The goals of this study are to determine the cis and trans acting factors which participate in the regulated recombination and expression of T cell receptor (TCR) variable region genes in thymocyte development and to determine how this process is arrested in mutant scid mice. Non-transformed scid thymocytes initiate recombination of the TCR delta locus, with biased recombination of D and J elements, and an apparent high incidence of normal recombination for these elements. Rearrangements detected contrast with aberrant rearrangements assayed in scid lymphomas, indicating distortions imposed on the scid phenotype by artificial recombination substrates and the transformed phenotype. Thus, the proposed studies of TCR gene recombination and recombinase activities are critical to understanding how the scid mutation mediates developmental arrest in vivo. Occurrence of recombination coding strand intermediates, and altered expression of recombinase associated genes (RAG-1, RAG-2) and proteins (TdT) in scid thymocytes indicate that the proposed studies are important to identification of the scid defect and to dissection of the mechanism of V(D)J recombination. V(D)J recombination of TCR genes in scid and wild-type thymocytes will be studied at two levels: (1) at the molecular mechanistic level, by analysis of TCR recombination intermediates and rearrangement; and (2) at the cellular level, by analysis of regulated expression of V(D)J recombinase activities and TCR gene elements.
Aim 1 studies will determine which TCR elements are actively recombined, and what the incidence of normal coding junction resolution is, throughout scid thymic ontogeny by PCR analysis of coding, signal and hybrid junction formation.
Aim 2 studies will determine the structure of hairpin modified TCR delta coding intermediates which are uniquely present in scid thymocytes, by direct cloning and PCR assisted approaches.
Aim 3 studies will examine constitutive and inducible expression of recombinase associated genes and proteins in scid thymocytes by cDNA PCR, ribonuclease protection, nuclear run-off and immunoprecipitation analyses. Evidence for altered expression of RAG genes and of TdT protein may provide important clues to the identity of the scid mutation.
Aim 4 studies will generate scid thymocytes of a more mature ( eg., CD4+/8+) phenotype and enhanced viability by generation of bone marrow chimeric scid mice, and scid mice with transgenic expression of bcl-2 in the T lineage. These studies will enable analysis of the association between surface differentiation phenotype and TCR gene recombination status. It will also enable determination of whether failed V element recombination in scid thymocytes is attributal to microenvironmental versus lymphoid intrinsic defect.
| Slack, J K; Lowry, C V; Carroll, A M (1994) HASTEN: a technique to identify the primary structure of terminal DNA hairpins. Nucleic Acids Res 22:1316-7 |
