The focus of this proposal is the characterization of IgM/IgG1 double producers induced by LPS/IL-4 in a normal murine B cell culture system. Initial characterization will be performed on the blast cells derived from B cells cultured with LPS/IL-4. Cell surface immunofluorescence and C'mediated killing will be employed to establish the existence of such cell population. Subsequently, sorted sIgM+/sIgG1+ cells will be immortalized by infection with a murine recombinant retrovirus containing two different oncogenes. Clones will be generated from immortalized cell lines by deposition of single cells into 96- well plates using a FACS lV. Clones will be assayed for expression and secretion of IgM and IgGl. Double producer clones will be selected and characterized at the cellular, biochemical and molecular levels. The surface 18 phenotype will be assayed by surface immunofluorescence and surface iodination followed by immunoprecipitation and SDS-PAGE analysis. Steady-state levels of mRNA will be assessed by Northern analysis. IgCH context will be analyzed by overlapping Southern analysis. The VDJ rearrangement utilized by Mu and gamma1 will be analyzed by direct sequencing of the V regions of MU and gamma1 mRNA and by genomic cloning and sequencing of the rearranged JHs. Furthermore, non-secretors will be isolated from the double producers to study what signal(s) will drive them to terminal IgG1 secretion. The level at which the signal exerts its effect will be examined by Southern analysis and run-on transcription. In this revision, we also attempt to immortalize the activated B lymphoblast and resting B cells. We will examine the surface and secreted (if any) Ig isotypes of the immortalized cells. In addition, we will also attempt to examine the methylation status of SMU, CMU, Sgamma1 and Cgamma1 regions, to determine whether the cells make any sterile GAMMA1 transcripts and to determine the nature of sterile gamma1 transcripts by primer extension and cloning and sequencing. We also attempt to induce or isolate the IgM/IgG1 double producers from the immortalized activated lymphoblast and """"""""resting"""""""" B cells and to characterize them at the cellular, biochemical, and molecular levels.
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