Gene expression vectors are useful and important tools that are commonly used in a variety of experiments, including expression of foreign genes, functional analysis of genes of interest and complementation experiments. In this study, a hybrid promoter, combining the adh1⁺ upstream activating sequence (UAS) of fission yeast and the GAL10 core promoter of budding yeast, was constructed to enable high level expression depending on the presence of zinc in culture medium for fission yeast. When the hybrid promoter was cloned on the multicopy plasmid, it was fully induced and repressed within 10 h in the presence and absence of zinc, respectively. The kinetics of induction and reduction were similar to those of the endogenous adh1⁺ mRNA. In contrast, native adh1⁺ promoter lost its tight repression in zinc‐depleted condition when it was cloned on the plasmid. Because adh1⁺ UAS‐specific transcription factors have not yet been identified, we identified UAS elements involved in zinc sensing by characterizing this hybrid promoter. We also found that the expression level increased by the TATA box mutation, GATAA, in the presence of zinc.

中文翻译：

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裂殖酵母锌诱导基因表达载体的构建与表征

基因表达载体是有用且重要的工具，常用于各种实验，包括外源基因的表达、目标基因的功能分析和互补实验。在这项研究中，构建了一个杂交启动子，结合了裂殖酵母的adh1 ⁺上游激活序列 (UAS) 和芽殖酵母的GAL10核心启动子，以根据裂殖酵母培养基中锌的存在实现高水平表达。当杂交启动子被克隆到多拷贝质粒上时，它在锌存在和不存在的情况下分别在 10 小时内被完全诱导和抑制。诱导和还原动力学与内源性adh1 ⁺mRNA。相比之下，天然adh1 ⁺启动子在缺锌条件下被克隆到质粒上时失去了它的严格抑制。由于尚未鉴定出adh1 ⁺ UAS 特异性转录因子，我们通过表征这种混合启动子鉴定了参与锌感应的 UAS 元件。我们还发现，在锌存在下，TATA 盒突变 GATAA 会增加表达水平。