Habib S, Pandey S, Chatterji U, Burma S, Ahmad R, Jain A, Hasnain S E
Eukaryotic Gene Expression Laboratory, National Institute of Immunology, New Delhi, India.
DNA Cell Biol. 1996 Sep;15(9):737-47. doi: 10.1089/dna.1996.15.737.
The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) homologous region sequence hr1 is a putative origin of replication (ori) sequence and can also function as a transcriptional enhancer for delayed-early genes. We demonstrate that this 750-bp sequence, carrying five 28-bp core palin-dromes, enhances expression from the very late polyhedrin promoter up to 11-fold in a classical enhancer fashion in transient expression assays. Enhancement is at the level of transcription, as evident from RNase protection assay analysis. It is mediated by an alpha-amanitin-insensitive RNA polymerase from the authentic polyhedrin promoter transcription start site and follows the temporal activation profile characteristic of the polyhedrin promoter. Three lines of evidence conclusively demonstrated that hr1 acts typically as an enhancer of polyhedrin gene transcription independent of its role as an ori: (i) linearized hr1-reporter plasmids, incapable of replicating in the host cell, could enhance transcription from the promoter; (ii) reporter plasmid copy number was not affected by the presence of aphidicolin during transfection; (iii) reporter plasmid DNA recovered from Sf9 cells was sensitive to Dpn I confirming its unreplicated state in the transfection regime followed by us. Molecular dissection of the hr1 sequence elements revealed that a core palindrome alone can function as an ori sequence whereas a palindrome along with flanking sequences is essential for the enhancer activity. Enhancement of luciferase expression from the polyhedrin promoter is a function of the number of core palindromes and flanking sequences. Our results demonstrate that hr1, which has several motifs for enhancer binding proteins and transcription factors, has a dual role associated with both DNA replication and transcriptional enhancement.
苜蓿银纹夜蛾多核衣壳核型多角体病毒（AcMNPV）的同源区域序列hr1是一个假定的复制起点（ori）序列，并且还可以作为延迟早期基因的转录增强子。我们证明，这个带有五个28bp核心回文序列的750bp序列，在瞬时表达试验中以经典增强子的方式将晚期多角体蛋白启动子的表达增强了11倍。从核糖核酸酶保护试验分析可以明显看出，增强作用发生在转录水平。它是由来自真实多角体蛋白启动子转录起始位点的α-鹅膏蕈碱不敏感RNA聚合酶介导的，并且遵循多角体蛋白启动子的时间激活特征。三条证据确凿地证明，hr1通常作为多角体蛋白基因转录的增强子，与其作为ori的作用无关：（i）无法在宿主细胞中复制的线性化hr1-报告质粒可以增强启动子的转录；（ii）转染过程中放线菌素D的存在不影响报告质粒的拷贝数；（iii）从Sf9细胞中回收的报告质粒DNA对Dpn I敏感，证实了其在我们采用的转染方案中的未复制状态。对hr1序列元件的分子剖析表明，单独一个核心回文序列就可以作为ori序列，而一个回文序列及其侧翼序列对于增强子活性是必不可少的。从多角体蛋白启动子增强荧光素酶表达是核心回文序列和侧翼序列数量作用的结果。我们的结果表明，具有多个增强子结合蛋白和转录因子基序序列的hr1，在DNA复制和转录增强方面具有双重作用。
