MOLECULAR CHARACTERIZATION OF ANTIGEN BINDING MUTANTS OF A MURINE MYELOMA CELL LINE (SOMATIC MUTATION, HYBRIDOMAS)
GIUSTI, ANGELA M.
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To study the contribution of somatic mutation to the generation of antibody diversity a family of antigen binding mutants and revertants of the S107.3.4 (TEPC 15 idiotype anti phosphocholine, IgA, (kappa)) mouse myeloma cell line were analyzed molecularly at the protein and DNA level.;Heavy and light chain reassociation experiments involving mutant and parental chains suggested that the lack of antigen binding was due to changes in the heavy chain. Protein sequencing of the heavy chain variable regions of these 1st generation mutants (U1 and U4) revealed single amino acid substitutions in each mutant scattered throughout the V region. Protein sequences of revertants isolated from one mutant (U1) revealed a return to the S107 sequence at the site of the mutation and in addition a second mutation at another residue which apparently did not affect binding. A 3rd generation mutant, U10, isolated from one revertant, did not bind antigen and carried a single amino acid substitution at the identical site as the 1st generation U1. In addition it was wild type in sequence at the second site. Therefore it appeared that loss and gain of reactivity of the immunoglobulin for its antigen can be due to one or two amino acid substitutions.;In order to understand the mechanism of these changes variable region cDNA was synthesized by primer extension from membrane associated PolyA('+) mRNA. The cDNA was cloned into the M13mp8 phage and sequenced according to the Sanger dideoxy nucleotide method. Each of the amino acid substitutions were caused by single base changes that were all A C or C A transversions. Also, there were no silent base changes in any mutant or revertant DNA in the part of the variable region gene sequenced.;Finally to analyze the effects of these amino acid substitutions on the 3-dimensional structure of the immunoglobulin molecules, rat and mouse monoclonal antibodies were generated against S107 and two antigen binding mutant proteins. The rat anti S107 monoclonals were either specific for the S107 heavy chain or required the presence of both heavy and light chains. They recognized this family of mutants and revertants with varying degrees of affinity. The mouse anti mutant monoclonals however were found to react with the antigen binding mutants and reacted less well or not at all with parental and revertant proteins. These amino acid substitutions have therefore been shown to generate new antigenic determinants in the immunoglobulin molecule. As a result the monoclonals reactive to these epitopes are useful reagents to search for and analyze heterogeneity in PC binding proteins.