Stable transformation and regulated expression of an inducible reporter construct in

Brown, D. H.; Slobodkin, I. V.; Kumamoto, Carol A.

doi:10.1007/s004380050142

Cited by 9 publications

(3 citation statements)

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“…The CaMAL2 promoter has recently been reported to be suitable for regulated gene expression in C. albicans (Brown et al, 1996 ;Cormack et al, 1997 ;Backen et al, 2000). Next, the promoter region was fused with the 3hUTR of the gene encoding the C. albicans elongation factor, CaEFB1 (Maneau et al, 1996).…”

Section: Construction Of C Albicans Strains Bearing Genetically Engimentioning

confidence: 99%

Genetically engineered human salivary histatin genes are functional in Candida albicans: development of a new system for studying histatin candidacidal activity

Baev

Edgerton

2001

Microbiology

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Histatins are a structurally related family of salivary proteins known as histidine-rich proteins that are produced and secreted by the human major salivary glands. In vitro, histatins are potent cytotoxic proteins with selectivity for pathogenic yeasts including Candida albicans. Studies that investigate the mechanism of action of histatin proteins upon this important human pathogen have used a candidacidal assay in which the histatin is applied extracellularly. In order to develop a model system to study the mechanism of histatin action independently from binding and translocation events, the authors constructed C. albicans strains that contain chromosomally encoded human salivary histatin genes under the control of a regulated promoter. Intracellular expression of either histatin 5 or histatin 3 induced cell killing and ATP release in parallel. Since histatin killing can be initiated solely from intracellular sites, extracellular binding and internalization are preceding transport events. Thus the mechanism of histatin-induced ATP release does not require extracellular binding, and intracellular targets alone can activate ATP release. By employing a codon-optimization strategy it was shown that expression of heterologous sequences in C. albicans can be a useful tool for functional studies.

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Section: Construction Of C Albicans Strains Bearing Genetically Engimentioning

confidence: 99%

Genetically engineered human salivary histatin genes are functional in Candida albicans: development of a new system for studying histatin candidacidal activity

Baev

Edgerton

2001

Microbiology

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“…Either wild-type LAC4 or the mutant derivatives were integrated into the C. albicans genome and expressed from the MAL2 (maltase) promoter. MAL2 is induced by maltose and repressed by glucose (Brown et al, 1996). No activity was detected in strains with the MAL2-LAC4 wild-type and MAL2-LAC4 mutant derivatives under inducing conditions using both liquid assays and qualitative plate assays (Fig.…”

Section: Assays Of β-Galactosidases In C Albicansmentioning

confidence: 99%

“…LAC4 from (Poch et al, 1992) and Strep. thermophilus lacZ (Schroeder et al, 1991) were placed under control of the maltose-inducible C. albicans MAL2 promoter (Brown et al, 1996) and integrated into the C. albicans genome. β-Galactosidase was scored by visual assays on medium containing X-Gal and maltose and by liquid assays on cells grown in medium with maltose (Ausubel et al, 1992).…”

Section: Assays Of β-Galactosidases In C Albicansmentioning

confidence: 99%

Development of Streptococcus thermophilus lacZ as a reporter gene for Candida albicans

Uhl

Johnson

2001

Microbiology

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The study of gene regulation in many organisms has been facilitated by the development of reporter genes. The authors report the use of lacZ from Streptococcus thermophilus, a gene encoding a β-galactosidase, as a reporter for the fungal pathogen Candida albicans. As test cases, Strep. thermophilus lacZ was placed under control of three different C. albicans promoters : MAL2 (maltase), inducible by maltose ; HWP1 (hyphal cell wall protein), induced by conditions that promote filamentous growth ; and ACT1 (actin). These constructs were each integrated into the C. albicans genome and β-galactosidase activity was readily detected from these strains, but only under the appropriate growth conditions. β-Galactosidase activity could be detected by several methods : quantitative liquid assays using permeabilized cells, colorimetric assays of colonies replicated to paper filters, and in situ coloration of colonies growing on medium containing the indicator X-Gal. These results show the usefulness of Strep. thermophilus lacZ as a monitor of gene regulation in this medically important yeast.

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Restriction enzyme-mediated insertional mutagenesis: an efficient method of Rosellinia necatrix transformation

2017

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Rosellinia necatrix: causing root rot disease is a very destructive pathogen of woody plants and is responsible for yield losses to a large number of fruit trees. The genetic analysis of this pathogen has not been picked up because of difficulty in generating mutations in Rosellinia necatrix for many reasons. A number of methods have been proposed for inducing mutations in Rosellinia necatrix but none of them proved worth because of very low transformation efficiencies. Here, we propose an efficient method for Rosellinia necatrix protoplast production, where protoplasts in the tune of 10 per ml can be easily generated. We also propose a restriction enzyme-mediated integration (REMI)-based methods for efficient transformation of Rosellinia necatrix. In the present study, an approximate of 800 transformants was obtained from 5 μg of linearized plasmid. Out of 47 single spored transformants analyzed, only 33 showed hygromycin gene amplification using PCR and only 19 transformants showed single gene integration in southern hybridization, which accounted for single gene integration percentage of 42%, highest amongst all the previous reports on Rosellinia necatrix transformations. Some of the transformants studied for pathogenicity phenotype also showed a marked reduction in pathogenicity. Thus, in the present investigation, 42% single gene integrations among the transformed colonies can be considered as excellent transformation efficiency.

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Stable transformation and regulated expression of an inducible reporter construct in

Cited by 9 publications

References 0 publications

Genetically engineered human salivary histatin genes are functional in Candida albicans: development of a new system for studying histatin candidacidal activity

Genetically engineered human salivary histatin genes are functional in Candida albicans: development of a new system for studying histatin candidacidal activity

Development of Streptococcus thermophilus lacZ as a reporter gene for Candida albicans

Restriction enzyme-mediated insertional mutagenesis: an efficient method of Rosellinia necatrix transformation

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