TY - JOUR
T1 - The recombination difference between mouse κ and λ segments is mediated by a pair-wise regulation mechanism
AU - Larijani, Mani
AU - Chen, Shuang
AU - Cunningham, Lesley A.
AU - Volpe, Joseph M.
AU - Cowell, Lindsay Grey
AU - Lewis, Susanna M.
AU - Wu, Gillian E.
N1 - Funding Information:
The authors wish to thank the members of the Wu and Lewis laboratories. We thank P. Cortes for the kind gift of RAG expression constructs. This research was supported by the Canadian Cancer Society and Canadian Institutes of Health research. LGC is supported by a Career Award at the Scientific Interface from the Burroughs Welcome Fund. M.L. is a fellow of the Leukemia and Lymphoma Society of Canada. M.L. acknowledges M.A.O.
PY - 2006/3
Y1 - 2006/3
N2 - In mice, κ light chains dominate over λ in the immunoglobulin repertoire by as much as 20-fold. Although a major contributor to this difference is the recombination signal sequences (RSS), the mechanism by which RSS cause differential representation has not been determined. To elucidate the mechanism, we tested κ and λ RSS flanked by their natural 5′ and 3′ flanks in three systems that monitor V(D)J recombination. Using extra-chromosomal recombination substrates, we established that a κ RSS and its flanks support six- to nine-fold higher levels of recombination than a λ counterpart. In vitro cleavage assays with these same sequences demonstrated that single cleavage at individual κ or λ RSS (plus flanks) occurs with comparable frequencies, but that a pair of κ RSS (plus flanks) support significantly higher levels of double cleavage than a pair of λ RSS (plus flanks). Using EMSA with double stranded oligonucleotides containing the same κ or λ RSS and their respective flanks, we examined RAG/DNA complex formation. We report that, surprisingly, RAG-1/2 form only modestly higher levels of complexes on individual 12 and 23 κ RSS (plus natural flanks) as compared to their λ counterparts. We conclude that the overuse of κ compared to λ segments cannot be accounted for by differences in RAG-1/2 binding nor by cleavage at individual RSS but rather could be accounted for by enhanced pair-wise cleavage of κ RSS by RAG-1/2. Based on the data presented, we suggest that the biased usage of light chain segments is imposed at the level of synaptic RSS pairs.
AB - In mice, κ light chains dominate over λ in the immunoglobulin repertoire by as much as 20-fold. Although a major contributor to this difference is the recombination signal sequences (RSS), the mechanism by which RSS cause differential representation has not been determined. To elucidate the mechanism, we tested κ and λ RSS flanked by their natural 5′ and 3′ flanks in three systems that monitor V(D)J recombination. Using extra-chromosomal recombination substrates, we established that a κ RSS and its flanks support six- to nine-fold higher levels of recombination than a λ counterpart. In vitro cleavage assays with these same sequences demonstrated that single cleavage at individual κ or λ RSS (plus flanks) occurs with comparable frequencies, but that a pair of κ RSS (plus flanks) support significantly higher levels of double cleavage than a pair of λ RSS (plus flanks). Using EMSA with double stranded oligonucleotides containing the same κ or λ RSS and their respective flanks, we examined RAG/DNA complex formation. We report that, surprisingly, RAG-1/2 form only modestly higher levels of complexes on individual 12 and 23 κ RSS (plus natural flanks) as compared to their λ counterparts. We conclude that the overuse of κ compared to λ segments cannot be accounted for by differences in RAG-1/2 binding nor by cleavage at individual RSS but rather could be accounted for by enhanced pair-wise cleavage of κ RSS by RAG-1/2. Based on the data presented, we suggest that the biased usage of light chain segments is imposed at the level of synaptic RSS pairs.
KW - Antibodies
KW - B lymphocytes
KW - Gene rearrangement
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U2 - 10.1016/j.molimm.2005.06.038
DO - 10.1016/j.molimm.2005.06.038
M3 - Article
C2 - 16054218
AN - SCOPUS:29144484171
SN - 0161-5890
VL - 43
SP - 870
EP - 881
JO - Molecular Immunology
JF - Molecular Immunology
IS - 7
ER -