Baru M, Shlissel M, Manor H
Department of Biology, Technion-Israel Institute of Technology, Haifa.
J Virol. 1991 Jul;65(7):3496-503. doi: 10.1128/JVI.65.7.3496-3503.1991.
We have replaced the polyomavirus (Py) enhancer, which is an essential component of the Py origin of DNA replication (ori), with five repeats of a 17-bp oligonucleotide including the yeast GAL4 upstream activating sequence (5xGAL4 sites). Plasmids containing this modified Py ori, designated test plasmids, and plasmids encoding either the GAL4 transcriptional activator protein or various derivatives of this protein were cotransfected into mouse cells which constitutively synthesize a temperature-sensitive Py large tumor antigen (T-Ag). Replication of the test plasmids was monitored by Southern blot determinations of the amounts of plasmid DNA that became resistant to cleavage by the enzyme DpnI. These studies showed that in the presence of a functional T-Ag, the GAL4 protein, and hybrid proteins including the GAL4 DNA-binding domain and the activating domain of the adenovirus E1a or herpesvirus VP16 protein transactivated the modified Py ori. A truncated protein including just the GAL4 DNA-binding domain was inactive in these assays. The authentic GAL4 protein was found to be a more efficient replication transactivator than the hybrid proteins. In contrast, chloramphenicol acetyltransferase assays showed that the hybrid proteins were more efficient transcriptional activators than the GAL4 protein. The extent of the GAL4-dependent replication of a plasmid in which the Py early promoter was deleted was 55% lower than that of a plasmid including the promoter. However, the extents of replication of plasmids including two tandem repeats of the remaining Py origin core and 5xGAL4 sites or two origin cores flanking a single cluster of 5xGAL4 sites were 4.8- and 1.6-fold higher than that of the plasmid including a single copy of each element. The replication of a plasmid including two clusters of 5xGAL4 sites flanking a single origin core was below the limit of detection of our assays. These results indicate that the GAL4 and hybrid transactivators do not activate the Py ori by virtue of their interactions with transcription factors that bind promoter elements. Rather, it appears that these activator proteins may interact with the replication initiation complexes, thereby facilitating or inhibiting the initiation of replication.
我们用包含酵母GAL4上游激活序列的17个碱基寡核苷酸的五个重复序列（5xGAL4位点）取代了多瘤病毒（Py）增强子，该增强子是Py DNA复制起点（ori）的重要组成部分。含有这种修饰的Py ori的质粒（称为测试质粒）以及编码GAL4转录激活蛋白或该蛋白各种衍生物的质粒，被共转染到组成型合成温度敏感型Py大T抗原（T-Ag）的小鼠细胞中。通过Southern印迹法测定对DpnI酶切割具有抗性的质粒DNA量，来监测测试质粒的复制情况。这些研究表明，在功能性T-Ag、GAL4蛋白以及包括GAL4 DNA结合结构域和腺病毒E1a或疱疹病毒VP16蛋白激活结构域的杂交蛋白存在的情况下，它们可反式激活修饰的Py ori。仅包含GAL4 DNA结合结构域的截短蛋白在这些测定中无活性。发现天然GAL4蛋白比杂交蛋白更有效地激活复制。相反，氯霉素乙酰转移酶测定表明，杂交蛋白比GAL4蛋白更有效地激活转录。Py早期启动子缺失的质粒中依赖GAL4的复制程度比包含该启动子的质粒低55%。然而，包含剩余Py起点核心和5xGAL4位点的两个串联重复序列的质粒，或单个簇状5xGAL4位点两侧的两个起点核心的质粒的复制程度，分别比包含每个元件单拷贝的质粒高4.8倍和1.6倍。单个起点核心两侧包含两个5xGAL4位点簇的质粒的复制低于我们测定的检测限。这些结果表明，GAL4和杂交反式激活因子并非通过与结合启动子元件的转录因子相互作用来激活Py ori。相反，这些激活蛋白似乎可能与复制起始复合物相互作用，从而促进或抑制复制起始。
