerevisiae. Special attention is given to the development and use of REMI for other lower eukaryotes (yeasts and mould fungi). Drawbacks of the method and perspectives of its use are discussed.

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References

  1. Smith, V., Chou, K.N., Lashkari, D., et al., Functional Analysis of the Genes of Yeast Chromosome V by Genetic Footprinting, Science, 1996, vol. 274, pp. 2069–2074.

    Article  PubMed  CAS  Google Scholar 

  2. Schiestl, R.H. and Petes, T.D., Integration of DNA Fragments by Illegitimate Recombination in Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. USA, 1991, vol. 88, pp. 7585–7589.

    Article  PubMed  CAS  Google Scholar 

  3. Burns, N., Grimwade, B., Ross-Macdonald, P.B., et al., Large-Scale Analysis of Gene Expression: Protein Localization. and Gene Disruption in Saccharomyces cerevisiae, Genes Dev., 1994, vol. 8, pp. 1087–1105.

    Article  PubMed  CAS  Google Scholar 

  4. Garfinkel, D.J. and Strathern, J.N., Ty Mutagenesis in Saccharomyces cerevisiae, Methods Enzymol., 1991, vol. 194, pp. 342–361.

    PubMed  CAS  Google Scholar 

  5. Devine, S.E., Boeke, J.D., Integration of the Yeast Retrotransposon Ty1 Is Targeted to Regions Upstream of Genes Transcribed by RNA Polymerase III, Genes Dev., 1996, vol. 10, pp. 620–633.

    Article  PubMed  CAS  Google Scholar 

  6. Ji, H., Moore, D.P., Blomberg, M.A., et al., Hotspots for Unselected Ty1 Transposition Events on Yeast Chromosome III Are Near tRNA Genes and LTR Sequences, Cell, 1993, vol. 73, pp. 1007–1018.

    Article  PubMed  CAS  Google Scholar 

  7. Manivasakam, P., and Schiestl, R.H., Nonhomologous End Joining During Restriction Enzyme-Mediated DNA Integration in Saccharomyces cerevisiae, Mol. Cell. Biol., 1998, vol. 18, pp. 1736–1745.

    PubMed  CAS  Google Scholar 

  8. Goffeau, A., Barrell, B.G., Bussey, H., et al., Life with 6000 Genes, Science, 1996, vol. 274, pp. 563–567.

    Article  Google Scholar 

  9. Lu, S., Lyngholm, L., Yang, G., et al., Tagged Mutations at the Tox1 Locus of Cochliobolus heterostrophus by Restriction Enzyme-Mediated Integration, Proc. Natl. Acad. Sci. USA, 1994, vol. 91, pp. 12649–12653.

    Article  PubMed  CAS  Google Scholar 

  10. Shi, Z. and Leung, H., Genetic Analysis of Sporulation in Magnaporthe grisea by Chemical and Insertional Mutagenesis, Mol. Plant Microbe Interact., 1995, vol. 8, pp. 949–959.

    CAS  Google Scholar 

  11. Bölker, M., Genin, S., Lehmler, C., and Kahmann, R., Genetic Regulation of Mating and Dimorphism in Ustilago maydis, Can. J. Bot., 1995, vol. 73, pp. 320–325.

    Article  Google Scholar 

  12. Xu, S.Y. and Schildkraut, I., Cofactor Requirements of BamHI Mutant Endonuclease E77K and Its Suppressor Mutants, J. Bacteriol., 1991, vol. 173, pp. 5030–5035.

    PubMed  CAS  Google Scholar 

  13. Kahmann, R. and Basse, C., REMI (Restriction Enzyme Mediated Integration) and Its Impact on the Isolation of Pathogenicity Genes in Fungi Attacking Plants, European J. Plant Pathol., 1999, vol. 105, pp. 221–229.

    Article  CAS  Google Scholar 

  14. Kwak, E., Gerald, N., Larochelle, D.A., et al., LvsA, a Protein Related to the Mouse Beige Protein Is Required for Cytokinesis in Dictyostelium, Mol. Cell Biol., 1999, vol. 10, pp. 4429–4439.

    CAS  Google Scholar 

  15. Kuspa, A. and Loomis, W.F., Tagging Developmental Genes in Dictyostelium by Restriction Enzyme-Mediated Integration of Plasmid DNA, Proc. Natl. Acad. Sci. USA, 1992, vol. 89, pp. 8803–8807.

    Article  PubMed  CAS  Google Scholar 

  16. Hastings, P.J., McGill, C., Shafer, B., and Strathern J.N. Ends-in vs Ends-out Recombination in Yeast, Genetics, 1993, vol. 135, pp. 973–980.

    PubMed  CAS  Google Scholar 

  17. Beyert, N., Reichenberger, S., Peters, M., et al., Nonhomologous DNA End Joining of Synthetic Hairpin Substrates in Xenopus laevis Egg Extracts, Nucleic Acids Res., 1994, vol. 22, pp. 1643–1650.

    Article  PubMed  CAS  Google Scholar 

  18. Lehman, C.W., Clemens, M., Worthylake, D.K., et al., Homologous and Illegitimate Recombination in Developing Xenopus Oocytes and Eggs, Mol. Cell Biol., 1993, vol. 13, pp. 6897–6906.

    PubMed  CAS  Google Scholar 

  19. Derbyshire, M.K., Epstein, L.H., Young, C.S., et al., Nonhomologous Recombination in Human Cells, Mol. Cell Biol., 1994, vol. 14, pp. 156–169.

    PubMed  CAS  Google Scholar 

  20. Roth, D.B. and Wilson, J.H., Nonhomologous Recombination in Mammalian Cells: Role for Short Sequence Homologies in the Joining Reaction, Mol. Cell Biol., 1986, vol. 6, pp. 4295–4304.

    PubMed  CAS  Google Scholar 

  21. Goedecke, W., Pfeiffer, P., and Vielmetter, W., Nonhomologous DNA End Joining in Schizosaccharomyces pombe Efficiently Eliminates DNA Double-Strand Breaks from Haploid Sequences, Nucleic Acids Res., 1994, vol. 22, pp. 2094–2101.

    Article  PubMed  CAS  Google Scholar 

  22. Schiestl, R.H., Zhu, J., and Petes, T.D., Effect of Mutations in Genes Affecting Homologous Recombination on Restriction Enzyme-Mediated and Illegitimate Recombination in Saccharomyces cerevisiae, Mol. Cell. Biol., 1994, vol. 14, pp. 4493–4500.

    PubMed  CAS  Google Scholar 

  23. Feldmann, H. and Winnacker, E.L., A Putative Homologue of the Human Autoantigen Ku from Saccharomyces cerevisiae, J. Biol. Chem., 1993, vol. 268, pp. 12895–12900.

    PubMed  CAS  Google Scholar 

  24. Tsukamoto, Y., Kato, J., and Ikeda, H., Hdf1, a Yeast Ku-Protein Homologue, Is Involved in Illegitimate Recombination, but not in Homologous Recombination, Nucleic Acids Res., 1996, vol. 24, pp. 2067–2072.

    Article  PubMed  CAS  Google Scholar 

  25. Brown, D.H., Jr., Slobodkin, I.V., and Kumamoto, C.A., Stable Transformation and Regulated Expression of an Inducible Reporter Construct in Candida albicans Using Restriction Enzyme-Mediated Integration, Mol. Gen. Genet., 1996, vol. 251, pp. 75–80.

    Article  PubMed  CAS  Google Scholar 

  26. Geber, A., Williamson, P.R., Rex, J.H., et al., Cloning and Characterization of a Candida albicans Maltase Gene Involved in Sucrose Utilization, J. Bacteriol., 1992, vol. 174, pp. 6992–6996.

    PubMed  CAS  Google Scholar 

  27. Kurtz, M.B., Cortelyou, M.W., Miller, S.M., et al., Development of Autonomously Replicating Plasmids for Candida albicans, Mol. Cell. Biol., 1987, vol. 7, pp. 209–217.

    PubMed  CAS  Google Scholar 

  28. Cannon, R.D., Jenkinson, H.F., and Shepard, M.G., Isolation and Nucleotide Sequence of an Autonomously Replicating Sequence (ARS) Element Functional in Candida albicans and Saccharomyces cerevisiae, Mol. Gen. Genet., 1990, vol. 21, pp. 210–218.

    Google Scholar 

  29. Hansenula polymorpha: Biology and Applications, Gellissen, G., Ed., Weinheim: Wiley-VCH, 2002.

    Google Scholar 

  30. Van Dijk, R., Faber, K.N., Hammond, A.T., et al., Tagging Hansenula polymorpha Genes by Random Integration of Linear DNA Fragments (RALF), Mol. Genet. Genomics, 2001, vol. 266, pp. 646–656.

    Article  PubMed  CAS  Google Scholar 

  31. Sato, T., Yaegashi, K., Ishii, S., et al., Transformation of the Edible Basidiomycete Lentinus edodes by Restriction Enzyme-Mediated Integration of Plasmid DNA, Biosci. Biotechnol. Biochem., 1998, vol. 62, pp. 2346–2350.

    Article  PubMed  CAS  Google Scholar 

  32. Gould, S.J., McCollum, D., Spong, A.P., et al., Development of the Yeast Pichia pastoris as a Model Organism for a Genetic and Molecular Analysis of Peroxisome Assembly, Yeast, 1992, vol. 8, pp. 613–628.

    Article  PubMed  CAS  Google Scholar 

  33. Johnson, M.A., Waterham, H.R., Ksheminska, G.P., et al., Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris, Genetics, 1999, vol. 4, pp. 1379–1391.

    Google Scholar 

  34. Tuttle, D.L. and Dunn, W.A.Jr., Divergent Modes of Autophagy in the Methylotropic Yeast Pichia pastoris, J. Cell Sci., 1995, vol. 108, pp. 25–35.

    PubMed  CAS  Google Scholar 

  35. Stasyk, O.V., Nazarko, T.Y., Stasyk, O.G., et al., Sterol Glucosyltransferases Have Different Functional Roles in Pichia pastoris and Yarrowia lipolytica, Cell. Biol. Int, 2003, vol. 11, pp. 947–952.

    Article  CAS  Google Scholar 

  36. Klionsky, D.J., Cregg, J.M., Dunn, W.A., Jr., et al., A Unified Nomenclature for Yeast Autophagy-Related Genes, Dev. Cell, 2003, vol. 5, pp. 539–545.

    Article  PubMed  CAS  Google Scholar 

  37. Dunn, W.A., Cregg, J.M., Kiel, J.A.K.W., et al., Pexophagy. The Selective Autophagy of Peroxisomes, Autophagy, 2005, vol. 1, pp. 75–83.

    PubMed  CAS  Google Scholar 

  38. Stromhaug, P.E., Bevan, A., and Dunn, W.A., Jr., GSA11 Encodes a Unique 208-KDa Protein Required for Pexophagy and Autophagy in Pichia pastoris, J. Biol. Chem., 2001, vol. 276, pp. 42422–42435.

    Article  PubMed  CAS  Google Scholar 

  39. Voronovsky, A., Abbas, C., Fayura, L., et al., Development of a Transformation System for the Flavinogenic Yeast Candida famata, FEMS Yeast Res., 2002, vol. 2, pp. 381–388.

    PubMed  CAS  Google Scholar 

  40. Dmytruk, K., Abbas, C., Voronovsky, A., et al., Cloning of Structural Genes Involved in Riboflavin Synthesis of the Yeast Candida famata, Ukr. Biokhim. Zh, 2004, vol. 76, pp. 78–87.

    PubMed  CAS  Google Scholar 

  41. Voronovsky, A.Y., Abbas, C.A., Dmytruk, K.V., et al., Candida famata (Debaryomyces hansenii) DNA Sequences Containing Genes Involved in Riboflavin Synthesis, Yeast, 2004, vol. 21, pp. 1307–1316.

    Article  PubMed  CAS  Google Scholar 

  42. Dmytruk, K.V., Voronovsky, A.Y., and Sibirny, A.A., Insertion Mutagenesis of Candida famata (Debaryomyces hansenii) by Random Integration of Linear DNA Fragments, Current Genet., 2006, vol. 50, pp. 183–191.

    Article  CAS  Google Scholar 

  43. Barth, G. and Gaillardin, C., Yarrowia lipolytica, in Nonconventional Yeasts in Biotechnology, Berlin: Springer, 1996, pp. 313–388.

    Google Scholar 

  44. Gunkel, K., van der Klei, I.J., Barth, G., and Veenhuis, M., Selective Peroxisome Degradation in Yarrowia lipolytica after a Shift of Cells from Acetate/Oleate/Ethylamine into Glucose/Ammonium Sulfate-Containing Media, FEBS Lett., 1999, vol. 451, pp. 1–4.

    Article  PubMed  CAS  Google Scholar 

  45. Mauersberger, S., Wang, H.J., and Gaillardin, C., Insertional Mutagenesis in the Nalkane-Assimilating Yeast Yarrowia lipolytica: Generation of Tagged Mutations in Genes Involved in Hydrophobic Substrate Utilization, J. Bacteriol., 2001, vol. 183, pp. 5102–5109.

    Article  PubMed  CAS  Google Scholar 

  46. Nazarko, T., Mala, M., Nicaud, J.-M., and Sibirny, A., Mutants of the Yeast Yarrowia lipolytica Deficient in the Inactivation of Peroxisomal Enzymes, Proc. Lviv Univ., 2004, vol. 35, pp. 128–136.

    Google Scholar 

  47. Nazarko, T.Y., Huang, J., Nicaud, J.-M., et al., Trs85 Is Required for Macroautophagy, Pexophagy and Cytoplasm to Vacuole Targeting in Yarrowia lipolytica and Saccharomyces cerevisiae, Autophagy, 2005, vol. 1, pp. 37–45.

    PubMed  CAS  Google Scholar 

  48. Yang, G., Turgeon, B.G., and Yoder, O.C., Toxin-Deficient Mutants from a Toxin-Sensitive Transformant of Cochliobolus heterostrophus, Genetics, 1994, vol. 137, pp. 751–757.

    PubMed  CAS  Google Scholar 

  49. Yang, G., Rose, M.S., Turgeon, B.G., and Yoder, O.C., A Polyketide Synthase Is Required for Fungal Virulence and Production of the Polyketide T-Toxin, Plant Cell, 1996, vol. 8, pp. 2139–2150.

    Article  PubMed  CAS  Google Scholar 

  50. Talbot, N.J., Having a Blast: Exploring the Pathogenicity of Magnaporthe grisea, Trends Microbiol., 1995, vol. 3, pp. 9–16.

    Article  PubMed  CAS  Google Scholar 

  51. Sweigard, J.A., Carroll, A.M., Farrall, L., et al., Magnaporthe grisea Pathogenicity Genes Obtained through Insertional Mutagenesis, Mol. Plant Microb. Interact., 1998, vol. 11, pp. 404–412.

    Article  CAS  Google Scholar 

  52. Mitchell, T.K. and Dean, R.A., The cAMP-Dependent Protein Kinase Catalytic Subunit Is Required for Appressorium Formation and Pathogenesis by the Rice Blast Pathogen Magnaporthe grisea, Plant Cell, 1995, vol. 7, pp. 1869–1878.

    Article  PubMed  CAS  Google Scholar 

  53. Maier, F.J. and Schafer, W., Mutagenesis via Insertionalor Restriction Enzyme-Mediated-Integration (REMI) as a Tool to Tag Pathogenicity-Related Genes in Plant Pathogenic Fungi, Biol. Chem., 1999, vol. 380, pp. 855–864.

    Article  PubMed  CAS  Google Scholar 

  54. Sanchez, O., Navarro, R.E., and Aguirre, J., Increased Transformation Frequency and Tagging of Developmental Genes in Aspergillus nidulans by Restriction Enzyme-Mediated Integration (REMI), Mol. Gen. Genet., 1998, vol. 258, pp. 89–94.

    Article  PubMed  CAS  Google Scholar 

  55. Karos, M. and Fischer, R., HymA (Hypha-Like Metulae), a New Developmental Mutant of Aspergillus nidulans, Microbiology, 1996, vol. 142, pp. 3211–3218.

    Article  PubMed  CAS  Google Scholar 

  56. Granado, J.D.., Kertesz-Chaloupkova, K., Aebi, M., and Kües, U., Restriction Enzyme-Mediated DNA Integration in Coprinus cinereus, Mol. Gen. Genet., 1997, vol. 256, pp. 28–36.

    Article  PubMed  CAS  Google Scholar 

  57. Gonzalez, R., Ferrer, S., Buesa, J., and Ramon, D., Transformation of the Dermatophyte Trichophyton mentagrophytes to Hygromycin B Resistance, Infect. Immun., 1989, vol. 57, pp. 2923–2925.

    PubMed  CAS  Google Scholar 

  58. Kaufman, G., Horwitz, B.A., Hadar, R., et al., Green Fluorescent Protein (GFP) as a Vital Marker for Pathogenic Development of the Dermatophyte Trichophyton mentagrophytes, Microbiology, 2004, vol. 150, pp. 2785–2790.

    Article  PubMed  CAS  Google Scholar 

  59. Rademacher, W. and Graebe, J.E., Gibberellin A4 Produced by Sphaceloma manihoticola, the Cause of the Superelongation Disease of Cassava Manihot esculenta, Biochem. Biophys. Res. Commun., 1979, vol. 91, pp. 35–40.

    Article  PubMed  CAS  Google Scholar 

  60. Kawanabe, Y., Yamane, H., and Murayama. T., Identification of Gibberellin A3 in Mycelia of Neurospora crassa, Agric. Biol. Chem., 1983, vol. 47, pp. 1693–1694.

    CAS  Google Scholar 

  61. Kawaide, H. and Sassa, T., Accumulation of Gibberellin A1 and the Metabolism of Gibberellin A9 to Gibberellin A1 in a Phaeosphaeria sp. L487 Culture, Biosci. Biotechnol. Biochem., 1993, vol. 57, pp. 1403–1405.

    Article  CAS  Google Scholar 

  62. Tudzynski, B., Fungal Phytohormones in Pathogenic and Mutualistic Associations, in The Mycota, Berlin: Springer, 1997, pp. 167–184.

    Google Scholar 

  63. Linnemannstons, P., Voss, T.., Hedden, P., et al., Deletions in the Gibberellin Biosynthesis Gene Cluster of Gibberella fujikuroi by Restriction Enzyme-Mediated Integration and Conventional Transformation-Mediated Mutagenesis, Appl. Environ. Microbiol., 1999, vol. 65, pp. 2558–2564.

    PubMed  CAS  Google Scholar 

  64. Guerin, N.A. and Larochelle, D.A., A User’s Guide to Restriction Enzyme-Mediated Integration in Dictyostelium, J. Muscle Res. Cell Motility, 2002, vol. 23, pp. 597–604.

    Article  CAS  Google Scholar 

  65. Barth, C., Fraser, D.J., and Fisher, P.R., A Rapid Small Scale Method for Characterization of Plasmid Insertions in the Dictyostelium Genome, Nucleic Acids Res., 1998, vol. 26, pp. 3317–3318.

    Article  PubMed  CAS  Google Scholar 

  66. Fey, P., Cox. E.C. Gene Trapping with GFP: The Isolation of Developmental Mutants in the Slime Mold Polysphondylium, Curr. Biol., 1997, vol. 7, pp. 909–912.

    Article  PubMed  CAS  Google Scholar 

  67. Hayashi, H., Czaja, I., Lubenow, H., et al., Activation of a Plant Gene by T-DNA Tagging: Auxin-Independent Growth in vitro, Science, 1992, vol. 258, pp. 1350–1353.

    Article  PubMed  CAS  Google Scholar 

  68. Walden, R., Fritze, K., Hayashi, H.J., et al., Activation Tagging: A Means of Isolating Genes Implicated as Playing a Role in Plant Growth and Development, Plant Mol. Biol, 1994, vol. 26, pp. 1521–1528.

    Article  PubMed  CAS  Google Scholar 

  69. Takeda, K., Saito, T., Tanaka, T., et al., A Novel Gene Trap Method Using Terminator-REMI-and 3′ Rapid Amplification of cDNA Ends (RACE) in Dictyostelium, Gene, 2003, vol. 312, pp. 321–333.

    Article  CAS  Google Scholar 

  70. Sparrow, D.B., Latinkic, B., and Mohun, T.J., A Simplified Method of Generating Transgenic Xenopus, Nucleic Acids Res., 2000, vol. 28, p. E12.

    Article  PubMed  CAS  Google Scholar 

  71. Shemesh, M., Gurevich, M., Harel-Markowitz, E., et al., Gene Integration into Bovine Sperm Genome and Its Expression in Transgenic Offspring, Mol. Reprod. Dev., 2000, vol. 56, pp. 306–308.

    Article  PubMed  CAS  Google Scholar 

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  1. Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, 79005, Ukraine

    K. V. Dmytruk & A. A. Sibirny

  2. Rzeszov University, Rzeszov, 35601, Poland

    A. A. Sibirny

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Original Russian Text © K.V. Dmytruk, A.A. Sibirny, 2007, published in Genetika, 2007, Vol. 43, No. 8, pp. 1013–1025.

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Dmytruk, K.V., Sibirny, A.A. Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi. Russ J Genet 43, 835–845 (2007). https://doi.org/10.1134/S1022795407080017

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