Tetracycline-Inducible Gene Expression and Gene Deletion in
            <i>Candida albicans</i>

Park, Yang-Nim; Morschhäuser, Joachim

doi:10.1128/ec.4.9.1603.2005

Cited by 42 publications

(70 citation statements)

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“…The resulting blunt-ended fragment was cloned into plasmid pNIM1 (from which CaGFP had been previously released by incubation with SalI and BglII) (Park and Morschhäuser 2005) to yield plasmid pTC46. The 243-bp region downstream of MBP1 was newly amplified from genomic DNA with primers MBP1-SacII and MBP1-SacI and cloned by blunt-end ligation in the MluI position of pTC46 to generate pTC47.…”

Section: Construction Of Disruption Cassettes and Gene Cloningmentioning

confidence: 99%

Phenotypic Consequences of a Spontaneous Loss of Heterozygosity in a Common Laboratory Strain of Candida albicans

et al. 2016

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By testing the susceptibility to DNA damaging agents of several Candida albicans mutant strains derived from the commonly used laboratory strain, CAI4, we uncovered sensitivity to methyl methanesulfonate (MMS) in CAI4 and its derivatives, but not in CAF2-1. This sensitivity is not a result of URA3 disruption because the phenotype was not restored after URA3 reintroduction. Rather, we found that homozygosis of a short region of chromosome 3R (Chr3R), which is naturally heterozygous in the MMS-resistant-related strains CAF4-2 and CAF2-1, confers MMS sensitivity and modulates growth polarization in response to MMS. Furthermore, induction of homozygosity in this region in CAF2-1 or CAF4-2 resulted in MMS sensitivity. We identified 11 genes by SNP/comparative genomic hybridization containing only the a alleles in all the MMS-sensitive strains. Four candidate genes, SNF5, POL1, orf19.5854.1, and MBP1, were analyzed by generating hemizygous configurations in CAF2-1 and CAF4-2 for each allele of all four genes. Only hemizygous MBP1a/mbp1b::SAT1-FLIP strains became MMS sensitive, indicating that MBP1a in the homo-or hemizygosis state was sufficient to account for the MMS-sensitive phenotype. In yeast, Mbp1 regulates G1/S genes involved in DNA repair. A second region of homozygosis on Chr2L increased MMS sensitivity in CAI4 (Chr3R homozygous) but not CAF4-2 (Chr3R heterozygous). This is the first example of sign epistasis in C. albicans.KEYWORDS Candida albicans; LOH; MMS susceptibility; MBP1; growth polarization T HE opportunistic fungal pathogen Candida albicans is often isolated as a highly heterozygous diploid; the genome of the reference strain SC5314 has 67,500 single nucleotide polymorphisms (SNPs) (Jones et al. 2004;Braun et al. 2005;Muzzey et al. 2013). SNPs found within regulatory regions can affect transcription levels between the alleles (Staib et al. 2002). Even synonymous SNPs residing in open reading frames (ORFS) can result in differences in the rate and efficiency of messenger RNA (mRNA) translation since poorly used codons delay protein synthesis. These delays may lead to misfolding of the nascent protein or formation of mRNA secondary structures (reviewed in Larriba and Calderone 2008). Recent genome-wide analysis of cis elements on gene expression showed that allele-specific effects are often due to mRNA levels and/or translation efficiency (Muzzey et al. 2014).While the majority of SNPs reside in intergenic regions, more than half of open reading frames contain one or more SNPs. The vast majority (78%) of these are nonsynonymous, implying that a significant fraction of ORFs encode proteins that differ in one or more amino acids (Jones et al. 2004) that may affect crucial properties. Nonconservative amino acid substitutions within an enzyme's catalytic domain could result in an inactive allele (Gómez-Raja et al. 2008). However, many SNPs will cause only minor or insignificant alterations in protein properties.Mitotic recombination or less frequently, chromosome truncation or loss, will reveal ...

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Section: Construction Of Disruption Cassettes and Gene Cloningmentioning

confidence: 99%

Phenotypic Consequences of a Spontaneous Loss of Heterozygosity in a Common Laboratory Strain of Candida albicans

et al. 2016

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“…The ARG4 gene fragment was then ligated into the YFP containing vector to create plasmid pYFP-ARG. Similarly, the SAT1 maker was PCR amplified from pNIM1 (Park and Morschhauser, 2005) by using oligos 13/14 and ligated into a Blunt II Topo vector. The SAT1 product was also digested with XhoI and XbaI and ligated with the YFP vector to create pYFP-SAT.…”

Section: Plasmidsmentioning

confidence: 99%

Stress-Induced Phenotypic Switching inCandida albicans

Alby

Bennett

2009

MBoC

112

108

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Candida albicans is both a common commensal and an opportunistic pathogen, being a prevalent cause of mucosal and systemic infections in humans. Phenotypic switching between white and opaque forms is a reversible transition that influences virulence, mating behavior, and biofilm formation. In this work, we show that a wide range of factors induces high rates of switching from white to opaque. These factors include different forms of environmental stimuli such as genotoxic and oxidative stress, as well as intrinsic factors such as mutations in DNA repair genes. We propose that these factors increase switching to the opaque phase via a common mechanism-inhibition of cell growth. To confirm this hypothesis, growth rates were artificially manipulated by varying expression of the CLB4 cyclin gene; slowing cell growth by depleting CLB4 resulted in a concomitant increase in white-opaque switching. Furthermore, two clinical isolates of C. albicans, P37005 and L26, were found to naturally exhibit both slow growth and high rates of white-opaque switching. Notably, suppression of the slow growth phenotype suppressed hyperswitching in the P37005 isolate. Based on the sensitivity of the switch to levels of the master regulator Wor1, we propose a model for how changes in cellular growth modulate white-opaque switching frequencies.

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“…Overexpression of candidate genes can therefore be used to complement studies of gene disruption [150]. A number of regulatable promoters have been developed to regulate gene expression, including the MET3 promoter [151], the MAL2 promoter [152] and tetracyclineregulated promoters (Tet-OFF and Tet-ON) [153,154]. A library of overexpression strains was constructed in a Tet-ON system for 107 putative transcription factors, most with a known biological role [155].…”

Section: Construction Of Systematic Strain Collectionsmentioning

confidence: 99%

Budding off: bringing functional genomics toCandida albicans

Anderson¹,

Bennett²

2015

Briefings in Functional Genomics

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Candida species are the most prevalent human fungal pathogens, with Candida albicans being the most clinically relevant species. Candida albicans resides as a commensal of the human gastrointestinal tract but is a frequent cause of opportunistic mucosal and systemic infections. Investigation of C. albicans virulence has traditionally relied on candidate gene approaches, but recent advances in functional genomics have now facilitated global, unbiased studies of gene function. Such studies include comparative genomics (both between and within Candida species), analysis of total RNA expression, and regulation and delineation of protein-DNA interactions. Additionally, large collections of mutant strains have begun to aid systematic screening of clinically relevant phenotypes. Here, we will highlight the development of functional genomics in C. albicans and discuss the use of these approaches to addressing both commensalism and pathogenesis in this species.

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Tetracycline-Inducible Gene Expression and Gene Deletion in Candida albicans

Cited by 42 publications

References 0 publications

Phenotypic Consequences of a Spontaneous Loss of Heterozygosity in a Common Laboratory Strain of Candida albicans

Phenotypic Consequences of a Spontaneous Loss of Heterozygosity in a Common Laboratory Strain of Candida albicans

Stress-Induced Phenotypic Switching inCandida albicans

Budding off: bringing functional genomics toCandida albicans

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