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裂殖酵母 Schizosaccharomyces pombe 中 nmt1 驱动基因的可控表达用化学化合物。

A chemical compound for controlled expression of nmt1-driven gene in the fission yeast Schizosaccharomyces pombe.

机构信息

Department of Microbiology, School of Medicine, Dokkyo Medical University, Mibu, Tochigi 321-0293, Japan.

出版信息

Anal Biochem. 2011 May 15;412(2):159-64. doi: 10.1016/j.ab.2011.01.039. Epub 2011 Feb 2.

Abstract

The fission yeast Schizosaccharomyces pombe is a useful model organism for studying a variety of eukaryotic cellular events such as the cell cycle control mechanisms. For inducible expression of exogenous genes in S. pombe, vectors carrying the nmt1 (no message in thiamine 1) promoter are most commonly used. Although nmt1 is a potent promoter, its transcription activity is drastically repressed in the presence of a low concentration of thiamine. Therefore, a combination of thiamine and nmt1 promoter is convenient for regulating gene expression in an all-or-none fashion. However, it has been difficult to adjust the nmt1 promoter activity in a controlled manner. Here we describe a chemical compound, designated as YAM2, whose repressive activity on the nmt1 promoter has a wider linear range than thiamine. Expression of exogenous proteins, such as human immunodeficiency virus type 1 Vpr and jellyfish green fluorescent protein, driven by the nmt1 promoter is gradually repressed by YAM2 in a dose-dependent manner. YAM2 does not exhibit a detectable level of cytotoxicity at a concentration required to fully repress the nmt1 promoter. The compound may serve as a useful tool for controlled expression of the nmt1-driven gene in S. pombe.

摘要

裂殖酵母 Schizosaccharomyces pombe 是一种研究真核细胞各种事件的有用模式生物,如细胞周期调控机制。为了在外源基因在 S. pombe 中的诱导表达,最常用的载体是携带 nmt1(硫胺素 1 无信息)启动子的载体。尽管 nmt1 是一个有效的启动子,但在低浓度硫胺素存在的情况下,其转录活性被严重抑制。因此,硫胺素和 nmt1 启动子的组合非常适合以全有或全无的方式调节基因表达。然而,很难以可控的方式调节 nmt1 启动子的活性。在这里,我们描述了一种化学化合物,命名为 YAM2,其对 nmt1 启动子的抑制活性具有比硫胺素更宽的线性范围。由 nmt1 启动子驱动的外源性蛋白质,如人类免疫缺陷病毒 1 型 Vpr 和水母绿色荧光蛋白的表达,被 YAM2 以剂量依赖的方式逐渐抑制。在完全抑制 nmt1 启动子所需的浓度下,YAM2 没有表现出可检测水平的细胞毒性。该化合物可能成为控制 S. pombe 中 nmt1 驱动基因表达的有用工具。

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