This project was initiated several years ago to identify genes of the Zebrafish that are homologous to immunoregulatory genes of higher organisms and to understand their, evolution, structure and function. Signifecant progress was made in identifying a number of such genes, and a novel one, the zebrafish invariant chain, has been analyzed in more detail. We have shown that this molecule functions in complementing the expression of mouse MHC-II chains and of directing their cellular pathway to endosomes, a finding with broad implications for the evolution of MHC-II function. These studies take advantage of a complex mixture of molecular biological techniques (for the cloning, sequencing, and identification of the zebrafish genes), molecular engineering techniques (for the expression of the zebrafish proteins in vitro), cell biological techniques (for the examination of the expression pathway of the Invariant chain assembly), and immunological techniques (for the demonstration of the ability of the MHC-I/peptide complexes to bind to appropriate antibodies). - Antigen presentation, evolutionary biology