Blazhenko O V, Kotliarchuk A B, Ubyĭvovk V M
Ukr Biochem J. 2014 Jan-Feb;86(1):75-84.
In a previous study we cloned GSH2 gene, encoding gamma-glutamylcysteine synthetase (gammaGCS) in the yeast Hansenula polymorpha. In this study an analysis of molecular organisation of the H. polymorpha GSH2 gene promoter was conducted and the potential binding sites of Yap1, Skn7, Creb/Atf1, and Cbfl transcription factors were detected. It was established that full regulation of GSH2 gene expression in the response to cadmium and oxidative stress requires the length of GSH2 promoter to be longer than 450 bp from the start of translation initiation. To study the transcriptional regulation of H. polymorpha GSH2 gene recombinant strain, harbouring a reporter system, in which 1.832 kb regulatory region of GSH2 gene was fused to structural and terminatory regions of alcohol oxidase gene, was constructed. It was shown that maximum increase in H. polymorpha GSH2 gene transcription by 33% occurs in the rich medium under four-hour incubation with 1 microM concentration of cadmium ions. In the minimal medium the GSH2 gene expression does not correlate with the increased total cellular glutathione levels under cadmium ion treatment. We assume that the increased content of total cellular glutathione under cadmium stress in the yeast H. polymorpha probably is not controlled on the level of GSH2 gene transcription.
在之前的一项研究中，我们克隆了编码多形汉逊酵母中γ-谷氨酰半胱氨酸合成酶（γGCS）的GSH2基因。在本研究中，对多形汉逊酵母GSH2基因启动子的分子组织进行了分析，并检测了Yap1、Skn7、Creb/Atf1和Cbfl转录因子的潜在结合位点。已确定，要在镉和氧化应激反应中完全调节GSH2基因表达，GSH2启动子从翻译起始点开始的长度需长于450 bp。为研究多形汉逊酵母GSH2基因重组菌株的转录调控，构建了一个报告系统，其中GSH2基因的1.832 kb调控区域与醇氧化酶基因的结构和终止区域融合。结果表明，在1 microM浓度的镉离子处理下孵育4小时，多形汉逊酵母GSH2基因转录在丰富培养基中最大增加33%。在基本培养基中，镉离子处理下GSH2基因表达与细胞内总谷胱甘肽水平的增加不相关。我们推测，多形汉逊酵母在镉胁迫下细胞内总谷胱甘肽含量的增加可能不是在GSH2基因转录水平上受到调控的。
