We isolated and characterized the germ-line counterpart of a DNA segment designated RS (for recombining sequence), that is frequently recombined in mouse X light chain-producing B lymphocytes. Using Southern blot analyses of myelomas and mouse-Chinese hamster fusion cell lines, we found that RS DNA sequences are located on mouse chromosome 6, evidently more than 15 kilobases downstream of the K light-chain locus. We find that a typical recognition site for Ig gene recombination is situated within germ-line RS sequences near the recombination points observed in at least two X chain-producing cell lines. This represents a complete and functional Ig recognition site that is not directly associated with Ig genes. We also characterized a recombined RS segment isolated from the cell line BM18-4.13.9. This recombined segment has a variable region K light chain gene (V,) joined directly to RS sequences. Our results suggest that the deletion of the K light chain constant region (C,) exon in many X chain-producing B cells is the result of RS recombination and that C, deletion may be mediated by the same processes as antibody gene V-J joining (J = joining segment gene). We discuss the potential biological significance of RS DNA recombination in B-cell maturation.We have reported that two X chain-producing cell lines, MOPC315 and CH2, display novel DNA recombinations within the K chain locus different from all previously observed K chain gene recombination events (7). In these two cell lines, a segment of DNA [designated RS for recombining sequence] has recombined into the Je-CK intron region and replaced the C,, exon (QK = K chain joining region gene; CK = K constant region gene). In addition to being recombined in MOPC315 and CH2, RS DNA also was found to be recombined frequently in X chain-producing, but not in K chain-producing, hybridomas. Although no functional role for RS DNA has yet been demonstrated, these results raised the possibility that RS recombinations might be involved in the switch from K to X chain gene recombination in maturing B cells.In this article we report that germ line RS sequences are located on mouse chromosome 6 and that, within germ-line RS DNA, an Ig gene recognition site is located contiguous to the sites of RS recombination observed in MOPC315 and CH2. We also have detected and characterized a recombined RS segment that contains a VK gene joined directly to RS.

The two Abelson murine leukemia virus (A-MuLV)-transformed cell lines, BM18-4 and ABC-1, undergo immunoglobulin L-chain gene recombination during passage in tissue culture. BM18-4 cells are capable of kappa gene recombination, whereas ABC-1 cells are capable of both kappa and lambda gene recombination. The expression of H chains is apparently not necessary for continuing L chain gene recombination in either of these cells, although H-chain expression may have been involved in the initiation of L-chain gene recombination. All ABC-1 cells that have lambda gene rearrangements also display recombined kappa alleles, supporting the hypothesis that kappa and lambda gene recombination are initiated in an ordered, developmentally regulated manner in maturing B cells. However, analyses of the ABC-1 line indicate that pre-B cells that have initiated lambda gene recombination do not terminate kappa gene rearrangement. The lambda gene recombinations that occur in the ABC-1 cell line indicate that the germline order of lambda gene segments is: 5' ... V lambda 2 ... J lambda 2C lambda 2-J lambda 4C lambda 4 ... V lambda 1 ... J lambda 3C lambda 3-J lambda 1C lambda 1 ... 3'. In addition, the frequencies of lambda 1, lambda 2, and lambda 3 gene recombinations among ABC-1 cells are quite different than the frequencies of B cells producing lambda 1, lambda 2, and lambda 3 L-chains in the mouse. RS DNA recombinations also occur in the BM18-4 and ABC-1 cell lines, supporting the notion that Ig gene recombinases are involved in RS rearrangement. Recombined RS segments are infrequent among BM 18-4 cells but common among ABC-1 cells, suggesting that RS recombinational events often occur in maturing pre-B cells just before initiation of lambda gene rearrangements. This developmental timing is consistent with the hypothesis that RS recombination may be involved in the initiation of lambda gene assembly.

We have used Abelson murine leukemia virus (A-MuLV) transformed pre-B cell lines to test the hypothesis that the rearrangement potential of a developing B-lymphocyte is dependent on an "opening" of the chromatin structure surrounding immunoglobulin (Ig) genes, thus allowing accessibility to an Ig gene recombinase.The chromatin structures surrounding heavy (H), kappa (K), and lambda (X) chain constant-region genes were assessed by DNase I sensitivity in A-MuLV transformed cell lines capable of H, K or X gene rearrangement. Our results indicate that DNase I-sensitive chromatin structures of these Ig constant-region genes correlate closely with the ability of the genes to undergo recombination.We also find that the chromatin structure of an Ig constantregion locus becomes DNase I sensitive before any DNA rearrangement events occur.

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The deletion of C kappa is a frequent event in lambda-producing B cells in both mice and humans. Deletions of the murine C kappa gene are mediated by recombination events that involve the RS (recombining segment) element located downstream of the C kappa gene. RS recombinations appear to be mediated by the same mechanisms involved in Ig and TCR gene rearrangement. It has been suggested that RS recombinations might activate a factor that is involved in the initiation of lambda gene rearrangement in maturing pre-B cells. We have identified a unique RNA transcript derived from the recombined RS element present in some pre-B cell lines. However, gene transfer studies indicate that this RS transcript is not sufficient to induce lambda gene recombination in pre-B cell lines. We also find that recombination of the RS element in pre-B cell lines is closely correlated with changes in chromatin structure and transcriptional activation. Thus, recombination of the RS element in pre-B cells appears to be regulated in a manner similar to the regulation of antibody gene VDJ joining.