Takano Y, Iuchi Y, Ito J, Otsu K, Kuzumaki T, Ishikawa K
Department of Biochemistry, Yamagata University School of Medicine, Yamagata, 990-9585, Japan.
Arch Biochem Biophys. 2000 May 1;377(1):58-64. doi: 10.1006/abbi.2000.1757.
Transcription of the aldolase B gene, AldB, in the liver is regulated by hormones such as insulin and glucagon. To characterize the elements that are responsive to these hormones in the upstream region of AldB, plasmids carrying various length of the upstream region of this gene were constructed and transfected to primary cultured rat hepatocytes. The promoter activities were gradually increased by progressive deletion of the 5'-upstream region, and high activities were observed for constructs carrying the sequence between -408 and -85 bp, suggesting the presence of suppressive element(s) in the upstream region of -409 bp. The transcription activities of the mutants containing the sequences between -228 and -85 bp were enhanced by insulin, and glucagon suppressed the transcription activities of those containing the sequence between -764 and -85 bp. Two sequence elements similar to the cAMP-responsive element (CRE), one from -89 to -82 bp and another from +13 to +20 bp, were found in the upstream sequence of the gene. The latter element is not functional because its deletion did not affect either the transcription efficiency or glucagon response. However, the deletion of the former element diminished both functions. A gel retardation assay showed that the nuclear factor binds to the former element, which was competitive with authentic CRE oligonucleotide but not with the mutant CRE one. These results suggest that the CRE-like element in the promoter region is prerequisite for both fundamental transcription efficiency of the gene and suppression by glucagon in hepatocytes.
肝脏中醛缩酶B基因（AldB）的转录受胰岛素和胰高血糖素等激素的调节。为了鉴定AldB基因上游区域中对这些激素有反应的元件，构建了携带该基因不同长度上游区域的质粒，并将其转染至原代培养的大鼠肝细胞。随着5'上游区域的逐步缺失，启动子活性逐渐增加，对于携带-408至-85 bp之间序列的构建体观察到高活性，这表明在-409 bp的上游区域存在抑制元件。含有-228至-85 bp之间序列的突变体的转录活性被胰岛素增强，而胰高血糖素抑制了含有-764至-85 bp之间序列的突变体的转录活性。在该基因的上游序列中发现了两个与环磷酸腺苷反应元件（CRE）相似的序列元件，一个位于-89至-82 bp，另一个位于+13至+20 bp。后一个元件没有功能，因为其缺失既不影响转录效率也不影响胰高血糖素反应。然而，前一个元件的缺失会使两种功能都减弱。凝胶阻滞试验表明，核因子与前一个元件结合，它与 authentic CRE寡核苷酸具有竞争性，但与突变型CRE寡核苷酸没有竞争性。这些结果表明，启动子区域中的CRE样元件对于该基因在肝细胞中的基本转录效率和胰高血糖素抑制作用都是必不可少的。
