THE ROLE OF RECOMBINATION SIGNAL SEQUENCES IN THE PREFERENTIAL JOINING BY DELETION IN D-H-J(H) RECOMBINATION AND IN THE ORDERED REARRANGEMENT OF THE IGH LOCUS

The bias favoring deletion over inversion in D-H-J(H) rearrangement ha s been known for years, but the underlying mechanism has yet to be ful ly defined. It has been suggested that the ratio of deletion/inversion is determined by the combined effect of two factors: (i) the relative strengths of 5' and 3' recombination signal sequences (RSS) of a D-H segment, and (ii) the efficiency with which the deletional product (on e joint) forms relative to the inversional product (two joints). In th is study, we analyzed for the first time the effect of factor 1 alone on the biased 3' RSS utilization in D-H-J(H) joining by using deletion al plasmids in an extrachromosomal substrate V(D)J recombination assay . It was found that the 3' RSS and associated coding end (12 bp) media te recombination more efficiently than the 5' RSS/coding end on D-H-J( H) plasmids. These results demonstrate that the effect of the RSS/codi ng end alone can account, at least partially, for the predominant dele tion in D-H-J(H) recombination. The potential effect of the relative s trength of RSS and associated coding end on the ordered rearrangement of D-H-J(H) followed by V-H to D-H-J(H) was also assessed. When recomb ination frequencies of D --> J (3' D-H to J3) were compared with frequ encies of V --> D (V(H)PJ14 to 3' D-H or V(H)OX2 to 3' D-H), it was fo und that V --> D joining was, if anything, more efficient than D --> J joining. Therefore, if all three segments were accessible, RSS/coding end effects would not contribute to the ordered rearrangement of the IgH locus.