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

1. 利用人rag-1 cDNA片段，分离出含有人rag-1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保护实验确定了其外显子/内含子组织和转录起始位点。类似的。我们利用从人类rag-2 cDNA片段中获得的探针，分离出含有人类rag-2第一或第二外显子的基因组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。这使我们首次揭示了人类基因组中rag-1和rag-2基因座的结构。rag转录调控的研究我们对rag- 1和rag-2的启动子区域进行了表征。通过对具有截断启动子片段或替换突变体的荧光素酶报告基因的瞬时表达试验，我们发现含有-110和-86之间的ccaat-box的5个启动子区域对于其基础启动子活性是必不可少的。相反，在rag-2启动子中，-63 ~ -107之间的序列既不含ccaat-box，也不含已报道的顺式元件，对其启动子活性至关重要，这表明rag-1和rag-2的转录调控可能由不同的转录机制控制。现在，我们正试图确定顺式元件，并分离与顺式元件结合并调节rag-2转录的转录因子。

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.