GENETIC ANALYSIS OF MEMBRANE IMMUNOGLOBULIN

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Membrane immunoglobulin M (IgM) is the receptor for a developmental signal that stimulates a small B lymphocyte to differentiate into an IgM secreting plasma cell. The structure of the membrane IgM glycoprotein has recently been determined and compared with secreted IgM by using B-lymphocyte tumors from inbred mice. These two forms are identical except for their carboxy termini. The membrane form has a hydrophobic "tail" for membrane interaction, and the secretable form has a hydrophilic "tail" that allows it to form multimers and to be secreted. One gene encodes the constant region of mu; separate gene segments encode the alternative carboxy termini. It is thought that the two forms of mu are produced by alternative RNA splicing of a single primary transcript. We have used mouse tumor cells that express both membrane (mIgM) and secretable (sIgM) forms. From these cells we have immunoselected more than 50 variants that no longer express the mIgM. Fourteen of these have been extensively characterized with respect to rate, amount, stability, and glycosylation of mum and mus protein and amount and rate of mRNA encoding mum and mus. One variant has suffered a large deletion of part of chromosome 12 and, therefore, synthesizes only the "sterile transcript" that initiates in the J-Cmu intron of the unrearranged homologue. All other variants have patterns on Southern blots that are identical to wildtype. Two variants that have altered proteins have been identified, but each affects both mum and mus. Three variants show no detectable mu protein synthesis and only "sterile" cytoplasmic mu transcripts. However, their rate of transcription in mu is only slightly lower than wildtype. The analysis of the RNA processing and DNA sequence of these variants and selection of new variants with specific phenotypes are currently in progress. We have begun to produce in vitro specific mutations of cloned mu genes. The two membrane-specific gene segments from membrane mu have been fused with the gene segments which encode the extracellular portions of a "reporter protein" (part of a murine major histocompatibility complex class I gene). This construction has been reintroduced into cultured cells to monitor its behavior as a membrane protein. It will be further modified to identify regions of the molecule critical for membrane expression. (CS)