Harel-Bellan A, Brini A T, Ferris D K, Robin P, Farrar W L
Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick, MD 21701.
Nucleic Acids Res. 1989 Jun 12;17(11):4077-87. doi: 10.1093/nar/17.11.4077.
In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also it was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer.
在各种研究中，增强子结合蛋白已通过插入质粒的竞争序列成功地被吸附去除，从而导致质粒表达受到抑制。理论上，使用未插入质粒的合成增强子序列也能实现这样的结果。在本研究中，化学合成了一段与人类热休克调节元件对应的双链DNA序列。通过体外阻滞分析，该合成序列显示能特异性结合人T细胞系Jurkat提取物中的一种蛋白质。当将合成增强子电穿孔导入Jurkat细胞时，不仅显示该增强子在细胞内长达2天未被降解，而且还显示它能在细胞内结合一种蛋白质。这种结合是特异性的，并且在热休克时受到调节。此外，通过紫外线交联显示结合蛋白具有预期的分子量。然而，当将合成增强子元件与HSP 70-CAT报告基因构建体共电穿孔时，在存在外源合成增强子的情况下，报告质粒的表达持续增强。
