RUI: Anaylsis of Somatic Mutation in Immunoglobulin V Genes Using a Reporter Gene Assay

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9419042 Pollock Somatic mutation of immunoglobulin variable (v) genes plays an important role in the generating antibody diversity. An assay is being developed to detect and isolate cells undergoing somatic mutation in vivo and in vitro. The assay is based on a DNA construct encoding a fusion protein composed of a rearranged V gene, containing a stop codon, linked to a lacZ gene. The V gene is 5' of the lacZ; its stop codon therefore prevents transltion of the lacZ gene, If somatic mutation revers the stop codon, the entire fusion protein is translated and lacZ expressed. LacZ expression can be readily detected by histochemical or fluorescence assays. This construct has been put into cell lines and a mutant cell line expressing lacZ isolated. Transgenic mice containing this construct are being made. Experiments are proposed to further study mutation in vitro and to address six basic questions using the transgenic mice: 1) where can somatic mutation be detected; 2) when is somatic mutation seen after immunization; 3) when does somatic mutation first occur during ontogeny; 4) which germinal center cells show active somatic mutation; 5) what is the phenotype of cells undergoing somatic mutation; and 6) what types of mutations occur. %%% Although it is know how the mammalian immune system generates much of the antibody diversity that is seen, genomic DNA rearrangement is not the whole answer. Somatic mutation of immunoglobulin variable genes plays an important role in generating such diversity. An investigation into six basic questions will be asked by this effort. Where can somatic mutation be detected ? When does somatic mutation first occur during ontogeny? Which germinal center cells show active somatic mutation? What is the phenotype of cells undergoing somatic mutation? What types of mutations occur? The answers to those questions have both basic and practical interest. ***