The genetic mechanisms and processes that underlie the evolutionary diversification of immunoglobulin (Ig), T cell antigen receptor (TCR) and other genes which encode inducible recognition factors (irf) will be studied as a means for gaining a further understanding of relationships of gene structure, organization and function in the immune system. Species that occupy significant positions in the phylogenetic development of vertebrates will serve as models. The structural, organizational and regulatory properties of Ig, TCR and irf genes which distinguish them relative to the corresponding systems found in higher vertebrates will be studied. In elasmobranchs, the multi-cluster TCR-like lg gene loci ore present at multiple sites on different chromosomes and exhibit extensive germline joining of segmental elements. In Heterodontus, the relationships of these functionally independent clusters will be examined in PI artificial chromosomes, a newly developed plasmid-based cloning system which stably integrates -300 kb inserts. The transcription of chromosomally-dispersed heavy and light chain gene clusters possessing unique isotypes will be examined in developmentally-staged Raja embryos. Relationships of gene transcription to gene rearrangement and precommitted germline-joining will be explored. Isotypic exclusion in Raja will be studied both by immunocytochemical and in situ hybridization methods as well as by single cell PCR analyses which will address lg mRNA complexity. The nature of an independent TCR system and its relationship to the TCR- like lg structures found in cartilaginous fish will be determined through analyses of mRNA transcripts and excision products in embryonic thymus. Conventional, PCR-generated and kinetically normalized cDNA libraries will be utilized. irf genes in jawless vertebrates will be characterized in Geotria, in which peptide sequence information is now available to derive gene-specific probes. The complexity of the germline irf gene family and the nature of somatic variation will be examined as will the possible presence of irf homologs in other Agnatha and more phylogenetically recent species. Information obtained for Ig, TCR and irf sequences in lower vertebrates will be used to develop conventional and PCR-based amplification approaches for identifying homologous structures in protochordates. Collectively, information obtained in these studies will lead to a better understanding of the mechanisms of diversification and developmental control of genes that encode molecules which function in adaptive immunity. Such studies are of profound significance in terms of our understanding the basis for immunological recognition and diseases of the immune system, including malignancy.
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