The long term goal of this project is to understand the molecular mechanisms involved in the regulation of IgE-specific immune responses. Immunoglobulin (Ig) heavy chain class-switch recombination allows a B lymphoid cell to change the function of the antibody it produces (which is determined by the heavy chain constant region of the molecule), while retaining the antigen binding specificity of the molecule (determined by the variable regions of the heavy and light chain). Recent studies have shown a correlation between the capacity of certain lymphokines and mitogens to modulate the isotypes of antibodies produced by B lymphoid cells with their capacity to affect the level of specific germline heavy chain transcripts. For instance, the culture of murine B cells or AMuLV- transformed pre-B cell lines with lipopolysaccharide (LPS) and interleukin-4 (IL-4) greatly increases the level of germline epsilon transcripts prior to the induction of switching to epsilon. It is unknown if germline epsilon transcription actually performs a function in controlling class-switching to epsilon. We have begun to address this issue by using homologous recombination to alter the endogenous Ig heavy chain epsilon locus in an AMuLV-transformed pre-B cell line. This mutation, which has placed a constitutive promoter at the epsilon locus, has both altered the transcriptional activity of the region and correspondingly changed the pattern of switching in this line. the specific aims of this project are to extend the studies of this mutation in order to define how it has effected the Ig heavy chain locus and the mechanism by which it induces switching. This analysis will provide information for new experiments to define the mechanism by which transcription of the epsilon locus targets the region for switching. In addition to work with the pre-B cell line, embryonic stem cell clones will be generated with a similar mutation for use in creating transgenic "knock-out" mice. These mice will be analyzed for the effect of constitutive transcription of the Ig epsilon locus in normal B cells. In addition, the mice will be used to address the role of other factors in controlling IgE levels after class-switch recombination. These experiments will provide important information on how the immune system controls production of IgE and may lead to new approaches for controlling the levels of IgE in conditions, such as allergic states, with deleteriously elevated levels.