Experiments are proposed to study early B cell development, particularly as it relates to the expression of immunoglobulin (Ig) genes. The following plans are described in this proposal: During the current grant period it was found that gamma2b heavy chain transgenes inhibit the development of B cells, because gamma2b causes strong feedback inhibition of H gene rearrangement, but cannot replace mu in nurturing preB cell maturation. The molecular and cellular basis of the roles of gamma2b and mu in B cell development will be studied by comparing gamma2b with mu in cell signaling; making new transgenic mice with gamma2b/mu hybrid genes to determine the domain of the mu molecule responsible for B cell maturation; comparing the numbers and distribution of apoptotic B cells in bone marrow of gamma2b transgenic and normal mice; crossing gamma2b transgenics with mice which express a Bcl-2 transgene in preB cells. While all gamma2b transgenic mice in several labs show the B cell defect, a unique gamma2b transgenic mouse line, the C line, arose in which mu is not required for preB cell development. Apparently, the transgene at its insertion site activates another gene whose function replaces mu in preB cell development. It is expected that this mouse line will provide novel insights into B cell development and the following experiments are proposed: the C line will be crossed with a RAG-1 knockout line to determine if T cells are required for early B cell development in the C line; the kinetics of pro/preB cell development, the levels of mRNAs important in early B cell development, and the rearrangement patterns of Ig genes will be compared with conventional gamma2b transgenic and normal mice; the molecules associated with gamma2b will be determined; feedback inhibition of VD and DJ rearrangement will be compared in C line and normal mice to determine if, as postulated, gamma2b has a strong heavy chain feedback effect; the V(D)J recombinase feedback inhibition by gamma2b/L chain will be compared to that by mu/L chain; the candidate gene postulated to be responsible for the phenotype of this mouse line will be cloned and characterized; new transgenic mice will be produced with the candidate gene to assess whether its expression under the control of the heavy chain enhancer can induce a C-phenotype; when a candidate gene has been proven to be responsible for the C phenotype, mice with homozygous deletion of this gene will be produced to determine if it is essential for B cell development. It is expected that these experiments will contribute to the understanding of the control of B cell development and Ig gene expression, and will provide new insights into immunological disorders with a B cell component.
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