The central focus for this proposal is to define the role of delta deletion in the formation of alpha beta T-cells. It contains four specific aims: 1) HPS1A directs the recombinase complex to deltaREC in alpha beta T-cells; 2) Elimination of murine deltaREC elements will block the development of alpha beta T-cells; 3) The utilization of the delta deleting elements is developmentally stage specific; and 4) the signal to undergo delta deletion is mediated through trans regulatory proteins recognizing HPS1A. Because of the chromosomal organization of the TCR alpha and delta chains, the rearrangement within the alpha/delta locus has profound effects upon the formation of T-cell type. Previous data implies a mechanism must exist that prevents incorporation of internal TCR delta chain segments in functional TCR alpha chains and inhibits TCR alpha chain rearrangement in gamma delta T-cells. This proposal's design is to define the molecular signals that initiate delta deletion and whether these signals are necessary for the formation of TCR alpha chains. Transgenic mice have been created that demonstrate alpha beta T- cell lineage specific utilization of transgenic delta deleting elements, and show that control for this lineage restriction is located within a 48 bp region near deltaREC , termed HPSIA. Additionally, mice containing the transgenic deltaREC construct, minus HPSIA, show a decreased incidence of delta deletion within the ore T-cell lineage. The experiments in this project propose to investigate whether the three endogenous murine deltaREC elements are controlled by HPSIA like elements, and determine if HPS1A has recombinase enhancing properties on minichromosomal substrates. Embryonic stem cell technology will be used to remove the three murine deltaREC elements and evaluate the formation of alpha beta T-cells in homozygous deficient mice. Furthermore, experiments will be performed that will define the stage in development within which the delta deleting elements are utilized and correlate their use with the rearrangement of all TCR chains. Finally, this proposal defines experiments to clone, sequence, and characterize DNA binding proteins recognizing the 48 bp signal sequence, HPSIA. Data generated from these studies will indicate the importance of delta deletion in those cells destined to become alpha beta bearing T-cells.
| Janowski, K M; Ledbetter, S; Mayo, M S et al. (1997) Identification of a DNA segment exhibiting rearrangement modifying effects upon transgenic delta-deleting elements. J Exp Med 186:91-100 |
| Shutter, J; Cain, J A; Ledbetter, S et al. (1995) A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages. Mol Cell Biol 15:7022-31 |
