Light Chain Recombination Research Articles

Secondary V(D)J rearrangements and B cell receptor-mediated down-regulation of recombination activating gene-2 expression in a murine B cell line.

It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD- murine B cell line, 38C-13, which has previously been found to undergo kappa light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vkappa-Jkappa and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD- B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination.

Mouse kappa light-chain recombination signal sequences mediate recombination more frequently than do those of lambda light chain.

Immunoglobulin and T-cell receptor genes are somatically rearranged by site-specific recombination. Recombination signal sequences (RSS) have been identified as the major targeting element of this process. Recent reports demonstrate that differences in RSS affect the frequency of recombination, suggesting a role for RSS in the development of the B-cell repertoire. Examination of mouse light-chain RSS indicates that kappa light-chain RSS consistently show a greater degree of similarity to a consensus sequence than do those of lambda light chain. To determine whether this difference in natural RSS could affect the patterns of light-chain gene rearrangement and expression, we have constructed recombination substrates containing both a typical mouse kappa RSS pair and a typical mouse lambda RSS pair. Experiments using these substrates demonstrate that the kappa RSS pair mediates recombination at a vastly higher frequency than does the lambda RSS pair. This result argues that RSS differences may contribute significantly to the patterns of mouse immunoglobulin light-chain rearrangement, ultimately resulting in a high proportion of kappa light chain relative to lambda.

Myosin heavy chain-light chain recombinations and interactions between the two classes of light chains.

Myosin subfragment 1 (S1) heavy chains were prepared from chicken muscle S1 by immunoadsorption in NH4Cl and from rabbit muscle S1 by ion exchange chromatography at 37 degrees C in MgATP. Both heavy chain preparations contained some intact S1, and the ATPase activities of both preparations were less than those of control S1. Recombinations of these heavy chains with alkali light chains prior to removing NH4Cl or MgATP lessened the decreases in ATPase activities. However, once NH4Cl or MgATP was removed, alkali light chain recombination did not result in any increase in ATPase activity. Thus, the lower activities appear to result from the instability of the S1 heavy chain in the absence of alkali light chain. When incubated with a 10-fold molar excess of radiolabeled alkali light chains for 1 h under approximately physiological conditions, almost all of the alkali light chains of S1 but only 8% of those of myosin or myofibrils were exchanged. Aggregation of myosin into filaments accounts for part of this difference in exchangeability. Removal of 30% of the 19,000-Da 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) light chains from myosin increased 3-fold the amount of alkali light chain exchanged. The exchange of the alkali light chains of S1 is inhibited by the presence of DTNB light chains, and cleavage of the DTNB light chains of heavy meromyosin to 17,000-Da fragments increased the rate of alkali light chain exchange. There was no detectable difference in the exchangeability of the two different alkali light chains.