The diversity of the antibody and T cell receptor (TCR) repertoires is achieved by the random combinatorial association of V, D and J segments and of the resulting two chains, and by significant diversity at the junctions of the gene segments. This junctional diversity is much more restricted in fetal and newborn Ig and TCR than in Ig and TCR generated in the adult. First, N regions are lacking early in ontogeny in all lymphoid populations. Second, in the absence of N regions, IgH and gammadeltaTCR junctions, but not alphabetaTCR junctions, often occur at regions of short sequence homology. This mechanism of homology-directed recombination results in significant junctional homogeneity in IgH and gammadeltaTCR receptors. Thus, the predominance of some junctions in vivo can be attributed to the mechanics of VDL recombination. In this application, we propose to analyze the process of homology-directed recombination through the use of recombination substrates and other experimental systems which avoid any possibility of cellular selection. The reason why homology-directed recombination does not create predominant junctions at all homologies will be investigated, as will the issue of whether the sequence surrounding the homology affects the rate with which the homology is used. We have observed that some gene segments often have P nucleotides while others seldom do. The hypothesis that the degree of nucleotide deletion and P nucleotide frequency is influenced by the specific sequence of the coding end will be addressed with recombination substrates containing actual V, D or J coding ends, or slightly modified ones, as well as by the analysis of partial D-J rearrangements made in vivo. Competition recombination substrates will be made to address the question of whether the recombination signal sequences or other coding region associated sequences can play a role in the observed overutilization of certain V, D and J segments. We will examine whether homology-directed recombination or non-standard spacer length plays a role in the non- random usage of Igk J segments, since use of short sequence homologies should preferentially generate nonproductive rearrangements for the underutilized Jk genes. The mechanics of VDJ recombination has the potential to put many biases into repertoire formation. One goal of these experiments is to determine which biases that may have previously been ascribed to cellular selection are actually due to the process of VDJ recombination and which are not. Finally, experiments are proposed to determine if extensive ligand induced expansion of B cells can occur early in differentiation.
Showing the most recent 10 out of 36 publications
