Evidence that immunoglobulin VH-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 VH germ line transcripts in proB lines from RAG-deficient mice. All 10 VH 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 VH region. However, not all VH 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 VH transcripts in individual lines. When V(D)J recombination was activated by restoration of RAG activity, recombinational activity of endogenous VH 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 VH 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.

FIG. 1

Organization of the murine V_H region. The lengths of the lines indicate the estimated numbers of V_H genes as described by reference .

FIG. 2

RT-PCR assay for germ line V_H transcripts. (A) Diagram of a typical V_H gene showing the location of V_H family-specific primers (5′CDR1 and 3′CDR2) and probes used in the assay. The primers were 15-base oligonucleotides complementary to the CDR1 and CDR2 regions conserved among family members. (B) Representative results from RT-PCR analysis of lines AR2, AR3, 1-1, 1-2, and AH7. Reactions were performed with 1 μg of total RNA with (+) or without (−) the addition of reverse transcriptase. A control RT-PCR with actin primers was performed for each sample.

FIG. 3

Specificity and sensitivity of the RT-PCR assay. (A) Duplicate blots of RT-PCR products derived from the amplification of nine different V_H families with 5′CDR1 and 3′CDR2 family-specific primers were hybridized individually with each family-specific V_H probe. (B) Dilutions (1,000 to 1 copies) of plasmid DNA encoding a 7183 V_H gene were added to a constant amount of cDNA, amplified with 7183 primers, and blotted with a 7183 probe.

FIG. 4

DNA sequences of V_H RT-PCR products. (A) Cycle sequencing was performed on RT-PCR products obtained from V-Gam3/8, Q52, SM7, and S107 V_H families. The consensus sequence of known gene segments is shown in the upper line; the lower line represents the sequence obtained from cycle-sequencing PCR. (B) Sequence from cloned RT-PCR products from the 7183 and J558 families. The consensus sequence for each family is shown in the upper line, and the sequences from individual clones are shown in the lower lines. “N” in the upper line represents nucleotides where members of the family are divergent.

FIG. 5

Transcription initiation site of the germ line V_H1 transcript. (A) RT-PCR strategy used to detect the transcript start site of the V_H1 germ line transcript. The 5′V_H1a and 5′V_H1b primers were designed to bracket the previously identified transcription start site for the rearranged V_H1 gene (14). These primers were used with the S107CDR2 3′ primer (Table 1); PCR products were blotted and probed with an S107CDR2 probe. In this case, the primers span the intron between leader and V exons; PCR products derived from the amplification of cDNA would be 328 and 307 bp for 5′V_H1a and 5′V_H1b, respectively; 471 bp and 492 bp are the expected sizes of PCR products derived from control amplification of genomic DNA. (B) RT-PCR performed on cDNA derived from the AR2 cell line with 5′V_H1a and with 5′V_H1b primers. C is a DNA-positive control for PCR.

FIG. 6

Analysis of lines 1-2, AH7, and 63-12 stably transfected with RAG expression plasmids. (A) Diagram of the PCR strategy used to detect the V-DJ rearrangement (21). The upper line represents a diagram of the unrearranged Ig heavy-chain locus (not drawn to scale). The primers used for the D-J and V-DJ PCR assays are shown by the short arrows. The probe used to hybridize PCR products after blotting is an oligonucleotide homologous to the J_H3 coding region. The lower lines represent the sizes of expected PCR products depending on whether J_H1, J_H2, or J_H3 gene segments were used during V-to-DJ joining. (B and C) Representative results showing the products of V-DJ rearrangement revealed by PCR analysis of DNA from the 1-2 (B) and AH7 (C) lines stably transfected with the RAG1 expression plasmid. For 1-2, three subcultures undergoing D-J recombination were tested for rearrangement of six V_H families. For AH7, nine subcultures undergoing DJ recombination were tested for rearrangement of the 3660 family. + indicates DNA from normal bone marrow cells used as a positive control; − indicates DNA from untransfected RAG1^−/− cells used as a negative control.

FIG. 7

Stability of the V_H germ line transcription pattern. (A) V_H germ line transcripts revealed by RT-PCR of cDNA from the 1-2 cell line before (1-2P) and after (1-2T) mock transfection with the drug-selectable plasmid. (B) 3660 and SM7 V_H germ line transcripts in the AH7 line before (AH7P) and after (AH7T) mock transfection with the drug-selectable plasmid. All reactions were carried out with (+) or without (−) reverse transcriptase.