ABSTRACT
Antigen receptor gene rearrangement is regulated by many factors in B and T lymphocytes. The sequences of the gene segments themselves, their associated recombination signal sequences (RSS), expression of the RAG genes and the chromatin accessibility of the particular gene segments to be rearranged all influence the outcome of recombination and thus antigen receptor diversity. In the present study, we have evaluated the effect of variations in RAG activity level on the junctional diversity of coding joint sequences. Using the pre-B-like 204-1-8 and the mature B DR3 cell lines under different transfection conditions, we were able to investigate recombination activity levels that varied 100-fold. We evaluated the sequences of the coding joints for junctional diversity resulting from nucleotide addition or deletion. Surprisingly, we found that the sequence of coding joints of these recombinants did not exhibit significant variation despite the large difference in recombination frequency. Our results indicate that the fidelity of the joining phase of V(D)J recombination is not jeopardized by varying RAG activity.
Subject(s)
B-Lymphocytes/immunology , Cell Differentiation/genetics , Cell Differentiation/immunology , Gene Rearrangement, B-Lymphocyte , Animals , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , Caffeine/pharmacology , Cell Line , Gene Rearrangement, B-Lymphocyte/drug effects , Genes, RAG-1 , Mice , Phosphodiesterase Inhibitors/pharmacologyABSTRACT
Unintended DNA rearrangements in a differentiating lymphocyte can have severe, oncogenic consequences, but the mechanisms for avoiding pathogenic outcomes in V(D)J recombination are not well understood. The first level at which fidelity is instituted is in discrimination by the recombination proteins between authentic and inauthentic recombination signal sequences. Nevertheless, this discrimination is not absolute and cannot fully eliminate targeting errors. To learn more about the basis of specificity during V(D)J recombination, we have investigated whether it is possible for the recombination machinery to detect an inaccurately targeted sequence subsequent to cleavage. These studies indicate that even postcleavage steps in V(D)J recombination are sequence specific and that noncanonical sequences will not efficiently support the resolution of recombination intermediates in vivo. Accordingly, interventions after a mistargeting event conceivably occur at a late stage in the joining process and the likelihood may well be crucial to enforcing fidelity during antigen receptor gene rearrangement.
