Sawado T, Hirose F, Takahashi Y, Sasaki T, Shinomiya T, Sakaguchi K, Matsukage A, Yamaguchi M
Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan.
J Biol Chem. 1998 Oct 2;273(40):26042-51. doi: 10.1074/jbc.273.40.26042.
Two mRNA species were observed for the Drosophila E2F (dE2F) gene, differing with regard to the first exons (exon 1-a and exon 1-b), which were expressed differently during development. A single transcription initiation site for mRNA containing exon 1-b was mapped by primer extension analysis and numbered +1. We found three tandemly aligned sequences, similar to the DNA replication-related element (DRE; 5'-TATCGATA), which is commonly required for transcription of genes related to DNA replication and cell proliferation, in the region upstream of this site. Band mobility shift analyses using oligonucleotides containing the DRE-related sequences with or without various base substitutions revealed that two out of three DRE-related sequences are especially important for binding to the DRE-binding factor (DREF). On footprinting analysis with Kc cell nuclear extracts and a glutathione S-transferase fusion protein with the N-terminal fragment (1-125 amino acid residues) of DREF, all three DRE-related sequences were found to be protected. Transient luciferase expression assays in Kc cells demonstrated that the region containing the three DRE-related sequences is required for high promoter activity. We have established transgenic lines of Drosophila in which ectopic expression of DREF was targeted to the eye imaginal disc cells. Overexpression of DREF in eye imaginal disc cells enhanced the promoter activity of dE2F. The obtained results indicate that the DRE/DREF system activates transcription of the dE2F gene.
在果蝇E2F（dE2F）基因中观察到两种mRNA，它们在首个外显子（外显子1-a和外显子1-b）方面存在差异，并且在发育过程中表达情况不同。通过引物延伸分析确定了含有外显子1-b的mRNA的单一转录起始位点，并将其编号为+1。我们在该位点上游区域发现了三个串联排列的序列，类似于DNA复制相关元件（DRE；5'-TATCGATA），DNA复制和细胞增殖相关基因的转录通常需要该元件。使用含有DRE相关序列且有或没有各种碱基替换的寡核苷酸进行的凝胶迁移率变动分析表明，三个DRE相关序列中的两个对于与DRE结合因子（DREF）的结合尤为重要。在用Kc细胞核提取物和带有DREF N端片段（1 - 125个氨基酸残基）的谷胱甘肽S - 转移酶融合蛋白进行足迹分析时，发现所有三个DRE相关序列均受到保护。在Kc细胞中进行的瞬时荧光素酶表达分析表明，含有三个DRE相关序列的区域对于高启动子活性是必需的。我们建立了果蝇转基因品系，其中DREF的异位表达靶向眼成虫盘细胞。眼成虫盘细胞中DREF的过表达增强了dE2F的启动子活性。所得结果表明，DRE/DREF系统激活dE2F基因的转录。
