RPG: The Isolation and Characterization of Recombination Activating Genes (RAGs) from a Lower Chordate

Bart1MCB-9629300重组激活基因(RAG-1和RAG-2)是哺乳动物基因重排所必需的。之前从护士鲨DNA中分离出的与哺乳动物RAG-1基因序列相似的PCR产物将用于筛选基因组文库。由于哺乳动物、鸟类和两栖动物的RAG-1和RAG-2紧密相连，鲨鱼的同源物可能也是如此。含有鲨鱼RAG-1的基因组克隆将使用聚合酶链式反应、异源探针或搜索开放阅读框架来检测连接的RAG-2同源物。在拟议的计划期之后，鲨鱼RAG-1和RAG-2同源物将被用来通过Northern杂交和原位杂交来检测不同年龄的哺乳鲨鱼淋巴器官中的表达。鲨鱼RAG蛋白的重组活性将使用人工底物在哺乳动物成纤维细胞中进行评估。高等脊椎动物的免疫系统是由细胞、器官和组织组成的复杂网络。这样一个紧密相连的系统的复杂运作和演变还没有得到很好的理解。免疫功能所需的一个基本过程是亚基因元件的重排以形成功能性抗原受体。阐明与哺乳动物有较远亲缘关系的生物体中的这种机制对于我们理解获得性免疫的进化至关重要。在拟议的研究计划中，之所以选择鲨鱼，是因为它们是与哺乳动物关系最远的分类单元，已有文献证明它们具有适应性免疫系统。

Bartl MCB-9629300 Recombination activating genes (RAG-1 and RAG-2) are essential for gene rearrangement in mammals. Previously isolated PCR products from nurse shark DNA that have sequence similarity to mammalian RAG-1 genes will be used to screen a genomic library. Since mammalian, avian and amphibian RAG-1 and RAG-2 are tightly linked, the same may be true of the shark homologs. Genomic clones containing shark RAG-1 will be assayed for linked RAG-2 homologs using PCR, heterologous probes or by searching for an open-reading frame. Following the proposed planning period, the shark RAG-1 and RAG-2 homologs will be used to assay expression in lymphoid organs from nurse sharks of different ages using Northern blots and in situ hybridization. Recombination activity of shark RAG proteins will be assessed in mammalian fibroblasts using artificial substrates. The immune system of higher vertebrates is a complex network of cells, organs and tissues. The intricate workings and the evolution of such a tightly linked system are not well understood. A basic process required for immune function is the rearrangement of subgenic elements to form functional antigen receptors. Elucidating the mechanism in organisms distantly related to mammals is crucial for our understanding of the evolution of adaptive immunity. For the proposed research plan, shark is selected because they are the taxon most distantly related to mammals in which an adaptive immune system has been documented.