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dc.contributor.authorCHIEN, NADINE CAROL
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 48-08, Section: B, page: 2252.
dc.description.abstractFamilies of monoclonal antibodies derived both in vivo and in vitro have provided substantial evidence that somatic diversification events occur in already rearranged and expressed immunoglobulin variable (V), diversity (D) and junctional (J) genes. Three genetic mechanisms have been proposed to be responsible for these diversification events, somatic point mutation, gene conversion, and V-region replacement. A series of variant cell lines were established to study the genetic mechanisms involved in antibody diversity and their impact on the structure and function of an antibody.;Two PC binding hybridomas representative of events early and late in the immune response were studied. The P28 cell line which produces an IgM antibody and thus represents events early in the immune response, was found to have 3 base pair changes in its heavy chain variable (V{dollar}\sb{lcub}\rm H{rcub}{dollar}) region with no changes in antibody affinity or specificity. The RP93 cell line which produces an IgG2 antibody and thus represents later events in the immune response, was found to have 10 base pair changes in its V{dollar}\sb{lcub}\rm H{rcub}{dollar} region resulting in decreased antibody affinity and altered specificity. The genetic mechanism responsible for the changes in both cell lines is almost certainly somatic point mutation.;Since the in vivo variants possessed multiple base pair changes, in vitro mutants were developed to assess the impact of diversification on antibody structure. Two mutants of PC binding antibodies were analyzed; U4, which had lost 80% of its PC binding; and U10, which had lost 100% of its PC binding. Each variant possessed a single amino acid substitution. In U4 the change was shown to be due to somatic point mutation while in U10 the change was thought to be due to gene conversion. Three-dimensional analysis of peptide structure revealed the substitutions affected the conformation of the binding pocket due to the loss of a hydrogen bond in U4 and the rotation of a residue in U10. Concomitant with the loss of PC binding, U4 acquired the ability to bind dsDNA. This lead to studies, both in vivo and in vitro, of the possible role of somatic point mutation in the formation of autoantibodies.
dc.publisherProQuest Dissertations & Theses

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