ABNORMAL RECOMBINATION ACTIVATING GENE (RAG) AND IMMUNO DEFICIENCY

1. 人Rag基因座；利用人RAG-1 cDNA片段，我们分离出含有人RAG-1第一或第二外显子的基因组DNA克隆。我们确定了它的外显子/内含子组织和转录起始位点。同样，我们分离了含有人类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基因座的组织结构。人RAG转录调控；我们对人类RAG-1和RAG-2的启动子区域进行了表征，发现RAG-1的5'启动子区域对于其基础启动子活性是必不可少的。相反，rag2的-63 ~ -107序列对其启动子活性至关重要，表明rag1和rag2的转录调控可能受到不同转录机制的控制。小鼠RAG-2的调控；我们发现小鼠RAG-2启动子活性的调控被赋予RAG-2上游80个核苷酸。我们还发现B细胞特异性转录蛋白Pax-5和T细胞特异性转录蛋白GATA-3分别结合到B细胞和T细胞的-80到- 17nt区域。这些结果表明，不同的DNA结合蛋白Pax-5和GATA-3可能分别调控小鼠B系和T系细胞中的RAG-2启动子。

1. 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. Similarly, we isolated genomic DNA clones that contains first or second exon of human RAG-2 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. Regulation of human RAG transcription ; We characterized promoter regions for human RAG-1 and RAG-2, and revealed that the 5' promoter region of RAG-lwas indispensable for its basal promoter activity. On the contrary, the sequences between -63 to -107 of RAG-2 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.3. Regulation of murine RAG-2 ; We found that the regulation of the mouse RAG-2 promoter activity is confered to 80 nucleotide upstream of RAG-2. We also found that a B cell-specific transcription protein, Pax-5, and a T cell-specific transcription protein, GATA-3, bind to the -80 to -17 nt region in B cells and T cells, respectively. These results indicate that distinct DNA binding proteins, Pax-5 and GATA-3, may regulate the murine RAG-2 promoter in B and T lineage cells, respectively.