Sequentially Derived Mutants of the Constant Region of the Heavy Chain of Murine Immunoglobulins

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Abstract
In an effort to define the mechanism of the γ2b → γ2a subclass switch observed in cultured cells, a series of constant region mutants has been isolated in the cell line (45.6) derived from the MPC-11 tumor. The investigation was begun with 10-1, a spontaneous variant of 45.6 which synthesizes a 47,000 dalton heavy chain with a deletion in CH1. From 10-1, two types of secondary variant was synthesizing a 55,000 dalton heavy chain that was γ2a serologically. Five independent variants synthesizing 55,000 dalton γ2a heavy chain were isolated from 10-1. All five were of the same apparent m.w. and four, but not the fifth, had identical isoelectric focusing patterns. The second type of secondary variant isolated from 10-1 was synthesizing a heavy chain even smaller than the heavy chain of 10-1. Two different variants of this type, I17 and G251, synthesized heavy chains of approximately 35,000 and 40,000 daltons, respectively, which did not react with any subclass-specific antiserum. From I17 and G251 it was possible to isolate tertiary variants that synthesized γ2a heavy chains of either 47,000 daltons or 55,000 daltons m.w. The 55,000 dalton γ2a heavy chains exhibited many different isoelectric focusing patterns. Peptide map analysis of two 47,000 dalton and two 55,000 dalton γ2a tertiary variants showed no differences from the Fc of MOPC-173, a naturally occurring γ2a myeloma. However, a third 55,000 dalton γ2a variant synthesized a heavy chain with a Fc that was apparently not identical to that of MOPC-173. These experiments show that it is possible to isolate γ2b → γ2a subclass switch mutants from variants synthesizing short heavy chains. It is not possible to define unequivocally the mechanism of the subclass switch. However, it clearly does not result from the translocation of an intact V region to an intact constant region. The resulting data are, however, compatible with the γ2b → γ2a switch resulting from a recombination event between tandem genes on homologous chromosomes or chromatids.