The planned experiments are concerned with the controls of expression of immunoglobulin (Ig) genes and the functional comparison of T cell receptor of Ig variable regions. Specifically, we have the following plans: 1) Mapping the orientation of mouse Lambda V- and C-genes. Previously we cloned, mapped and partially sequenced large regions containing all of the mouse Lambda V- and C-genes. We propose now to use a new method, pulse field electrophoresis, to determine how the V- and C-genes are organized with respect to each other. If necessary, the genes will be linked by "walking" or "hopping" the chromosome. The experiments will aid the understanding of how rearrangement of Ig genes is controlled. 2) Analysis of Ig gene rearrangement. Synthetic genes will be used to determine when Ig gene-like rearrangement has occurred after introduction into pre B cell lines and transgenic mice. We hope to exactly determine the DNA sequences, proteins and mechanism involved in Ig gene rearrangement. 3) Hybrids between immunoglobulin and T cell receptor genes. The functional divergence of the two antigen receptor gene families immunoglobulin (Ig) and T cell receptors (TCR) will be evaluated. Hybrid genes composed of the variable domains of Igs spliced to the constant, transmembrane, and intracellular gene segments of TCRs will be expressed both by cell transfection and in transgenic mice. A hapten (PC)-binding antibody will be used first, to determine which portions of the TCR genes are required for correct expression of the hybrid genes. It will then be studied if a TCR without MHC specificity can be involved in T cell activation and allelic exclusion. A second antibody specificity, anti murine class II MHC molecules will be used to test a model for the determination of T cell MHC restriction. It is predicted that the ability of thymocytes bearing hybrid T cell receptors to mature and emigrate from the thymus will be dictated by the relative affinity of these receptors for host MHC. The various plans are closely connected by the need for mutual exchange of technical and conceptual advances.

R01AI024780

1986-09-30

1991-11-30