Firak T A, Subramanian K N
Mol Cell Biol. 1986 Nov;6(11):3667-76. doi: 10.1128/mcb.6.11.3667-3676.1986.
We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.
我们检测了猿猴病毒40（SV40）72碱基对（bp）重复增强子区域的片段在SV40早期启动子控制下激活基因表达的能力，以及在猴CV - 1或人HeLa细胞体内竞争有限的反式作用增强子结合因子的能力。在这些检测中，细菌氯霉素乙酰转移酶基因和单纯疱疹病毒1型胸苷激酶基因被用作报告基因。在之前的研究中，位于SV40核苷酸179和272之间的一段94 bp序列，包括一个72 bp重复序列的拷贝，被称为最小增强子。在本研究中，我们发现位于核苷酸179和198之间的20 bp起源近端区域是可有可无的，因为去除该区域只会导致增强子活性略有降低。然而，再删除直至核苷酸202的另外4 bp则会消除增强子活性。我们提出最小增强子是一个位于核苷酸199和272之间的74 bp序列，包括72 bp重复序列一个拷贝的52 bp以及直至272处PvuII位点的22 bp相邻序列。我们将九聚体5'-AAGT/CATGCA-3'称为K核心，它在核苷酸200处的SphI位点周围以串联重复的形式出现，并且我们发现这种重复对于增强和因子结合活性至关重要。一个先前鉴定出的异源核心元件（我们称为C核心），5'-GTGGA/TA/TA/TG-3'，也以重复形式出现，其中一个拷贝位于72 bp重复序列之外、靠近核苷酸272的22 bp序列内。我们提供了直接证据表明这个22 bp序列能显著增强增强子活性。我们还发现，除了最小增强子内K核心和C核心之间正常发生的异源相互作用外，只要元件之间有适当的间隔，某些同源相互作用也会发生。
