1887

Abstract

The study of gene regulation in many organisms has been facilitated by the development of reporter genes. The authors report the use of from , a gene encoding a β-galactosidase, as a reporter for the fungal pathogen . As test cases, was placed under control of three different promoters: (maltase), inducible by maltose; (hyphal cell wall protein), induced by conditions that promote filamentous growth; and (actin). These constructs were each integrated into the genome and β-galactosidase activity was readily detected from these strains, but only under the appropriate growth conditions. β-Galactosidase activity could be detected by several methods: quantitative liquid assays using permeabilized cells, colorimetric assays of colonies replicated to paper filters, and coloration of colonies growing on medium containing the indicator X-Gal. These results show the usefulness of as a monitor of gene regulation in this medically important yeast.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-5-1189
2001-05-01
2020-01-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/5/1471189a.html?itemId=/content/journal/micro/10.1099/00221287-147-5-1189&mimeType=html&fmt=ahah

References

  1. Alex L. A., Korch C., Selitrennikoff C. P., Simon M. I. 1998; COS1, a two-component histidine kinase that is involved in hyphal development in the opportunistic pathogen Candida albicans. Proc Natl Acad Sci USA95:7069–7073[CrossRef]
    [Google Scholar]
  2. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1992; Current Protocols in Molecular Biology New York: Greene Publishing Associates and Wiley-Interscience;
    [Google Scholar]
  3. Blanco C., Ritzenthaler P., Mata-Gilsinger M. 1985; Nucleotide sequence of a regulatory region of the uidA gene in Escherichia coli K12. Mol Gen Genet199:101–105[CrossRef]
    [Google Scholar]
  4. Braun B. R., Johnson A. D. 1997; Control of filament formation in Candida albicans by the transcriptional repressor TUP1. Science277:105–109[CrossRef]
    [Google Scholar]
  5. Brown A. J., Gow N. A. 1999; Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol7:333–338[CrossRef]
    [Google Scholar]
  6. Brown D. H. Jr, Slobodkin I. V., Kumamoto C. A. 1996; Stable transformation and regulated expression of an inducible reporter construct in Candida albicans using restriction enzyme-mediated integration. Mol Gen Genet251:75–80
    [Google Scholar]
  7. Burns N., Grimwade B., Ross-Macdonald P. B., Choi E. Y., Finberg K., Roeder G. S., Snyder M. 1994; Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. Genes Dev8:1087–1105[CrossRef]
    [Google Scholar]
  8. Calera J. A., Zhao X. J., Calderone R. 2000; Defective hyphal development and avirulence caused by a deletion of the SSK1 response regulator gene in Candida albicans. Infect Immun68:518–525[CrossRef]
    [Google Scholar]
  9. Cormack B. P., Bertram G., Egerton M., Gow N. A. R., Falkow S., Brown A. J. 1997; Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology 143:303–311[CrossRef]
    [Google Scholar]
  10. Csank C., Makris C., Meloche S., Dignard D., Thomas D. Y., Whiteway M, Schröppel K., Röllinghoff M.. 1997; Derepressed hyphal growth and reduced virulence in a VH1 family-related protein phosphatase mutant of the human pathogen Candida albicans. Mol Biol Cell. 82539–2551[CrossRef]
  11. Csank C., Schroppel K., Leberer E., Harcus D., Mohamed O., Meloche S., Thomas D. Y., Whiteway M. 1998; Roles of the Candida albicans mitogen-activated protein kinase homolog, Cek1p, in hyphal development and systemic candidiasis. Infect Immun66:2713–2721
    [Google Scholar]
  12. Delbruck S., Ernst J. F. 1993; Morphogenesis-independent regulation of actin transcript levels in the pathogenic yeast Candida albicans. Mol Microbiol. 10859–866[CrossRef]
  13. Fonzi W. A., Irwin M. Y. 1993; Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717–728
    [Google Scholar]
  14. Gale C. A., Bendel C. M., McClellan M., Hauser M., Becker J. M., Berman J., Hostetter M. K. 1998; Linkage of adhesion, filamentous growth, and virulence in Candida albicans to a single gene, INT1. Science279:1355–1358[CrossRef]
    [Google Scholar]
  15. Gietz R. D., Schiestl R. H., Willems A. R., Woods R. A. 1995; Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast11:355–360[CrossRef]
    [Google Scholar]
  16. Gilbert W., Maxam A. 1973; The nucleotide sequence of the lac operator. Proc Natl Acad Sci USA70:3581–3584[CrossRef]
    [Google Scholar]
  17. Gimeno C. J., Ljungdahl P. O., Styles C. A., Fink G. R. 1992; Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS . Cell68:1077–1090[CrossRef]
    [Google Scholar]
  18. Guarente L. 1983; Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol101:181–191
    [Google Scholar]
  19. Guarente L., Ptashne M. 1981; Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae. Proc Natl Acad Sci USA78:2199–2203[CrossRef]
    [Google Scholar]
  20. Guthrie C., Fink G. R. 1991; Guide to Yeast Genetics and Molecular Biology San Diego: Academic Press;
    [Google Scholar]
  21. Hill J., Donald K. A., Griffiths D. E., Donald G. 1991; DMSO-enhanced whole cell yeast transformation. [An erratum appears in Nucleic Acids Res 11, 6688.]. Nucleic Acids Res19:5791[CrossRef]
    [Google Scholar]
  22. Jacobson R. H., Zhang X. J., DuBose R. F., Matthews B. W. 1994; Three-dimensional structure of beta-galactosidase from E. coli. Nature 369:761–766[CrossRef]
    [Google Scholar]
  23. Kwon-Chung K. B. 1992; Medical Mycology Philadelphia: Lea & Febinger;
    [Google Scholar]
  24. Leberer E., Harcus D., Broadbent I. D.. 7 other authors 1996; Signal transduction through homologues of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans. Proc Natl Acad Sci USA93:13217–13222[CrossRef]
    [Google Scholar]
  25. Leberer E., Ziegelbauer K., Schmidt A., Harcus D., Dignard D., Ash J., Johnson L., Thomas D. Y. 1997; Virulence and hyphal formation of Candida albicans require the Ste20p-like protein kinase CaCla4p. Curr Biol7:539–546[CrossRef]
    [Google Scholar]
  26. Leuker C. E., Hahn A. M., Ernst J. F. 1992; β-Galactosidase of Kluyveromyces lactis (Lac4p) as reporter of gene expression in Candida albicans and C. tropicalis. Mol Gen Genet235:235–241[CrossRef]
    [Google Scholar]
  27. Leuker C. E., Sonneborn A., Delbruck S., Ernst J. F. 1997; Sequence and promoter regulation of the PCK1 gene encoding phosphoenolpyruvate carboxykinase of the fungal pathogen Candida albicans. Gene 192:235–240[CrossRef]
    [Google Scholar]
  28. Liu H., Kohler J., Fink G. R. 1994; Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. Science266:1723–1726[CrossRef]
    [Google Scholar]
  29. Lo H. J., Kohler J. R., DiDomenico B., Loebenberg D., Cacciapuoti A., Fink G. R. 1997; Nonfilamentous C. albicans mutants are avirulent. Cell90:939–949[CrossRef]
    [Google Scholar]
  30. Myers K. K., Sypherd P. S., Fonzi W. A. 1995; Use of URA3 as a reporter of gene expression in C. albicans. Curr Genet27:243–248[CrossRef]
    [Google Scholar]
  31. Newport G., Agabian N. 1997; KEX2 influences Candida albicans proteinase secretion and hyphal formation. J Biol Chem272:28954–28961[CrossRef]
    [Google Scholar]
  32. Odds F. C. 1988; Candida and Candidosis , 2nd edn. London: Baillière Tindall;
    [Google Scholar]
  33. Ohama T., Suzuki T., Mori M., Osawa S., Ueda T., Watanabe K., Nakase T. 1993; Non-universal decoding of the leucine codon CUG in several Candida species. Nucleic Acids Res21:4039–4045[CrossRef]
    [Google Scholar]
  34. Poch O., L’Hote H., Dallery V., Debeaux F., Fleer R., Sodoyer R. 1992; Sequence of the Kluyveromyces lactis beta-galactosidase: comparison with prokaryotic enzymes and secondary structure analysis. Gene118:55–63[CrossRef]
    [Google Scholar]
  35. Rose M., Casadaban M. J., Botstein D. 1981; Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast. Proc Natl Acad Sci USA78:2460–2464[CrossRef]
    [Google Scholar]
  36. Schroeder C. J., Robert C., Lenzen G., McKay L. L., Mercenier A. 1991; Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus β-galactosidase sequences. J Gen Microbiol137:369–380[CrossRef]
    [Google Scholar]
  37. Sreekrishna K., Dickson R. C. 1985; Construction of strains of Saccharomyces cerevisiae that grow on lactose. Proc Natl Acad Sci USA82:7909–7913[CrossRef]
    [Google Scholar]
  38. Srikantha T., Klapach A., Lorenz W. W., Tsai L. K., Laughlin L. A., Gorman J. A., Soll D. R. 1996; The sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differential gene expression in Candida albicans. J Bacteriol178:121–129
    [Google Scholar]
  39. Srikantha T., Tsai L. K., Soll D. R. 1997; The WHI1 gene of Candida albicans is regulated in two distinct developmental programs through the same transcription activation sequences. J Bacteriol179:3837–3844
    [Google Scholar]
  40. Staab J. F., Ferrer C. A., Sundstrom P. 1996; Developmental expression of a tandemly repeated, proline- and glutamine-rich amino acid motif on hyphal surfaces of Candida albicans. J Biol Chem271:6298–6305[CrossRef]
    [Google Scholar]
  41. Stoldt V. R., Sonneborn A., Leuker C. E., Ernst J. F. 1997; Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans , is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi. EMBO J16:1982–1991[CrossRef]
    [Google Scholar]
  42. Swoboda R. K., Bertram G., Delbruck S., Ernst J. F., Gow N. A. R., Gooday G. W., Brown A. J. 1994; Fluctuations in glycolytic mRNA levels during morphogenesis in Candida albicans reflect underlying changes in growth and are not a response to cellular dimorphism. Mol Microbiol13:663–672[CrossRef]
    [Google Scholar]
  43. Timpel C., Strahl-Bolsinger S., Ziegelbauer K., Ernst J. F. 1998; Multiple functions of Pmt1p-mediated protein O-mannosylation in the fungal pathogen Candida albicans. J Biol Chem273:20837–20846[CrossRef]
    [Google Scholar]
  44. Wirsching S., Michel S., Kohler G., Morschhauser J. 2000; Activation of the multiple drug resistance gene MDR1 in fluconazole-resistant, clinical Candida albicans strains is caused by mutations in a trans -regulatory factor. J Bacteriol182:400–404[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-5-1189
Loading
/content/journal/micro/10.1099/00221287-147-5-1189
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error