The Evolution of Lymphocyte Recognition

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9406249 Warr Although the structure and pathways of expression of the gene encoding the heavy chain of the IgM antibody of the channel catfish (Ictalurus punctatus) have been defined in previous studies, many aspects of the function of this gene remain unresolved. The evolution of the unusual pre-mRNA processing pathways which produce the antigen receptor, membrane-bound form of the IgM is not understood, and neither is the nature of the transcriptional control elements (particularly the enhancer) that drive the expression of this gene. As the major objective of this application, it is proposed to define the core enhancer element in the catfish heavy chain gene in terms of its precise location, structure and specificity of cell-type expression. An understanding of both the structure and expression of the catfish heavy chain gene could permit the successful design of genetic approaches to manipulating the antibody response in a commercially important fish. The catfish IgH enhancer will be investigated initially by functional criteria: the core enhancer will be defined as that minimal segment of DNA from the catfish Ig heavy chain locus that will drive expression of a reporter gene (e.g. chloramphenicol acetyltransferase) in a B cell-specific manner. Reporter constructs will be transfected into cloned, in vitro grown channel catfish cell lines of B cell, T cell and macrophage lineage and their transcriptional activity measured in transient expression assays. Once the core enhancer region of the catfish IgH locus has been located, then the specific sequence motifs that bind the transcription factors will be defined by a combination of mobility- shift, footprinting and functional analyses. The functional analysis will consist of utilizing fragments of the enhancer, in various combinations, in reporter constructs in which it is attempted to reconstruct the tissue specific activity of the native IgH enhancer. %%% These molecular level studies of the regulati on of catfish antibody gene expression will provide insight into the evolution of immune function and generate fundamental information that will lay the groundwork for our future ability to enhance fish immune responses by genetic means. ***