J. Mol. Biol. (1996) 261, 309–314 COMMUNICATION V(D)J Recombination is Regulated Similarly in RAG -transfected Fibroblasts and Pre-B Cells Udo Do ¨ bbeling, Reinhard Hobi, Martin W. Berchtold and Clive C. Kuenzle* Here we compare the regulation of V(D)J recombination in the fibroblast Institut fu ¨r Veterina ¨rbiochemie der cell line L4 and the pre-B cell line 38B9. The former has been rendered Universita ¨t Zu ¨ rich recombination-competent by stable transfection of a genomic fragment Winterthurerstrasse 190 comprising the recombination activating genes, RAG-1 and RAG-2 , along with some of their flanking sequences. We show that V(D)J recombination CH-8057 Zu ¨ rich, Switzerland is similarly regulated in these two cell lines. Activating signals are transmitted through the protein kinase A (PKA) pathway, and inhibitory signals through the protein kinase C (PKC) and the calcium signalling pathways. In both cell lines, recombinational activity reflects steady state levels of mRNA transcribed from the RAG-1 and RAG-2 genes. This suggests that transcription of the RAG genes is a major determinant regulating V(D)J recombination. A comparison of RAG-1 and RAG-2 mRNA levels within each cell line reveals almost identical response patterns indicating that RAG-1 and RAG-2 transcription is coordinately regulated. Together, these results imply that the RAG-containing fragment in L4 fibroblasts carries most, if not all, control regions regulating the transcription of the RAG-1 and RAG-2 genes.  1996 Academic Press Limited Keywords: intracellular signalling; molecular immunology; RAG genes; *Corresponding author regulation; V(D)J recombination Regulation of V(D)J recombination Functional immunoglobulin genes are assembled from individual gene segments by a process known as V(D)J recombination. This event is restricted to early stages of B-lymphocyte maturation and has been shown to respond to chemical effectors acting on signal transduction pathways (Menetski & Gellert, 1990). Activation proceeds through the protein kinase A (PKA) pathway with concomitant increase in the level of mRNA transcribed from the RAG-1 and RAG-2 genes; in contrast, V(D)J recombination is inhibited through the protein kinase C (PKC) and the calcium signalling pathways. The capacity to perform V(D)J recombination has been transferred to fibroblasts by stable transfection of genomic DNA containing the recombination activating genes, RAG-1 and RAG-2 (Schatz & Baltimore, 1988; Schatz et al ., 1989; Oettinger et al ., 1990). The present work was initiated to compare the regulation of V(D)J recombination in RAG- transfected fibroblasts and pre-B cells. Here we show that similar patterns of regulation prevail in the RAG-transfected fibroblast cell line L4 and the pre-B cell line 38B9, implying that the same signalling pathways are involved. V(D)J recombination is stimulated through the protein kinase A pathway in both cell lines In Figure 1, the relative recombination frequen- cies of L4 fibroblasts and 38B9 cells are shown in the absence and presence of modulators and com- ponents of the protein kinase A pathway. Caffeine, an activator of PKA, stimulated V(D)J recombi- nation in both cell lines. Under optimal caffeine Present address: Udo Do ¨ bbeling, Dermatologische Klinik, Universita ¨tsspital Zu ¨ rich, Gloriastrasse 31, CH-8090 Zu ¨ rich, Switzerland. Abbreviations used: CREB, cAMP response element binding factor; ELISA, enzyme-linked immunosorbent assay; IPTG, isopropyl thiogalactoside; PCR, polymer- ase chain reaction; PKA, protein kinase A; PKA cat.s., PKA catalytic subunit; PKC, protein kinase C; RAG, recombination activating gene; RT, reverse transcriptase; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA response element; s.d., standard deviation. 0022–2836/96/330309–06 $18.00/0  1996 Academic Press Limited