Pearson B E, Nasheuer H P, Wang T S
Department of Pathology, Stanford University School of Medicine, California 94305.
Mol Cell Biol. 1991 Apr;11(4):2081-95. doi: 10.1128/mcb.11.4.2081-2095.1991.
We have investigated the DNA polymerase alpha promoter sequence requirements for the expression of a heterologous gene in actively cycling cells and following serum addition to serum-deprived cells. An 11.4-kb genomic clone that spans the 5' end of this gene and includes 1.62 kb of sequence upstream from the translation start site was isolated. The transcription start site was mapped at 46 +/- 1 nucleotides upstream from the translation start site. The upstream sequence is GC rich and lacks a TATA sequence but has a CCAAT sequence on the opposite strand. Analysis of a set of deletion constructs in transient transfection assays demonstrated that efficient expression of the reporter in cycling cells requires 248 bp of sequence upstream from the cap site. Clustered within these 248 nucleotides are sequences similar to consensus sequences for Sp1-, Ap1-, Ap2-, and E2F-binding sites. The CCAAT sequence and the potential E2F- and Ap1-binding sites are shown to be protected from DNase I digestion by partially purified nuclear proteins. The DNA polymerase alpha promoter can confer upon the reporter an appropriate, late response to serum addition. No single sequence element could be shown to confer serum inducibility. Rather, multiple sequence elements appear to mediate the full serum response.
我们研究了DNA聚合酶α启动子序列对于异源基因在活跃增殖细胞中以及血清添加到血清饥饿细胞后表达的要求。分离出一个11.4kb的基因组克隆，其跨越该基因的5'端，并包括翻译起始位点上游1.62kb的序列。转录起始位点定位于翻译起始位点上游46±1个核苷酸处。上游序列富含GC，缺乏TATA序列，但在相反链上有一个CCAAT序列。在瞬时转染实验中对一组缺失构建体的分析表明，报告基因在增殖细胞中的有效表达需要帽位点上游248bp的序列。在这248个核苷酸内聚集着与Sp1、Ap1、Ap2和E2F结合位点的共有序列相似的序列。CCAAT序列以及潜在的E2F和Ap1结合位点被部分纯化的核蛋白保护而免受DNase I消化。DNA聚合酶α启动子可使报告基因对血清添加产生适当的晚期反应。没有单个序列元件能赋予血清诱导性。相反，多个序列元件似乎介导了完整的血清反应。
