This project addresses the molecular and genetic bases of diversification of the recognition molecules that are essential for the generation of specific immunity in the adaptive/combinatorial immune system of jawed vertebrates. The focus is upon the immune system of sharks because these are the most primitive vertebrates to share with humans the combinatorial immune response. Sharks arose approximately 450 million years ago and, like humans, have immunoglobulins (antibodies), T cell receptors (TCRs), major histocompatibility antigens (MHC) and recombination activating genes (RAGs) that are necessary to allow rearrangements of the gene segments making up the antigen-specific antibodies and T cell receptors. The project includes two major areas of focus, with particular emphasis upon the T cell receptors a/b and g/d because it is likely that the T cell receptor g chain is the "primitive immunoglobulin" in evolution. The first major aim is to map the genomic organization of the TCR genes. Two strategies will be used: direct sequence analysis in the individual clusters, where possible, and by utilizing a Bacterial Artificial Chromosome (BAC) library for the nurse shark. The second aim is to construct recombinant single chain molecules containing complete Va/Vb or Vg/Vd gene products, possibly in association with the constant region segments, to obtain information on structural and antigen binding capacities. The project would start with the g/d constructs because mammalian homologs of the shark molecules bind small ligands such as bacterial pyrophosphates in the absence of presentation by MHC molecules. Overall, these studies are directed towards applying molecular and protein chemical technology in a functional genomic approach to obtain precise answers to fundamental problems of the combinatorial immune system as it obtains in vertebrates, with implications for defense against invading pathogens, cancers and immunological dysfunctions in autoimmunity. Graduate, undergraduate, and high school students including those from underrepresented groups will be involved in this project. Moreover, Dr. Marchalonis is also involved in several outreach activities including giving lectures to high school students and teachers.
