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Original Article |
1 Locus in Scid Mice
MJ_Bosma{at}fccc.edu
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Abstract |
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1–J1 rearrangement is routinely detectable in scid fetal liver, adult bone marrow, and spleen in the apparent absence of completed VH–DJH and V
–J rearrangements. Particularly surprising, we found the level of V1–J1 rearrangement in scid fetal liver to be comparable to that in fetal liver of wild-type mice. The majority of scid V1–J1 rearrangements contained abnormal deletions at the VJ junction, consistent with the known effect of scid. However, 
15% of V1–J1 rearrangements lacked abnormal deletions. Productive 1 transcripts resulting from in-frame rearrangements were readily detectable in scid adult bone marrow and spleen, consistent with our ability to detect 1-expressing cells by flow cytometry in the spleens of bcl-2–transgenic scid mice. Strikingly, 1 transcripts from individual scid mice often showed VJ junctional sequences with the same recurring palindromic (P) additions of three, four, or five nucleotides. To account for these findings, we suggest that (a) nonhomologous end joining of V1 and J1 coding ends in fetal B lineage cells may not be (severely) impaired by scid; (b) recurring P additions in scid 1 transcripts may reflect certain molecular constraints imposed by scid on the resolution of V1 and J1 hairpin coding ends; and (c), scid lymphocytes with productively rearranged V1 and J1 elements may differentiate into recombinase-inactive cells and emigrate from bone marrow to spleen.
Key Words: B cell differentiation • pro-B cells • premature Ig recombination • VJ junctional diversity • P additions
Consistent with the stochastic model of L chain gene rearrangement, we report here a low frequency of V
Although a low frequency of V
Nonetheless, as shown here, V
Our findings raise several puzzling issues: (a) Why is the level of V
Flow Cytometric Analysis.
Oligonucleotides.
Genomic PCR.
RT-PCR.
Quantitation.
Sequence Analysis.
Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
The rearrangement of Ig genes proceeds in an ordered fashion (for review see references 1 and 2). It begins with rearrangement of the H chain gene elements, DH and JH, followed by VH–DJH rearrangement 3. H chain gene rearrangement is initiated at the pro-B cell stage and is generally followed by L chain gene rearrangement at the pre-B cell stage 456, with V–J rearrangement occurring later 78910 and/or less frequently 111213 than V–J rearrangement. The order of H and L chain gene rearrangement is not absolute, however, as studies with B lineage cell lines 141516 and sorted pro-B cells 56 indicate that rearrangement at the or locus may precede or occur independently of H chain gene rearrangement. Also, inactivation of the locus by gene targeting has shown that rearrangement of the locus does not require prior rearrangement of the locus 1718, consistent with earlier evidence for independent rearrangement of and loci in various cell lines 16192021 and in -transgenic mice 22. Independent rearrangement of and loci is in agreement with a stochastic model of L chain gene rearrangement 1123 and contrary to a strictly regulated model of L chain gene rearrangement (for review see reference 2). 1–J1 rearrangement in severe combined immunodeficient (scid) and in wild-type (wt) mice with a targeted deletion of the JH locus (JHT mice) 24. As JHT mice lack a functional H chain locus, V1–J1 rearrangement in these mice must occur independently of H chain rearrangement. Furthermore, as both scid and JHT mice lack pre-B cells, the observed V1–J1 rearrangement is inferred to occur at the pro-B cell stage and independently of V–J rearrangement. 1–J1 rearrangement at the pro-B cell stage in JHT mice could have been predicted based on the stochastic model of L chain gene rearrangement, the regular occurrence of such rearrangement in scid pro-B cells would not have been predicted. In scid mice, V(D)J recombination is severely impaired as a result of a DNA repair defect 252627. The defect is due to a nonsense mutation in the gene coding for the catalytic subunit of DNA protein kinase (DNA-PKcs) 282930. Because of this mutation, developing scid lymphocytes cannot efficiently join V, D, and J coding ends resulting from the initiation of V(D)J recombination 31323334. Consequently, most developing scid lymphocytes are thought to die prematurely with persisting DNA breaks. Therefore, given that V1–J1 rearrangement generally follows DH–JH and VH–DJH rearrangement and is much less frequent than V–J rearrangement, we would not expect to detect V1–J1 rearrangement in scid mice. 1–J1 rearrangement is routinely detectable in fetal liver, adult bone marrow, and spleen of individual scid mice. Moreover, in scid fetal liver we found the level of V1–J1 rearrangement to be comparable to that in wt fetal liver. Although most scid V1–J1 rearrangements showed abnormal deletions of V1 and/or J1 nucleotides at the VJ junction, 15% lacked abnormal deletions. Productive 1 transcripts resulting from in-frame V1–J1 rearrangements were clearly evident in bone marrow and spleens of individual scid mice. Most scid 1 transcripts displayed the same recurring palindromic (P) additions of three, four, or five nucleotides. P addition 3536 is thought to result from asymmetric nicking of a hairpin coding end 37383940, followed by fill-in of the overhang and joining to another coding end. 1–J1 rearrangement comparable in fetal liver of scid and wt mice; (b) Why do most 1 transcripts of individual scid mice show the same recurring P additions; and (c) How do 1 expressing scid cells survive the deleterious effect of scid and apparently emigrate from bone marrow to spleen? These three issues are discussed.
Materials and Methods
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Mice.
C.B-17 mice homozygous and heterozygous for the scid mutation 41 are here denoted as scid and scid/+ mice, respectively. C.B-17 scid mice hemizygous for the bcl-2-36 transgene (bcl-2 scid mice; reference 42) were obtained from S. Cory (The Walter and Eliza Hall of Medical Research, Melbourne, Australia). Genotyping of bcl-2 mice was done by PCR using DNA from tail snips 43 and oligonucleotide primers for the SV40 sequence included in the transgene 44. Mice with both of their recombination activation gene (RAG)1 loci inactivated by gene targeting (RAG–/– mice; reference 45) and mice with their JH elements deleted by gene targeting 24 were provided by R. Hardy (Fox Chase Cancer Center). The targeted JHT allele was backcrossed onto C.B-17 mice for three backcross generations (N3). N3F1 mice were intercrossed to generate N3F2 mice homozygous for the JHT allele (JHT mice). JHT mice were crossed with scid mice to obtain JHT/+, scid/+ mice; these were then intercrossed to obtain JHT scid mice. Genotyping for the wt and inactivated JH allele was done by PCR using tail DNA and primers specific for the wt and inactivated JH locus (see JH1 and JHT oligonucleotides below). All of the above mice were bred and maintained at the Fox Chase Cancer Center and were analyzed between the ages of 6 and 12 wk.
Flow cytometry was used to test for the presence of cells with surface Ig1 (1+ cells) in scid and bcl-2 scid mice. In brief, spleen cells of individual scid, scid/+, bcl-2 scid, bcl-2 scid/+, and RAG1–/– mice were stained with biotin-conjugated anti-CD8 (53.6), allophycocyanin (PharMingen)-conjugated anti-CD45 (B220), and FITC-conjugated anti-1 (R11-153–FITC; PharMingen) in the manner previously described 46. Cells were analyzed by three-color flow cytometry using a dual laser FACStarPLUSTM (Becton Dickinson). Binding of biotinylated antibodies was revealed by Texas Red–conjugated streptavidin (Southern Biotechnology). Dead cells were identified by propidium iodide staining and excluded from analysis. Gates were set to score 1+ cells based on the distribution of 1 staining of spleen cells in the scid/+ positive controls. Due to the paucity of 1+ cells in scid and bcl-2 scid mice, between 0.5 and 1.0 x 107 spleen cells were analyzed per mouse. Cells were simultaneously stained for the B and T specific markers B220 and CD8, respectively, to ensure that cells scored as 1+ were indeed B lineage cells (i.e., 1+B220+CD8–). Spleen cells from RAG1–/– mice served as negative controls for background staining of 1.
Oligonucleotides were synthesized by an Applied Biosystems 394 DNA/RNA Synthesizer. Oligonucleotides used as primers for PCR or reverse transcriptase (RT)-PCR were as follows: VH (#91), 5'-GCCGGATCCGTGCAGCTGGTGGAGTCTGG-3'; DH (#285), 5'-ACTGCTACCTCTGGCCCCACCAG-3'; JH4 (#361R), 5'-AGATAATCTGTCCTAAAGGCTC-3'; Cµ (#289), 5'-ATGCAGATCTCTGTTTTTGCC-TCC-3'; V (#68), 5'-GGCTGCAGGACATTGTGCTGACCCAATCTCCAGCTTCT-3'; J2 (#367), 5'-GGTAGACAATTATCCCTCTTCCCCTAGT-3'; C (#130), 5'-ATGGATCCAGTTGGTGCAGCATC-3'; V1ext (#282), 5'-TCTCCT-GGCTCTCAGCTCAG-3'; V1int (#294), 5'-AGGAATCTGCACTCACCACATC-3'; J1 (#271), 5'-GCACCTCAAGTC-TTGGAGAG-3'; C1 (#283), 5'-GAGGAAGGTGGAAACAG-GGTG-3'; β2ML (#229), 5'-GAATGGGAAGCCGAACATAC-TGAACTG-3'; β2MR (#230), 5'-TGCTGATCACATGTCTC-GATCC-3'; SV40L (#355), 5'-GGAACTGATGAATGGGAGC-AGTGG-3'; SV40R (#356), 5'-GCAGACACTCTATGCCTGTG-3'; JHTL (#370), 5'-CCTTGCGCAGCTGTGCTCGACGTTG-3'; JHTR (#371), 5'-GCCGCATTGCATCAGCCATGATGGA-3'; JH1L (#368), 5'-GGACCAGGGGGCTCAGGTC-ACTCAGG-3'; and JH1R (#369), 5'-GAGGAGACGGTGACCGTGGTGCCTGC-3'.
5 x 106 bone marrow or spleen cells were used to prepare genomic DNA by PureGene Kit (Gentra Systems). PCR was carried out in 50 µl with 106 cell genome equivalents of DNA. Controls for nonspecific amplification of PCR products included the use of RAG–/– DNA and no DNA template in the reaction. Reactants included oligonucleotide primers for DH and JH, VH and JH, V and J, V1 and J1 elements or for the β2 microglobulin (β2M) locus along with 220 µM each of dATP, dGTP, dCTP, and dTTP, 0.4 µM primers, 20 µM Tris-HCl, pH 8.4, 50 µM KCl, 1.5 µM MgCl2, and 2.5 U of AmpliTaq DNA polymerase. The cycling reaction consisted of an initial denaturation for 4 min at 95°C, with 23 cycles of 1 min at 94°C, 45 s at 68°C, and 1 min at 72°C, and a final elongation step for 5 min at 72°C. 1/10 of each PCR reaction (1/20 for β2M control) was electrophoresed through 1.5% LE agarose (FMC Bioproducts) in 1x Tris-acetate-EDTA buffer, turboblotted by alkaline transfer onto maximum strength Nytran Plus membranes (Schleicher & Schuell) and hybridized in Denhardt's solution with the appropriate probes. Radioactive 
-[32P]dCTP labeling was done by random priming using the Prime-It II Kit (Stratagene). Hybridization probes included pJH6.3 47, pEC 11, and a PCR-amplified and gel-purified (QiaexII; Qiagen) VJ1 gene fragment to score for DH–JH (or VH–DJH), V–J, and V1–J1 rearrangements, respectively. As a control for the amount of input DNA, a portion of the nonrearranging β2M gene was PCR amplified and hybridized to a β2M-specific probe. Blots were exposed to X-Omat (Eastman Kodak Co.) autoradiographic film and also to a PhosphorImaging plate for quantitation by a BAS1000Mac Bio-Imaging Analyzer (Fuji Photo Film Co.).
Total RNA from 5 x 106 bone marrow or spleen cells was obtained by using RNEasy (Qiagen) as prescribed by the manufacturer. RNA was eluted into DEPC-treated H2O and stored at –73°C. RNA from the equivalent of 1.5 x 106 bone marrow cells or 3.0 x 106 spleen cells was used to synthesize first strand cDNA using SuperscriptII RT and 100 ng of random hexamers (Amersham Pharmacia Biotech) as directed by the manufacturer (GIBCO BRL). A portion of this cDNA (equivalent to 3 x 105 bone marrow cells or 6 x 105 spleen cells) was amplified by PCR using 220 µM each of dATP, dGTP, dCTP, and dTTP, 0.4 µM primers, 20 µM Tris-HCl, pH 8.4, 50 µM KCl, 1.5 µM MgCl2, and 2.5 U of AmpliTaq DNA polymerase (PerkinElmer) in a reaction volume of 50 µl. Controls for nonspecific amplification of PCR products included RAG–/– cDNA and no cDNA template in the reaction. Semiquantitative PCR using a PTC-100 Thermal Controller (MJ Research) was carried out after an initial denaturation for 4 min at 95°C, with 23 cycles of 1 min at 94°C, 45 s at 65°C, and 1 min at 72°C, and a final elongation step for 5 min at 72°C. Southern blotting and hybridization was carried out as described above. The hybridization probes included pCµ3741 48, pEC, and gel-purified VJ1 and β2M PCR-amplified gene fragments.
Conditions for semiquantitative PCR were determined by varying cycle number and the amount of input DNA (or cDNA). Filters were exposed to a Fuji imaging plate to quantify the amount of -32P–hybridized probe in experimental samples relative to that in control (reference) samples using a BAS1000Mac Bio-Imaging Analyzer (Fuji Photo Film Co.). We found that with 23 cycles of amplification, the amount of PCR product was proportional to the amount of input DNA (from 106 cells) at several different dilutions. Similarly, at 23 cycles, the amount of RT-PCR product was found to be proportional to the amount of input cDNA (from 3–6 x 105 cells) at several different dilutions.
To ensure sufficient PCR product for cloning, one microliter from the primary PCR or RT-PCR reaction was subjected to an additional 15 cycles of PCR using conditions as above. The Vlint primer was used with J1 or C1 for recovery of junctional sequences from genomic DNA or cDNA, respectively. PCR products were electrophoresed through 1.5% LE agarose, purified using QiaexII, and cloned into pCR2.1 for transformation of INVF' bacteria (Invitrogen). Recombinant colonies were randomly chosen for plasmid recovery by Perfect Prep (5Prime-3Prime, Inc.). Plasmids were submitted for cycle sequencing using the ABI Prism Dye Terminator Reaction Kit and an ABI 377 DNA Sequencer (PerkinElmer).
Results
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Evidence for V1–J1 Rearrangement before the Pre-B cell Stage.
To test whether the 1 locus can rearrange early in B cell differentiation, we assayed for the presence of nongermline 1 transcripts in scid mice and also in bcl-2 scid mice. As shown in Fig. 1 A, 1 transcripts resulting from V1–J1 rearrangement (1 transcripts) were clearly evident in the bone marrow of scid mice and more so in the bone marrow of bcl-2 scid mice. The higher abundance of 1 transcripts in the latter mice presumably reflects the greater longevity of B lineage cells in bcl-2 scid mice than in scid mice 42. Fig. 1 A also illustrates that transcripts resulting from V–J rearrangement ( transcripts) were routinely detectable in bone marrow of bcl-2 scid but not scid mice, whereas transcripts resulting from VH–DJH rearrangement (µ transcripts) were barely detectable in some bcl-2 scid mice and not at all in scid mice (Fig. 1 A). Consistent with previous reports of detectable DH–JH rearrangement in scid mice 4950, Dµ transcripts resulting from DH–JH rearrangement were readily detectable in scid and bcl-2 scid bone marrow.
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1 transcripts in scid mice suggests that V1–J1 rearrangement may occur before the pre-B cell stage. To test whether this is indeed true and whether the 1 locus can rearrange independently of the H chain locus, we assayed for 1 transcripts in bone marrow of JHT mice. In these mice, B cell differentiation is completely arrested at the pro-B cell stage as a result of gene-targeted inactivation of the JH locus 24. Fig. 1 B shows that 1 transcripts were readily detectable in JHT bone marrow. The abundance of 1 transcripts in JHT mice was 40-fold less than in wt mice but 25-fold greater than in JHT scid mice (Table ). We conclude that V1–J1 rearrangement can occur at the pro-B cell stage and independently of H chain gene rearrangement.
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1–J1 Rearrangement in scid and wt Embryos.1–J1 rearrangement with that at other Ig loci, we tested genomic DNA from pooled livers of scid and wt embryos for DH–JH, VH–DJH, V–J, and V1–J1 rearrangement (Fig. 2). In wt embryos, we found that DH–JH rearrangement could be detected as early as day 12, whereas VH–DJH and V–J rearrangements were not detectable until day 14. These results are in general agreement with earlier reports on the time course of H and chain gene rearrangement in fetal mice 5152. In scid embryos, DH–JH rearrangement was not clearly evident until day 14, and VH–DJH and V–J rearrangements were not detected except for a VH–DJH rearrangement of aberrant size in the day 15 sample. In contrast, V1–J1 rearrangement was evident as early as day 12 in both scid and wt embryos. These results indicate that V1–J1 rearrangement is initiated early in development and may developmentally precede VH–DJH and V–J rearrangement.
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1–J1 rearrangement and 1 transcript in day 13–15 scid fetal liver remains relatively constant and appears comparable to that in the day 13 and 14 wt fetal liver. Indeed, quantitation of the amount of 1 hybridizing signal for V1–J1 rearrangement and 1 transcript in the day 13 and 14 scid samples showed this to be 80% of that in the corresponding wt samples (Table ). It should be noted that the observed level of V1–J1 rearrangement in DNA from 106 fetal liver cells was about two orders of magnitude less than in control DNA samples from 106 adult bone marrow cells of wt mice (data not shown). This is not surprising, as day 13–14 fetal liver lacks detectable pre-B cells and contains 
1% pro-B cells (reference 53 and our unpublished results).
V1–J1 Rearrangements from scid Mice Contain Abnormal Deletions at the VJ Junction.
PCR-amplified V1–J1 rearrangements were detectable not only in scid fetal liver and adult bone marrow, but also in scid adult spleen. In most of these rearrangements, the V1 and/or J1 coding segments were abnormally truncated by >20 nucleotides. Deletions of this magnitude were not observed in V1–J1 rearrangements from wt mice. Representative results are illustrated in Fig. 3 and Fig. 4 for adult bone marrow and spleen, respectively. More than 70% of scid V1–J1 rearrangements (121/147 distinct sequences analyzed) contained abnormal deletions; those lacking such deletions often showed unusually long P additions, as illustrated in Fig. 4.
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1–J1 rearrangements from bone marrow of JHT mice, in contrast to those from bone marrow of scid mice, showed nontemplated (N) additions and comparatively small deletions at the VJ junction (Fig. 3). N addition is dependent on terminal deoxynucleotidyl transferase (TdT) 5455. This enzyme is expressed at the pro-B cell stage 56, the stage at which B cell differentiation is arrested in JHT mice 24. The absence of N additions in V1–J1 junctions from wt mice (Fig. 3 and Fig. 4) is in agreement with earlier reports 5758 and consistent with the occurrence of most L chain rearrangement at the late pre-B cell stage 56, when TdT expression is dramatically downregulated 56. As scid and JHT mice both show an arrest of B cell differentiation at the pro-B stage, the abnormal loss of nucleotides in scid V1–J1 junctions must reflect the effect of the scid mutation and not a peculiarity of premature V1–J1 rearrangement.
1 Transcripts in scid Adult Bone Marrow and Spleen Lack Abnormal Deletions at Their VJ Junction.
Most 1 transcripts from scid adult bone marrow and spleen corresponded to in-frame V1–J1 rearrangements with frequent P additions (illustrated in Fig. 5). The P additions consisted of three to five nucleotides (cag, ccag, and gaaat) and were found repeatedly in individual mice. The most common recurring sequence consisted of two P additions separated by an AT dinucleotide (gaaat-AT-ccag). Interestingly, the AT dinucleotide is palindromic to the last two nucleotides of the (gaaat) P addition. Similar restricted VJ junctional sequences and recurring P additions were also observed in 1 transcripts recovered from bcl-2 scid spleen (data not shown). It is important to note that each PCR amplification of bone marrow and splenic cDNA from scid mice was done in parallel with PCR amplification of splenic cDNA from wt and RAG1–/– mice. We found that 1 transcripts from three individual wt mice lacked N/P additions (illustrated in Fig. 5). No 1 transcripts were recovered from cDNA of RAG1–/– mice. Thus, the observed recurring P additions appear to be a unique property of 1 transcripts in the bone marrow and spleens of scid mice.
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1 transcripts in bone marrow and/or spleens of five individual scid mice. We found that 115 clones contained P additions. In Fig. 6, each distinct junctional sequence (denoted A–I) among the 115 clones is listed according to its representation in individual mice and overall frequency (see histogram). Sequences B and C are treated as one in the histogram, as are sequences F and G, because each of these pairs is identical except for the substitution of T (underlined) for C in the V1 germline codon, AGC. Note that (a) sequences A–G, comprising most of the clones (110/115), corresponded to in-frame V1–J1 rearrangements; (b) sequences A, B, and C accounted for 70% (83/115) of the V1–J1 rearrangements; and (c), the gaaat-AT-ccag P addition was present in all mice analyzed and represented nearly 40% of the clones (45/115).
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1 transcripts from JHT scid mice lacked P additions and showed abnormal deletions at their VJ junctions similar to scid genomic V1–J1 rearrangements; furthermore, most corresponded to out-of-frame V1–J1 rearrangements (illustrated in Fig. 5). These results suggest, as discussed later, that survival of 1-expressing scid cells could depend on the coexpression of a Dµ (or µ) chain. Consistent with this possibility, Dµ and 1 transcripts were the only Ig gene transcripts routinely detectable in scid bone marrow (illustrated in Fig. 1). Moreover, most scid Dµ transcripts (10/16 analyzed) corresponded to DH–JH rearrangements in reading frame 2 (data not shown), which would be expected to result in the expression of a Dµ chain.
Cell Surface Expression of 1 Chains Is Detectable in Spleens of bcl-2 scid Mice.
The presence of 1-expressing scid cells in the spleen prompted us to test for possible cell surface expression of 1 chains (1+ cells) in the spleens of scid and bcl-2 scid mice. The latter mice were included because survival of scid B lineage cells is known to be enhanced in the presence of the bcl-2 transgene 42. Large numbers (5–10 x 106) of cells were analyzed by three-color flow cytometry for expression of cell surface 1 and the B and T cell markers CD45 (B220) and CD8, respectively. Mice with an inactivated RAG1 locus (RAG1–/– mice; reference 45) served as a negative control for background staining of 1. As illustrated in Fig. 7, there were no detectable 1+B220+CD8– cells in scid and RAG1–/– mice. However, in bcl-2 scid mice, 1+B220+CD8– cells were detectable at a frequency of 0.02–0.05% versus 0.5–1.5% in the wt controls. The distribution of B220 staining for 1 gated cells is shown in the histograms on the right side of Fig. 7. Note that bcl-2 scid spleen cells stained less bright for B220 and 1 than scid/+ or bcl-2 scid/+ spleen cells. These results indicate that a low frequency of scid cells express 1 chains on their cell surfaces and can be detected in the spleen of bcl-2 scid mice.
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locus does not require prior rearrangement at the H or chain locus. Our detection of 1 transcripts in bone marrow of scid and JHT mice indicates that V1–J1 rearrangement can occur before the pre-B cell stage and independently of H chain gene rearrangement. Moreover, the detection of V1–J1 rearrangement as early as day 12 in wt fetal liver, in which there is no genetic impairment of Ig gene rearrangement, suggests that the onset of V1–J1 rearrangement may developmentally precede VH–DJH and V–J rearrangement. The latter rearrangements were not detectable before day 14.
The most novel aspect of our findings is the regular detection of V1–J1 rearrangement in scid mice. This would not have been predicted, particularly the comparable level of V1–J1 rearrangement in scid and wt fetal liver. Also unexpected are the recurring P additions in scid 1 transcripts and the presence of scid cells with in-frame V1–J1 rearrangements in the spleen. We discuss the implications of these findings below.
Levels of V1–J1 Rearrangement and 1 Transcript in scid Mice.
We were surprised to find that the level of V1–J1 rearrangement in scid fetal liver was comparable to that in wt fetal liver (Table ), despite the abnormal loss of nucleotides in the VJ junction of most scid V1–J1 rearrangements. Thus, in scid fetal liver, abnormally truncated V1 and J1 coding ends appear to be joined as efficiently as V1 and J1 coding ends in wt fetal liver. This is unexpected because previous studies have clearly shown that V(D)J rearrangement is severely impaired in B and T lineage cells of adult scid mice 31323334. Indeed, as discussed below, V1–J1 rearrangement in scid adult bone marrow cells appears to be much less efficient than in wt bone marrow cells. The basis for the comparable level of V1–J1 rearrangement in scid and wt fetal liver is unexplained. Possibly, the machinery available for nonhomologous end joining in fetal liver differs from that in adult bone marrow and is able to compensate for the scid deficiency in DNA-PKcs.
Whereas the level of 1 transcripts was comparable in scid and wt fetal liver, in scid adult bone marrow (from JHT scid mice), 1 transcript levels were 25-fold less than in control adult bone marrow of JHT mice (Table ). The latter comparison is valid because B cell differentiation is arrested at the same stage (pro-B cell stage) in both JHT and JHT scid mice. Moreover, neither JHT nor JHT scid mice can make Dµ or µ chains, which could potentially affect the selection (or survival) of cells with productive 1 transcripts.
Two explanations can be considered for the 25-fold difference in levels of 1 transcript in JHT and JHT scid adult bone marrow. The first postulates that the scid 1 transcripts are much less stable than those generated in wt bone marrow, possibly owing to the abnormal nucleotide deletions accompanying scid V1–J1 rearrangement. We would expect such instability to be manifest in scid fetal liver as well, and yet in this tissue the level of 1 transcript was comparable to that in wt fetal liver. A second explanation is that the 25-fold difference in abundance of 1 transcripts in JHT and JHT scid mice primarily reflects a lower efficiency of V1–J1 rearrangement in scid adult pro-B cells than wt adult pro-B cells. This explanation is consistent with previous studies showing a 10–20-fold lower level of DH–JH rearrangement in scid than in wt adult bone marrow 4950.
The question arises as to why we did not obtain evidence of premature V–J rearrangement in scid bone marrow, given that a low level of such rearrangement has been previously observed in the pro-B cell fraction of wt mice 56. One possible reason is that initiation of V–J rearrangement invariably results in cell death. Consistent with this possibility, transcripts were readily detectable in the bone marrow of bcl-2 scid mice. The bcl-2 transgene is known to promote the longevity of scid B lineage cells 42 and their differentiation beyond the pro-B cell stage 4259. As reasoned elsewhere 60, primary and secondary initiation of V–J rearrangement in scid cells lacking the bcl-2 transgene would be expected to result in persisting chromosomal breaks and cell death.
VJ Junctions of scid 1 Transcripts Show Recurring P Additions.
Most 1 transcripts recovered from scid adult mice corresponded to in-frame V1–J1 rearrangements, and >60% of these transcripts contained recurring P additions. Whereas in wt mice most P additions are one or two nucleotides 61, in scid mice they are often longer 6263. In the present case, all of the scid P additions at V1 were five nucleotides in length (gaaat), and those at J1 were either three or four nucleotides in length (cag or ccag). This may reflect a strong bias in the resolution of hairpin coding ends imposed by the scid DNA-PKcs deficiency, such that J1 and V1 coding ends are frequently nicked three to four and five nucleotides from the hairpin tip, respectively, and then joined without further modification. In addition, cells expressing 1 transcripts with these junctions may be strongly selected, as discussed later.
Of particular interest is the recurring V1–J1 junctional sequence of two P additions separated by an AT dinucleotide (gaaat-AT-ccag), which was present in 40% of the 1 transcripts with P additions (Fig. 6). The basis for the AT dinucleotide is unclear. Given that scid V1–J1 rearrangements occur at the pro-B cell stage in the presence of high TdT levels, the AT dinucleotide could represent a nontemplated addition mediated by TdT. Another possibility is that the AT dinucleotide, which is palindromic to the last two nucleotides of the gaaat P addition, corresponds to a secondary P addition. In this scenario, one could postulate two successive recombination events. The generation of the gaaat P addition would result from an open and shut recombination 64 at V1. This would be followed by secondary cleavage at the V1 signal/coding border, asymmetric nicking of the V1 hairpin coding end, and joining to a J1 coding end with a ccag overhang. Regardless of how the AT dinucleotide is generated, we suggest that cells with the gaaat-AT-ccag junctional sequence are strongly selected to account for the repeated occurrence of this sequence in 1 transcripts of individual scid mice.
Evidence for 1-Expressing scid Cells.
Scid 1 transcripts were not only detected in bone marrow but also in spleen. Moreover, 1 transcripts with the same VJ junctional sequences were often found to recur in both of these tissues. This implies a strong selection for cells with in-frame V1–J1 rearrangements containing particular VJ junctional sequences. Such selection was not evident in JHT scid mice. 1 transcripts from JHT scid bone marrow contained abnormal deletions at the VJ junction, lacked P additions, and in most cases corresponded to out-of-frame V1–J1 rearrangements. These findings suggest that survival and selection of 1-expressing cells requires a functional H chain locus.
How might 1-expressing scid cells be dependent on a functional H chain locus? One possibility is that Dµ and 1 chains, resulting from expression of in-frame Dµ and 1 transcripts in scid bone marrow, pair and associate with the Ig and β signal-transducing chains 6566 to form a B cell–like receptor (BCRDµ/1). Although Dµ chains cannot pair efficiently with chains 67, they can pair with the surrogate L chain 6869 and might be expected to pair with 1 chains as well because the latter share some homology with the surrogate L chain (for review see reference 70). Expression of BCRDµ/1 in this proposed scenario would signal rapid (or direct) progression of scid pro-B cells to the recombinase-inactive B cell stage and allow these cells to survive and migrate to the periphery. But as BCRDµ/1 would lack a VH region, we would not expect cells bearing this receptor to persist or expand in response to naturally occurring antigens. This could in part explain the very low frequency of cells with surface 1 in bcl-2 scid spleen (0.05% of the cells examined). What is experimentally missing in support of the above scenario, however, is evidence for bcl-2 scid cells with surface 1 and µ chains. Despite reported evidence for intracellular expression of µ (or Dµ) chains in B220+CD22+ spleen cells of bcl-2 scid mice 59, cells with surface µ chains have not been detected in bcl-2 scid mice 4259.
In conclusion, we suggest that (a) joining of V1 and J1 coding ends after the initiation of V1–J1 rearrangement may not be impaired in B lineage cells of scid fetal liver because of the developmental time at which such rearrangement occurs; (b) recurring P additions in scid 1 transcripts may reflect a strong bias in the resolution of V1 and J1 hairpin coding ends imposed by the scid defect as well as possible strong selection for cells expressing these 1 transcripts; and (c) pro-B cells with in-frame V1–J1 rearrangements may express a pre-BCR–like receptor and differentiate into recombinase-inactive cells and emigrate from bone marrow to spleen.
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Grants from the National Institutes of Health (CA06927 and CA04946) and an appropriation from the Commonwealth of Pennsylvania supported this work.
Submitted: 4 February 2000
Revised: 31 March 2000
Accepted: 7 April 2000
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