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Microbiology 143 (1997), 303-311; DOI  10.1099/00221287-143-2-303
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Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans

Brendan P. Cormack1,5, Gwyneth Bertram2, Mark Egerton3, Neil A. R. Gow2, Stanley Falkow1,4 and Alistair J. P. Brown2,6

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5402, USA
Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
Zeneca Pharmaceuticals, Alderley Park, Macclesfield SK10 4TG, UK
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840, USA

6(European requests for plasmid to Alistair J. P. Brown, e-mail: al.brownt@abdn.ac.uk; all other requests to Brendan P. Cormack)

5Author for correspondence: Brendan P. Cormack. e-mail: cormack@cmgm.stanford.edu

ABSTRACT

The green fluorescent protein (GFP) of Aequorea victoria has been developed here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed in C. albicans, it was not possible to detect fluorescence or a translation product for the wt protein. Since this was probably due in part to the presence of the non-canonical CTG serine codon in the Aequorea sequence, this codon was changed to the leucine codon TTG. C. albicans cells expressing this construct contained GFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized GFP gene was constructed in which all of the 239 amino acids are encoded by optimal codons for C albicans. In this gene were also incorporated two previously identified mutations in the chromophore that increase GFP fluorescence. C. albicans cells expressing this yeast-enhanced GFP gene (yEGFP3) are fluorescent and contain GFP protein. yEGFP3 can be used as a versatile reporter of gene expression in C. albicans and Saccharomyces cerevisiae and the optimized GFP described here should have broad applications in these and other fungal species.

Keywords: enhanced GFP, green fluorescent protein, Candida albicans., Saccharomyces cerevisiae




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Control of Microtubule Dynamics by Stu2p Is Essential for Spindle Orientation and Metaphase Chromosome Alignment in Yeast
Mol. Biol. Cell, September 1, 2001; 12(9): 2870 - 2880.
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 Mol. Biol. CellHome page
F. Schaerer, G. Morgan, M. Winey, and P. Philippsen
Cnm67p Is a Spacer Protein of the Saccharomyces cerevisiae Spindle Pole Body Outer Plaque
Mol. Biol. Cell, August 1, 2001; 12(8): 2519 - 2533.
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 Mol. Cell. Biol.Home page
S. Dupre and R. Haguenauer-Tsapis
Deubiquitination Step in the Endocytic Pathway of Yeast Plasma Membrane Proteins: Crucial Role of Doa4p Ubiquitin Isopeptidase
Mol. Cell. Biol., July 15, 2001; 21(14): 4482 - 4494.
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 J. Bacteriol.Home page
A. Strauß, S. Michel, and J. Morschhäuser
Analysis of Phase-Specific Gene Expression at the Single-Cell Level in the White-Opaque Switching System of Candida albicans
J. Bacteriol., June 15, 2001; 183(12): 3761 - 3769.
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 J. Bacteriol.Home page
F. Rodrigues, M. van Hemert, H. Y. Steensma, M. Côrte-Real, and C. Leão
Red Fluorescent Protein (DsRed) as a Reporter in Saccharomyces cerevisiae
J. Bacteriol., June 15, 2001; 183(12): 3791 - 3794.
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 Appl. Environ. Microbiol.Home page
J. M. Lorang, R. P. Tuori, J. P. Martinez, T. L. Sawyer, R. S. Redman, J. A. Rollins, T. J. Wolpert, K. B. Johnson, R. J. Rodriguez, M. B. Dickman, et al.
Green Fluorescent Protein Is Lighting Up Fungal Biology
Appl. Envir. Microbiol., May 1, 2001; 67(5): 1987 - 1994.
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 MicrobiologyHome page
M. A. Uhl and A. D. Johnson
Development of Streptococcus thermophilus lacZ as a reporter gene for Candida albicans
Microbiology, May 1, 2001; 147(5): 1189 - 1195.
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 JCBHome page
M. Farkasovsky and H. Kuntzel
Cortical Num1p Interacts with the Dynein Intermediate Chain Pac11p and Cytoplasmic Microtubules in Budding Yeast
J. Cell Biol., January 22, 2001; 152(2): 251 - 262.
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 J. Virol.Home page
B. D. Price, M. Roeder, and P. Ahlquist
DNA-Directed Expression of Functional Flock House Virus RNA1 Derivatives in Saccharomyces cerevisiae, Heterologous Gene Expression, and Selective Effects on Subgenomic mRNA Synthesis
J. Virol., December 15, 2000; 74(24): 11724 - 11733.
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 Mol. Cell. Biol.Home page
J. Chen, S. Zhou, Q. Wang, X. Chen, T. Pan, and H. Liu
Crk1, a Novel Cdc2-Related Protein Kinase, Is Required for Hyphal Development and Virulence in Candida albicans
Mol. Cell. Biol., December 1, 2000; 20(23): 8696 - 8708.
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 GeneticsHome page
N. Marcoux, S. Cloutier, E. Zakrzewska, P.-M. Charest, Y. Bourbonnais, and D. Pallotta
Suppression of the Profilin-Deficient Phenotype by the RHO2 Signaling Pathway in Saccharomyces cerevisiae
Genetics, October 1, 2000; 156(2): 579 - 592.
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 MicrobiologyHome page
J. F. Ernst
Transcription factors in Candida albicans - environmental control of morphogenesis
Microbiology, August 1, 2000; 146(8): 1763 - 1774.
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 Nucleic Acids ResHome page
E. B. Vervoort, A. v. Ravestein, N. N. M. E. v. Peij, J. C. Heikoop, P. J. M. v. Haastert, G. F. Verheijden, and M. H. K. Linskens
Optimizing heterologous expression in Dictyostelium: importance of 5' codon adaptation
Nucleic Acids Res., May 15, 2000; 28(10): 2069 - 2074.
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 ScienceHome page
N. Ramanan and Y. Wang
A High-Affinity Iron Permease Essential for Candida albicans Virulence
Science, May 12, 2000; 288(5468): 1062 - 1064.
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J. Chen and P. Ahlquist
Brome Mosaic Virus Polymerase-Like Protein 2a Is Directed to the Endoplasmic Reticulum by Helicase-Like Viral Protein 1a
J. Virol., May 1, 2000; 74(9): 4310 - 4318.
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 MicrobiologyHome page
T.-W. Soong, T.-F. Yong, N. Ramanan, and Y. Wang
The Candida albicans antiporter gene CNH1 has a role in Na+ and H+ transport, salt tolerance, and morphogenesis
Microbiology, May 1, 2000; 146(5): 1035 - 1044.
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 GeneticsHome page
C. R. DeMattei, C. P. Davis, and J. B. Konopka
Point Mutations Identify a Conserved Region of the Saccharomyces cerevisiae AFR1 Gene That Is Essential for Both the Pheromone Signaling and Morphogenesis Functions
Genetics, May 1, 2000; 155(1): 43 - 55.
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 Clin. Microbiol. Rev.Home page
A. Alvarez-Barrientos, J. Arroyo, R. Canton, C. Nombela, and M. Sanchez-Perez
Applications of Flow Cytometry to Clinical Microbiology
Clin. Microbiol. Rev., April 1, 2000; 13(2): 167 - 195.
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 Proc. Natl. Acad. Sci. USAHome page
Z. Weissman, I. Berdicevsky, B.-Z. Cavari, and D. Kornitzer
The high copper tolerance of Candida albicans is mediated by a P-type ATPase
PNAS, March 28, 2000; 97(7): 3520 - 3525.
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 MicrobiologyHome page
C. L. Gordon, V. Khalaj, A. F. J. Ram, D. B. Archer, J. L. Brookman, A. P. J. Trinci, D. J. Jeenes, J. H. Doonan, B. Wells, P. J. Punt, et al.
Glucoamylase::green fluorescent protein fusions to monitor protein secretion in Aspergillus niger
Microbiology, February 1, 2000; 146(2): 415 - 426.
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 J. Bacteriol.Home page
S. V. Avery, S. Malkapuram, C. Mateus, and K. S. Babb
Copper/Zinc-Superoxide Dismutase Is Required for Oxytetracycline Resistance of Saccharomyces cerevisiae
J. Bacteriol., January 1, 2000; 182(1): 76 - 80.
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 J. Gen. Virol.Home page
L. Rubino, A. Di Franco, and M. Russo
Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology
J. Gen. Virol., January 1, 2000; 81(1): 279 - 286.
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 J. Biol. Chem.Home page
J. L. Urbanowski and R. C. Piper
The Iron Transporter Fth1p Forms a Complex with the Fet5 Iron Oxidase and Resides on the Vacuolar Membrane
J. Biol. Chem., December 31, 1999; 274(53): 38061 - 38070.
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 Mol. Biol. CellHome page
A. Madania, P. Dumoulin, S. Grava, H. Kitamoto, C. Schärer-Brodbeck, A. Soulard, V. Moreau, and B. Winsor
The Saccharomyces cerevisiae Homologue of Human Wiskott-Aldrich Syndrome Protein Las17p Interacts with the Arp2/3 Complex
Mol. Biol. Cell, October 1, 1999; 10(10): 3521 - 3538.
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 J. Bacteriol.Home page
Y. C. Chang and K. J. Kwon-Chung
Isolation, Characterization, and Localization of a Capsule-Associated Gene, CAP10, of Cryptococcus neoformans
J. Bacteriol., September 15, 1999; 181(18): 5636 - 5643.
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 Infect. Immun.Home page
A. B. Herrero, M. C. Lopez, S. Garcia, A. Schmidt, F. Spaltmann, J. Ruiz-Herrera, and A. Dominguez
Control of Filament Formation in Candida albicans by Polyamine Levels
Infect. Immun., September 1, 1999; 67(9): 4870 - 4878.
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 ScienceHome page
C. M. Hull and A. D. Johnson
Identification of a Mating Type-Like Locus in the Asexual Pathogenic Yeast Candida albicans
Science, August 20, 1999; 285(5431): 1271 - 1275.
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 JCBHome page
J. S. Tirnauer, E. O'Toole, L. Berrueta, B. E. Bierer, and D. Pellman
Yeast Bim1p Promotes the G1-specific Dynamics of Microtubules
J. Cell Biol., May 31, 1999; 145(5): 993 - 1007.
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 Infect. Immun.Home page
M. del Poeta, D. L. Toffaletti, T. H. Rude, S. D. Sparks, J. Heitman, and J. R. Perfect
Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein
Infect. Immun., April 1, 1999; 67(4): 1812 - 1820.
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 JCBHome page
A. Nern and R. A. Arkowitz
A Cdc24p-Far1p-G{beta}{gamma} Protein Complex Required for Yeast Orientation during Mating
J. Cell Biol., March 22, 1999; 144(6): 1187 - 1202.
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 Proc. Natl. Acad. Sci. USAHome page
H. K. Edskes, V. T. Gray, and R. B. Wickner
The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments
PNAS, February 16, 1999; 96(4): 1498 - 1503.
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 J. Cell Sci.Home page
A Balguerie, P Sivadon, M Bonneu, and M Aigle
Rvs167p, the budding yeast homolog of amphiphysin, colocalizes with actin patches
J. Cell Sci., January 8, 1999; 112(15): 2529 - 2537.
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 JCBHome page
H. J. Chial, M. P. Rout, T. H. Giddings Jr., and M. Winey
Saccharomyces cerevisiae Ndc1p Is a Shared Component of Nuclear Pore Complexes and Spindle Pole Bodies
J. Cell Biol., December 28, 1998; 143(7): 1789 - 1800.
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