In this application, it is planned to identify molecular mechanisms of VH recombinatorial events involved in generation of the Ig heavy chains. Based on the application of FACS and PCR technology to the analysis of gene segment rearrangements in single cells isolated at various stages of B-cell development, the investigator will investigate the hypothesis that because H chains differ from clone to clone, B-cell development is clone specific. More to the point, while most of the molecular machinery responsible for B-cell differentiation is common to all B-cells, individual physicochemical characteristics of mu heavy chains can elicit positive or negative selective effects on the life of a B-cell. He proposes that a spectrum of heavy chains, even those which result from functional rearrangements, are dysfunctional and fail to mediate clonal maturation and allelic exclusion, particularly based on their ability to assemble with surrogate light chains. This hypothesis will be tested by transfection of a variety of genetically manipulated mu heavy chains into in vitro cell lines followed by analysis of their ability to assemble with surrogate light chains. Transgenic mice expressing so-called functional and dysfunctional heavy chains will be constructed to assess where these blocks in development occur.
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