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.
