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Volume 151, Issue 4

Flanking direct repeats of alter marker expression at the locus of Free

Abstract

encodes an adhesin of and has been implicated in filamentation and virulence. , an often-used transformation selection marker, is apparently incorrectly expressed when integrated at the locus, which results in an attenuated virulence phenotype. Expression of is compromised by ectopic integration at other loci as well. In contrast, prior studies from the authors' laboratory had demonstrated that the filamentation deficiency and attenuated virulence of Δ mutants were fully restored in rescued strains in which was integrated at the locus. This discrepancy prompted a reinvestigation of these mutants. A series of congenic strains were constructed which demonstrated that the filamentation and virulence defects of a homozygous Δ mutant could be rescued without introduction of a functional allele. Despite the absence of detectable differences in expression, analysis of suppressor mutations suggested that reduced expression gave rise to the mutant phenotypes. Several independent spontaneous suppressor mutations that restored filamentation to strains of genotype Δ : : /Δ : :  had acquired a tandem duplication of the marker cassette. The null mutant and rescued strains differed by the presence or absence of flanking sequence. Substitution of the insert of the null mutant with alone largely rescued the filamentation and virulence phenotypes. The presence of a single copy of adjacent to had no effect. It is concluded that flanking direct repeats of , present as part of a recyclable disruption cassette, negatively influenced expression and are responsible for the previously reported phenotypes of the mutants.

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Keyword(s): OMPase, orotidine-5′-monophosphate decarboxylase
SGM
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2005-04-01
2024-04-19
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References

  1. AVMA 2001; 2000 Report of the AVMA Panel on Euthanasia. J Am Vet Med Assoc 218:669–696 [CrossRef]
     [Google Scholar]
  2. Brand A., Maccallum D. M., Brown A. J., Gow N. A., Odds F. C. 2004; Ectopic expression of URA3 can influence the virulence phenotypes and proteome ofCandida albicansbut can be overcome by targeted reintegration of URA3 at the RPS10 locus. Eukaryot Cell 3:900–909 [CrossRef]
     [Google Scholar]
  3. Brody R. S., Westheimer F. H. 1979; The purification of orotidine-5′-phosphate decarboxylase from yeast by affinity chromatography. J Biol Chem 254:4238–4244
     [Google Scholar]
  4. Campbell R. C. 1989 Statistics for Biologists, 3rd edn. Cambridge, UK: Cambridge University Press;
     [Google Scholar]
  5. Cheng S., Nguyen M. H., Zhang Z., Jia H., Handfield M., Clancy C. J. 2003; Evaluation of the roles of four Candida albicans genes in virulence by using gene disruption strains that express URA3 from the native locus. Infect Immun 71:6101–6103 [CrossRef]
     [Google Scholar]
  6. Daniels K. J., Lockhart S. R., Staab J. F., Sundstrom P., Soll D. R. 2003; The adhesin Hwp1 and the first daughter cell localize to the a/a portion of the conjugation bridge during Candida albicans mating. Mol Biol Cell 14:4920–4930 [CrossRef]
     [Google Scholar]
  7. Dawson B., Trapp R. G. 2001 Basic and Clinical Biostatistics, 3rd edn. New York: Lange Medical Books;
     [Google Scholar]
  8. Fonzi W. A., Irwin M. Y. 1993; Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717–728
     [Google Scholar]
  9. Fonzi W. A., Saporito-Irwin S., Chen J.-Y., Sypherd P. S. 1993; Genetic basis for dimorphism and pathogenicity in Candida albicans. In Dimorphic Fungi in Biology and Medicine pp 37–50 Edited by Bossche H. Vanden, Odds F. C., Kerridge D. New York: Plenum;
     [Google Scholar]
  10. Garcia M. G., O'Connor J. E., Garcia L. L., Martinez S. I., Herrero E., Agudo L. 2001; Isolation of a Candida albicans gene, tightly linked to URA3, coding for a putative transcription factor that suppresses a Saccharomyces cerevisiae aft1 mutation. Yeast 18:301–311 [CrossRef]
     [Google Scholar]
  11. Gillum A. M., Tsay E. Y. H., Kirsch D. R. 1984; Isolation of the Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation ofS. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198:179–182 [CrossRef]
     [Google Scholar]
  12. Greger I. H., Proudfoot N. J. 1998; Poly(A) signals control both transcriptional termination and initiation between the tandem GAL10 and GAL7 genes of Saccharomyces cerevisiae. EMBO J 17:4771–4779 [CrossRef]
     [Google Scholar]
  13. Kirsch D. R., Whitney R. R. 1991; Pathogenicity of Candida albicans auxotrophic mutants in experimental infections. Infect Immun 59:3297–3300
     [Google Scholar]
  14. Kurtz M. B., Cortelyou M. W., Miller S. M., Lai M., Kirsch D. R. 1987; Development of autonomously replicating plasmids for Candida albicans. Mol Cell Biol 7:209–217
     [Google Scholar]
  15. Lay J., Henry L. K., Clifford J., Koltin Y., Bulawa C. E., Becker J. M. 1998; Altered expression of selectable marker URA3 in gene-disrupted Candida albicans strains complicates interpretation of virulence studies. Infect Immun 66:5301–5306
     [Google Scholar]
  16. Mason M. M., Lasker B. A., Riggsby W. S. 1987; Molecular probe for identification of medically important Candida species and Torulopsis glabrata. J Clin Microbiol 25:563–566
     [Google Scholar]
  17. Murad A. M., Lee P. R., Broadbent I. D., Barelle C. J., Brown A. J. 2000; CIp10, an efficient and convenient integrating vector for Candida albicans. Yeast 16:325–327 [CrossRef]
     [Google Scholar]
  18. Odds F. C., Van Nuffel L., Gow N. A. 2000; Survival in experimental Candida albicans infections depends on inoculum growth conditions as well as animal host. Microbiology 146:1881–1889
     [Google Scholar]
  19. Porta A., Ramon A. M., Fonzi W. A. 1999; PRR1, a homolog ofAspergillus nidulans palF, controls pH-dependent gene expression and filamentation in Candida albicans. J Bacteriol 181:7516–7523
     [Google Scholar]
  20. Ramon A. M., Porta A., Fonzi W. A. 1999; Effect of environmental pH on morphological development of Candida albicans is mediated via the PacC-related transcription factor encoded byPRR2. J Bacteriol 181:7524–7530
     [Google Scholar]
  21. Russel P. J., Welsch J. A., Rachlin E. M., Mccloskey J. A. 1987; Different levels of DNA methylation in yeast and mycelial forms of Candida albicans. J Bacteriol 169:4393–4395
     [Google Scholar]
  22. Saporito-Irwin S. M., Birse C. E., Sypherd P. S., Fonzi W. A. 1995; PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis. Mol Cell Biol 15:601–613
     [Google Scholar]
  23. Saxe D., Datta A., Jinks-Robertson S. 2000; Stimulation of mitotic recombination events by high levels of RNA polymerase II transcription in yeast. Mol Cell Biol 20:5404–5414 [CrossRef]
     [Google Scholar]
  24. Selker E. U. 1997; Epigenetic phenomena in filamentous fungi: useful paradigms or repeat-induced confusion?. Trends Genet 13:296–301 [CrossRef]
     [Google Scholar]
  25. Selker E. U. 1998; Trichostatin A causes selective loss of DNA methylation in Neurospora. Proc Natl Acad Sci U S A 95:9430–9435 [CrossRef]
     [Google Scholar]
  26. Sharkey L. L., Mcnemar M. D., Saporito-Irwin S. M., Sypherd P. S., Fonzi W. A. 1999; HWP1 functions in the morphological development ofCandida albicansdownstream of EFG1, TUP1 and RBF1. J Bacteriol 181:5273–5279
     [Google Scholar]
  27. Sherman F., Fink G. R., Hicks J. B. 1986 Methods in Yeast Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratories;
     [Google Scholar]
  28. 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 Chem 271:6298–6305 [CrossRef]
     [Google Scholar]
  29. Staab J. F., Bradway S. D., Fidel P. L., Sundstrom P. 1999; Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1. Science 283:1535–1538 [CrossRef]
     [Google Scholar]
  30. Sundstrom P., Cutler J. E., Staab J. F. 2002a; Reevaluation of the role of HWP1 in systemic candidiasis by use of Candida albicans strains with selectable marker URA3 targeted to the ENO1 locus. Infect Immun 70:3281–3283 [CrossRef]
     [Google Scholar]
  31. Sundstrom P., Balish E., Allen C. M. 2002b; Essential role of the Candida albicans transglutaminase substrate, hyphal wall protein 1, in lethal oroesophageal candidiasis in immunodeficient mice. J Infect Dis 185:521–530 [CrossRef]
     [Google Scholar]
  32. Thomas B. J., Rothstein R. 1989; Elevated recombination rates in transcriptionally active DNA. Cell 56:619–630 [CrossRef]
     [Google Scholar]
  33. Tsuchimori N., Sharkey L. L., Fonzi W. A., French S. W., Filler S. G, Edwards J. E. Jr 2000; Reduced virulence of HWP1-deficient mutants of Candida albicans and their interactions with host cells. Infect Immun 68:1997–2002 [CrossRef]
     [Google Scholar]
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Flanking direct repeats of hisG alter URA3 marker expression at the HWP1 locus of Candida albicans
Microbiology 151, 1061 (2005); https://doi.org/10.1099/mic.0.27487-0
/content/journal/micro/10.1099/mic.0.27487-0
/content/journal/micro/10.1099/mic.0.27487-0
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