Tomarev S I, Duncan M K, Roth H J, Cvekl A, Piatigorsky J
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892.
J Mol Evol. 1994 Aug;39(2):134-43. doi: 10.1007/BF00163802.
Previous experiments have shown that the minimal promoters required for function of the squid SL20-1 and SL11 crystallin genes in transfected rabbit lens epithelial cells contain an overlapping AP-1/antioxidant responsive element (ARE) upstream of the TATA box. This region resembles the PL-1 and PL-2 elements of the chicken beta B1-crystallin promoter which are essential for promoter function in transfected primary chicken lens epithelial cells. Here we demonstrate by site-directed mutagenesis that the AP-1/ARE sequence is essential for activity of the squid SL20-1 and SL11 promoters in transfected embryonic chicken lens cells and fibroblasts. Promoter activity was higher in transfected lens cells than in fibroblasts. Electrophoretic mobility shift and DNase protection experiments demonstrated the formation of numerous complexes between nuclear proteins of the embryonic chicken lens and the AP-1/ARE sequences of the squid SL20-1 and SL11 crystallin promoters. One of these complexes comigrated and cross-competed with that formed with the PL-1 element of the chicken beta B1-crystallin promoter. This complex formed with nuclear extracts from the lens, heart, brain, and skeletal muscle of embryonic chickens and was eliminated by competition with a consensus AP-1 sequence. The nonfunctional mutant AP-1/ARE sequences did not compete for complex formation. These data raise the intriguing possibility that entirely different, nonhomologous crystallin genes of the chicken and squid have convergently evolved a similar cis-acting regulatory element (AP-1/ARE) for high expression in the lens.
先前的实验表明，枪乌贼SL20-1和SL11晶状体蛋白基因在转染的兔晶状体上皮细胞中发挥功能所需的最小启动子，在TATA框上游含有一个重叠的AP-1/抗氧化反应元件（ARE）。该区域类似于鸡βB1-晶状体蛋白启动子的PL-1和PL-2元件，这两个元件对于转染的原代鸡晶状体上皮细胞中的启动子功能至关重要。在这里，我们通过定点诱变证明，AP-1/ARE序列对于枪乌贼SL20-1和SL11启动子在转染的胚胎鸡晶状体细胞和成纤维细胞中的活性至关重要。转染的晶状体细胞中的启动子活性高于成纤维细胞。电泳迁移率变动和DNase保护实验表明，胚胎鸡晶状体的核蛋白与枪乌贼SL20-1和SL11晶状体蛋白启动子的AP-1/ARE序列之间形成了大量复合物。其中一种复合物与鸡βB1-晶状体蛋白启动子的PL-1元件形成的复合物迁移率相同且存在交叉竞争。这种复合物由胚胎鸡的晶状体、心脏、大脑和骨骼肌的核提取物形成，并通过与AP-1共有序列竞争而被消除。无功能的突变AP-1/ARE序列不竞争复合物的形成。这些数据提出了一种有趣的可能性，即鸡和枪乌贼完全不同的、非同源的晶状体蛋白基因已经趋同进化出一个相似的顺式作用调控元件（AP-1/ARE），以便在晶状体中高表达。
