One of the central issues in immunology concerns the nature of antibody variability. We are studying a closely related group of antibodies that have been generated within our own laboratory with specificity for the p-azophenylarsonate hapten. Five of these molecules are being subjected to complete amino acid sequence analysis. Fifteen more have been subjected to partial analysis. The present proposal has three goals. The first concerns expressed anti-arsonate antibodies. We intend to determinate the total primary structure of several of these anti-arsonate hybridomas but by sequencing the mRNA (which can be isolated from tumor cells) by the Sanger dideoxy technique. We estimate that the C-terminal half of the immunoglobulin heavy chain variable region can be sequenced by this technique. N-terminal protein sequencing can reach residue 60 thereby providing an appropriate overlap. The second portion of this proposal concerns a study of the genomic sequences in both the A/J and Balb/c mouse that are encoding the rare VH subgroup that encodes these molecules. Preliminary data indicates a limited number of germ-line genes and we wish to study their sequences and relate them to the expressed products studies above. Finally, we propose to study the relationship between the germline and expressed genes in anti-arsonate hybridomas. Here we intend to analyze specific hybridomas with DNA probes which distinguish between germline and rearranged genes. These studies should provide insights into the genetic origin of antibody diversity and a molecular understanding of idiotypy.
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