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Induced Receptor Editing Shows No Allelic Preference in a Mouse Pre-B Cell Line¹

Xiangdong Liu^2,*, Michael Linden and Brian Van Ness^3,

^* Department of Biochemistry, Medical School, Genetics, Cell Biology, and Development Department, and the Cancer Center, University of Minnesota, Minneapolis, MN 55455

	Abstract

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B cell Ag receptor editing is a process that can change antigen recognition specificity of a B cell receptor through secondary gene rearrangements on the same allele. In this study we used a model mouse pre-B cell line (38B9) to examine factors that might affect allelic targeting of secondary rearrangements of the locus. We isolated clones that showed both productive and nonproductive rearrangements of one allele, while retaining the other allele in the germline configuration (⁺/° or ^-/°). In the absence of any selective pressures, subsequent rearrangement of the germline alleles occurred at the same frequency as secondary rearrangement of the productive or nonproductive rearranged alleles. Because 38B9 cells lack Ig heavy chains, we stably expressed µ heavy chain protein in 38B9 cells to determine whether heavy-light pairing might affect allelic targeting of secondary rearrangements. Although the expression of heavy chain was found to both pair with and stabilize protein in these cells, it had no effect on preferential targeting V-J receptor editing compared with rearrangement of a germline allele. These studies suggest that in the absence of selection to eliminate autoreactive V-J genes, there is no allelic preference for secondary rearrangement events in 38B9 cells.

	Introduction

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B cell development is a highly ordered process involving positive and negative selection (1). Although numerous B cells are produced in bone marrow, few survive the many checkpoints to become mature circulating B cells, and even fewer are able to reach the germinal center and participate in Ag-driven reactions. Virtually all B cells express a single heavy and light chain to form the B cell receptor (BCR).⁴ The process of BCR gene rearrangements contributes to BCR diversification, but is imprecise, leading to frequent nonfunctional V(D)J joints. We (2) and others (3, 4, 5, 6, 7) have demonstrated that secondary recombinations, in which new V genes rearrange to a downstream J segment, can serve to replace nonfunctional V-J joints, or replace functional, autoreactive encoding V-J alleles.

The replacement of productive V-J alleles has been referred to as receptor editing (4). A physiologic model of receptor editing has made use of genetically engineered Ig loci in which single autoreactive encoding V-J genes are inserted into the endogenous locus in the mouse through embryonic stem cell targeting (4, 5). These studies directly show that receptor editing is an important mechanism of B cell tolerance. The changes in BCR specificity not only remove self-reactive Abs but also serve to diversify the B cell Ag receptor repertoire.

Several factors likely influence the targeting of a locus for secondary recombinations. A number of studies have linked transcriptional activation of the locus to induction of rearrangement (8, 9). The expression of recombination activating gene (RAG)1 and RAG2 are critical in reinitiating rearrangement events (10, 11). A recent study of sorted primary B cells correlated demethylation to rearrangement of the alleles (12). Finally, there is evidence that receptor editing may require heavy-light chain pairing and Ag engagement (13, 14, 15, 16, 17, 18).

Studies using gene targeting in the mouse strongly suggest that secondary rearrangement occurs preferentially on the previously rearranged allele that codes for the V-J with autoreactivity (4). However, given the significant selective pressure to eliminate autoreactivity, it might not be possible to conclude that the recombination machinery has allelic preference based on the B cell populations that grow out in the mouse. Indeed, other mechanisms have been shown to eliminate autoreactive B cells through clonal deletion (19, 20, 21).

In this study, we used a murine pre-B model cell culture system to follow the sequential rearrangement of germline, productive, and nonproductive alleles. Without the immunologic pressure to preferentially expand B cells that undergo receptor editing of the autoreactive encoding allele, we were able to directly determine allelic frequency of primary and secondary V-J rearrangements. Our results show that in the absence of positive or negative selection, there is no preferential targeting for secondary rearrangements of productive or nonproductive alleles compared with the germline allele.

	Materials and Methods

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Cell culture and cloning

The 38B9 mouse pre-B cell line was provided by Dr. Eugene Oltz (Vanderbilt University, Nashville, TN). Cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies, Rockville, MD). 2-ME (50 µM) was added to the media. For LPS induction, cells were treated with LPS (Difco, Detroit, MI) at 1 µg/ml. Cloning of single cells was conducted in the absence of LPS by limited dilution of cells into 96-well plates with 25–30 cells/plate. PCR analyses of rearrangements were conducted after 3–4 wk of LPS induction.

Stable expression of an Igµ gene

A plasmid containing an Igµ gene (VH12.CH27-Cµ) was a kind gift from Dr. Chris Pennell (University of Minnesota, Minneapolis, MN; Ref. 22). To stably transfect cells, SfiI-linearized plasmid DNA was added to 4 x 10⁶ cells in RPMI 1640 media. The mixture was transferred to an electrocuvette, and the cells were then electroporated at 300 volts and 960 µF in a GenePulser apparatus connected to a capacitance extender (Bio-Rad, Richmond, CA). Cells were allowed to recover for 10 min at room temperature, transferred to 10 ml supplemented RPMI 1640 media, and incubated for 24–48 h at 37°C in a 7% CO₂ incubator before undergoing drug selection in 800 mg/ml Geneticin (Life Technologies). Old media were replaced by fresh selective media every 2–3 days for 2–3 wk, at which time the anti-Geneticin clones were selected and expanded. Igµ expression was detected by Ab staining of transfected cells and FACS analysis.

Isolation of genomic DNA samples

To isolate genomic DNA, cells (>10⁷) were harvested and lysed in 3 ml of lysis buffer (10 mM Tris-HCl (pH 8.0), 25 mM EDTA, 0.5% sodium dodecyl sulfate, 2 µg/µl RNase A) at 37°C for 30 min. Proteins were precipitated by the addition of 1 ml of 5 M ammonium acetate followed by vigorous vortexing for 30 s. The precipitate was pelleted by centrifugation at room temperature for 10 min at 3000 rpm in a clinical centrifuge. The supernatant was transferred to a fresh tube, and DNA was precipitated by mixing with an equal volume of isopropanol. The DNA was pelleted by centrifugation at 3000 rpm, washed once with 70% ethanol, and rocked at 4°C overnight in 50–100 µl TE (10 mM Tris (pH 8.0), 0.5 mM EDTA) to be dissolved. Concentration of the DNA sample was determined with a DU-52 spectrophotometer (Beckman Coulter, Fullerton, CA).

PCR analysis of rearrangements

One microgram of genomic DNA sample of each cell clone was used as a template in a 50 µl PCR mixture containing 200 µM each deoxynucleoside triphosphate, 200 nM each oligonucleotide, 1x PCR buffer II (Perkin-Elmer, Norwalk, CT), 1.5 mM MgCl₂, and 2.5 U AmpliTaq Gold polymerase (Perkin-Elmer). To detect DNA remaining in the unrearranged germline configuration, the reactions were heated at 94°C for 7 min and then were cycled 40 times at 94°C for 30 s, 60°C for 45 s, and 72°C for 3 min. Sequences of oligonucleotides used for the detection are as follows: 5' primer, 5'-CTACCCACTGCTCTGTTCCTCTTCAGT-3'; and 3' primer, 5'-TTTGATCTGCGCTGTTTCATCCTCTGGGTCATTC-3'. A pair of oligonucleotides complementary to the constant region were used as an internal control for template loading and amplification efficiency. Their sequences are: 5' primer, 5'-CCACGGACGAGTATGAACGACATAACAGCTATAC-3'; and 3' primer, 5'-GTGTAATCTCACGGTATAGAGGTCTCTTGAAG-3'. To detect already rearranged substrates, the same conditions as above were used, except the following primer set was substituted to amplify the rearranged allele(s): 5' primer, 5'-GTCCCTGCCAGGTTYAGTGGCAGTGGRTCWRGGAC-3' (where Y = C or T, R = A or G, and W = T or A); and 3' primer, 5'-TTAGTGGCTCTGTTCCTATCACTGTGTCCTCAGG-3'. The same internal control as described above was included.

Detection of µ heavy chain and light chain proteins by cytoplasmic and cell surface Ab staining

A FITC-conjugated mAb used to detect µ protein was purchased from PharMingen (San Diego, CA), and a FITC-conjugated polyclonal antiserum used to detect protein was obtained from Southern Biotechnology Associates (Birmingham, AL). Cytoplasmic staining was performed at room temperature as follows: 5 x 10⁶ cells were harvested after 48 h of LPS induction and washed in 1x PBS three times; the cells were then resuspended in 100 µl 1x PBS and transferred into a 12 x 75 mm FACS tube; 2 ml of 1x Ortho PermeaFix solution (Ortho Diagnostics, Raritan, NJ) was mixed with the cells and incubated for 40 min; the cells were pelleted by centrifugation at 1500 rpm for 5 min in a clinical centrifuge, resuspended, and incubated in 2 ml wash buffer (1x PBS, 5% FCS, 1.5% BSA, and 0.0055% EDTA(w/v)) for 10 min; and the cells were pelleted again and resuspended in 50 µl FACS buffer (1x PBS, 2% FCS) supplemented with 1–2 µg anti- light chain or anti-heavy chain Ab. After incubation for 40 min, the cells were washed with 2 ml wash buffer, pelleted, and resuspended in 1 ml of wash buffer for FACS analysis.

Cell surface staining was performed as follows: 5 x 10⁵ cells were harvested after 48 h of LPS induction, washed in 500 µl FACS buffer three times, resuspended, and incubated in 50 µl FACS buffer supplemented with 1–2 µg anti- light chain or anti-heavy chain Ab at 4°C in the dark for 30 min. The cells were then washed in FACS buffer three times and resuspended in 1 ml FACS buffer containing 0.01% sodium azide for FACS analysis.

FACS analysis was performed according to manufacturer’s recommendations with a FACScalibur, and a CellQuest software package (Becton Dickinson, San Jose, CA). The data obtained at each experimental condition represent counts of 25,000 cells, falling within a specified gate determined by forward light scatter.

Coimmunoprecipitation and protein detection by Western blotting

A FITC-conjugated mAb used to immunoprecipitate the light chain was purchased from Southern Biotechnology Associates. Five micrograms of this Ab were bound to 20 µl of protein G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) in 400 µl of radioimmunoprecipiation assay (RIPA) lysis solution (50 mM Tris-HCl (pH 7.9), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, 1% aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na₃VO₄, and 1 mM NaF). All immunoprecipitation procedures were performed in a cold room. Cell lysates were made from 1 x 10⁷ cells stimulated by culturing them overnight in RPMI 1640 media with 10 mg/ml LPS. All cells were spun down, their supernatant was decanted, and then resuspended in 500 µl RIPA. Agarose beads were allowed to bind to Ab by rocking the mixture for 1 h. Cell lysates were then added to the Ab/agarose bead mixtures, and these immunoprecipitation products were rocked in the cold room overnight. The samples were then washed with RIPA three times the following day in preparation for electrophoresis.

Immunoprecipitation samples were mixed with an equal volume of 2x sample loading buffer containing 10% 2-ME. All samples, as well as 15 µl of a Bio-Rad high range prestained molecular marker, were heated to 100°C for 5 min, and then loaded onto a 10% SDS-polyacrylamide gel. Samples were electrophoresed for 1 h at 40 mA, then transferred to a Bio-Rad polyvinylidene fluoride membrane, using an Integrated Separation Systems (Hyde Park, MA) electroblotter for 1 h at 50 mA. The electroblotted membrane was then blocked overnight in a TN-TBN solution (10 mM Tris (pH 7.7), 150 mM NaCl, 0.1% Tween 20, 2% BSA, and 5% dry nonfat milk). It was then washed four times (two times quick rinse, two times 10') with 1x PBS. The membrane was transferred to a solution containing 25 ml TN-TBN and a 1:1000 dilution of a rat anti-mouse IgM FITC Ab (PharMingen) and then rocked for 1 h at room temperature. It was washed four times in the same manner as before, and was rocked for 30 min in a solution containing 25 ml TN-TBN and a 1:1500 dilution of a sheep anti-rat Ig HRP Ab (Amersham, Cleveland, OH). A final wash was performed, and the membrane was introduced to the Amersham enhanced chemoluminescence-protein detection kit reagents for 1 min. The membrane was subsequently exposed to Kodak O-MAT radiographic film for 15 min and developed.

	Results

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The study of sequential rearrangements and BCR editing in a mouse pre-B cell line

To examine the relative frequency of secondary recombinations vs primary germline rearrangements, we used the 38B9 pre-B cell line (9). We and others have shown that this line expresses RAG1 and RAG2 and contains two alleles in germline (°) configuration (9). These cells can be induced to initiate V-J rearrangements by mitogens such as LPS. In this system, there is a strong correlation of gene rearrangements and germline transcription (9), and both alleles show equal probability of initiating a primary rearrangement of the germline locus. Notably, these cells do not express a heavy chain. Our strategy to follow the course of primary and secondary rearrangements is shown in Fig. 1. 38B9 pre-B cells were induced with LPS for 2 wk. Single-cell subclones were then derived to establish populations reflecting different genotypes of the two alleles as follows, as determined by PCR assays and cytoplasmic staining: l) clonal populations that contained one productively rearranged allele and one germline allele (⁺/°); 2) clonal populations that contained one nonproductive V-J allele and a germline allele (^-/°); and 3) clonal populations derived from populations 1 and 2 into which we stably transfected an Igµ expressing construct (⁺/°/µ⁺ and ^-/°/µ⁺).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. The strategy to study the regulation of secondary rearrangements/BCR editing in a model 38B9 pre-B cell line. For the primary screen, single-cell clones were selected after LPS induction and subjected to PCR analysis of rearrangements. Clones were selected that have one rearranged allele and one germline allele. The productivity of rearrangements was determined by Ab staining for light chain protein and FACS analysis (see Fig. 3). Clones were then subjected to a secondary induction with LPS, before or after stable transfection of an Igµ expression vector. Secondary rearrangement by receptor editing of the initial rearrangement or rearrangement of the germline allele was determined as described in Materials and Methods (shown in Fig. 2).

Two allele-specific PCR approaches were employed (Fig. 2). One assay detects unrearranged, germline alleles by using a unique primer upstream of the J cluster. Rearrangement of this allele results in the loss of the germline PCR product. The second assay is based on the assay first described by Schlissel, et al. (8) and is described in our characterization of 38B9 cells (9). This assay uses a consensus V-region primer, paired with a primer downstream of the J cluster, and detects V-J products whose size is characteristic of V-J 1,2,3, or 4 rearrangements.

View larger version (60K): [in this window] [in a new window]   FIGURE 2. Detection of rearrangements by PCR-based approaches. A schematic illustration of the two different PCR assays of rearrangements is presented at the top. One assay detects rearrangements by measuring the loss of unrearranged germline alleles, and the other assay detects subsequent V-J alleles using a consensus V region primer paired with a intron primer. A, A PCR screen of single-cell clones after initial LPS induction. Clones that contained both a germline allele and one rearranged allele (J1, J2, or J3; J4s could not undergo further rearrangement and were not used for further study) were subjected to further LPS induction to assess allelic preference. B, A PCR screen of the number of single-cell clones derived after inducing a VJ3/° with LPS, and alterations in the VJ3 allele or germline allele were monitored and scored as shown.

Secondary rearrangements occur on both alleles in 38B9 pre-B cells without preference, regardless of the productivity of their initial rearrangements

Both alleles were in the unrearranged germline configuration (°/°) in the initial 38B9 pre-B cell population. Rearrangements were induced with LPS for 2 wk and individual subclones were isolated and characterized for rearrangements. As shown in Fig. 2A, several clones containing one V-J rearrangement and one germline allele were identified. The productivity of initial rearrangements was determined by cytoplasmic staining and flow cytometry (Fig. 3). To support the efficacy of FACS analysis to assess productivity, the VJ joining regions of three clones, one ⁺/° and two ^-/°, were amplified by PCR and then sequenced. Sequencing results were consistent with cytoplasmic staining in that the ⁺/° produced a sequence with an open reading frame in the VJ region, while the ^-/° clones had termination codons in all three reading frames in the same region (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 3. FACS analysis of µ heavy chain and light chain expression in Igµ-transfected 38B9 cell clone 1-37. Cytoplasmic staining of Igµ-transfected 38B9 cell clones carrying initial rearrangements with either FITC-conjugated anti-Igµ Ab or FITC-conjugated anti- light chain antiserum was performed and analyzed by FACS. A. Cytoplasmic staining and FACS profile of µ protein in nontransfected (top), Igµ transfected (middle), and Igµ transfected and LPS-induced (bottom) cells. B, Cytoplasmic staining of nontransfected 1-37 cells ± LPS (top two panels) and Igµ transfected cells ± LPS (bottom two panels). C, Coimmunoprecipitation shows heavy-light chain pairing. Cell extracts were prepared from LPS-induced 1-37 and 2-48 clones as well as a positive control (WEHI-231); immunoprecipitated with goat anti- Ab (lanes 1, 3, and 5), or nonspecific Ab (NSAb), goat anti-rabbit-Ig (lanes 2, 4, and 6); and Western-blotted with anti-µ Ab as described in Materials and Methods.

J

J

J

Although more complex rearrangement scenarios could be envisioned, we recorded the minimal number of rearrangement events necessary to generate the observed PCR patterns. The data collected and results are summarized in Table I. Statistical analyses using 95% confidence intervals show no statistically significant evidence that there is a bias in allele usage, and because the variation in individual experiments occurs on both sides of 50%, the probability of recombination events occurring on the germline or rearranged allele likely converges at 50%. As expected, some of these initial rearrangements were found to be productive, capable of producing light chain protein; others were assumed to be nonproductive based on the lack of any cytoplasmic staining.

View this table: [in this window] [in a new window]   Table I. Secondary rearrangements of the locus in 38B9 cells shows no allelic preference

Expression of Igµ does not affect the allelic preference of secondary rearrangements in the cell clones that had either productive or nonproductive initial rearrangements

38B9 cells do not produce Ig heavy chain protein. However, signals from functional complete heavy-light chain pairs have been shown to play an important role in the regulation of subsequent light chain gene rearrangements during B cell development (14, 15, 16, 17, 18). To test whether the lack of allelic preference of the secondary rearrangements noted above was due to the lack of functional heavy chains in 38B9 cells, we stably introduced an Igµ expression vector in the cell clones that were initially isolated as ⁺/° or ^-/°. Expression of Igµ was determined by anti-Igµ Ab staining and FACS analysis (Fig. 3), and was found to be present in all transfected cells. Notably, we could also detect stabilization of light chain protein in cytoplasm of some but not all of the transfected cell clones, further demonstrating that not all initial rearrangements resulted in chain production.

To address whether µ heavy chain protein and productive initial rearrangements have any effect on the allelic preference of secondary rearrangements, secondary LPS induction and single-cell cloning were again conducted with the Igµ⁺ cell clones. After examining secondary rearrangements in newly selected single-cell clones, we found that expression of Igµ did not affect the allelic preference of secondary rearrangements in the cells that had either productive or nonproductive initial rearrangements (Table II). Heavy-light chain pairs were formed as demonstrated by coimmunoprecipitation (Fig. 3C). These results suggest that the presence of both heavy chain µ and light chain proteins did not influence the allelic preference of secondary rearrangements in 38B9 pre-B cells.

View this table: [in this window] [in a new window]   Table II. Expression of µ heavy chain does not affect allelic preference of secondary rearrangements

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Both RAG1/RAG2 activity and transcriptional activation of the Ig loci have been shown to be involved in the regulation of rearrangements (23, 24, 25). In all the 38B9 cell populations we examined, RAG1/RAG2 expression was maintained, but rearrangement was directly correlated with the induction of transcription (Ref. 9 and data not shown). Both germline alleles and rearranged alleles showed this correlation. Previously, we found that no cytokines could induce rearrangement in the absence of transcriptional activation (9). Therefore, transcriptional activation and rearrangement are coordinately regulated in these cells. In another pre-B model cell culture system, we have shown that 70Z cells transfected with RAG1/RAG2 can also be induced to undergo primary and secondary recombinations of the locus (Ref. 9 and data not shown).

Numerous previous studies suggest that a feedback regulatory mechanism of Ag-receptor engagement might be involved in the regulation of receptor editing, and signals transduced by a functional BCR might trigger receptor editing (13, 15, 16, 17, 18). Thus, it was important to determine whether productive or nonproductive alleles influenced the target of secondary rearrangements. We studied the effect of functional/nonfunctional status of initial rearrangements and presence of µ heavy chain protein on secondary rearrangements. Our analysis of secondary rearrangements in this cell line shows that secondary rearrangements can occur on both alleles with no apparent preference, regardless of whether there were productive initial rearrangements or not, indicating that initial rearrangements, per se, do not affect secondary rearrangements. However, the initial -expressing subclones showed very weak protein production in the absence of heavy chain expression, possibly due to destabilization of the unpaired light chain protein. Thus, productive rearrangements in 38B9 cells would not result in the formation of BCR complexes capable of signaling and/or influencing the allelic preference of secondary rearrangements. Although protein expression may influence signaling to re-initiate rearrangement events, it is difficult to imagine how such protein would influence allelic choice (i.e., how it would preferentially stimulate secondary rearrangements of V-J alleles compared with primary rearrangement of germline alleles). Nevertheless, when we stably expressed Ig heavy chain µ protein in the 38B9 cell clones, both and µ heavy chain protein were readily detected. We found that expression of Igµ stabilized the expression of the light chain, likely by heavy-light chain pairing. Even in the presence of stable heavy-light chain pairs, we found no allelic preference of secondary rearrangements in the cells that carried either productive or nonproductive initial rearrangements.

Another model for Ag receptor gene rearrangement using computer-derived statistical analyses concluded that a stochastic, ordered rearrangement process supports targeting of the previously rearranged allele for further recombination events (26). This model shows that for mice to produce a B cell repertoire in which greater than 70% of the cells are ^R/°, rearrangement events preferentially occur on the previously rearranged allele; i.e., the probability of rearrangement of the germline allele is 0 (26). Although this model provides a potential explanation for Ag receptor gene rearrangement, the empirical statistics it is based on are derived from in vivo murine studies, where populations of cells are subject to positive and negative selection. Again, we find that that our ex vivo model gives us the opportunity to study gene rearrangement without selective bias.

Accessibility of the gene loci has been thought to play a role in rearrangement events. We and others (27) have examined methylation status in LPS-inducible pre-B cell lines, and we find that demethylation is very slow and does not distinguish the two alleles. Additionally, previous studies have shown that the promoter strength of the germline promoter is significantly weaker than most V region promoters (28). Because transcriptional activation has been linked to accessibility, V gene promoter proximity and strength might influence allelic choice, and V region proximity may influence secondary rearrangements, as rearranged alleles would likely bring V region gene segments closer to the J locus. Despite establishing these conditions in the 38B9 line, we did not observe an allelic bias for secondary rearrangements, suggesting that promoter proximity and strength do not play a role in allelic choice. Nevertheless, it is certainly possible that in vivo primary progenitor B cells regulate accessibility differently than this single transformed cell line, as previous studies have demonstrated that activation of the Ig locus in 38B9 cells relies on the intronic enhancer (9), while knockout studies suggest a role for the 3' enhancer (29), which is inactive in these cells.

Although our model cell line lacks some of the physiologic signaling available in vivo, we could address several important potential regulators of allelic choice in the absence of immune selection. Indeed, one of the limitations of this study is the apparent failure to obtain surface expression of the complete BCR in 38B9 cells. All detection was by cytoplasmic staining, and we could not detect surface expression. In addition, the BCR specificities were unknown so that we were unable to regulate secondary rearrangements by receptor engagement. Although receptor engagement may be important in vivo to signal reactivation of RAG-mediated rearrangements, there is no evidence in any receptor-mediated system that shows locus activation can be allele specific.

Based on our demonstration of equivalent allelic targeting, the apparent allelic preference in mouse models could be due to the selective expansion of B cell populations that successfully replace the autoreactive allele. Despite the limitations of our cell line, locus accessibility dictated by a productive or nonproductive rearrangement does not appear to provide sufficient inherent bias for secondary rearrangements. Alternatively, receptor-mediated allelic selection requires the identification of novel mechanisms of selective Ig gene targeting that have not been defined.

	Acknowledgments

 
We are grateful to Dr. Chris Pennell (University of Minnesota) for providing the Igµ expression vector, Dr. Eugene Oltz (Vanderbilt University) for the 38B9 pre-B cell line, Dr. Darin O’Brien for initial characterizations of rearrangements in these cells, Dr. Lap Che for statistical advice, and Dr. Martin Weigert for stimulating discussions.

	Footnotes

 
¹ This work is supported in part by funds from the University of Minnesota.

² Current address: Sugen, Inc., 230 East Grand Avenue, South San Francisco, CA 94080.

³ Address correspondence and reprint requests to Dr. Brian Van Ness, Cancer Center Research Building, Box 806 FUMC, 425 East River Road SE, University of Minnesota, Minneapolis, MN 55455.

⁴ Abbreviations used in this paper: BCR, B cell receptor; RAG, recombination activating gene; RIPA, rapid immunoprecipitation assay.

Received for publication July 18, 2000. Accepted for publication September 26, 2000.

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