DISTINCT ROLES FOR RAG-1 IN THE INITIATION OF V(D)J RECOMBINATION ANDIN THE RESOLUTION OF CODING ENDS

Although RAG-1 and RAG-2 have been shown to be indispensible for V(D)J recombination, their exact role in this reaction remains unclear. Co- transfecting RAG-1 and RAG-2 expression vectors into NIH3T3 fibroblast s confers V(D)J recombination activity to these otherwise recombinatio nally inactive cells. In this report we have found that in transient t ransfections of mouse NIH3T3 fibroblasts with RAG-1 and RAG-2 and the appropriate recombination substrates, one RAG-1 expression vector, pRA G-1A, is capable of yielding both signal joints and coding joints, whi le another RAG-1 expression vector, pRAG-1B, yields only signal joints . The RAG-1 open reading frame for these two expression vectors is int erchangeable, indicating that the inability to resolve coding joints i s due to the 45-base pair difference found in the 5'-untranslated regi ons of these constructs. Differences in this region result in a 15-fol d difference in gene expression when the luciferase coding region is s ubstituted for the RAG-1 cDNA. This report provides evidence that RAG- 1 may have a role in both the initiation of V(D)J recombination as wel l as the resolution of coding ends. The data also suggest that these R AG-1 activities may be dependent on different levels of RAG-1 expressi on.