ANALYSIS OF TRANSCRIPTIONAL REGULATION OF RECOMBINATION ACTIVATING GENE (RAG) DURING PROCESS OF LYMOHOID DEVEROPMENT

重组激活基因(RAG)在淋巴样发育过程中的转录调控分析

基本信息

批准号：
09836004
负责人：
MURAGUCHI Atsushi
金额：
$ 1.92万
依托单位：
TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

1. Reseach for human rag genomic locus Using a fragment of human rag-1 cDNA, we isolated genomic DNA clones that contained either first or second exon of human rag-1 . We determined its exon/intron organization and the transcriptional start site by the primer extenstion analysis and RNA protection assay. Similarly. we isolated genomic DNA clones that contains first or second exon of human rag-2, using a probe obtained from a fragment of human rag-2 cDNA, and determined its exon/intron structure and the transcription initiation site for rag-2. These led us to reveal, for the first time, the organization of human genomic rag-1 and rag-2 locus.2. Research for regulation of rag transcription ; We characterized promoter regions for both rag- 1 and rag-2. By transient expression assays using a luciferase reporter gene with truncated promoter fragments, or substitution mutants, we showed that the 5 promoter region containing the ccaat-box between -110 and -86 was indispensable for its basal promoter activity. On contrary, in case of rag-2 promoter, the sequences between -63 to -107, which contained neither ccaat-box or reported cis-element, were shown to be essential for its promoter acticity, suggesting the regulation of rag-1 and rag-2 transcription may be controled by different transcriptional mechanisms. Now, we are trying to determine the cis-element and isolate the transcription factor that binds to the cis- element and regulates the transcription of rag-2.

1.人类rag基因组位点的研究使用人类rag-1 cDNA片段，我们分离了包含人类rag-1的第一或第二外显子的基因组DNA克隆。我们通过引物延伸分析和RNA保护测定确定了其外显子/内含子组织和转录起始位点。相似地。我们使用从人 rag-2 cDNA 片段获得的探针分离了包含人 rag-2 第一个或第二个外显子的基因组 DNA 克隆，并确定了其外显子/内含子结构和 rag-2 的转录起始位点。这些使我们首次揭示了人类基因组rag-1 和rag-2 位点的组织。2。 rag转录调控研究；我们对 rag-1 和 rag-2 的启动子区域进行了表征。通过使用具有截短的启动子片段或取代突变体的荧光素酶报告基因的瞬时表达测定，我们表明包含-110和-86之间的ccaat盒的5启动子区域对于其基础启动子活性是不可或缺的。相反，在 rag-2 启动子的情况下，-63 至 -107 之间的序列既不包含 ccaat-box 也不包含报道的顺式元件，但被证明对其启动子活性至关重要，这表明 rag-1 和rag-2转录可能受到不同转录机制的控制。现在，我们正在尝试确定顺式元件并分离与顺式元件结合并调节rag-2转录的转录因子。