This research plan addresses three problems in immunobiology: 1) the diversification and regulation of immunoglobulin v-genes, 2) the location, structure and functioning of T-cell receptor v-genes, and 3) the detailed mechanisms governing cell-cell interactions which regulate the self-nonself discrimination and the class of the immune response. With regard to the first problem, we have manufactured the Kappa-suppressed (i.e. """"""""Lambda-only"""""""") mouse as a tool that will allow us to a) test the functional significance of an idiotype network, b) analyze the total pattern of the somatically driven variability of a single germline v-gene, and c) determine the Kappa/Lambda ratio in virgin B-cells as a key parameter in evaluating mechanisms of allelic exclusion. Central to this latter question is our discovery of the regulatory mutation r(lo/Lambda I) which affects the frequency of successful v-j fusion to form a functional transcription unit. The next step requires that we know the relationship between this regulatory gene and the structural vLambdaI-gene it controls. To this end we manufactured a new set of recombinant inbred mice (BALB/cxSJL)RI which are important also in analyzing many other immune phenomena, e.g. IgE expression. In order to confront the mechanism of somatic diversification, we introduced the concept of """"""""genetic dissection"""""""" and tested it with the somatically derived Kappa anti-Alpha(1,3) dextran response in animals unable to respond in the germline encoded Lambda I class. This is the only system available today to analyze this phenomenon. With regard to the second problem, we deeeeloped two systems to locate the T-cell receptor: a) responsiveness genetics and b) cloning DNA sequences which are deleted when v-j fusions occur, i.e. """"""""deletion hybridization."""""""" This latter should permit us to isolate regions of the genome encoding the T-cell receptor, and compare it with the findings of conventional responsiveness genetics. With regard to the third problem, we concentrate on cloning immune related cells with the aim of reconstructing partial immune responses. We are applying DNA transfection technics using conditional lethal (e.g. ts) transforming plasmids which allow us to grow large amounts of cells in their undifferentiated state and at will switch them to the functioning differentiated non-growing state for analysis.
