MOLECULAR GENETICS OF LYMPHOCYTE DIFFERENTIATION

Molecular analysis of the genes and/or control regions affecting expression of B and T lymphocyte products will be undertaken as a step toward understanding the way in which gene expression is controlled during the development of these cell lineages. In the proposed studies, we will seek a molecular explanation for the observed instability of Ig gene expression in myeloma cells. The goal is to learn how this phenomenon and the molecular events responsible for it are related to those that modulate Ig gene expression during normal B cell development. In particular, we will ask whether the cessation of Ig production which occurs at high frequency in myeloma cultures is a consequence of spontaneous DNA rearrangement within the structural genes for these molecules. If there are cases where loss of Ig production is not accompanied by a change in Ig gene structure, we will begin genetic and functional characterization (by DNA transfection and cell fusion) of these variants to identify genes or control regions capable of modulating immunoglobulin gene expression "at a distance". In this proposal, we also describe a method for the physical isolation of genes and/or DNA regions essential for the expression of T lymphocyte surface antigens. This method involves insertional mutation of such genes through physical disruption by an integrating retroviral provirus. Characterization of the provirus integration site in resulting antigen-loss variants will include genetic linkage studies between the interrupted DNA region and the genetically defined antigen locus. Using this strategy, regions of functional importance to any number of lymphocyte surface alloantigens can be physically isolated and used to study their contribution to the molecular events that accompany normal lymphocyte development.