The overall goal of these studies is to gain further understanding of the mechanisms involved in regulating or generating antibody diversity. The approach is to examine the switch in the spectrum of antibody diversity that occurs during development. Early in ontogeny, in the fetal/neonatal mammal or in the Xenopus tadpole, the antibody pool is very limited. The sources of this restriction have to do, in part, with the enzymatic machinery governing Ig assembly: there seems to be a paucity of junctional diversity manifested by Lack of N-region sequence insertion and a more precisely defined site of recombination by use of regions containing homology. Antibody diversity increases during development. In Xenopus, the more heterogeneous adult-type antibodies appear only with metamorphosis and whenever the process of metamorphosis is contrived. This clear separation of larval and adult immunological states in Xenopus is a unique advantage that allows more incisive examination of the differential regulation of the genes involved in antibody repertoire generation. The specific aims of the proposed projects are: 1) to isolate Xenopus homologs of mammalian genes involved in B lymphocyte differentiation (TdT, RAG1, RAG2, Oct2); 2) to study the ontogeny of lymphopoiesis in Xenopus using these markers, with focus on the onset and course of expression in the larva and changes at metamorphosis; and 3) to construct transgenic Xenopus carrying TdT in order to alter the normal pattern of Ig diversification during development. The ability of the immune system to survey and recognize a broad spectrum of different antigens is based on the huge repertoire of different immunoglobulin (antibody) variable region amino acid sequences and the binding specificities that result. The Nobel Prize-winning discovery, just a few years ago, that this diversity is generated by a complex somatic gene recombination mechanism has raised the fundamental question of how this recombination mechanism is regulated. In mammals, there is a difference in the amount of recombination (and therefore in the diversity of the antibody pool) that occurs in adults vs. newborns, with newborns having a much more limited repertoire of specificities than adults. This difference seems to be based on differences in the enzymatic machinery needed to effect the recombination events. In frogs, this difference is reflected by a change in the ability to generate diversity at metamorphosis: tadpoles have a very limited pool of antibodies, while post-metamorphosis adult frogs have the more heterogeneous spectrum of antibodies. This project will take advantage of this distinct difference in immunological states in tadpoles vs. adult frogs, in order to study the differential regulation of expression of the genes involved in the somatic recombination events that result in the generation of antibody diversity.
