The functional diversity of the molecules recognizing foreign antigens in the immune system (immunoglobulins (Ig) and T cell receptors) is generated by the DNA rearrangement of their gene families during lymphocyte differentiation. The investigators will study the regulation and mechanism of the rearrangement process of Ig genes by a combined molecular biological and biochemical approach.
The specific aims of the proposed experiments are: (i) to determine the role of the Ig mu heavy chain in allelic exclusion, by development of a rearrangement frequency assay of pre-B tissue culture cells following DNA transfections with rearranged mu genes. (ii) to study the direct or indirect role of mu chains in allelic exclusion and activation of kappa gene rearrangement by characterizing specific protein interactions and intracellular localization of mu chains during the B cell stage where Ig rearrangement events occur. We will further analyze these properties by mutation and substitution of mu gene fragments and DNA transfection analysis in pre-B cells. (iii) to characterize the proteins involved in activation of kappa gene rearrangement. We will study the roles of two B cell- specific DNA binding proteins that bind the kappa gene region: KLP and a kappa-enhancer binding protein (NF-kappa beta). We will study whether transcriptional activation may trigger kappa gene rearrangement. (iv) to describe the altered mechanism of Ig rearrangement in scid mice, defective in lymphopoiesis. We will characterize by Southern blots, genomic cloning, and DNA sequencing, the junctions of the abberant rearrangements. We will examine continuing Ig rearrangement in scid pre-beta cell lines by using retroviral constructs containing Ig gene elements. We will clone the SCID (wildtype) gene by functional complementation of scid pre-beta cells or by subtractive cDNA hybridization experiments. (v) to characterize proteins involved in DNA rearrangement by identifying DNA binding proteins that recognize the conserved heptamer/nonamer recognition sequences for rearrangement.
| Hendrickson, E A; Schatz, D G; Weaver, D T (1988) The scid gene encodes a trans-acting factor that mediates the rejoining event of Ig gene rearrangement. Genes Dev 2:817-29 |
