THE RECOMBINATION ACTIVATING GENE-1 (RAG1) OF RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) - CLONING, EXPRESSION, AND PHYLOGENETIC ANALYSIS
THE RECOMBINATION ACTIVATING GENE-1 (RAG1) OF RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) - CLONING, EXPRESSION, AND PHYLOGENETIC ANALYSIS
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DOI：
10.1007/bf00191224
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发表时间：
1995-07-01
期刊：
影响因子：
3.2
通讯作者：
KAATTARI, SL
中科院分区：
文献类型：
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作者：
HANSEN, JD;KAATTARI, SL
参与免疫库生成的基因表征是低等脊椎动物分类群研究的一个活跃领域。重组激活基因（RAG）已被证明是t细胞抗原受体（TCR）和免疫球蛋白（Ig）基因V (D) J重组所必需的，导致初级库的产生。由于RAG1对前b细胞和t细胞的分化至关重要，因此其在相关的原发性淋巴器官中的表达可作为发育标志物。为了研究虹鳟淋巴细胞的个体发育，我们从鳟鱼中克隆了RAG1，并检测了其组织和淋巴细胞特异性表达。利用聚合酶链反应，结合简并寡核苷酸引物，从虹鳟鱼基因组DNA中扩增出一个同源探针[（633碱基对）（bp）]，进而分离出一个lambda基因组克隆，该基因组克隆的序列分析证实了该基因的RAGI性质（3888 bp），并发现了一个666 bp的内含子。与先前报道的其他RAGI序列相比，预测的氨基酸翻译（1073 aa）在完整序列上的相似性至少为78%，在保守区域（aa 417-1042）上的相似性为89%。通过northern blot分析，我们发现RAGI的表达仅限于胸腺表面的Ig-n淋巴细胞。这一数据为以下观点提供了基础：硬骨鱼的胸腺在淋巴形成中起着重要的发育作用，因此可以被视为原发性淋巴器官。
The characterization of genes involved in the generation of the immune repertoire is an active area of research in lower vertebrate taxa. The recombination activating genes (RAG) have been shown to be essential for V (D) J recombination of T-cell antigen receptor (TCR) and immunoglobulin (Ig) genes, leading to the generation of the primary repertoire. As RAG1 is critical to the differentiation of pre-B and -T cells, its expression within an associated primary lymphoid organ can serve as a developmental marker. To examine the ontogeny of lymphocytes in Oncorhynchus mykiss, we cloned RAG1 from trout and ex amined its tissue- and lymphocyte-specific expression. The polymerase chain reaction, coupled with degenerate oligonucleotide primers, was used to amplify a homologous probe [(633 base pairs) (bp)] from rainbow trout genomic DNA, which in turn was used to isolate a lambda genomic clone, Sequence analysis of this genomic clone confirmed the RAGI nature of this gene (3888 bp) and revealed an internal intron of 666 bp. When compared with other previously reported RAGI sequences, the predicted amino acid translation (1073 aa) displayed a minimum of 78% similarity for the complete sequence and 89% similarity in the conserved region (aa 417-1042). Using northern blot analysis, we found the expression of RAGI to be limited to surface Ig-n lymphocytes within the thymus. This data forms the basis for a proposal that the thymus of teleost species plays an essential developmental role in lymphopoiesis and thus can be regarded as a primary lymphoid organ.