Westin G, Gerster T, Müller M M, Schaffner G, Schaffner W
Institut für Molekularbiologie II, Universität Zürich, Switzerland.
Nucleic Acids Res. 1987 Sep 11;15(17):6787-98. doi: 10.1093/nar/15.17.6787.
We have developed a vector, OVEC ("oligonucleotide vector") to study DNA sequences involved in the regulation of transcription in mammalian cells. This vector is equally suitable for studying expression in vivo after transfection into cells, or for transcription studies in vitro with cell-free extracts. Putative cis-acting DNA segments from enhancers or promoters can be inserted at a position immediately upstream of the TATA box and coding sequence of the rabbit beta-globin gene. A regulatory DNA segment can be tested by itself or in conjunction with an enhancer located either in an adjacent upstream position, or downstream of the beta-globin gene. S1 nuclease mapping can be used to study transcription from circular and linear templates and run-off transcription in vitro is also feasible. Transcripts from a reference globin gene with a small deletion around the transcription initiation site can be measured with the same S1 nuclease probe and thus serve as an internal standard. We demonstrate the usefulness of OVEC by inserting either short oligonucleotides comprising a metal responsive enhancer element, or the SV40 enhancer, directly upstream of the TATA box. Both constructs yield high levels of correctly initiated transcripts in a transient expression assay in HeLa cells. In a HeLa cell nuclear extract the SV40 enhancer stimulates transcription 40-fold.
我们开发了一种载体，即OVEC（“寡核苷酸载体”），用于研究参与哺乳动物细胞转录调控的DNA序列。该载体同样适用于转染细胞后在体内研究表达情况，或用于利用无细胞提取物进行体外转录研究。来自增强子或启动子的假定顺式作用DNA片段可插入兔β-珠蛋白基因TATA盒和编码序列紧邻上游的位置。一个调控DNA片段可单独进行测试，也可与位于β-珠蛋白基因相邻上游位置或下游的增强子联合进行测试。S1核酸酶图谱分析可用于研究环状和线性模板的转录情况，体外连续转录也是可行的。利用相同的S1核酸酶探针可测量转录起始位点周围有小缺失的参考珠蛋白基因的转录本，从而作为内标。我们通过在TATA盒紧邻上游插入包含金属反应增强子元件的短寡核苷酸或SV40增强子，证明了OVEC的实用性。在HeLa细胞的瞬时表达试验中，这两种构建体均产生高水平的正确起始转录本。在HeLa细胞核提取物中，SV40增强子可刺激转录40倍。
