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 goal is to understand not only how the immune system functions, but also how it developmentally arises and how in evolution it arrived at its current level of complexity. The zebrafish, Brachydanio rerio, is an organism that represents a 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 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, beta2-microglobulin, and immunoglobulin constant region genes. We have also identified the invariant chain associated with antigen processing, Ii. Attempts are underway to evaluate whether any of a number of candidate genes are indeed T cell receptors, and protein expression of beta2-microglobulin and invariant chain genes has allowed us to begin to examine the conservation of function of these molecules over millions of years. We expect that studies underway on the assembly of zebrafish beta2-microglobulin with mouse MHC class I, as well as studies on the function of zebrafish invariant chain and its assembly with MHC class II molecules, will provide insight into the general question of how multimeric proteins coevolve. Such studies can also help to identify structural features that are important for the assembly and stabilization of multimeric proteins in general.
