The long term goals of this application are to define the molecular requirements and regulation of immunoglobulin heavy chain constant region gene switch (S) recombination in B lymphoid cell development. Our strategy is largely based on the introduction of retroviral vectors harboring immunoglobulin gene recombination sequences into lymphoid and non-lymphoid cell lines. Recombinant DNA techniques will be employed to determine the molecular constraints and minimal requirements for S region recombination. Appropriate vectors will also be prepared to screen for putative CH isotope specific switch-recombinases. The presence of CH switch-recombinase activity will be assayed in cell lines representative of various stages of B cell development and the effects of lipopolysaccharide (LPS) and interleukins which cause B cell differentiation, such as IL-4 will also be determined. In a second specific aim, we will study B cell specific nuclear factors, which bind to S region sequences, to determine their potential role(s) in switch recombination. In a third objective, the nuclear run-on transcription technique will be employed to directly determine if transcriptional activation of CH genes predetermines CH switch-recombination in differentiating B cells. We will also investigate the regulation of sense and anti-sense transcripts of unrearranged CH loci and their potential significance for the switch recombination mechanism. Finally, we plan to clone Igh switch-recombinase gene(s) from a mammalian expression vector cDNA library by a convenient, selectable rescue strategy. Here, thymidine kinase negative fibroblast cell lines harboring stably integrated, though recombinationally inert, retroviral Igh gene recombination substrates will be transfected with a mammalian expression vector cDNA library prepared from the mRNA of an Igh recombination competent pre B cell line. The loss of a thymidine kinase marker gene inserted in between our retrovector's Igh recombination sequences will be selected for in bromodeoxyuridine (BudR) media. By this selection strategy, cDNA clones which could encode a switch recombinase responsible for TK gene deletion will be identified and characterized. These objectives will enable us to dissect the molecular basis and developmental control of a key process in immune response maturation.
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