We have used extrachromosomal substrates carrying immunoglobulin heavy-chain S,u and Sy3 switch region sequences to study activation and targeting of recombination by a transcriptional enhancer element. Substrates are transiently introduced into activated primary murine B cells, in which recombination involving S-region sequences deletes a conditionally lethal marker, and recombination is measured by transformation of Escherichia coli in the second step of the assay. Previously we found that as many as 25% of replicated substrates recombined during 40-h transfection of lipopolysaccharide (LPS)-stimulated primary cells and that efficient recombination was dependent on the presence of S-region sequences as well as a transcriptional activator region in the constructs (H. Leung and N. Maizels, Proc. Natl. Acad. Sci. USA 89:4154-4158, 1992). Here we show that recombination of the switch substrates is threefold more efficient in LPS-cultured primary B cells than in the T-cell line EL4; the activities responsible for switch substrate recombination thus appear to be more abundant or more active in cells which can carry out chromosomal switch recombination. We test the role of the transcriptional activator region and show that the immunoglobulin heavy-chain intron enhancer (E,u) alone stimulates recombination as well as E,L combined with a heavy-chain promoter and that mutations that diminish enhancer-dependent transcription 500-fold diminish recombinational activation less than 2-fold. These observations suggest that the enhancer stimulates recombination by a mechanism that does not depend on transcript production or that is insensitive to the level of transcript production over a very broad range.Furthermore, we find that E,u stimulates recombination when located either upstream or downstream of SFL but that the position of the recombinational activator does affect the targeting of recombination junctions, suggesting that the relatively imprecise targeting of switch junctions in vivo may reflect the availability of many potential activator sites within each switch region.Immunoglobulin isotype switch recombination is a regulated recombination event which joins an expressed heavychain variable [V(D)J] region to a new downstream constant (C) region, deleting the DNA between (for reviews, see references 5, 6, and 8). Isotype switching occurs in B cells which have accomplished V(D)J joining and have been activated by antigen. Recombination involves G-rich switch or S regions, from 1 to 8 kb in length, which are found in the intron upstream of each C region that undergoes switching-C,u, Cy, Ca, and Ce; expression of Ca is regulated by alternative splicing, not recombination, and there is no G-rich S region upstream of Ca. In contrast to V(D)J joining, in which heptamer-nonamer recognition elements precisely target recombination (13), switch recombination is notably imprecise. No conserved sequences comparable to the heptamer-nonamer elements are apparent within S regions or at switch recombination sites, and chromosomal swi...
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