A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae.
A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae.
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DOI：
10.1002/cfg.85
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发表时间：
2001
影响因子：
--
通讯作者：
Klis, F M
中科院分区：
文献类型：
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作者：
de Groot, P W;Ruiz, C;Vazquez de Aldana, C R;Duenas, E;Cid, V J;Del Rey, F;Rodriquez-Pena, J M;Perez, P;Andel, A;Caubin, J;Arroyo, J;Garcia, J C;Gil, C;Molina, M;Garcia, L J;Nombela, C;Klis, F M
使用分层方法，系统地筛选了由EUROFAN I产生的620个非必需单基因酵母缺失体，以寻找与细胞壁相关的表型。通过分析对生长介质中存在的硫代荧光白或十二烷基硫酸钠的敏感性改变、对超声波的敏感性改变或形态异常，选择了145个(23%)至少具有一种细胞壁相关表型的突变体。进一步对这些基因进行筛选，以确定可能参与细胞壁大分子的生物合成、重塑或偶联的基因，或者参与细胞壁结构整体调控的基因，并消除那些具有更普遍的多效性效应的基因。从初步试验中选择的突变体中，有90%表现出与细胞壁相关的额外表型。当推算到整个酵母基因组时，这些数据表明，超过1200个基因可能直接或间接影响细胞壁的形成及其调节。发现了21个β1，3-葡聚糖合成酶活性改变的突变体和5个甲壳素合成酶活性改变的抗氯氟白色突变体，表明相应的基因影响β1，3-葡聚糖或甲壳素的合成。通过选择生长介质中特定细胞壁成分水平的增加，我们鉴定了可能与细胞壁组装的不同步骤有关的13个基因。此外，14个突变体表现出细胞壁完整性途径的结构性激活，这表明它们要么直接作为信号成分参与该途径的调节，要么通过触发依赖于SLT2的补偿机制来参与调节。总之，我们的筛选方法代表了对参与真菌细胞壁形成的各个方面的基因产物的基因组规模的全面功能分析。
Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of β1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect β1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.