The long term goals of this research are to understand the structure, organization, and expression of immunoglobulin (Ig) heavy chain genes in the goldfish, Carassius auratus. A genomic library has been constructed in lambda phage; initial sequencing of three VH.related genes has been completed, and putative JH regions have been identified by oligonucleotide probe hybridization. These studies will be expanded in three areas. First, representative VH, DH, JH, and CH regions will be identified and sequenced to elucidate their germline organization. Standard DNA cloning, mapping, and sequencing strategies will be used. To identify CH regions, peptides of goldfish IgM heavy chains will be microsequenced, and specific mixed oligonucleotide probes will be synthesized to correspond to their coding sequences. Second, computer assisted analyses of goldfish VH gene sequences and comparison of these with each other and with published VH sequences for other vertebrates will be undertaken in an attempt to infer the relative contributions of selection, mutation, drift, and non.reciprocal exchange in the evolution of these genes. Third, the function of putative regulatory regions of the goldfish Ig heavy chain genes will be studied. Appropriate expression vectors will be constructed to test whether the goldfish heavy chain promoter (in association with a mammalian Ig enhancer region) permits gene expression in mammalian lymphoid cells. The mechanisms of immune function in animals other than mammals are as yet poorly understood. Basic knowledge of the structure, organization and regulation of expression of immunoglobulin genes in fish is of importance not only for its intrinsic interest, but also because understanding of immune function in fish is fundamental for the capability to grow and maintain of fish in commercial culture and for understanding the factors involved in the success or failure of fish to survive in natural environments. Knowledge of the evolution of immune function obtained from these studies may also shed light on the function of immune systems in higher vertebrates.
