The Evolution of Lymphocyte Recognition

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The proposed studies focus on the expression of the immunoglobulin (Ig) heavy chain gene in the channel catfish, Ictalurus punctatus. The specific issues addressed are: 1) Regulation of the differential expression of B-cell membrane receptor and secreted forms of the IgM heavy (mu) chain. This laboratory has found that the pattern of alternate exon splicing involved in the production of the mRNAs encoding the membrane and secreted forms of the mu chain in the channel catfish is different from that used by mammals, elasmobranchs and amphibia. Expression vectors containing relevant regions of the channel catfish mu chain gene will be transfected into mouse pre-B or plasma cells to investigate the factors influencing splice-site choice in the catfish mu chain transcript. 2) Assembly of hybrid catfish/mouse Ig heavy chains in mouse B cells. This laboratory has observed that the assembly and secretion of Igs in which the heavy chain consists of a mouse VH domain fused to the channel catfish mu chain constant region domains is blocked. It is proposed to identify the sites of this blockage in the biosynthetic pathway. Potential sites of blockade to be investigated include defects in disulfide bond formation improper glycosylation, abnormal association of the heavy chain with the immunoglobulin binding protein (BiP), and failure to associate correctly with the endogenous mouse light chains. 3) Identification of the putative enhancer driving expression of the channel catfish Igh locus. Regions of the channel catfish Igh locus will be put into appropriate expression vectors, and B-cell specific enhancer activity in heterologous (mouse) B lymphocytes will be sought. These studies should result in greater understanding of the expression of the antibody mu chain gene and the function of its encoded protein in the channel catfish. In addition, they will contribute to our overall understanding of the evolution of the immunoglobulins and their genes in the vertebrates.