The research in our labortory is directed toward understanding the molecular events in the differentiation of a B lymphocyte to an IgM-secreting blast. During the next period of support, the work will focus on the mechanisms that (1) activate the expression of the J chain gene and (2) regulate the synthesis of pentamer IgM. 1. The promoter region of the J chain gene will be characterized with the aim of identifying the changes responsible for transcriptional activity. First, the sequences critical to transcriptional competence will be determined by systematically modifying the promoter region and measuring the effects on expression in transfection experiments. Second, the requirement for protein interaction(s) will be investigated by examining the ability of the promoter sequences to depress transcription of the J chain gene in transfected cells and to form a functional structure in vitro in the presence of appropriate nuclear extracts. Third, the requirement for signals from other regions of the J chain gene will be explored by measurements of the enhancer activity of J chain gene subfragments. Finally, the requirement for membrane signals will be pursued by examining the stimulation by defined T cell lymphokines and characterizing the expression of lymphokine receptors during the pentamer IgM response. 2. The regulation of pentamer IgM synthesis will be investigated with the aim of defining the mechanisms responsible for the dramatic increases in the mRNA abundance of the component J and lambda s chains. The role of message stability in J chain RNA amplification will be explored by modifying the 3' untranslated sequence and evaluating the effects on message half-life. The role of chromatin changes in lambda-s RNA amplification will be investigated by methylating specific sites in the lambda chain gene and measuring the changes produced in transription rate and message level. The role of similar mechanisms in the synthesis of the enzymes required for pentamer IgM assembly will be determined by studies of the kinetics of enzyme RNA expression. Because the pentamer IgM response of the B lymphocyte is particularly amenable to analysis at the molecular level, the information from these studies may well yield clues of significance to the general process of eukaryotic development and to the rational control of the immune system in disease.
