Chapter 10 : Genetic and Genomic Approaches to Environmental and Host Responses

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $15.00

Preview this chapter:
Zoom in

Genetic and Genomic Approaches to Environmental and Host Responses, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555816858/9781555815011_Chap10-1.gif /docserver/preview/fulltext/10.1128/9781555816858/9781555815011_Chap10-2.gif


Infection with species is often considered an AIDS-defining illness, the outbreak of on Vancouver Island underscores the broad potential of the species complex to evade host immune defenses and cause disease. There are several simple developments that need to be made to maximize the use of genomes. One development is the publication of the genomes of the serotype A and strains and a finalized nomenclature for each gene in each strain sequenced, preferably as similar to other designations as possible. Examples of larger-scale screens include a series on melanin, morphology, suppressor screens of light sensitivity of mating , and growth under hypoxic conditions. In the first, "alternative" hosts have been used, with the most success obtained thus far in the nematode . Importantly, both genes were found to be required for maximal virulence in murine inhalation models. Protein-protein interactions are important in a number of regulatory events involved in cryptococcal virulence and have been detected in by using a variety of methods. Single gene interactions have been detected by coimmunoprecipitation studies of the nuclear heat shock transcription factor, Hsf1, with a coactivating heat shock homolog, Ssa1, during laccase induction. A major experimental advantage of , uncommon among the human pathogenic fungi, is the established complete sexual cycle that enables classical Mendelian crosses and genetics.

Citation: Idnurm A, Williamson P. 2011. Genetic and Genomic Approaches to Environmental and Host Responses, p 127-137. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch10
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


Image of FIGURE 1

Overlap PCR for generating gene disruption constructs. The overlap PCR process first generates three products (A): a 5’ (primers 1 and 2) and 3’ (primers 3 and 4) region of 1.0–1.5 kb flanking the gene to be mutated []) and one for the marker (primers 5 and 6) used to select for transformed strains. All primers are standard 18 nt in length, with the exception of numbers 2 and 3, which are 36 nt long and composed of a chimera of the gene and the selectable marker. (B) The three pieces are mixed in equimolar amounts and amplified a second time using primers 1 and 4. (C) This product is transformed into using a biolistic apparatus, where the construct in many cases undergoes a double homologous recombination event at the locus. (D) The specific targeting events are screened using primers ( and and ) that only amplify when the marker is inserted, and gene disruption and the presence of only one copy of the construct in the genome is subsequently confirmed by Southern blot analysis. Primers 7 and 10 are distal to 1 and 4. Primers 5, 6, 8, and 9 can be a common set used for multiple gene disruption events.

Citation: Idnurm A, Williamson P. 2011. Genetic and Genomic Approaches to Environmental and Host Responses, p 127-137. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch10
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 2

Example of a forward genetic screen, using T-DNA insertions delivered by and temperature sensitivity as the phenotype. (A) strains are replicated on yeast extract peptone dextrose medium and incubated overnight at 30°C or 37°C. One strain (circled) shows impaired growth at 37°C. (B) The insertional mutant strain (IMα) is crossed to the wild-type strain of opposite mating type (WT), and progeny are isolated. Here a subset of eight progeny from a genetic analysis show no growth at 37°C if they are also nourseothricin resistant, or show wild-type growth at 37°C and nourseothricin sensitivity, indicating linkage between the T-DNA insertion event and the temperature-sensitive phenotype. Mating type ( or α adjacent to the progeny number) can be used as an independent marker to demonstrate genetic recombination in the population of the progeny. (C) Genomic DNA is extracted, digested with restriction enzyme, and selfligated, and the regions flanking the T-DNA insertion are obtained by inverse PCR using the ligation as a template. (D) The inverse PCR product is sequenced, defining the junctions between T-DNA (gray) and DNA. (E) Sequence comparison against the genome database reveals the nature of the gene affected. In this case the T-DNA insertion is 321 bp upstream of the start codon of a gene previously characterized with a role in growth at 37°C [, which encodes an α(1–3)glucan synthase] ( ).

Citation: Idnurm A, Williamson P. 2011. Genetic and Genomic Approaches to Environmental and Host Responses, p 127-137. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch10
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 3

Overview of three alternative methods to create insertional mutants of for use in forward genetic screens. Tagged DNA constructs containing the transformation markers such as the cryptococcal gene or the synthetic antibiotic-resistant cassettes Nat, Hyg, and Neo, conveying resistance to nourseothricin, hygromycin, and neomycin, respectively, are inserted within the cryptococcal genome using at least three methods: electroporation, biolistics, and . Electroporation uses a high-voltage pulse delivered within a metal-coated cuvette. Biolistic transformation uses gold beads coated with transforming DNA that is delivered by a helium gas pulse directly on a thin layer of fungal cells inoculated on agar plates. methods use the transforming bacterium to incorporate a specialized Ti plasmid containing the selected cryptococcal transformation marker and tagged elements into the cryptococcal DNA chromosome. Transformants are then isolated using the selective media appropriate for the transformation marker.

Citation: Idnurm A, Williamson P. 2011. Genetic and Genomic Approaches to Environmental and Host Responses, p 127-137. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch10
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 4

Genes required for melanin biosynthesis in serotype A . Those that have emerged from forward genetic screens are underlined. Laccase is the central enzyme for catalyzing the polymerization of phenolic substrates into melanin. Laccase is a copper-dependent enzyme; copper metabolism is therefore required for melanization. Other genes play regulatory roles for laccase directly or through copper metaliation. Laccase localizes to the cell wall, and thus mutations affecting cell wall integrity may have altered melanin profiles, such as chitin synthase (Chs3) and its regulator (Csr2). Additional genes that are known to regulate melanization ( ), but through unknown mechanisms, have been omitted.

Citation: Idnurm A, Williamson P. 2011. Genetic and Genomic Approaches to Environmental and Host Responses, p 127-137. In Heitman J, Kozel T, Kwon-Chung K, Perfect J, Casadevall A (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555816858.ch10
Permissions and Reprints Request Permissions
Download as Powerpoint


1. Baba, T. 1988. Electron microscopic cytochemical analysis of hepatic granuloma induced by Cryptococcus neoformans. Mycopathologia 104:3746.
2. Bahn, Y.-S., S. Geunes-Boyer,, and J. Heitman. 2007. Ssk2 mitogen-activated protein kinase kinase kinase governs divergent patterns of the stress-activated Hog1 signaling pathway in Cryptococcus neoformans. Eukaryot. Cell 6:22782289.
3. Blakely, G.,, J. Hekman,, K. Chakraburtty, and, P. R. Williamson. 2001. Evolutionary divergence of an elongation factor 3 from Cryptococcus neoformans. J. Bacteriol. 183:22412248.
4. Casadevall, A., and, J. Perfect. 1998. Cryptococcus neoformans. ASM Press, Washington, DC.
5. Chang, Y. C.,, C.M. Bien,, H. Lee, P. J. Espenshade,, and K. J. Kwon-Chung. 2007. Sre1p, a regulator of oxygen sensing and sterol homeostasis, is required for virulence in Cryptococcus neoformans. Mol. Microbiol. 64:614629.
6. Chang, Y. C., and, K. J. Kwon-Chung. 1994. Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol. Cell. Biol. 14:49124919.
7. Clarke, L., and, J. Carbon. 1976. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell 9:9199.
8. Cohen, J., J. R. Perfect, and, D. T. Durack. 1982. Cryptococcosis and the basidiospore. Lancet 1:1301.
9. Cruz, M. C.,, D.S. Fox, and, J. Heitman. 2001. Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans. EMBO J. 20:10201032.
10. Davidson, R. C.,, J.R. Blankenship,, P.R. Kraus,, M. de Jesus Berrios,, C. M. Hull,, C. D’Souza,, P. Wang,, and J. Heitman. 2002. A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148:26072615.
11. Dhaese, P.,, H. De Greve,, H. Decraemer,, J. Schell,, and M. Van Montagu. 1979. Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens. Nucleic Acids Res. 7:18371849.
12. Edman, J. C., and, K. J. Kwon-Chung. 1990. Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation. Mol. Cell. Biol. 10:45384544.
13. Erickson, T.,, L. Liu,, A. Gueyikian,, X. Zhu,, J. Gibbons,, and P. R. Williamson. 2001. Multiple virulence factors of Cryptococcus neoformans are dependent on VPH1. Mol. Microbiol. 42:11211131.
14. Fox, D. S.,, G.M. Cox, and, J. Heitman. 2003. Phospholipid-binding protein Cts1 controls septation and functions coordinately with calcineurin in Cryptococcus neoformans. Eukaryot. Cell 2:10251035.
15. Fraser, J. A.,, R. L. Subaran,, C. B. Nichols,, and J. Heitman. 2003. Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada. Eukaryot. Cell 2:10361045.
16. Görlach, J.,, D. S. Fox,, N. S. Cutler,, G. M. Cox,, J. R. Perfect,, and J. Heitman. 2000. Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans. EMBO J. 19:36183629.
17. Heitman, J.,, A. Casadevall,, J. K. Lodge, and, J. R. Perfect. 1999. The Cryptococcus neoformans genome sequencing project. Mycopathologia 148:17.
18. Hull, C. M.,, M.-J. Boily, and, J. Heitman. 2005. Sexspecific homeodomain proteins Sxi1a and Sxi2 a coordinately regulate sexual development in Cryptococcus neoformans. Eukaryot. Cell 4:526535.
19. Idnurm, A., and, J. Heitman. 2005. Light controls growth and development via a conserved pathway in the fungal kingdom. PLoS Biol. 3:615626.
20. Idnurm, A.,, J. L. Reedy,, J.C. Nussbaum,, and J. Heitman. 2004. Cryptococcus neoformans virulence gene discovery through insertional mutagenesis. Eukaryot Cell 3:420429.
21. Idnurm, A.,, F. J. Walton,, A. Floyd, J. L. Reedy,, and J. Heitman. 2009. Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis. Eukaryot. Cell 8:315326.
22. Ingavale, S. S.,, Y.C. Chang,, H. Lee, C. M. McClelland, M. L. Leong,, and K. J. Kwon-Chung. 2008. Importance of mitochondria in survival of Cryptococcus neoformans under low oxygen conditions and tolerance to cobalt chloride. PLoS Pathog. 4:e1000155.
23. Jeon, J.,, S.-Y. Park,, M.-H. Chi,, J. Choi,, J. Park,, H.-S. Rho,, S. Kim,, J. Goh,, S. Yoo,, J. Choi,, J.-Y. Park,, M. Yi,, S. Yang,, M.-J. Kwon,, S.-S. Han,, B. R. Kim,, C. H. Khang,, B. Park,, S.-E. Lim,, K. Jung,, S. Kong,, M. Karunakaran,, H.-S. Oh,, H. Kim,, S. Kim,, J. Park,, S. Kang,, W.-B. Choi,, S. Kang,, and Y.-H. Lee. 2007. Genome-wide functional analysis of pathogenicity genes in the rice blast fungus. Nat. Genet. 39:561565.
24. Kontoyiannis, D. P.,, R. E. Lewis,, B. D. Alexander,, O. Lortholary,, F. Dromer,, K. L. Gupta,, G. T. John,, R. del Busto,, G. B. Klintmalm,, J. Somani,, G. M. Lyon,, K. Pursell,, V. Stosor,, P. Munoz,, A. P. Limaye,, A. C. Kalil,, T. L. Pruett,, J. Garcia-Diaz,, A. Humar,, S. Houston,, A. A. House,, D. Wray,, S. Orloff,, L. A. Dowdy,, R. A. Fisher,, J. Heitman,, N. D. Albert,, M. M. Wagener,, and N. Singh. 2008. Calcineurin inhibitor agents interact synergistically with antifungal agents in vitro against Cryptococcus neoformans isolates: correlation with outcome in solid organ transplant recipients with cryptococcosis. Antimicrob. Agents Chemother. 52:735738.
25. Kwon-Chung, K. 1998. Gene disruption to evaluate the role of fungal candidate virulence genes. Curr. Opin. Microbiol. 1:381389.
26. Kwon-Chung, K. J. 1976. A new species of Filobasidiella, the sexual state of Cryptococcus neoformans B and C serotypes. Mycologia 68:943946.
27. Kwon-Chung, K. J., J. C. Edman, and, B. L. Wickes. 1992. Genetic association of mating types and virulence in Cryptococcus neoformans. Infect. Immun. 60:602605.
28. Lengeler, K. B.,, P. Wang,, G. M. Cox,, J. R. Perfect,, and J. Heitman. 2000. Identification of the MATa mating-type locus of Cryptococcus neoformans reveals a serotype A MATa strain thought to have been extinct. Proc. Natl. Acad. Sci. USA 97:1445514460.
29. Liautard, J.-P.,, V. Jubier-Maurin,, R.-A. Boigegrain,, and S. Köhler. 2006. Antimicrobials: targeting virulence genes necessary for intracellular multiplication. Trends Microbiol. 14:109113.
30. Lin, X.,, J. C. Huang,, T.G. Mitchell,, and J. Heitman. 2006. Virulence attributes and hyphal growth of C. neoformans are quantitative traits and the MATα allele enhances filamentation. PLoS Genet. 2:e187.
31. Litvintseva, A. P.,, R.E. Marra,, K. Nielsen,, J. Heitman,, R. Vilgalys,, and T. G. Mitchell. 2003. Evidence of sexual recombination among Cryptococcus neoformans serotype A isolates in sub-Saharan Africa. Eukaryot. Cell 2:11621168.
32. Litvintseva, A. P., and, T. G. Mitchell. 2009. Most environmental isolates of Cryptococcus neoformans var. grubii (serotype A) are not lethal for mice. Infect. Immun. 77:31883195.
33. Liu, M., and, A. Gelli. 2008. Elongation factor 3, EF3, associates with the calcium channel Cch1 and targets Cch1 to the plasma membrane in Cryptococcus neoformans. Eukaryot. Cell 7:11181126.
34. Liu, O. W.,, C.D. Chun,, E.D. Chow,, C. Chen,, H. D. Madhani,, and S. M. Noble. 2008. Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans. Cell 135:174188.
35. Loftus, B. J.,, E. Fung,, P. Roncaglia,, D. Rowley,, P. Amedeo,, D. Bruno,, J. Vamathevan,, M. Miranda,, I. J Anderson,, J.A. Fraser,, J. E Allen,, I.E. Bosdet,, M. R Brent,, R. Chiu,, T. L Doering,, M.J. Donlin,, C. A. D’Souza,, D. S Fox,, V. Grinberg,, J. Fu,, M. Fukushima,, B. J Haas,, J.C. Huang,, G. Janbon,, S. J Jones,, H.L. Koo,, M. I Krzywinski,, J. K. Kwon-Chung,, K. B Lengeler,, R. Maiti,, M. A Marra,, R.E. Marra,, C. A Mathewson,, T.G. Mitchell,, M. Pertea,, F. R Riggs,, S.L. Salzberg,, J. E Schein,, A. Shvartsbeyn,, H. Shin,, M. Shumway,, C. A Specht,, B.B. Suh,, A. Tenney,, T. R Utterback,, B.L. Wickes,, J. R Wortman,, N.H. Wye,, J. W Kronstad,, J.K. Lodge,, J. Heitman,, R. W Davis,, C. M. Fraser,, and R. W. Hyman. 2005. The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 307:13211324.
36. Marra, R. E.,, J.C. Huang,, E. Fung,, K. Nielsen,, J. Heitman,, R. Vilgalys,, and T. G. Mitchell. 2004. A genetic linkage map of Cryptococcus neoformans variety neoformans serotype D (Filobasidiella neoformans). Genetics 167:619631.
37. McClelland, C. M., Y. C. Chang, and, K. J. Kwon-Chung. 2005. High frequency transformation of Cryptococcus neoformans and Cryptococcus gattii by Agrobacterium tumefaciens. Fungal Genet. Biol. 42:904913.
38. McCluskey, K. 2003. The Fungal Genetics Stock Center: from molds to molecules. Adv. Appl. Microbiol. 52:245262.
39. Michielse, C. B.,, R. van Wijk,, L. Reijnen,, B. J. Cornelissen,, and M. Rep. 2009. Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis. Genome Biol. 10:R4.
40. Mitchell, T. G., and, J. R. Perfect. 1995. Cryptococcosis in the era of AIDS: 100 years after the discovery of Cryptococcus neoformans. Clin. Microbiol. Rev. 8:515548.
41. Moyrand, F., T. Fontaine, and, G. Janbon. 2007. Systematic capsule gene disruption reveals the central role of galactose metabolism on Cryptococcus neoformans virulence. Mol. Microbiol. 64:771781.
42. Mylonakis, E., A. Casadevall, and, F. M. Ausubel. 2007. Exploiting amoeboid and non-vertebrate animal model systems to study the virulence of human pathogenic fungi. PLoS Pathog. 3:e101.
43. Mylonakis, E.,, A. Idnurm,, R. Moreno, J. El Khoury,, J.B. Rottman,, F. M Ausubel,, J. Heitman,, and S. B. Calderwood. 2004. Cryptococcus neoformans Kin1 protein kinase homologue, identified through a Caenorhabditis elegans screen, promotes virulence in mammals. Mol. Microbiol. 54:407419.
44. Nelson, R. T.,, J. Hua,, B. Pryor, and, J. K. Lodge. 2001. Identification of virulence mutants of the fungal pathogen Cryptococcus neoformans using signature-tagged mutagenesis. Genetics 157:935947.
45. Nielsen, K.,, G. M. Cox,, A. P. Litvintseva,, E. Mylonakis,, S. D. Malliaris,, D. K. Benjamin, Jr.,, S. S. Giles,, T. G. Mitchell,, A. Casadevall,, J. R. Perfect,, and J. Heitman. 2005. Cryptococcus neoformans α strains preferentially disseminate to the central nervous system during coinfection. Infect. Immun. 73:49224933.
46. Nielsen, K.,, G. M. Cox,, P. Wang,, D. L. Toffaletti,, J. R. Perfect,, and J. Heitman. 2003. Sexual cycle of Cryptococcus neoformans var. grubii and virulence of congenic a and α isolates. Infect. Immun. 71:48314841.
47. Nielsen, K.,, R. E. Marra,, F. Hagen,, T. Boekhout,, T. G. Mitchell,, G. M. Cox,, and J. Heitman. 2005. Interaction between genetic background and the mating-type locus in Cryptococcus neoformans virulence potential. Genetics 171:975983.
48. Noverr, M. C.,, P.R. Williamson,, R.S. Fajardo,, and G. B. Huffnagle. 2004. CNLAC1 is required for extrapulmonary dissemination of Cryptococcus neoformans but not pulmonary persistence. Infect. Immun. 72:16931699.
49. Olszewski, M. A.,, M.C. Noverr,, G.H. Chen,, G. B Toews,, G.M. Cox,, J. R. Perfect,, and G. B. Huffnagle. 2004. Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion. Am. J. Pathol. 164:17611771.
50. Palmer, D. A.,, J.K. Thompson,, L. Li,, A. Prat,, and P. Wang. 2006. Gib2, a novel Gβ-like/RACK1 homolog, functions as a Gβ subunit in cAMP signaling and is essential in Cryptococcus neoformans. J. Biol. Chem. 281:3259632605.
51. Panepinto, J.,, L. Liu,, J. Ramos,, X. Zhu,, T. Valyi-Nagy,, S. Eksi,, J. Fu,, H. A. Jaffe,, B. Wickes,, and P. Williamson. 2005. The DEAD-box RNA helicase Vad1 regulates multiple virulence-associated genes in Cryptococcus neoformans. J. Clin. Invest. 115:632641.
52. Panepinto, J. C.,, K.W. Komperda,, M. Hacham,, S. Shin,, X. Liu,, and P. R. Williamson. 2007. Binding of serum mannan binding lectin to a cell integrity-defective Cryptococcus neoformans ccr4Δ mutant. Infect. Immun. 75:47694779.
53. Park, B. J.,, K.A. Wannemuehler,, B.J. Marston,, N. Govender,, P. G. Pappas,, and T. M. Chiller. 2009. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23:525530.
54. Perfect, J. R. 2005. Cryptococcus neoformans: a sugarcoated killer with designer genes. FEMS Immunol. Med. Microbiol. 45:395404.
55. Reese, A. J., and, T. L. Doering. 2003. Cell wall α-1,3-glucan is required to anchor the Cryptococcus neoformans capsule. Mol. Microbiol. 50:14011409.
56. Reese, A. J.,, A. Yoneda,, J. A. Breger,, A. Beauvais,, H. Liu,, C. L Griffith,, I. Bose,, M.-J. Kim,, C. Skau,, S. Yang,, J. A Sefko,, M. Osumi,, J.-P. Latge,, E. Mylonakis,, and T. L. Doering. 2007. Loss of cell wall α(1–3) glucan affects Cryptococcus neoformans from ultrastructure to virulence. Mol. Microbiol. 63:13851398.
57. Salas, S. D.,, J.E. Bennett,, K. J. Kwon-Chung, J. R. Perfect,, and P. R. Williamson. 1996. Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J. Exp. Med. 184:377386.
58. Singh, N.,, O. Lortholary,, B. D. Alexander,, K. L. Gupta,, G. T. John,, K. Pursell,, P. Munoz,, G. B. Klintmalm,, V. Stosor,, R. del Busto,, A. P. Limaye,, J. Somani,, M. Lyon,, S. Houston,, A. A. House,, T. L. Pruett,, S. Orloff,, A. Humar,, L. Dowdy,, J. Garcia-Diaz,, A. C. Kalil,, R. A. Fisher,, and S. Husain. 2005. An immune reconstitution syndrome-like illness associated with Cryptococcus neoformans infection in organ transplant recipients. Clin. Infect. Dis. 40:17561761.
59. Tang, R. J.,, J. Breger,, A. Idnurm,, K. J. Gerik,, J. K. Lodge,, J. Heitman,, S. B. Calderwood,, and E. Mylonakis. 2005. Cryptococcus neoformans gene involved in mammalian pathogenesis identified by a Caenorhabditis elegans progeny-based approach. Infect. Immun. 73:82198225.
60. Tenney, A. E.,, R.H. Brown,, C. Vaske,, J. K Lodge,, T. L. Doering,, and M. R. Brent. 2004. Gene prediction and verification in a compact genome with numerous small introns. Genome Res. 14:23302335.
61. Toffaletti, D. L.,, T.H. Rude,, S. A. Johnston, D. T. Durack,, and J. R. Perfect. 1993. Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J. Bacteriol. 175:14051411.
62. Viviani, M. A.,, M.C. Esposto,, M. Cogliati, M. T. Montagna,, and B. L. Wickes. 2001. Isolation of a Cryptococcus neoformans serotype A MAT a strain from the Italian environment. Med. Mycol. 39:383386.
63. Walton, F. J.,, J. Heitman, and, A. Idnurm. 2006. Conserved elements of the RAM signaling pathway establish cell polarity in the basidiomycete Cryptococcus neoformans in a divergent fashion from other fungi. Mol. Biol. Cell 17:37683780.
64. Walton, F. J.,, A. Idnurm, and, J. Heitman. 2005. Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans. Mol. Microbiol. 57:13811396.
65. Waterman, S. R.,, M. Hacham,, G. Hu,, X. Zhu,, Y. D Park,, S. Shin,, J. Panepinto,, T. Valyi-Nagy,, C. Beam,, S. Husain,, N. Singh,, and P. R. Williamson. 2007. Role of a CUF1-CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans. J. Clin. Invest. 117:794802.
66. Williamson, P. R. 1994. Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase. J. Bacteriol. 176:656664.
67. Xue, C.,, Y.-S. Bahn,, G.M. Cox,, and J. Heitman. 2006. G protein-coupled receptor Gpr4 senses amino acids and activates the cAMP-PKA pathway in Cryptococcus neoformans. Mol. Biol. Cell 17:667679.
68. Xue, C.,, Y. P. Hsueh,, L. Chen,, and J. Heitman. 2008. The RGS protein Crg2 regulates both pheromone and cAMP signalling in Cryptococcus neoformans. Mol. Microbiol. 70:379395.
69. Yeh, Y. L.,, Y.S. Lin,, B.J. Su,, and W. C. Shen. 2009. A screening for suppressor mutants reveals components involved in the blue light-inhibited sexual filamentation in Cryptococcus neoformans. Fungal Genet. Biol. 46:4254.
70. Zerhouni, E. 2005. Translational and clinical science: time for a new vision. N. Engl. J. Med. 353:16211623.
71. Zhang, S.,, M. Hacham,, J. Panepinto,, G. Hu,, S. Shin,, X. Zhu,, and P. R. Williamson. 2006. The Hsp70 member, Ssa1 acts as a DNA-binding transcriptional co-activator in Cryptococcus neoformans. Mol. Microbiol. 62:10901101.
72. Zhu, X., and, P. R. Williamson. 2003. A CLC-type chloride channel gene is required for laccase activity and virulence in Cryptococcus neoformans. Mol. Microbiol. 50:12711281.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error