1887

Abstract

is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.

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2002-08-01
2020-01-18
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References

  1. Alspaugh J. A., Perfect J. R., Heitman J. 1997; Cryptococcus neoformans mating and virulence are regulated by the G-protein α subunit GPA1 and cAMP. Genes Dev11:3206–3217[CrossRef]
    [Google Scholar]
  2. Alspaugh J. A., Cavallo L. M., Perfect J. R., Heitman J. 2000a; RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans . Mol Microbiol36:352–365[CrossRef]
    [Google Scholar]
  3. Alspaugh J. A., Davidson R. C., Heitman J. 2000b; Morphogenesis of Cryptococcus neoformans . In Dimorphism in Human Pathogenic and Apathogenic Yeasts pp217–238 Edited by Ernst J. F., Schmidt A.. Basel: Karger;
    [Google Scholar]
  4. Baudin A., Ozier-Kalogeropoulos O., Denouel A., Lacroute F., Cullin C. 1993; A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae . Nucleic Acids Res21:3329–3330[CrossRef]
    [Google Scholar]
  5. Casadevall A., Perfect J. R. 1998; Cryptococcus neoformans Washington: ASM Press;
    [Google Scholar]
  6. Chen S. C., Wright L. C., Golding J. C., Sorrell T. C. 2000; Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans . Biochem J347:431–439[CrossRef]
    [Google Scholar]
  7. Clarke D. L., Woodlee G. L., McClelland C. M., Seymour T. S., Wickes B. L. 2001; The Cryptococcus neoformans STE11 α gene is similar to other fungal mitogen-activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific. Mol Microbiol40:200–213[CrossRef]
    [Google Scholar]
  8. Cox G. M., Mukherjee J., Cole G. T., Casadevall A., Perfect J. R. 2000; Urease as a virulence factor in experimental cryptococcosis. Infect Immun68:443–448[CrossRef]
    [Google Scholar]
  9. Cox G. M., McDade H. C., Chen S. C. A.. 8 other authors 2001; Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans . Mol Microbiol39:166–175[CrossRef]
    [Google Scholar]
  10. Cruz M. C., Cavallo L. M., Görlach J. M., Cox G., Perfect J. R., Cardenas M. E., Heitman J. 1999; Rapamycin antifungal action is mediated via conserved complexes with FKBP12 and TOR kinase homologs in Cryptococcus neoformans . Mol Cell Biol19:4101–4112
    [Google Scholar]
  11. Cruz M. C., Sia R. A. L., Olson M., Cox G. M., Heitman J. 2000; Comparison of the roles of calcineurin in physiology and virulence in serotype D and serotype A strains of Cryptococcus neoformans . Infect Immun68:982–985[CrossRef]
    [Google Scholar]
  12. Davidson R. C., Cruz M. C., Sia R. A. L., Allen B. M., Alspaugh J. A., Heitman J. 2000; Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans . Fungal Genet Biol29:38–48[CrossRef]
    [Google Scholar]
  13. Eberhardt I., Hohmann S. 1995; Strategy for deletion of complete open reading frames in Saccharomyces cerevisiae . Curr Genet27:306–308[CrossRef]
    [Google Scholar]
  14. Edman J. C., Kwon-Chung K. J. 1990; Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation. Mol Cell Biol10:4538–4544
    [Google Scholar]
  15. Fox D. S., Cruz M. C., Sia R. A. L., Ke H., Cox G. M., Cardenas M. E., Heitman J. 2001; Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans . Mol Microbiol39:835–849[CrossRef]
    [Google Scholar]
  16. Goldman D., Lee S. C., Casadevall A. 1994; Pathogenesis of pulmonary Cryptococcus neoformans infection in the rat. Infect Immun62:4755–4761
    [Google Scholar]
  17. Gorlach J. M., McDade H. C., Perfect J. R., Cox G. M. 2002; Antisense repression in Cryptococcus neoformans as a laboratory tool and potential antifungal strategy. Microbiology148:213–219
    [Google Scholar]
  18. Heitman J., Casadevall A., Lodge J. K., Perfect J. R. 1999; The Cryptococcus neoformans genome sequencing project. Mycopathologia148:1–7[CrossRef]
    [Google Scholar]
  19. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. 1989; Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene77:51–59[CrossRef]
    [Google Scholar]
  20. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. 1989; Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene77:61–68[CrossRef]
    [Google Scholar]
  21. Hua J., Meyer J. D., Lodge J. K. 2000; Development of positive selectable markers for the fungal pathogen Cryptococcus neoformans . Clin Diagn Lab Immunol7:125–128
    [Google Scholar]
  22. Kwon-Chung K. J. 1975; A new genus, Filobasidiella , the perfect state of Cryptococcus neoformans . Mycologia67:1197–1200[CrossRef]
    [Google Scholar]
  23. Kwon-Chung K. J. 1976; Morphogenesis of Filobasidiella neoformans , the sexual state of Cryptococcus neoformans . Mycologia68:821–833[CrossRef]
    [Google Scholar]
  24. Kwon-Chung K. J., Bennett J. E. 1992; Cryptococcosis. In Medical Mycology pp397–446 Malvern, PA: Lea & Febiger;
    [Google Scholar]
  25. Kwon-Chung K. J., Polacheck I., Popkin T. J. 1982; Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice. J Bacteriol150:1414–1421
    [Google Scholar]
  26. Liu H., Cottrell T. R., Pierini L. M., Goldman W. E., Doering T. L. 2002; RNA interference in the pathogenic fungus Cryptococcus neoformans . Genetics160:463–470
    [Google Scholar]
  27. Lorenz M. C., Muir R. S., Lim E., McElver J., Weber S. C., Heitman J. 1995; Gene disruption with PCR products in Saccharomyces cerevisiae. Gene 158:113–117[CrossRef]
    [Google Scholar]
  28. McDade H. C., Cox G. M. 2001; A new dominant selectable marker for use in Cryptococcus neoformans . Med Mycol39:151–154[CrossRef]
    [Google Scholar]
  29. Mitchell T. G., Perfect J. R. 1995; Cryptococcosis in the era of AIDS – 100 years after the discovery of Cryptococcus neoformans . Clin Microbiol Rev8:515–548
    [Google Scholar]
  30. Moore T. D. E., Edman J. C. 1993; The α-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol Cell Biol13:1962–1970
    [Google Scholar]
  31. Nelson R. T., Hua J., Pryor B., Lodge J. K. 2001; Identification of virulence mutants of the fungal pathogen Cryptococcus neoformans using signature-tagged mutagenesis. Genetics157:935–947
    [Google Scholar]
  32. Nelson R. T., Pryor B. A., Lodge J. K. 2002; Sequence length required for homologous recombination in Cryptococcus neoformans . Fungal Genet Biol in press
    [Google Scholar]
  33. Odom A., Muir S., Lim E., Toffaletti D. L., Perfect J., Heitman J. 1997; Calcineurin is required for virulence of Cryptococcus neoformans . EMBO J16:2576–2589[CrossRef]
    [Google Scholar]
  34. Perfect J. R., Lang S. D. R., Durack D. T. 1980; Chronic cryptococcal meningitis: a new experimental model in rabbits. Am J Pathol101:177–194
    [Google Scholar]
  35. Pitkin J. W., Panaccione D. G., Walton J. D. 1996; A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant-pathogenic fungus Cochliobolus carbonum . Microbiology142:1557–1565[CrossRef]
    [Google Scholar]
  36. Sambrook J., Fritsch E. F., Maniatis T. 1989; Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  37. Sanfelice F. 1894; Contributo alla morfologia e biologia dei blastomiceti che si sviluppano nei succhi di alcuni frutti. Ann Igien4:463–495
    [Google Scholar]
  38. Sherman F. 1991; Getting started with yeast. Methods Enzymol194:3–21
    [Google Scholar]
  39. Sia R. A., Lengeler K. B., Heitman J. 2000; Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Fungal Genet Biol29:153–163[CrossRef]
    [Google Scholar]
  40. Sudarshan S., Davidson R. C., Heitman J., Alspaugh J. A. 1999; Molecular analysis of the Cryptococcus neoformans ADE2 gene, a selectable marker for transformation and gene disruption. Fungal Genet Biol27:36–48[CrossRef]
    [Google Scholar]
  41. Toffaletti D. L., Perfect J. R. 1994; Biolistic DNA delivery for Cryptococcus neoformans transformation. In Molecular Biology of Pathogenic Fungi: a Laboratory Manual pp303–308 Edited by Maresca B., Kobayashi G. S.. New York: Telos Press;
    [Google Scholar]
  42. Toffaletti D. L., Rude T. H., Johnston S. A., Durack D. T., Perfect J. R. 1993; Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J Bacteriol175:1405–1411
    [Google Scholar]
  43. Tong A. H. Y., Evangelista M., Parsons A. B.. 10 other authors 2001; Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science294:2364–2368[CrossRef]
    [Google Scholar]
  44. Wach A. 1996; PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae . Yeast12:259–265[CrossRef]
    [Google Scholar]
  45. Wach A., Brachat A., Pohlmann R., Philippsen P. 1994; New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae . Yeast10:1793–1808[CrossRef]
    [Google Scholar]
  46. Wang P., Perfect J. R., Heitman J. 2000; The G-protein β subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans . Mol Cell Biol20:352–362[CrossRef]
    [Google Scholar]
  47. Wilson R. B., Davis D., Mitchell A. P. 1999; Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol181:1868–1874
    [Google Scholar]
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