Currently little is known about the mechanisms and locations of lymphocyte development in teleosts. In this study several aspects of the underlying factors which govern B lymphocyte development in trout were investigated which included: the isolation and characterization of immunoglobulin heavy chain (IgH) genes, the recombination activating genes 1 and 2 (RAG1 and RAG2) and the use of cellular markers to identify tissues harboring precursor B-cells. Immunoglobulin heavy chains are part of the structural components which make up antibody molecules produced by B-cells. We isolated various full-length IgH cDNA clones, some of which contained the secreted while others contained the membrane bound form of IgH. Upon characterization of the membrane bound forms, typical features common to all IgH cDNAs were found including a leader peptide, a variable region and constant domain containing transmembrane (TM) segments as well. Further sequence analysis of this region revealed that the TM domains were spliced directly to the CH3 domains which results in the loss of the entire CH4 region. Our results support previous observations of unusual splicing events in fish IgH genes. RAG1 and -2 in mammals have been shown to be essential for carrying out V (D) J recombination of lymphocyte receptors and are found to be expressed within primary lymphoid tissues and precursor lymphocytes. We isolated the RAG locus from a rainbow trout genomic library and characterized their conservation and expression. Overall the complete amino acid sequences of RAG1 and RAG2 displayed 78% and 75% similarity when compared to RAG genes from higher vertebrates thus demonstrating the highly conserved nature of these genes. Tissue specific expression of both genes was primarily associated with the thymus and pronephros in both juvenile and adult trout. Based upon these observation we conclude that the thymus and pronephros likely serve as the tissue sites for V (D) J recombination in trout and are thus primary lymphoid organs. Finally we addressed the question as to where B-cell lymphopoiesis occurs in trout. Our results using both immunofluorescence and confocal microscopy putatively demonstrate that the thymus harbors precursor B-cells and thus alludes to a dual function for both B and T-cell development in trout. / Graduation date: 1996

The ABF-1 gene encodes for a protein that belongs to the basic helix-loop-helix family of transcription factors. ABF-1 mRNA molecules have been detected in the lymphoid tissues, which include the bone marrow, lymph nodes, and appendix, as well as transformed B cells lines infected with Epstein-Barr virus. This study investigates the role of ABF-1 in regulating downstream target genes in the human mature B cell line RAJI, as well as the plasma cell line, ARH-77. Quantitative real-time polymerase chain reaction and DNA microarray technology was used to investigate target genes that are subjected to transcriptional regulation by ABF-1. Using ABF-1 inducible cell lines or B cell lines that overexpress ABF -1 by transient transfection experiments, we discovered many cellular genes that change in their transcriptional profiles in response to ABF -1 expression. Based upon the analysis of genes being affected following ABF-1 induction, our results support the hypothesis that ABF-1 primarily functions as a transcriptional repressor in vivo. Many genes that regulate the cellular processes of apoptosis, as well as the cell cycle, were repressed following ABF-1 expression. Because EBV has been reported to control ABF-1 gene expression, the identification of downstream target genes regulated by ABF-1 may provide insight into the molecular events that follow after EBV infection.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy