Kurioka H, Kishi H, Isshiki H, Tagoh H, Mori K, Kitagawa T, Nagata T, Dohi K, Muraguchi A
Department of Immunology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Japan.
Mol Immunol. 1996 Sep;33(13):1059-66. doi: 10.1016/s0161-5890(96)00062-4.
Human recombination activating gene-1 (RAG-1) genomic DNA clones containing the first exon coding for the 5' untranslated region and the second exon coding for the remaining 5' untranslated region, coding region, and 3' untranslated region were cloned. Primer extension analysis and RNase protection analysis demonstrated the multiple RAG-1 transcription start sites, clustered in a 31 nucleotide (nt) region. Sequence analysis showed that the RAG-1 promoter lacked a TATA box as well as an initiator sequence. Transient expression assays using a luciferase reporter gene with truncated promoter fragments and substitution mutants, showed that the 5' promoter region containing the CCAAT box between -110 and -86, is indispensable for its basal promoter activity in RAG-1 expressing Nalm 6 cell line. Comparative transient expression assays in various cell lines revealed that the 854 nt upstream promoter region was active, not only in RAG-1 expressing cell lines but also in RAG-1 non-expressing cell lines. These data indicate that the 854 nt upstream region of RAG-1 gene confer basal promoter activity, and that the tissue- and stage-specific expression of RAG-1 is controlled by elements present outside of the promoter region and/or differential chromatin structure(s) of the individual cells.
克隆了包含编码5'非翻译区的第一个外显子以及编码其余5'非翻译区、编码区和3'非翻译区的第二个外显子的人类重组激活基因-1（RAG-1）基因组DNA克隆。引物延伸分析和核糖核酸酶保护分析证实了多个RAG-1转录起始位点，它们聚集在一个31个核苷酸（nt）的区域内。序列分析表明，RAG-1启动子缺乏TATA框以及起始序列。使用带有截短启动子片段和替代突变体的荧光素酶报告基因进行的瞬时表达分析表明，在表达RAG-1的Nalm 6细胞系中，位于-110至-86之间包含CCAAT框的5'启动子区域对于其基础启动子活性是不可或缺的。在各种细胞系中进行的比较瞬时表达分析表明，854 nt的上游启动子区域不仅在表达RAG-1的细胞系中具有活性，在不表达RAG-1的细胞系中也具有活性。这些数据表明，RAG-1基因的854 nt上游区域赋予基础启动子活性，并且RAG-1的组织和阶段特异性表达受启动子区域之外存在的元件和/或单个细胞的差异染色质结构控制。
