This proposal describes experiments to analyze genetic variation at the immunoglobulin loci during the ontogeny of B lymphocytes. After the appropriate gene segments have been joined, the complete, functional immunoglobulin gene does not necessarily remain unaltered: (i) The rearranged gene segments encoding the immunoglobulin variable (V) region are usually further diversified by somatic hypermutation. (ii) The gene segments encoding the constant (C) region of the heavy (H) chain are often switched, i.e., replaced by segments encoding another C region. Both events, hypermutation and H chain class switching, are thought to be catalyzed by enzyme systems.
The aim i s to characterize the two putative systems, the so-called immunoglobulin mutator system (mutator) and the switch recombinase. Heavy chain class switching is the underlying cause for a major health problem; the difference between switching to IgG and switching to IgB, for example, is the difference between immunity and allergy. If the genetic instability implied by hypermutation affects other loci in the cell, even at a small fraction of the rated at which the immunoglobulin loci mutate, hypermutation may be an important source of leukemias and lymphomas. One set of experiments aims to study the spectrum of mutations produced by the mutator. Another set attempts to define the recognition sequences for the mutator by two complementary approaches: (i) Cells with an active mutator will be stably transfeced with vectors carrying a reporter gene and, in its neighborhood, various sequences to be tested for their ability to confer hypermutability on the reporter gene. (ii) Transfected candidate recognition sequences will be amplified and tested for their ability to suppress hypermutability of the endogenous immunoglobulin gene by competing for the binding component of the mutator. The sequences to be identified by these two approaches are, in principle, the same sequences; their positive or negative effect on mutability is a result of the experimental assay system, not of any intrinsic difference. A third set of experiments aims to clone (parts of) the switch recombinase by two approaches: (i) searching for proteins binding to a particular switch region -- the putative target sequences of the recombinase -- by gel shift assays and affinity chromotography as well as screening a cDNA expression library made from cells undergoing switching. (ii) cloning genes that upon transfection are able to recombine switch substrates stably transfected into yeast.
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