Fig. 5. Igk-RSSs enhance diffusional D-to-Jκ joining in the Igh–Igk hybrid locus and activate inversional V_H-to-DJ_H joining in the Igh locus.

a,b, Absolute level of individual D-to-Jκ joins in the Igh–Igk hybrid-D line (a) and the Igh–Igk hybrid-D-VκRSS line in which the DQ52 upstream 12RSS was replaced with a Vκ12-44 12RSS (b), assayed with Jκ5 bait. Deletional DQ52-to-Jκ5 joining in b is 114-fold higher than that in a (P = 0.0008). c–e, Absolute level of individual inversional V_H-to-DJ_H joins in the DJ_H-inv line (c), the DJ_H-inv-VκRSS line, in which DQ52 upstream 12RSS was replaced with a Vκ11-125 12RSS (d) and the DJ_H-inv-VκRSS-JκRSS line, in which V_H5-2 23RSS was replaced with a Jκ1-23RSS (e), assayed with J_H4 bait. Total rearrangement level in d is 13-fold higher than that in c (P = 0.0153). Inversional V_H5-2 usage level in e is 35-fold higher than that in d (P = 0.0005) and 383-fold higher than that in c (P = 0.0007). In a–e, red arrows show inversional joins and blue arrows show deletional joins. Corresponding junction numbers are shown. Arrow thickness represents relative amounts of junctions. f, Comparison of relative Vκ usage in the single Jκ5 allele v-Abl cells with Vκ-RSS RIC scores calculated using the Recombination Signal Sequences Site³³ (http://www.itb.cnr.it/rss). Vκ segments are colour-coded according to the three Vκ domains with names indicated for highly used Vκ segments. g, Comparison of relative V_H usage in primary pro-B cells⁷ with V_H-RSS RIC scores. V_H segments are colour-coded according to the four V_H domains, and square black outlines indicate V_H segments with CBEs within 20 bp of their RSSs. The circled V_H5-1, V_H5-2 and V_H2-2 have been shown to depend on associated CBEs for robust utilization³. D and V_H utilization data are presented as mean ± s.e.m. from 4 (a,d), 6 (b) and 3 (c,e) biological repeats. P values were calculated with unpaired, two-sided Welch’s t-test.

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