Takaoka Naohisa, Fukuzawa Masashi, Kato Atsushi, Saito Tamao, Ochiai Hiroshi
Division of Biological Sciences, Graduate School of Science, Hokkaido University, 060-0810, Sapporo, Japan.
Biochim Biophys Acta. 2002 Apr 12;1574(3):304-10. doi: 10.1016/s0167-4781(02)00227-0.
gp64 mRNA in Polysphondylium pallidum is expressed extensively during vegetative growth, and begins to rapidly decrease at the onset of development. To examine this unique regulation, 5' deletion analysis of the gp64 promoter was undertaken, and two growth-phase activated elements have been found: a food-dependent, upstream regulatory region (FUR, -222 to -170) and a vegetatively activated, downstream region (VAD, -110 to -63). Here we concentrate our analysis on an A1 and A2 sequences in the FUR region: A1 consists of a GATTTTTTTA sequence called a corresponding sequence and A2 consists of the direct repeat TTTGTTGTG. The cells carrying a combined construct of A1 and A2 acted synergistically in a reporter activity. A point mutation analysis in A1 indicates that a G residue is required for the activation of A1. From analyses of promoter regulation in a liquid or a solid medium, the promoter activity of the cells fed on bacteria in A-medium (axenic medium for Polysphondylium) or grown in A-medium alone was only one fourth of that of the cells fed on bacteria. By the gel retardation, we detected a protein bound to the A1 sequence.
盘基网柄菌中的gp64 mRNA在营养生长期间广泛表达，并在发育开始时迅速减少。为了研究这种独特的调控，对gp64启动子进行了5'缺失分析，发现了两个生长阶段激活元件：一个食物依赖性上游调控区域（FUR，-222至-170）和一个营养激活下游区域（VAD，-110至-63）。在这里，我们将分析集中在FUR区域的A1和A2序列上：A1由一个称为对应序列的GATTTTTTTA序列组成，A2由直接重复序列TTTGTTGTG组成。携带A1和A2组合构建体的细胞在报告基因活性中具有协同作用。A1中的点突变分析表明，G残基是A1激活所必需的。通过对液体或固体培养基中启动子调控的分析，在A培养基（盘基网柄菌的无菌培养基）中以细菌为食或仅在A培养基中生长的细胞的启动子活性仅为以细菌为食的细胞的四分之一。通过凝胶阻滞实验，我们检测到一种与A1序列结合的蛋白质。
