SOMATIC IMMUNOGLOBULIN GENE INSTABILITY IN A CULTURED MOUSE MYELOMA CELL LINE (ANTIBODY DIVERSITY, GENETICS, ANTIGEN BINDING)
ZACK, DONALD J.
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Cultured mouse myeloma cells generate variant clones with changes in antibody expression and structure at a high rate. In an attempt to further understand the generation of antibody diversity and the mechanisms of animal cell mutation, the mutation rates and spectrum of variants from a particular antibody secreting mouse myeloma cell line, S107, were categorized and mutants of particular interest have been further analyzed at the molecular level. The motivation behind studying the spectrum of variants rather than concentrating on individual mutants was the hypothesis that all the mutants would reflect the operation of a single underlying mechanism and that the study of the mechanism's diverse manifestations might help provide insights into its mysteries. In order to facilitate these studies, new or modified methods for the detection of variant clones were developed. A rapid approach to the sequencing of variable region mutants utilizing synthetic oligonucleotide primed cDNA synthesis with M13 cloning and dideoxy sequencing was also developed.;Variants were shown by fluctuation analysis to arise at rates as high as 1.5 x 10('-4) per cell per generation. Phenotypically, the mutants isolated included ones with changes in antigen binding, expressed idiotype, assembly, secretion, constant region structure, and heavy and/or light chain expression. The antigen binding variants were shown to contain single and double base substitutions, all of which were A to C transversions or C to A reversions; the constant region mutants had nonsense mutations; and the heavy chain non-producing mutants had specific deletions of their expressed variable region gene. These results are discussed in terms of the utility of myeloma and hybridoma cells as a somatic cell genetic system and also in terms of the insights and constraints they place on the nature of the mutational process and its possible relation to the in vivo generation of antibody diversity. (').