The recognition of pathogens by the adaptive immune system relies on a diverse antigen receptor repertoire on the surface of B and T lymphocytes. The genes encoding these receptors are assembled from individual gene segments by a process named V(D)J recombination. The key factors in this process are the products of the recombination activating genes 1 and 2. Studies during this fiscal year led to the identification of homologs of RAG1 and RAG2, named spRAG1L and spRAG2L respectively, in the genome of the purple sea urchin, strongylocentrotus purpuratus. This is unexpected as, according to the current literature, echinoderms lack a diversified antigen receptor repertoire and are devoid an adaptive immune system. We expressed the minimal central domain of the spRAG1L protein and showed that it has DNA binding activities similar to the respective sub-domain of mouse RAG1. This provided first experimental data the sea urchin spRAG1L/2L gene may indeed represent a functional homolog of the vertebrate RAG1. To further support this hypothesis of we performed pull-down assays to show interactions between sea urchin and vertebrate RAG1 proteins. In addition we expressed and purified recombinant spRAG2L that will be used in subsequent assays to determine the functional properties of this protein. Our studies have major implications for the current model of how adaptive immunity evolved in jawed vertebrates and will help to illuminate conserved features of how V(D)J recombination is tightly controlled to avoid potentially dangerous modifications of the genome. During an immune response of the immunoglobulins (Ig) genes are further diversified in order to improve the specificity of the encoded antibodies by two closely related mechanisms: somatic hypermutation and immunoglobulin gene conversion (IgGC). During this fiscal years studies were initiated to characterize the mechanisms that limited these processes specifically to the Ig gene loci. We determined that the IgGC machinery is closely confined to the Ig genes as even the immediately neighbouring genes remain unaltered. We hypothesized that insulator elements might be involved and thus generated cell lines containing targeted integrations of heterologous insulator elements at defined positions within the Ig locus. An analysis of these cells will shed a light on the mechanism that protects the rest of the genome from potentially deleterious modifications.
| Kothapalli, Nagarama; Norton, Darrell D; Fugmann, Sebastian D (2008) Cutting edge: a cis-acting DNA element targets AID-mediated sequence diversification to the chicken Ig light chain gene locus. J Immunol 180:2019-23 |
| Gopal, Anjali R; Fugmann, Sebastian D (2008) AID-mediated diversification within the IgL locus of chicken DT40 cells is restricted to the transcribed IgL gene. Mol Immunol 45:2062-8 |
| Yang, Shu Yuan; Fugmann, Sebastian D; Gramlich, Hillary S et al. (2007) Activation-induced cytidine deaminase-mediated sequence diversification is transiently targeted to newly integrated DNA substrates. J Biol Chem 282:25308-13 |
| Fugmann, Sebastian D; Messier, Cynthia; Novack, Laura A et al. (2006) An ancient evolutionary origin of the Rag1/2 gene locus. Proc Natl Acad Sci U S A 103:3728-33 |
