Haas M W, Ramanujam P, Chandrasekharappa S C, Subramanian K N
Department of Microbiology and Immunology, University of Illinois College of Medicine 60612.
Virology. 1991 Jan;180(1):41-8. doi: 10.1016/0042-6822(91)90007-x.
Previous studies have demonstrated that the 21- or the 72-bp repeat transcriptional control elements enhance the efficiency of SV40 DNA replication in vivo, provided either of these repeats is located near the end of the core replication origin containing the 17-bp A + T-containing sequence. Using two sets of point mutants we have investigated the contributions of the various sequence motifs present in the 21- or the 72-bp repeats toward activation of replication. Regarding the contribution of the six GC motif components of the 21-bp repeats, we find that GC motif I, located closest to the core origin, is dispensable for activation of replication. A mutation in GC-I in fact causes an increase in replication efficiency. We also find that GC motifs I and II present in the nontandem copy of the 21-bp repeats are not sufficient to activate replication. Our present study indicates that a combination of three GC motifs such as II, III, and IV (including one of the two perfect, tandem copies of the 21-bp repeats) is important for activation of replication. Regarding the 72-bp repeat transcriptional enhancer region, we find mutations in a number of its individual motifs to have a negative consequence on replication, with mutations in the GT-I*/TC-II and Sph-II/octamer motifs exhibiting the most negative effects. Overall, we find that the replication activation effects of the 21- and the 72-bp repeats require the participation of multiple motifs present in them. Cellular factors binding to these motifs are expected to mediate their replication activation effects. For the most part, the motifs required for activation of replication are the same as those reported in earlier studies to be important for efficient early and late viral mRNA transcription.
先前的研究表明，21 碱基对或 72 碱基对的重复转录控制元件可提高 SV40 DNA 在体内的复制效率，前提是这些重复序列中的任何一个位于包含 17 碱基对富含 A+T 序列的核心复制起点末端附近。我们使用两组点突变体研究了 21 碱基对或 72 碱基对重复序列中存在的各种序列基序对复制激活的贡献。关于 21 碱基对重复序列的六个 GC 基序成分的贡献，我们发现最靠近核心起点的 GC 基序 I 对于复制激活是可有可无的。事实上，GC-I 中的突变会导致复制效率增加。我们还发现，21 碱基对重复序列的非串联拷贝中存在的 GC 基序 I 和 II 不足以激活复制。我们目前的研究表明，三个 GC 基序（如 II、III 和 IV，包括 21 碱基对重复序列的两个完美串联拷贝之一）的组合对于复制激活很重要。关于 72 碱基对重复转录增强子区域，我们发现其许多单个基序中的突变对复制有负面影响，其中 GT-I*/TC-II 和 Sph-II/八聚体基序中的突变表现出最负面的影响。总体而言，我们发现 21 碱基对和 72 碱基对重复序列的复制激活效应需要其中存在的多个基序的参与。预计与这些基序结合的细胞因子会介导它们的复制激活效应。在很大程度上，复制激活所需的基序与早期研究中报道的对有效的早期和晚期病毒 mRNA 转录很重要的基序相同。
