MOLECULAR BASIS OF THE SOMATIC INSTABILITY OF S107 IMMUNOGLOBULIN GENES

Abstract

Immunoglobulin variable region genes undergo a high rate of somatic diversification during B cell differentiation. In order to study the molecular mechanisms responsible for the instability of immunoglobulin genes, the S107 cell line was examined for antigen binding mutants. This cell line makes the dominant T15 antibody produced by BALB/c mice in response to a number of bacterial polysaccharides. The studies described in this thesis reveal that S107 has duplicated its heavy chain variable (V) region gene so that it contains two productively rearranged and expressible T15 V1 genes. One has the germline sequence and one has an A-C transversion in the codon for residue 83. Oligonucleotide probes representing mutant and wild type sequences in Northern and Southern analysis demonstrated that the V region and its associated constant (C) region underwent further mutations in culture. These mutations arose spontaneously at frequencies of 10{dollar}\sp{lcub}-3{rcub}{dollar}-10{dollar}\sp{lcub}-4{rcub}{dollar}. The V region mutations were single base substitutions and the C region mutations were the result of 1 or 2 base deletions leading to premature terminations. The sequence differences in the V and C regions of the duplicated heavy chain gene have been used as markers to determine if these mutations occur in the wild type gene or the 83 mutant gene.;In summary, variable region mutations always arose through A-C transversions in the wild type gene and not in the 83 mutant gene, which had itself been generated by duplication and somatic mutation. One or two base deletions in the constant region also arose in the wild type gene and not in the 83 mutant gene. This thesis showed that both variable and constant region genes underwent somatic mutations in cultured cells. Furthermore, somatic point mutation and gene conversion are probably occurring. Finally, even within a single cell, there are differences in the instability of two copies of the same variable and constant region genes.