The long term objective of this proposal is to understand functional differentiation in B lymphocytes. The experimental model is the murine response to phosphocholine (PC) a ubiquitous molecule found in the cell walls of many microorganisms. The specific goal of this project is to understand the molecular basis of the antibody heterogeneity that occurs to PC during the development of immunological memory. Hybridomas that produce anti-PC antibodies will be analyzed at the molecular level through recombinant DNA technology and protein sequencing. Preliminary data indicate that IgG antibodies characteristic of the secondary immune response (Group II) may utilize VH genes distinct from the VH1 gene employed by the dominant T15 family of anti-PC antibodies. In addition, Group II antibodies can use different light chain genes from those of the T15 family, in particular Vkappa1-3 and lambda1 and lambda2, as is evident from a library of hybridomas that has been created for this study. Group II hybridomas using unique VH gene(s) as determined by restriction digest analysis will serve as a source for molecular cloning of a VH coding sequence for Group II. Should this sequence prove unique, it will be used to define the gene family involved. When members of other characterized gene families are identified as exemplified by the lambda system, mRNA sequencing will be used to determine whether the antibodies in question utilize the germline sequence or whether somatic mutation is required to derive anti-PC combining sites; depending on the particular chain in question, amino acid sequencing may also be employed. Molecular recombination experiments between heavy and light chains of selected hybridomas will be employed in order to gain information regarding the role of particular V regions or mutations on the formation of Group II-like combining sites. The ontogeny and precursor frequency of B cells expressing the Group II phenotype during neonatal development will be determined.
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