Candida glabrata shuttle vectors suitable for translational fusions to lacZ and use of β-galactosidase as a reporter of gene expression

Barkani, Abdelmalic El; Haynes, Ken; Mösch, Hans‐Ulrich; Frosch, Matthias; Mühlschlegel, Fritz A.

doi:10.1016/s0378-1119(00)00065-2

Cited by 16 publications

(10 citation statements)

References 17 publications

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“…This indicates that CgOpi1p acts as a repressor of CgINO1. We were also able to demonstrate the repression of CgINO1 expression by CgOpi1p using the C. glabrata copper-inducible MTII promoter (El Barkani et al, 2000) as well (E. K. Bethea & T. B. Reynolds, unpublished).…”

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confidence: 80%

The inositol regulon controls viability in Candida glabrata

et al. 2010

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Inositol is essential in eukaryotes, and must be imported or synthesized. Inositol biosynthesis in Saccharomyces cerevisiae is controlled by three non-essential genes that make up the inositol regulon: ScINO2 and ScINO4, which together encode a heterodimeric transcriptional activator, and ScOPI1, which encodes a transcriptional repressor. ScOpi1p inhibits the ScIno2-ScIno4p activator in response to extracellular inositol levels. An important gene controlled by the inositol regulon is ScINO1, which encodes inositol-3-phosphate synthase, a key enzyme in inositol biosynthesis. In the pathogenic yeast Candida albicans, homologues of the S. cerevisiae inositol regulon genes are ‘transcriptionally rewired’. Instead of regulating the CaINO1 gene, CaINO2 and CaINO4 regulate ribosomal genes. Another Candida species that is a prevalent cause of infections is Candida glabrata; however, C. glabrata is phylogenetically more closely related to S. cerevisiae than C. albicans. Experiments were designed to determine if C. glabrata homologues of the inositol regulon genes function similarly to S. cerevisiae or are transcriptionally rewired. CgINO2, CgINO4 and CgOPI1 regulate CgINO1 in a manner similar to that observed in S. cerevisiae. However, unlike in S. cerevisiae, CgOPI1 is essential. Genetic data indicate that CgOPI1 is a repressor that affects viability by regulating activation of a target of the inositol regulon.

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confidence: 80%

The inositol regulon controls viability in Candida glabrata

et al. 2010

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“…A 699-bp DNA fragment containing the 5′ untranslated region (UTR) and the first 17 codons of the C. glabrata GAS2 gene was amplified from the genomic DNA of CBS138 and fused to the lacZ reporter in pEM14 [86] to construct pEM14-GAS2 as described in Table S8. Logarithmic-phase cells of the C. glabrata wild-type and Δ ire1 strains containing pEM14-GAS2 were grown in SC-ura broth, adjusted to 1×10 7 cells/ml, and then incubated in the presence and absence of 3 mM DTT for 2 h. β-galactosidase assay was performed as described previously [59].…”

Section: Methodsmentioning

confidence: 99%

Dissection of Ire1 Functions Reveals Stress Response Mechanisms Uniquely Evolved in Candida glabrata

et al. 2013

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Proper protein folding in the endoplasmic reticulum (ER) is vital in all eukaryotes. When misfolded proteins accumulate in the ER lumen, the transmembrane kinase/endoribonuclease Ire1 initiates splicing of HAC1 mRNA to generate the bZIP transcription factor Hac1, which subsequently activates its target genes to increase the protein-folding capacity of the ER. This cellular machinery, called the unfolded protein response (UPR), is believed to be an evolutionarily conserved mechanism in eukaryotes. In this study, we comprehensively characterized mutant phenotypes of IRE1 and other related genes in the human fungal pathogen Candida glabrata. Unexpectedly, Ire1 was required for the ER stress response independently of Hac1 in this fungus. C. glabrata Ire1 did not cleave mRNAs encoding Hac1 and other bZIP transcription factors identified in the C. glabrata genome. Microarray analysis revealed that the transcriptional response to ER stress is not mediated by Ire1, but instead is dependent largely on calcineurin signaling and partially on the Slt2 MAPK pathway. The loss of Ire1 alone did not confer increased antifungal susceptibility in C. glabrata contrary to UPR-defective mutants in other fungi. Taken together, our results suggest that the canonical Ire1-Hac1 UPR is not conserved in C. glabrata. It is known in metazoans that active Ire1 nonspecifically cleaves and degrades a subset of ER-localized mRNAs to reduce the ER load. Intriguingly, this cellular response could occur in an Ire1 nuclease-dependent fashion in C. glabrata. We also uncovered the attenuated virulence of the C. glabrata Δire1 mutant in a mouse model of disseminated candidiasis. This study has unveiled the unique evolution of ER stress response mechanisms in C. glabrata.

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“…27 This episomal plasmid was transformed into Cgnce103D and Cgrca1D to generate Cgnce103D + pCgNCE103-LacZ and Cgrca1D + pCgNCE103-LacZ, respectively.pCgNCE103-LacZ-MUT carrying a mutation in the NCE103 promoter was obtained after digestion of pCgNCE103-LacZ with AatII, blunting with Klenow fragment (Fermentas) and re-ligation. Sequencing confirmed that the original 'tcTGACGTCAac' sequence present in position À236 from the ATG was mutated to 'tcAac'.…”

Section: Plasmids Constructionmentioning

confidence: 99%