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Genome-Wide Transposon Mutagenesis in Saccharomyces cerevisiae and Candida albicans

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Strain Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 765))

Abstract

Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by chemical/UV treatment, transposon insertions can be easily identified in mutants with phenotypes of interest. The construction of transposon insertion mutants is also less labor-intensive on a genome-wide scale than methods for targeted gene replacement, although transposon insertions are not precisely targeted to a specific residue, and thus coverage of the target DNA can be problematic. The collective advantages of transposon mutagenesis have been well demonstrated in studies of the budding yeast Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans, as transposon mutagenesis has been used extensively for phenotypic screens in both yeasts. Consequently, we present here protocols for the generation and utilization of transposon-insertion DNA libraries in S. cerevisiae and C. albicans. Specifically, we present methods for the large-scale introduction of transposon insertion alleles in a desired strain of S. cerevisiae. Methods are also presented for transposon mutagenesis of C. albicans, encompassing both the construction of the plasmid-based transposon-mutagenized DNA library and its introduction into a desired strain of Candida. In total, these methods provide the necessary information to implement transposon mutagenesis in yeast, enabling the construction of large sets of identifiable gene disruption mutations, with particular utility for phenotypic screening in nonstandard genetic backgrounds.

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References

  1. Hensel M., Shea J. E., Gleeson C., Jones M. D., Dalton E., and Holden D. W. (1995) Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400–403.

    Google Scholar 

  2. Way J. C., Davis M. A., Morisato D., Roberts D. E., and Kleckner N. (1984) New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene 32, 369–379.

    Google Scholar 

  3. Jacobs M. A., Alwood A., Thaipisuttikul I., Spencer D., Haugen E., Ernst S., Will O., Kaul R., Raymond C., Levy R., Chun-Rong L., Guenthner D., Bovee D., Olson M. V., and Manoil C. (2003) Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U.S.A. 100, 14339–14344.

    Google Scholar 

  4. Hoekstra M. F., Burbee D., Singer J., Mull E., Chiao E., and Heffron F. (1991) A Tn3 derivative that can be used to make short in-frame insertions within genes, Proc. Natl. Acad. Sci. U.S.A. 88, 5457–5461.

    Google Scholar 

  5. Smith V., Botstein D., and Brown P. O. (1995) Genetic footprinting: a genomic strategy for determining a gene’s function given its sequence. Proc. Natl. Acad. Sci. U.S.A. 92, 6479–6483.

    Google Scholar 

  6. Devine S., and Boeke J. (1994) Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing, and genetic analysis. Nucleic Acids Res. 22, 3765–3772.

    Google Scholar 

  7. Long D., Martin M., Sundberg E., Swinburne J., Puangsomlee P., and Coupland G. (1993) The maize transposable element system Ac/Ds as a mutagen in Arabidopsis: identification of an albino mutation induced by Ds insertion. Proc. Natl. Acad. Sci. U.S.A. 90, 10370–10374.

    Google Scholar 

  8. Karess R. E., and Rubin G. M. (1984) Analysis of P transposable element functions in Drosophila. Cell 38, 135–146.

    Google Scholar 

  9. Spradling A. C., Stern D. M., Kiss I., Roote J., Laverty T., and Rubin G. M. (1995) Gene disruptions using P transposable elements: An integral component of the Drosophila genome project. Proc. Natl. Acad. Sci. U.S.A. 92, 10824–10830.

    Google Scholar 

  10. Ivics Z., Hackett P. B., Plasterk R. H., and Izsvak Z. (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501–510.

    Google Scholar 

  11. Burns N., Grimwade B., Ross-Macdonald P. B., Choi E.-Y., Finberg K., Roeder G. S., and Snyder M. (1994) Large-scale characterization of gene expression, protein localization and gene disruption in Saccharomyces cerevisiae. Genes Dev. 8, 1087–1105.

    Google Scholar 

  12. Ross-Macdonald P., Coelho P. S., Roemer T., Agarwal S., Kumar A., Jansen R., Cheung K. H., Sheehan A., Symoniatis D., Umansky L., Heidtman M., Nelson F. K., Iwasaki H., Hager K., Gerstein M., Miller P., Roeder G. S., and Snyder M. (1999) Large-scale analysis of the yeast genome by transposon tagging and gene disruption. Nature 402, 413–418.

    Google Scholar 

  13. Kumar A., Seringhaus M., Biery M., Sarnovsky R. J., Umansky L., Piccirillo S., Heidtman M., Cheung K.-H., Dobry C. J., Gerstein M., Craig N., and Snyder M. (2004) Large-Scale Mutagenesis of the Yeast Genome Using a Tn7-Derived Multipurpose Transposon. Genome Res. 14, 1975–1986.

    Google Scholar 

  14. Seringhaus M., Kumar A., Hartigan J., Snyder M., and Gerstein M. (2006) Genomic analysis of insertion behavior and target specificity of mini-Tn7 and Tn3 transposons in Saccharomyces cerevisiae. Nucleic Acids Res. 34, e57.

    Google Scholar 

  15. Winzeler E. A., Shoemaker D. D., Astromoff A., Liang H., Anderson K., Andre B., Bangham R., Benito R., Boeke J. D., Bussey H., Chu A. M., Connelly C., Davis K., Dietrich F., Dow S. W., Bakkoury M. E., Foury F., Friend S. H., Gentalen E., Giaever G., Hegemann J. H., Laub T. J. M., Liao H., Liebundguth N., Lockhart D. J., Lucau-Danila A., Lussier M., M’Rabet N., Menard P., Mittmann M., Pai C., Rebischung C., Revuelta J. L., Riles L., Roberts C. J., Ross-MacDonald P., Scherens B., Snyder M., Sookhai-Mahadeo S., Storms R. K., Véronneau S., Voet M., Volckaert G., Ward T. R., Wysocki R., Yen G. S., Yu K., Zimmermann K., Philippsen P., Johnston M., and Davis R. W. (1999) Fuctional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906.

    Google Scholar 

  16. Ross-Macdonald P., Sheehan A., Roeder G. S., and Snyder M. (1997) A multipurpose transposon system for analyzing protein production, localization, and function in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 94, 190–195.

    Google Scholar 

  17. Kumar A., DesEtages S., Coelho P., Roeder G., and Snyder M. (2000) High-throughput methods for the large-scale analysis of gene function by transposon tagging. Methods Enzymol. 328, 550–574.

    Google Scholar 

  18. Kumar A., Vidan S., and Snyder M. (2002) Insertional mutagenesis: transposon-insertion libraries as mutagens in yeast. Methods Enzymol. 350, 219–229.

    Google Scholar 

  19. Davis D. A., Bruno V. M., Loza L., Filler S. G., and Mitchell A. P. (2002) Candida albicans Mds3p, a conserved regulator of pH responses and virulence identified through insertional mutagenesis. Genetics 162, 1573–1581.

    Google Scholar 

  20. Uhl M. A., Biery M., Craig N., and Johnson A. D. (2003) Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the diploid human fungal pathogen C.albicans. EMBO J. 22, 2668–2678.

    Google Scholar 

  21. Nobile C. J., and Mitchell A. P. (2009) Large-scale gene disruption using the UAU1 cassette. Methods Mol. Biol. 499, 175–194.

    Google Scholar 

  22. Smith V., Chou K. N., Lashkari D., Botstein D., and Brown P. O. (1996) Functional analysis of the genes of yeast chromosome V by genetic footprinting. Science 1996 274, 2069–2074.

    Google Scholar 

  23. Blankenship J. R., Fanning S., Hamaker J. J., and Mitchell A. P. An extensive circuitry for cell wall regulation in Candida albicans. PLoS Pathog. 6, e1000752.

    Google Scholar 

  24. Homann O. R., Dea J., Noble S. M., and Johnson A. D. (2009) A phenotypic profile of the Candida albicans regulatory network. PLoS Genet. 5, e1000783.

    Google Scholar 

  25. Noble S. M., French S., Kohn L. A., Chen V., and Johnson A. D. Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nat. Genet. 42, 590–598.

    Google Scholar 

  26. Jin R., Dobry C. J., McCown P. J., and Kumar A. (2008) Large-scale analysis of yeast filamentous growth by systematic gene disruption and overexpression. Mol. Biol. Cell 19, 284–296.

    Google Scholar 

  27. Kumar A. (2008) Multipurpose transposon insertion libraries for large-scale analysis of gene function in yeast. Methods Mol. Biol. 416, 117–129.

    Google Scholar 

  28. Stellwagen A. E., and Craig N. L. (1997) Avoiding self: two Tn7-encoded proteins mediate target immunity in Tn7 transposition. EMBO J. 16, 6823–6834.

    Google Scholar 

  29. Ochman H., Gerber A. S., and Hartl D. L. (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623.

    Google Scholar 

  30. Riley J., Butler R., Ogilvie D., Finniear R., Jenner D., Powell S., Anand R., Smith J. C., and Markham A. F. (1990) A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res. 18, 2887–2890.

    Google Scholar 

  31. Richard M. L., Nobile C. J., Bruno V. M., and Mitchell A. P. (2005) Candida albicans biofilm-defective mutants. Eukaryot. Cell 4, 1493–1502.

    Google Scholar 

  32. Craig N. L. (1991) Tn7: a target site-specific transposon. Mol. Microbiol. 5, 2569–2573.

    Article  PubMed  CAS  Google Scholar 

  33. Biery M., Stewart F., Stellwagen A., Raleigh E., and Craig N. (2000) A simple in vitro Tn7-based transposition system with low target site selectivity for genome and gene analysis. Nucleic Acids Res. 28, 1067–1077.

    Google Scholar 

  34. Stellwagen A., and Craig N. (1997) Gain-of-Function Mutations in TnsC, an ATP-Dependent Transposition Protein That Activates the Bacterial Transposon Tn7. Genetics 145, 573–585.

    Google Scholar 

  35. Wilson R. B., Davis D., Enloe B. M., and Mitchell A. P. (2000) A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions. Yeast 16, 65–70.

    Google Scholar 

  36. Boeke J. D., Trueheart J., Natsoulis G., and Fink G. R. (1987) 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154, 164–175.

    Google Scholar 

  37. Slutsky B., Staebell M., Anderson J., Risen L., Pfaller M., and Soll D. R. (1987) “White-opaque transition”: a second high-frequency switching system in Candida albicans. J. Bacteriol. 169, 189–197.

    Google Scholar 

  38. Baldari C., and Cesareni G. (1985) Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences. Gene 35, 27–32.

    Google Scholar 

  39. Bensen E. S., Clemente-Blanco A., Finley K. R., Correa-Bordes J., and Berman J. (2005) The mitotic cyclins Clb2p and Clb4p affect morphogenesis in Candida albicans. Mol. Biol. Cell 16, 3387–3400.

    Google Scholar 

  40. Walther A., and Wendland J. (2003) An improved transformation protocol for the human fungal pathogen Candida albicans. Curr. Genet. 42, 339–343.

    Google Scholar 

  41. McNemar M. D., and Fonzi W. A. (2002) Conserved serine/threonine kinase encoded by CBK1 regulates expression of several hypha-associated transcripts and genes encoding cell wall proteins in Candida albicans. J. Bacteriol. 184, 2058–2061.

    Google Scholar 

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Acknowledgments

Research in the Kumar laboratory was supported by grant RSG-06-179-01-MBC from the American Cancer Society and National Institutes of Health grant 1R21A1084539-01.

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Correspondence to Anuj Kumar .

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Xu, T., Bharucha, N., Kumar, A. (2011). Genome-Wide Transposon Mutagenesis in Saccharomyces cerevisiae and Candida albicans . In: Williams, J. (eds) Strain Engineering. Methods in Molecular Biology, vol 765. Humana Press. https://doi.org/10.1007/978-1-61779-197-0_13

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  • DOI: https://doi.org/10.1007/978-1-61779-197-0_13

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-196-3

  • Online ISBN: 978-1-61779-197-0

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