The long term goals of this research are to understand the regulation of immunoglobulin heavy chain mRNA processing and expression during normal and neoplastic B cell development. Mouse myeloma tumor cells and lymphoma cells are being used, respectively, as models of plasma cells and earlier B cell stages and memory cells. Differential production of membrane and secretory encoding mRNAs from a single IgG gene has been shown to be regulated by post-transcriptional processing events, differential polyadenylation and/or splicing. We propose to study the mechanism of this regulation by making altered gene constructs and transfecting them back into the myeloma and lymphoma cells to test the role of various cis-acting sequences. Areas of DNA near the membrane encoding poly A sites will be sequenced and compared with the already sequenced secretory poly A sites. In vitro polyadenylation systems from myeloma and lymphoma cells will be established. Requirements for cis-acting sequences and putative trans-acting, stage-specific factors will be investigated in the vitro system. The role of transcription termination sites in regulated membrane and secretory mRNA production will be investigated by transcription run-on assays, analysis of nulcease resistant species, DNA sequencing and tests for biological functionality. Differences in transcription rate alone cannot account for the tremendous differences observed in immunoglobulin mRNA abundancies during B cell development. We propose to measure immunoglobulin heavy chain mRNA turnover in cells representing different B cell stages and correlate these with abundancy, transcription rate and nuclear transport and stability.
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