This project is directed at identifying genes that are critical to the immune response in an animal species that allows careful study of both developmental biology and of evolutionary genetics. The long term goal is to understand not only how a primitive immune system functions, but also how it developmentally arises and the evolutionary path that was taken to arrive at the level of complexity seen in contemporary mammals. The zebrafish, Brachydanio rerio, is an organism that represents a branch point of evolution of at least 200 million years ago, can be grown cheaply in laboratories, is a species that undergoes its developmental stages in a visible transparent embryo, and is a species for which a large number of genetic markers throughout its genome have been identified. We have begun our efforts by identifying genes of the zebrafish that clearly have similar structure to those of mammals: we have cloned MHC class I genes, b2-microglobulin, and immunoglobulin constant region genes. We have also identified the invariant chain associated with antigen processing, Ii. Amino acid sequence comparisons of zebrafish invariant chain with mouse Ii show 24% sequence identity. The greatest conservation of sequence is seen in the transmembrane and thyroglobulin domains, with 63% and 54% identity, respectively. In addition, the amino terminal lumenal domain of the encoded protein shows amino acid residues that align with the two "dileucine" motifs of mouse and human Ii. These similarities suggest that strong evolutionary forces have acted to preserve these regions of the molecule relative to other parts of the molecule. A putative CLIP region, known for mouse and human Ii to bind and protect the MHC class II molecule?s peptide binding site from inappropriate acquisition of peptides, has been identified. This region shows limited identity to mouse CLIP, although several proline residues and the C-terminal PLL motif are conserved. Surprisingly, despite limited sequence conservation overall with mouse Ii, zebrafish Ii can facilitate the assembly and intracellular transport of murine I-Ad molecules in a transient transfection assay using COS cells. In addition, synthetic peptides representing the zebrafish CLIP peptide are capable of binding the mouse IAd. The identification of Ii at the phylogenetic level of zebrafish indicates that components of the class II antigen processing and presentation machinery arose early in vertebrate evolution. The surprising observation that zebrafish Ii can substitute for murine Ii for a cellular function suggests stringent evolutionary demands to preserve interaction with phylogenetically conserved regions of the MHC class II structure. Similarly, we have studied the ability of the zebrafish b2- microglobulin chain to assemble with mouse MHC-I molecules. For these experiments we have expressed, refolded, and purified the zebrafish b2-microglobulin from a bacterial expression system, and have assembled it in vitro with mouse MHC-I molecules and antigenic peptides. Surprisingly, the zebrafish b2-microglobulin serves as a good subunit for the assembly of the complete murine MHC-I molecule. Studies of the binding of this MHC-I/peptide/ zebrafish b2-microglobulin complex to both T cell receptors and to NK cell receptors are now underway. 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).