The long term goal of this physician-scientist grant proposal is to develop the skills required to understand the mechanisms by which B lymphocytes undergo and regulate immunoglobulin (Ig) gene somatic hypermutation. This process allows for generation of greater antibody diversity and development of more effective higher affinity antibodies. Previous attempts to study the molecular mechanisms and detailed organization of this process have been hampered by the lack of an appropriate B cell model for this process. Evidence exists for high frequency mutations of the variable region heavy chain Ig gene of a subclone of 18.81, an Abelson-transformed murine pre-B cell line. During Phase I of this project, in addition to coursework in molecular biology, will be the confirmation that the 18.81 cell line undergoes frequent and spontaneous point mutations in the variable (V) region of its endogenous immunoglobulin gene and that the frequency of V region mutation is greater than constant region mutation. Practical serological and molecular assays will be established to identify mutant genes. Comparisons will be performed of the rate and nature of mutations that occur in transfected and endogenous Ig genes. In Phase II, transfected constructs of an Ig gene will be used to determine DNA sequences in the flanking and/or coding sequences which are required for mutations to occur and to see if specific sequences are targeted for these events. Those sequences shown to be important for this process will be analyzed by site directed mutagenesis and will be used to search for proteins involved in the regulation of this process by gel retardation assay. Their relevance in vivo will be examined using transgenic mice. Genes for these proteins will be cloned for determination of sequence, expression, and in this in vitro system. Determination of the mechanism and regulation of this process would resolve one of the major mechanisms for generating antibody diversity. It would also allow the generation of higher affinity monoclonal antibodies such as those used for anti- neoplastic or infectious processes and for diagnostic purposes. Mutations in the Ig gene are important in the oncogenesis of malignancies such as Burkitt's lymphoma. Improved understanding of this process may lead to novel treatments for these and other lymphoid tumors, and for manipulation of lymphoid cell differentiation.
