Single-Copy
 IMH3
 Allele Is Sufficient To Confer Resistance to Mycophenolic Acid in
 Candida albicans
 and To Mediate Transformation of Clinical
 Candida
 Species

Beckerman, Janna L.; Chibana, Hiroji; Turner, Joshua; Magee, Patrick

doi:10.1128/iai.69.1.108-114.2001

Cited by 51 publications

(37 citation statements)

References 34 publications

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“…In vitro populations: To generate in vitro populations, strain AF7 was streaked out on synthetic galactose medium and grown 2 days at 30°to obtain single colonies, which were removed and transferred to each of 20, 5 ml YEPD liquid cultures (Beckerman et al 2001;Spell and Jinks-Robertson 2004;Mookerjee and Sia 2006). These were grown for 16 hr in a roller tube incubator at 30°.…”

Section: Methodsmentioning

confidence: 99%

“…Primers gal1-detF, ura3-detR, and 2020 were used for upstream and primers 2278, 2279, and 2280 were used for downstream diagnostic PCR (Table S1). Total genomic DNA extractions were carried out as described previously (Beckerman et al 2001). PCRs were carried out in a total volume of 25 ml with 10 mm Tris-HCl (pH 8.0); 50 mm KCl; 3 mm MgCl 2 ; 100 mm each dATP, dCTP, dGTP, and dTTP; 2.5 units Taq polymerase (rTaq, TAKARA); either 5 or 10 mmol of each primer (see Table S1); and 30 ng of genomic DNA under the following conditions: initial denaturation for 3 min at 94°, 30 cycles of a denaturation step for 1 min at 94°, a primer annealing step for 30 sec at 54°, and an extension step for 1 min at 72°.…”

Section: Methodsmentioning

confidence: 99%

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Evolution inCandida albicansPopulations During a Single Passage Through a Mouse Host

et al. 2009

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The mechanisms and rates by which genotypic and phenotypic variation is generated in opportunistic, eukaryotic pathogens during growth in hosts are not well understood. We evaluated genomewide genetic and phenotypic evolution in Candida albicans, an opportunistic fungal pathogen of humans, during passage through a mouse host (in vivo) and during propagation in liquid culture (in vitro). We found slower population growth and higher rates of chromosome-level genetic variation in populations passaged in vivo relative to those grown in vitro. Interestingly, the distribution of long-range loss of heterozygosity (LOH) and chromosome rearrangement events across the genome differed for the two growth environments, while rates of short-range LOH were comparable for in vivo and in vitro populations. Further, for the in vivo populations, there was a positive correlation of cells demonstrating genetic alterations and variation in colony growth and morphology. For in vitro populations, no variation in growth phenotypes was detected. Together, our results demonstrate that passage through a living host leads to slower growth and higher rates of genomic and phenotypic variation compared to in vitro populations. Results suggest that the dynamics of population growth and genomewide rearrangement contribute to the maintenance of a commensal and opportunistic life history of C. albicans.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Evolution inCandida albicansPopulations During a Single Passage Through a Mouse Host

et al. 2009

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“…Resistance to mycophenolic acid (MPA) was used as a marker to distinguish the progenitor from the evolved populations in competition assays designed to measure relative fitness (see below). We encountered a spontaneous mutant that was resistant to MPA while attempting to transform the progenitor strain to resistance to MPA with a cloned putative inosine 5-monophosphate dehydrogenase (IMH3) allele (kindly provided by P. T. Magee and J. Beckerman, University of Minnesota; materials and conditions are available from us upon request), which has been demonstrated to confer resistance to MPA in C. albicans (5). This mutant satisfied our requirements for (i) stability during growth in the absence of MPA and (ii) the absence of any detectable impact on fitness, the MIC of fluconazole, doubling time, or stationary-phase cell density.…”

Section: Methodsmentioning

confidence: 99%

Divergence in Fitness and Evolution of Drug Resistance in Experimental Populations of Candida albicans

2001

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“…In C. albicans, the expression level of URA3 affects filament development and virulence (3) and is apparently influenced by position effects (2). We wished to determine the effect of CaNAT1 expression on filamentous growth.…”

Section: Fig 2 Cells Expressing Canat1 Do Not Exhibit a Growth Disamentioning

confidence: 99%

CaNAT1 , a Heterologous Dominant Selectable Marker for Transformation of Candida albicans and Other Pathogenic Candida Species

2005

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A dominant selectable marker for Candida albicans and other Candida species, which confers resistance to nourseothricin, was characterized. In a heterologous promoter system and a recyclable cassette, the marker efficiently permitted deletion and complementation of C. albicans genes. Neither growth nor filamentous development was affected in strains expressing this marker.Candida albicans is the most commonly isolated invasive fungal pathogen. Molecular genetic analysis of this fungus to date is principally based on the marker URA3, a biosynthetic gene that complements uridine auxotrophy (5), and on a dominant selectable marker, MPA r or IMH3 r , developed from a mutant C. albicans gene (2, 9).We developed a heterologous dominant marker to investigate morphogenesis of C. albicans. We chose a gene conferring resistance to nourseothricin, since wild-type C. albicans is susceptible to moderate nourseothricin concentrations (250 to 450 g/ml). Codon usage of the Streptomyces noursei nat1 gene, encoding nourseothricin acetyltransferase, was adapted to that of C. albicans to generate CaNAT1 (6, 8). De novo synthesis was performed by Bionexus (Oakland, Calif.). CaNAT1 was placed under the control of the ACT1 promoter in vector pAU34, which contains URA3 as a selectable marker (15), generating pJK850. We transformed strain CAI4 (ura3/ura3) (5) with pJK850 to be uridine prototrophic. The resulting strains JKC435 and JKC436 were resistant to 250 g of nourseothri-FIG. 1. CaNAT1 confers nourseothricin resistance on C. albicans. The ura3/ura3 strain CAI4 was transformed with the empty vector pAU34, which bears the selectable marker URA3, giving rise to JKC347 and JKC348, and with pAU34 containing CaNAT1, giving rise to JKC345 and JKC346. Transformants were streaked onto medium to select for uridine prototrophy (sc-ura). They were then replica plated onto YPD containing 250 g of nourseothricin/ml (YPDϩnat250). 1, JKC437; 2, JKC438; 3, JKC436; 4, JKC435. FIG. 2. Cells expressing CaNAT1 do not exhibit a growth disadvantage. (A)Competition between wild-type SC5314 and the DLH1/dlh1:: CaNAT1 strain JKC336. Fresh cultures of the two strains were inoculated in equal parts into rich medium at a density of ca. 1 cell/ml. The numbers of total colonies and nourseothricin-resistant colonies were determined at 48 h, and the proportion of CaNAT1-expressing cells to wild-type cells was calculated. The cultures were diluted in fresh medium after 48 h to the starting density of ca. 1 cell/ml. This process was repeated three times. Two separate experiments were performed. Per time point, three samples were taken to determine numbers of CFU. Bars represent standard deviations. (B) Competition between strains CAF2-1 (URA3/ura3) and CAI4 (ura3/ura3). To control for the sensitivity of the competition experiment protocol, the ura3/ura3 homozygote CAI4 was tested in competition with the URA3/ura3 CAF2-1 heterozygote. For colony counts, cultures were plated onto YPD. They were replica plated onto medium to select for uridine prototrophy to determin...

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Single-Copy IMH3 Allele Is Sufficient To Confer Resistance to Mycophenolic Acid in Candida albicans and To Mediate Transformation of Clinical Candida Species

Abstract: Parasexual genetic analysis of

Cited by 51 publications

References 34 publications

Evolution inCandida albicansPopulations During a Single Passage Through a Mouse Host

Evolution inCandida albicansPopulations During a Single Passage Through a Mouse Host

Divergence in Fitness and Evolution of Drug Resistance in Experimental Populations of Candida albicans

CaNAT1 , a Heterologous Dominant Selectable Marker for Transformation of Candida albicans and Other Pathogenic Candida Species

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