Widen S G, Papaconstantinou J
Proc Natl Acad Sci U S A. 1986 Nov;83(21):8196-200. doi: 10.1073/pnas.83.21.8196.
We have identified cis-acting regulatory elements in the 5' flanking region of the mouse alpha-fetoprotein (Afp) gene, using the expression of the bacterial gene for chloramphenicol acetyltransferase (CAT) in a transient expression assay. Tissue-specific enhancer activity was determined by transfection of mouse hepatoma (BWTG3) and fibroblast cells (C127, NIH 3T3) with various DNA fragments linked to the CAT gene. A 5.4-kilobase restriction fragment was shown to have characteristics typical of enhancers, including the ability to function independent of orientation and position and the ability to enhance transcription from a heterologous promoter. The enhancer activity was greatest in the hepatoma cells, which express Afp. By deletion analysis, it was demonstrated that enhancer activity is present in several subfragments, indicating the presence of more than one element in this fragment. An additional regulatory element within 950 base pairs of the Afp transcription initiation site has been identified and shown to confer tissue-specific expression on the CAT gene. This fragment, which lacks enhancer activity, contains the Afp promoter region and mediates the tissue-specific expression of the CAT gene when driven by nonspecific viral enhancers. We conclude from our studies that there are several types of regulatory elements in the 5' flanking region of the Afp gene that help mediate tissue-specific expression.
我们利用氯霉素乙酰转移酶（CAT）细菌基因在瞬时表达试验中的表达，鉴定了小鼠甲胎蛋白（Afp）基因5'侧翼区域的顺式作用调控元件。通过用与CAT基因相连的各种DNA片段转染小鼠肝癌细胞（BWTG3）和成纤维细胞（C127、NIH 3T3）来确定组织特异性增强子活性。一个5.4千碱基的限制性片段显示出具有增强子的典型特征，包括能够独立于方向和位置发挥功能以及增强来自异源启动子的转录的能力。增强子活性在表达Afp的肝癌细胞中最强。通过缺失分析表明，增强子活性存在于几个亚片段中，这表明该片段中存在不止一个元件。在Afp转录起始位点的950个碱基对内还鉴定出了另一个调控元件，它能赋予CAT基因组织特异性表达。这个缺乏增强子活性的片段包含Afp启动子区域，并在非特异性病毒增强子驱动下介导CAT基因的组织特异性表达。我们从研究中得出结论，Afp基因5'侧翼区域存在几种类型的调控元件，它们有助于介导组织特异性表达。
