Dobens L, Rudolph K, Berger E M
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755.
Mol Cell Biol. 1991 Apr;11(4):1846-53. doi: 10.1128/mcb.11.4.1846-1853.1991.
A synthetic, 23-bp ecdysterone regulatory element (EcRE), derived from the upstream region of the Drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (CAT). Hybrid constructs were transfected into Drosophila S3 cells and assayed for ecdysterone-inducible CAT expression. In the absence of ecdysterone a tandem pair of EcREs repressed the high constitutive level of CAT activity found after transfection with the parent reporter plasmid alone. After hormone addition very high levels of CAT activity were observed. Insertion of the EcRE pair 3' of the CAT gene also led to high levels of ecdysterone-induced CAT expression, but the repression of high constitutive levels of CAT activity failed to occur. The EcRE-CAT construct was cotransfected with plasmids containing tandem 10-mers or 40-mers of the EcRE but lacking a reporter gene. These additional EcREs led to a reduced level of ecdysterone-induced CAT activity and to an elevation of basal CAT activity in the absence of hormone. The data suggest that the receptor binds to the EcRE in the absence of hormone, blocking basal transcription from a constitutive promoter. In the presence of ecdysterone, receptor-hormone binding to the EcRE leads to greatly enhanced transcription.
从黑腹果蝇hsp27基因上游区域衍生而来的一个合成的23个碱基对的蜕皮激素调节元件（EcRE），被插入到与氯霉素乙酰转移酶（CAT）细菌基因融合的单纯疱疹病毒胸苷激酶启动子附近。将杂种构建体转染到果蝇S3细胞中，并检测蜕皮激素诱导的CAT表达。在没有蜕皮激素的情况下，一对串联的EcRE抑制了仅用亲本报告质粒转染后发现的高水平组成型CAT活性。添加激素后，观察到非常高的CAT活性水平。在CAT基因的3'端插入EcRE对也导致高水平的蜕皮激素诱导的CAT表达，但未能抑制高水平的组成型CAT活性。将EcRE-CAT构建体与含有串联10聚体或40聚体EcRE但缺乏报告基因的质粒共转染。这些额外的EcRE导致蜕皮激素诱导的CAT活性水平降低，并且在没有激素存在的情况下基础CAT活性升高。数据表明，在没有激素的情况下，受体与EcRE结合，阻断了组成型启动子的基础转录。在有蜕皮激素存在的情况下，受体-激素与EcRE的结合导致转录大大增强。
