Evidence that Immunoglobulin V_H-DJ Recombination Does Not Require Germ Line Transcription of the Recombining Variable Gene Segment

ABSTRACT

The importance of V(D)J recombination for generating diversity in the immune system is well established, but the mechanisms which regulate V(D)J recombination are still poorly understood. Although transcription of unrearranged (germ line) immunoglobulin and T-cell receptor gene segments often precedes V(D)J recombination and has been implicated in its control, the actual role of germ line transcripts in V(D)J recombination is not known. We used a sensitive reverse transcription-PCR assay to study immunoglobulin V_H germ line transcripts in proB lines from RAG-deficient mice. All 10 V_H families analyzed were germ line transcribed, and germ line transcription was found in all of the cell lines examined, indicating that active chromatin was present in the V_Hregion. However, not all V_H families were germ line transcribed in every cell line, and there was a surprising lack of uniformity in the number and family distribution of germ line V_H transcripts in individual lines. When V(D)J recombination was activated by restoration of RAG activity, recombinational activity of endogenous V_H genes for which germ line transcription was observed could be compared with those of genes for which it was not observed. This analysis revealed multiple examples of endogenous V_H gene segments which were rearranged in cells where their germ line transcription was not detectable prior to RAG expression. Thus, our data provide strong support for the idea that V-(D)J recombination does not require germ line transcription of the recombining variable gene segment.

ACKNOWLEDGMENTS

We are grateful to A. Henderson, P. Rothman, and C. Tunyaplin for critically reading the manuscript and to members of the Calame laboratory for many helpful discussions. We thank K. Merrell for lines AR2, AR3, AR8, and AR19; C. Roman and D. Baltimore for lines 1-1, 1-2, and AH7; and G. Rathbun and F. Alt for lines 63-12 and RAG expression vectors PDR1 and PDR2.

This work was supported by grant RO1 GM29361 to K.C.