Cowie A, Myers R M
Department of Physiology, University of California, San Francisco 94143.
Mol Cell Biol. 1988 Aug;8(8):3122-8. doi: 10.1128/mcb.8.8.3122-3128.1988.
We have developed a transient assay in murine erythroleukemia (MEL) cells to analyze the cis-acting sequence requirements for transcriptional regulation of the mouse beta-major-globin promoter. From deletion analysis, a fragment of the promoter region, from -106 to +26 relative to the RNA cap site, was found to be sufficient for regulated transcription in MEL cells following induction of differentiation by dimethyl sulfoxide. Single-base mutational analysis of this 132-base-pair promoter fragment identified three sequence elements required for transcription in MEL cells. These are the ATATAA sequence at -31 to -26, the CCAATC sequence between -77 and -72, and the GCCACACCC sequence between -95 and -87. In addition, we found a requirement for sequences adjacent to the CCAAT and ATATAA consensus motifs. Point mutations within the promoter did not abolish transcriptional regulation following induction of differentiation by dimethyl sulfoxide. However, mutations that resulted in reduced transcription levels in uninduced MEL cells gave similarly decreased levels in induced MEL cells.
我们已经在小鼠红白血病（MEL）细胞中开发了一种瞬时分析方法，以分析小鼠β-珠蛋白主要启动子转录调控的顺式作用序列要求。通过缺失分析，发现相对于RNA帽位点从-106至+26的启动子区域片段，在经二甲基亚砜诱导分化后的MEL细胞中足以进行调控转录。对这个132个碱基对的启动子片段进行单碱基突变分析，确定了MEL细胞转录所需的三个序列元件。它们是位于-31至-26的ATATAA序列、位于-77至-72之间的CCAATC序列以及位于-95至-87之间的GCCACACCC序列。此外，我们发现需要与CCAAT和ATATAA共有基序相邻的序列。启动子内的点突变在经二甲基亚砜诱导分化后并未消除转录调控。然而，导致未诱导的MEL细胞中转录水平降低的突变，在诱导的MEL细胞中也同样使转录水平降低。
