Promoter-dependent disruption of genes: simple, rapid, and specific PCR-based method with application to three different yeast
Promoter-dependent disruption of genes: simple, rapid, and specific PCR-based method with application to three different yeast
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DOI：
10.1007/s00294-005-0008-3
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发表时间：
2005-08-01
期刊：
影响因子：
2.5
通讯作者：
Katiyar, SK
中科院分区：
文献类型：
--
作者：
Edlind, TD;Henry, KW;Katiyar, SK
基于PCR产物的基因破坏极大地加快了酿酒酵母的分子分析。该方法包括扩增标记基因（例如，URA 3），包括其侧翼调节（启动子和多聚腺苷酸化）区，使用在其5'端包括与靶基因同源的约50个碱基的引物。不幸的是，这种方法已被证明在非同源重组率较高的生物体中不太有用;例如，在光滑念珠菌酵母中，所需的重组体比使用传统的基于PCR的基因破坏的已发表结果高出六倍。所有5 C。glabrata和C.白念珠菌突变体具有预测的氟钙超敏性（slt 2 δ和RH 0 1/rho 1 δ）、放线菌酮超敏性（pdr 1 δ）或米替福新耐药性（lem 3 δ和lem 3 δ/lem 3 δ）的表型。PRODIGE应用于S.在含有和不含该基因的Pdr 1-Pdr 3转录激活因子的菌株中对酿酒酵母PDR 5基因的研究证实，转化体产量和生长速率取决于启动子强度。使用这种PDR 5启动子-URA 3重组体，我们进一步证明了通过5-氟乳清酸选择产生调节突变体的方法的简单扩展。PRODIGE保证在其他酵母，霉菌和超越测试。
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 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 (slt2 Delta and RHO1/rho1 Delta), cycloheximide hypersensitivity (pdr1 Delta), or miltefosine resistance (lem3 Delta and lem3 Delta/lem3 Delta). 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.