Memory B cells that express high affinity surface immunoglobulin (sIg) receptors encoded by somatically mutated Ig variable (V) region genes play a major role in protection against infectious agents and contribute to autoimmunity as well. In spite of this, and over three decades of intensive investigation, many gaps exist in our understanding of the cellular origins and affinity dependent selection of memory B cells, and the molecular mechanisms that generate somatic mutations. Both the pattern of accumulated mutations and the possibility that double strand DNA breaks are an intermediate in somatic mutation, have led to the hypothesis that error-prone DNA repair may be responsible for introducing base substitutions in V region DNA. Because of its known role as an error-prone polymerase, DNA polymerase zeta (pol zeta) has been suggested as a key mediator in this process. Mice that express transgene encoded antisense RNA specific for pol zeta RNA display very low levels of pol zeta RNA. Although upon immunization these mice generate vigorous GC reactions, normal titers of isotype switched antibodies, and normal numbers of memory B cells, their antibodies affinity-mature slowly and their antibody V regions accumulate fewer than normal somatic mutations with a marked reduction in affinity enhancing mutations. This is a application to study further these antisense transgenic mice to define precisely the impact of low pol zeta levels on somatic mutation and affinity maturation with time after immunization with a variety of antigens. Because in vivo selection could greatly bias the quantity and quality of accumulated mutations, cells from these mice will also be evaluated at the clonal level in vitro for their immune responsiveness, tolerance susceptibility and their accumulation of somatic mutations. Finally, adoptive transfer studies of mixtures of precursor cells from conventional, green fluorescence protein transgenic, and antisense p01 zeta transgenic mice will be carried out to evaluate the cellular origins of memory B cells and any competitive advantage of normally mutating vs. sub-normally mutating cells in the selection of cells into the memory B cell pool. If successful, these experiments will provide important advances in understanding the molecular and cellular phenomena responsible for B cell memory.
| Diaz, Marilyn; Watson, Nicholas B; Turkington, Gene et al. (2003) Decreased frequency and highly aberrant spectrum of ultraviolet-induced mutations in the hprt gene of mouse fibroblasts expressing antisense RNA to DNA polymerase zeta. Mol Cancer Res 1:836-47 |
