Journal article
Promoter-dependent disruption of genes: simple, rapid, and specific PCR-based method with application to three different yeast
Current genetics, v 48(2), pp 117-125
Aug 2005
PMID: 16078083
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
PCR product-based gene disruption has greatly accelerated molecular analysis of Saccharomyces cerevisiae. This approach involves amplification of a marker gene (e.g., URA3) including its flanking regulatory (promoter and polyadenylation) regions using primers that include at their 5′ ends about 50 bases of homology to the targeted gene. Unfortunately, this approach has proved less useful in organisms with higher rates of non-homologous recombination; e.g., in the yeast Candida glabrata, desired recombinants represent ≤2% of transformants. We modified the PCR-based approach by eliminating marker-flanking regions and precisely targeting recombination such that marker expression depends on the regulatory sequences of the disrupted gene. Application of this promoter-dependent disruption of genes (PRODIGE) method to three C. glabrata genes (SLT2, LEM3, and PDR1) yielded desired recombinants at frequencies of 20, 31, and 11%, the latter representing a weakly expressed gene. For Candida albicans
LEM3 and RHO1, specificity was 79–95% for one or both alleles, >sixfold higher than the published results with conventional PCR-based gene disruption. All 5 C. glabrata and C. albicans mutants had predicted phenotypes of calcofluor hypersensitivity (slt2Δ and RHO1/rho1Δ), cycloheximide hypersensitivity (pdr1Δ), or miltefosine resistance (lem3Δ and lem3Δ/lem3Δ). PRODIGE application to the S. cerevisiae
PDR5 gene in strains with and without the Pdr1–Pdr3 transcriptional activators of this gene confirmed that transformant yield and growth rate depend on promoter strength. Using this PDR5 promoter-URA3 recombinant, we further demonstrate a simple extension of the method that yields regulatory mutants via 5-fluoroorotic acid selection. PRODIGE warrants testing in other yeast, molds, and beyond.
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Details
- Title
- Promoter-dependent disruption of genes: simple, rapid, and specific PCR-based method with application to three different yeast
- Creators
- Thomas Edlind - Department of Microbiology and Immunology Drexel University College of Medicine 2900 Queen Lane Philadelphia PA 19129 USAKarl Henry - Department of Microbiology and Immunology Drexel University College of Medicine 2900 Queen Lane Philadelphia PA 19129 USAJohn-Paul Vermitsky - Department of Microbiology and Immunology Drexel University College of Medicine 2900 Queen Lane Philadelphia PA 19129 USAMerritt Edlind - Department of Microbiology and Immunology Drexel University College of Medicine 2900 Queen Lane Philadelphia PA 19129 USAShriya Raj - Department of Microbiology and Immunology Drexel University College of Medicine 2900 Queen Lane Philadelphia PA 19129 USASantosh Katiyar - Department of Microbiology and Immunology Drexel University College of Medicine 2900 Queen Lane Philadelphia PA 19129 USA
- Publication Details
- Current genetics, v 48(2), pp 117-125
- Publisher
- Springer-Verlag; Berlin/Heidelberg
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Microbiology and Immunology
- Web of Science ID
- WOS:000231957800005
- Scopus ID
- 2-s2.0-27644475881
- Other Identifier
- 991014878044904721
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- Web of Science research areas
- Genetics & Heredity