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Volume 146, Issue 2

Single allele knock-out of leads to unexpected resistance to hygromycin B and elevated temperature Free

Abstract

Almost all eukaryotic mRNAs are capped at their 5′-terminus. Capping is crucial for stability, processing, nuclear export and efficient translation of mRNA. We studied the phenotypic effects elicited by depleting a strain of mRNA 5′-guanylyltransferase (mRNA capping enzyme; CGT1). Construction of a Cgt1-deficient mutant was achieved by URA-blaster-mediated genetic disruption of one allele of the gene, which was localized on chromosome III. The resulting heterozygous mutant exhibited an aberrant colony morphology resembling the ‘irregular wrinkle’ phenotype typically obtained from a normal strain upon mild UV treatment. Its level of mRNA was reduced two- to fivefold compared to the parental strain. Proteome analysis revealed a large number of differentially expressed proteins confirming the expected pleiotropic effect of disruption. The disrupted strain was significantly more resistant to hygromycin B, an antibiotic which decreases translational fidelity, and showed increased resistance to heat stress. Proteome analysis revealed a 50-fold overexpression of Ef-1αp and a more than sevenfold overexpression of the cell-wall heat-shock protein Ssa2p. Compared to a reference strain, the / heterozygote was equally virulent for mice and guinea pigs when tested in an intravenous infection model of disseminated candidiasis.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): Candida albicans , CGT1 , DIG, digoxigenin , GTase, 5′ guanylyltransferase , MALDI, matrix-assisted laser desorption/ionization , mRNA capping enzyme , proteome , real-time RNA quantitation , TEF, translation elongation factor and TPase, 5′ triphosphatase
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2000-02-01
2024-04-24
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Single allele knock-out of Candida albicans CGT1 leads to unexpected resistance to hygromycin B and elevated temperature
Microbiology 146, 353 (2000); https://doi.org/10.1099/00221287-146-2-353
/content/journal/micro/10.1099/00221287-146-2-353
/content/journal/micro/10.1099/00221287-146-2-353
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