| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Research Paper |
Departments of Medicine and Microbiology, Duke University Medical Center, Durham, NC, USA1
Author for correspondence: Gary M. Cox. Tel: +1 919 681 5055. Fax: +1 919 684 8902. e-mail: gary.cox{at}duke.edu
Antisense repression was used as a method to alter gene function in the human-pathogenic fungus Cryptococcus neoformans. The calcineurin A gene (CNA1) and the laccase gene (LAC1) were targeted since disruption of these loci results in phenotypes that are easy to screen (temperature sensitivity and lack of melanin, respectively). Serotype D yeasts were transformed with a plasmid containing the CNA1 cDNA in an antisense orientation under the control of the inducible GAL7 promoter, and serotype A yeasts were transformed with a plasmid containing the LAC1 cDNA in an antisense orientation under the control of the constitutive actin promoter. The calcineurin transformants demonstrated a temperature-sensitive phenotype only when grown on galactose, and the laccase transformants had decreased melanin production. Northern blot analysis of the calcineurin antisense transformants confirmed that the inducible phenotype was associated with a decrease in the native CNA1 transcript levels. Furthermore, it was possible to modestly impair growth of C. neoformans at 37 °C by using a 30 bp antisense oligonucleotide targeting CNA1. Antisense repression is now available as a tool for molecular studies in this organism, and may be applicable to other human-pathogenic fungi that have less amenable genetic systems.
Keywords: calcineurin, antisense oligonucleotide, yeast, fungus, pathogenesis
Abbreviations: RACE, rapid amplification of cDNA ends; 5-FOA, 5-fluoroorotic acid
This article has been cited by other articles:
|
|
 |
|
  M. Liu and A. Gelli Elongation Factor 3, EF3, Associates with the Calcium Channel Cch1 and Targets Cch1 to the Plasma Membrane in Cryptococcus neoformans Eukaryot. Cell, July 1, 2008; 7(7): 1118 - 1126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Palmer, J. K. Thompson, L. Li, A. Prat, and P. Wang Gib2, A Novel Gbeta-like/RACK1 Homolog, Functions as a Gbeta Subunit in cAMP Signaling and Is Essential in Cryptococcus neoformans J. Biol. Chem., October 27, 2006; 281(43): 32596 - 32605. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Walti, C. Villalba, R. M. Buser, A. Grunler, M. Aebi, and M. Kunzler Targeted Gene Silencing in the Model Mushroom Coprinopsis cinerea (Coprinus cinereus) by Expression of Homologous Hairpin RNAs. Eukaryot. Cell, April 1, 2006; 5(4): 732 - 744. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Loftus, E. Fung, P. Roncaglia, D. Rowley, P. Amedeo, D. Bruno, J. Vamathevan, M. Miranda, I. J. Anderson, J. A. Fraser, et al. The Genome of the Basidiomycetous Yeast and Human Pathogen Cryptococcus neoformans Science, February 25, 2005; 307(5713): 1321 - 1324. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. Heung, C. Luberto, A. Plowden, Y. A. Hannun, and M. Del Poeta The Sphingolipid Pathway Regulates Pkc1 through the Formation of Diacylglycerol in Cryptococcus neoformans J. Biol. Chem., May 14, 2004; 279(20): 21144 - 21153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. T. Magee, C. Gale, J. Berman, and D. Davis Molecular Genetic and Genomic Approaches to the Study of Medically Important Fungi Infect. Immun., May 1, 2003; 71(5): 2299 - 2309. [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Davidson, J. R. Blankenship, P. R. Kraus, M. de Jesus Berrios, C. M. Hull, C. D'Souza, P. Wang, and J. Heitman A PCR-based strategy to generate integrative targeting alleles with large regions of homology Microbiology, August 1, 2002; 148(8): 2607 - 2615. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
