Construction and characterization of bidirectional expression vectors in Saccharomyces cerevisiae
Construction and characterization of bidirectional expression vectors in Saccharomyces cerevisiae
复制标题
DOI：
10.1111/j.1567-1364.2007.00335.x
复制
发表时间：
2008-02-01
影响因子：
3.2
通讯作者：
Deng, Xuming
中科院分区：
文献类型：
--
作者：
Li, Aimin;Liu, Zengshan;Deng, Xuming
酿酒酵母的 GAL1 和 GAL10 基因的转录方式不同，这两种基因的转录均可被半乳糖诱导并被葡萄糖抑制。本研究描述了 8 个双向表达载体的构建和表征。这些载体在一个方向上携带修饰的诱导型 GAL 启动子，在相反方向上携带组成型 GPD 启动子。当将基因编码的α-半乳糖苷酶克隆到修饰的GAL1和GAL10载体中时，GAL1启动子的启动子活性分别为野生型的85%，GAL10启动子的启动子活性分别为野生型的90%。修饰的 GAL 启动子和 GPD 启动子在双向载体中不会相互干扰。此外，在双向载体中修饰的GAL I或修饰的GAL 10的控制下酵母过表达人Bax赋予致死表型，通过在同一载体中GPD启动子的控制下共表达人Bcl-2来挽救这种致死表型。这八个载体可用于表达致死基因并筛选将酵母从致死基因产物中拯救出来的基因。
The GAL1 and GAL10 genes of Saccharomyces cerevisiae are transcribed divergently and transcription of both genes can be induced by galactose and repressed by glucose. This study describes the construction and characterization of 8 bidirectional expression vectors. These vectors carry both a modified inducible GAL promoter in one direction and a constitutive GPD promoter in the reverse direction. When the gene-encoded alpha-galactosidase was cloned into the modified GAL1 and GAL10 vectors, promoter activity was 85% of wild-type for the GAL1 promoter and 90% of wild-type for the GAL10 promoter, respectively. The modified GAL promoters and GPD promoter did not interfere with one another in the bidirectional vectors. Furthermore, yeast overexpressing human Bax under the control of either modified GAL I or modified GAL 10 in a bidirectional vector conferred a lethal phenotype that was rescued by coexpression of human Bcl-2 under the control of the GPD promoter in the same vector. These eight vectors can be used to express lethal genes and screen for genes that rescue the yeast from the lethal gene product.