GENERATION OF SWITCH HYBRID DNA BETWEEN IG HEAVY CHAIN-MU AND DOWNSTREAM SWITCH REGIONS IN B-LYMPHOCYTES

Ig heavy chain isotype switching in B lymphocytes is known to be prece ded by transcription of a portion of the particular heavy chain gene s egment that is targeted for recombination. Here, we describe an active role for these transcripts in the switch recombination process. Using an in vitro assay that exposes an artificial switch-mu (S mu) minisub strate to switch region transcripts in the presence of nuclear extract s from switching cells, we demonstrate that free 3' ends of the S mu s equence are extended onto switch region transcripts by reverse transcr iption. The activity was induced in splenic B lymphocytes upon activat ion with LPS or CD40 ligand, This in vitro process is thought to be re levant to in vivo class switching for two reasons: 1) although only on e-third of the S mu minisubstrate actually contains S mu sequence, all crossovers between snitch regions occurred in the S mu portion; and 2 ) treatment of B lymphocytes with IL-4, which enriches for switching t o S gamma 1, increases the ratio of S mu-S gamma 1 to S mu-S gamma 3 h ybrids by 16% after LPS treatment and by 37% after CD40 ligand activat ion, implicating this S mu-primed reverse transcription of switch regi on transcripts as a novel mechanism of regulating the specificity of i sotype switching. Further evidence for an active role of switch region transcripts was obtained by expressing S alpha RNA in trans in the Bc l(1)B(1) B lymphoma line. Endogenous S mu-S alpha switch circles were detected in Bcl(1)B(1) cells expressing exogenous S alpha RNA but not in mock-transfected cells.