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首页> 外文期刊>Molecular and Cellular Biology >Genetic manipulation of Saccharomyces cerevisiae by use of the LYS2 gene.
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Genetic manipulation of Saccharomyces cerevisiae by use of the LYS2 gene.

机译：通过使用LYS2基因进行酿酒酵母的遗传操作。

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The structural gene for alpha-aminoadipate reductase (LYS2) was isolated from a Saccharomyces cerevisiae genomic DNA library by complementation of a lys2 mutant. Both genetic and biochemical criteria confirmed that the DNA obtained corresponds to the LYS2 locus on chromosome II. Subcloning and deletion analysis showed that a functional LYS2 gene is contained within a 4.6-kilobase (kb) EcoRI-HindIII fragment of the original insert, and the slightly larger EcoRI-ClaI segment (4.8 kb) was used to construct a series of cloning vehicles, including integrating, episomal, replicative, and centromeric vectors. The cloned DNA was also used to generate a genomic deletion that lacks all LYS2 coding sequences on chromosome II. The level of the LYS2 transcript (4.2 kb) was 10-fold higher in cells grown on minimal medium than in cells grown on complete medium and was not repressed by the presence of lysine alone. Gene disruption, gene replacement, and promoter analysis of the major alpha-factor structural gene (MF alpha 1) were performed to illustrate the utility of the LYS2 gene for the genetic manipulation of yeasts. Because all fungi synthesize lysine via the alpha-aminoadipate pathway, the techniques developed here for using the S. cerevisiae LYS2 gene should be directly applicable to other fungal systems.
机译：通过lys2突变体的互补，从酿酒酵母基因组DNA库中分离出α-氨基己二酸还原酶（LYS2）的结构基因。遗传和生化标准均确认获得的DNA对应于II号染色体上的LYS2基因座。亚克隆和缺失分析表明，功能性LYS2基因包含在原始插入片段的4.6 kb（kb）EcoRI-HindIII片段内，并且使用稍大的EcoRI-ClaI片段（4.8 kb）构建了一系列克隆载体，包括整合型，附加型，复制型和着丝粒载体。克隆的DNA也用于产生缺少染色体II上所有LYS2编码序列的基因组缺失。在基本培养基上生长的细胞中，LYS2转录本的水平（4.2 kb）比在完全培养基上生长的细胞高10倍，并且单独存在赖氨酸并不能抑制。进行了基因破坏，基因替换和主要α因子结构基因（MF alpha 1）的启动子分析，以说明LYS2基因在酵母基因操作中的实用性。因为所有真菌都通过α-氨基己二酸途径合成赖氨酸，所以此处开发的使用酿酒酵母LYS2基因的技术应直接适用于其他真菌系统。

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