Kumar R, Yoon K P, Subramanian K N
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60680.
Mol Cell Biol. 1988 Apr;8(4):1509-17. doi: 10.1128/mcb.8.4.1509-1517.1988.
In a previous study in our laboratory, the effect of the reiteration frequency of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer on transcription from the proximal SV40 early promoter was investigated (R. Kumar, T. A. Firak, C. T. Schroll, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 83:3199-3203, 1986). Increasing the enhancer copy number to four increased transcription proportionately; further increments in enhancer copy number reversed this effect, resulting in a decrease in the transcriptional activation. In the present study, the effect of enhancer reiteration on the replication efficiency of plasmids containing the SV40 origin of replication was investigated in transient replication assays in vivo in COS-1 monkey kidney cells producing the SV40 large tumor antigen required for replication. A plasmid containing the SV40 core origin and three copies of the replication-activating, G+C-rich 21-bp repeat promoter element replicated efficiently. Plasmids containing multiple copies of the 72-bp repeat enhancer cloned in head-to-tail linkage adjacent to the 21-bp repeat and the core origin replicated less efficiently; the decrease in replication efficiency could be correlated with the number of copies of the 72-bp repeat; replication was severely curtailed when 10 or more copies of the 72-bp repeat were present. Replication was not significantly inhibited by an increase in the number of copies of the 21-bp repeat to 15 or by the presence of three copies of a 360-bp pBR322 sequence in the immediate vicinity. Multiple copies of the 72-bp enhancer in cis were unable to inhibit replication from a second SV40 origin of replication situated 2 kilobase pairs away from the enhancer reiteration. Replication of four different test plasmids was not inhibited in trans by cotransfection of an excess of a potential competitor plasmid containing a 24-copy reiteration of the 72-bp enhancer. These results indicate that multiple tandem reiterations of the 72-bp enhancer inhibit replication only when they are present in cis adjacent to the origin of replication. Possible explanations for this inhibitory effect, such as an unfavorable local chromatin structure induced by the multimeric enhancer region or reduced or improper communications between factors bound to the multimeric region and the adjacent replication origin, are discussed.
在我们实验室先前的一项研究中，研究了猿猴病毒40（SV40）72碱基对（bp）重复增强子的重复频率对近端SV40早期启动子转录的影响（R.库马尔、T.A.菲拉克、C.T.施罗尔和K.N.苏布拉马尼亚姆，《美国国家科学院院刊》83:3199 - 3203，1986年）。将增强子拷贝数增加到四个会使转录成比例增加；增强子拷贝数的进一步增加则逆转了这种效应，导致转录激活降低。在本研究中，在体内瞬时复制试验中，于产生复制所需的SV40大肿瘤抗原的COS - 1猴肾细胞中，研究了增强子重复对含有SV40复制起点的质粒复制效率的影响。一个含有SV40核心起点和三个复制激活的、富含G + C的21 bp重复启动子元件的质粒能高效复制。含有多个首尾相连克隆在21 bp重复序列和核心起点附近的72 bp重复增强子的质粒复制效率较低；复制效率的降低与72 bp重复的拷贝数相关；当存在10个或更多72 bp重复拷贝时，复制会严重受限。将21 bp重复的拷贝数增加到15个或在紧邻位置存在三个360 bp的pBR322序列，均不会显著抑制复制。顺式排列的多个72 bp增强子拷贝无法抑制来自距离增强子重复2千碱基对处的第二个SV40复制起点的复制。通过共转染过量的含有24拷贝72 bp增强子重复的潜在竞争性质粒，不会反式抑制四种不同测试质粒的复制。这些结果表明，只有当72 bp增强子多个串联重复顺式存在于复制起点附近时，才会抑制复制。讨论了这种抑制效应的可能解释，例如多聚体增强子区域诱导的不利局部染色质结构，或与多聚体区域结合的因子和相邻复制起点之间的通讯减少或不当。
