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Asymmetric Contribution to Ig Repertoire Diversity by V Exons: Differences in the Utilization of V10 Exons

Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mouse has approximately 140 germline V genes, and functional V exons are expressed at roughly equivalent levels in the preimmune repertoire. We have examined the expression of individual members of the V10 family. V10A and V10B genes have been utilized in numerous hybridomas and myelomas, while V10C has not. In this study, we have cloned the V10C gene and shown that it is structurally functional, has the expected promoter elements and recombination signal sequences, and that it is capable of recombination. V10C mRNA, however, is present at levels at least 1000-fold lower than V10A and V10B in adult spleens. While there are no sequence differences in the octamer or TATA box between V10C and V10A, there are three nucleotide changes in the promoter region. These promoters equally drive the expression of a reporter gene in B cells or plasma cells, but the V10A promoter is able to drive expression in pre-B cell lines significantly better than the V10C promoter (p < 0.05). V10C rearrangements can be detected in bone marrow and splenic DNA. Therefore, the lack of V10C expression may reflect the inability of V10C-rearranged cells to undergo positive or negative selection. Our results suggest that the available Ab repertoire is shaped not only by the number of structurally functional genes, but also by the ability of assembled genes to be expressed at critical points during B cell maturation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The recombination of an estimated 140 germline V genes (1) with multiple J genes and 100 to 200 V_H (2, 3) genes with multiple D_H and J_H genes in the mouse confers an enormous potential preimmune repertoire of Ab specificities. Diversity is further enriched by the contributions of junctional additions or deletions of nucleotides, the combinatorial pairing of individual heavy and light chain proteins, and finally, somatic mutation that occurs in the periphery. By these mechanisms it has been estimated that 10⁹ to 10¹¹ distinct Ab specificities are theoretically possible. The primary Ab repertoire is more limited in size because it is shaped by the number of functional V genes in the genome, the frequency of individual V gene recombination, the effect of promoter efficiency on transcription (4, 5, 6), the ability of specific V_H and V_L chains to pair, and positive or negative selection of B cells during ontogeny (7, 8, 9).

The precise mechanisms governing the selection of individual V_H and V exons for rearrangement and expression are unclear. Several studies have examined the use of V_H or V genes at the family level. In some instances, the utilized repertoire was reported to be nonrandom. For example, early in ontogeny, V_H gene families that lie proximal to DJ genes are utilized to a greater extent than those distal to DJ (10, 11, 12, 13), while in the adult mouse, V_H utilization does not appear to be positionally biased, but correlates with the size of a given family (12, 14, 15, 16, 17, 18). A positional bias is not evident at the locus during early development, and furthermore, the frequency of V utilization in adult mice is not dependent on family size (19, 20, 21). That approximately 40% of V genes lie in an opposite transcriptional orientation relative to J and rearrange by inversion (22) may explain some of the observed differences in V and V_H utilization. As a byproduct of inversional recombination, reciprocal products consisting of the fused VJ recombination signal sequences (RSS)² and the VJ intervening DNA are retained on the chromosome, allowing secondary recombinations between what were once distant V genes with J gene segments. Rearrangement at the locus does not shut down immediately after VJ recombination and, thus, secondary rearrangements may mask detection of any inherent positional bias in the locus.

Several investigators have reported the preferential use of certain V families early in ontogeny. Medina and Teale showed that the early repertoire is dominated by V families that undergo inversion-type rearrangements (23). It was shown that the repertoires of day 18 fetal liver and day 15 fetal omentum were restricted to five and six families, respectively, with a predominant usage of the V4,5, V9, and V10 families, all of which undergo recombination by inversion. Kaushik et al. (19) reported a preference for the V1 and V9 families in B cell colonies derived from splenic B cells of 6- to 8-day-old neonatal mice. In contrast, Ramsden et al. demonstrated that V usage from day 14 and 16 fetal livers represented 14 of the 18 known V families (24).

In the preimmune repertoire of B lymphocytes, the precise mechanisms favoring selection of one V gene over another for recombination and expression are not known. Several factors, however, are known to be necessary for recombination and transcription of V genes. For example, Ig gene rearrangement has been correlated with DNA hypomethylation and chromatin accessibility (25, 26, 27). Often germline transcripts from V genes (4, 28, 29, 30) or the C region locus (31, 32) are detected. It is not known whether these germline transcripts play a role in locus accessibility or if they are a byproduct of an open chromatin configuration. Expression of a V gene requires a functional RSS and a functional promoter. It has been shown that RSS strengths can directly affect the frequency of recombination (33, 34). In addition, differences in promoters of both V_H and V genes are known to influence transcription efficiencies (4, 6, 35).

Previous studies have compared levels of expression of different V_H or V_L families, or have examined the functionality of the promoters or RSS of individual genes. In this study, we have compared the utilization of individual, but closely related, V genes and the ability of their regulatory sequences to drive efficient recombination or expression.

Members of the same V family share >80% homology at the DNA level (36) and, for the most part, lie close together within the locus on chromosome 6 of the mouse. We chose to study the V10 family, which is small, containing three members (37), V10A,³ V10B, and V10C; resides in the middle of the locus (1); and has been shown to rearrange by inversion (22). V10A and V10B are utilized in response to a wide variety of T-dependent and T-independent Ags and have been isolated and sequenced from numerous hybridomas and myelomas derived from several inbred mouse strains. The third family member, V10C, has not been seen in functional Abs and has been isolated only once as a reciprocal product of an aberrant VJ recombination (22, 38). Since V10A and V10B are expressed in response to a wide variety of Ags and appear to contribute to early repertoire diversity (20), while V10C is not utilized, it was of interest to determine the factors limiting a V gene’s contribution to diversity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of germline V10C gene

The V10C germline gene (GenBank accession AF029261) was isolated from a genomic library constructed from BALB/c kidney DNA in the dash II vector (Stratagene, La Jolla, CA). Plaques were screened by hybridization with a ³²P-labeled 0.9-kb PC3386 EcoRI/HindIII fragment (22) on nitrocellulose. Hybridizing fragments from EcoRI and BamHI digests of positive plaque DNAs were subcloned into pBluescript (Stratagene) for sequencing. Genomic clones were cycle sequenced with primers produced in Core Facility for Biotechnology Resources at the Center for Biologics Evaluation and Research, Food and Drug Adminstration, and a dsDNA cycle sequencing kit (Life Technologies, Gaithersburg, MD).

Primer specificities

Oligonucleotides were synthesized in the Core Facility for Biotechnology Resources facility at the Center for Biologics Evaluation and Research and are listed in Table I. All PCR reactions were performed in a DNA thermal cycler 480 (Perkin-Elmer, Norwalk, CT) in 100-µl reactions with 1.5 mM MgCl₂, 0.05 mM dNTPs, and 50 pmol of each primer. Specific PCR conditions were determined for the V10A, V10B, and V10C primers in cross-priming experiments using hybridoma 226.1 (39) cDNA (V10A), hybridoma H24C2 (40) cDNA (V10B), and a V10C spleen clone 4C17 (see below) as templates. V10A and V10B primers were specific for their templates under the following conditions: 95°C, 5 min; 73°C, 2 min/95°C, 1 min (30 cycles); and 73°C, 10 min, 4°C hold. The V10C primer was specific under identical conditions, but with an annealing/extension temperature of 74°C. As shown in Figure 1, the V10C primer differs from V10A and V10B template by 2 and 4 bases, respectively, V10B primer differs from V10A and V10C template by 2 and 3 bases, respectively, and V10A primer differs from V10B and V10C template by 5 and 6 bases, respectively. The C region primers 5'KC and 3'KC were specific for their target under the following conditions: 95°C, 5 min; 65°C, 1 min/72°C, 1 min/95°C, 1 min (30 cycles); and 72°C, 10 min, 4°C hold. PCR products obtained from a BALB/c spleen cDNA with V10A, V10B, V10C, and C region primers under specific conditions were cloned into the PCRII vector (Invitrogen, San Diego, CA). Twenty clones each for V10A, V10B, and V10C were cycle sequenced using Sp6 and T7 primers under the following conditions: 95°C, 3 min; 55°C, 30 s/70°C, 30 s/95°C, 30 s (20 cycles); and 58°C, 1 min/95°C, 30 s (10 cycles), 4°C hold. Clones A5, 4B16, 4C17, and K7 were used as standards in V10A, V10B, V10C, and C semiquantitation assays, respectively.

View larger version (53K): [in this window] [in a new window]   FIGURE 1. Alignment of V10 germline sequences. V10A and V10B sequences were obtained from GenBank (42). The V10C germline sequence was obtained from a BALB/c kidney genomic clone (GenBank accession no. AF029261). Regulatory regions within the promoter are underlined. Proceeding in the 5' to 3' direction, starting within framework 1, the underlined primers are Gen7, V10B, V10C, V10A, and A3. Nucleotide substitutions in the V10A and V10B germline that result in amino acid changes are in bold type. Gaps resulting from alignment are depicted by dots (.). Splice sites are represented by forward slashes (/).

Semiquantitation of V10 and total mRNA

Total RNA from four BALB/c spleens was isolated using the Trizol method (Life Technologies), according to the manufacturer’s instructions. RNA was reverse transcribed with oligo(dT) primer and the superscript preamplification system (Life Technologies), according to the manufacturer’s instructions. A total of 1 µl of the cDNAs and 1 µl of log dilutions of A5, 4B16, 4C17, and K7 standards (10^-1-10^-8 for A5, 4B16, and 4C17; 10^-1-10^-10 for K7) was amplified using the specific PCR conditions described above. constant 3'KC primer was the 3' primer for all PCRs, while V10A, V10B, and total PCRs utilized V10A-specific, V10B-specific, and constant 5'KC as 5' primers, respectively. Thirty-five-microliter samples from each reaction tube were electrophoresed in 3% agarose gels at 170 V in 1x TAE for 3 to 4 h. Gels were stained with ethidium bromide (0.5 µg/ml) in 1x TAE for 30 min, and band intensities were quantitated on a fluorimeter. Band intensities of standards were plotted versus number of standard molecules at each dilution. The number of target structures in each cDNA sample was estimated by interpolating its band intensity into the standard curve. V10C PCR sensitivity was determined by performing V10C PCRs on serially diluted 4C17 standard.

Nested PCRs were performed as described above, with the following exceptions: the 3'KC and Gen7 primers were used to amplify 1 µl of cDNA in the primary PCR, as follows: 95°C, 5 min; 55°C, 1 min/72°C, 1 min/95°C, 1 min (30 cycles). Primary PCR products were purified with Wizard PCR preps columns (Promega, Madison, WI), and 1 µl was used as the template for a secondary PCR with the V10A-, B-, and C-specific primers, and the 3'KC2 primer under the cycling conditions described for V10A, B, and C PCRs above.

pGL3 enhancer/promoter reporter vector construction

Volumes for all PCR reactions were 100 µl with 1.5 mM MgCl₂, 0.05 mM dNTPs, and 50 pmol each primer. The V10AS (142-bp) promoter fragment was PCR amplified from a BALB/c liver DNA V10A genomic clone (22) with the 5'AJ1/10C short HindIII and AJ13' HindIII primers. The V10CS (142-bp) promoter fragment was PCR amplified from the dash II (Stratagene) BALB/c kidney DNA V10C genomic clone 91-3 with the 5'AJ1/10C short HindIII and 10C3'HindIII primers. The intronic enhancer (537 bp) was PCR amplified from pECK DNA (containing the intronic enhancer and germline constant DNA) with the Enhanc.5'KpnI+Enhanc.3'KpnI primers. V10AS, V10CS, and enhancer PCRs were performed, as follows: 95°C, 5 min; 50°C, 1 min/72°C, 1 min/95°C, 1 min (30 cycles); and 72°C, 10 min, 4°C hold. V10AS and V10CS PCR products were digested with HindIII, and enhancer product was digested with KpnI. The enhancer fragment was ligated into KpnI-linearized pGL3 basic vector (Promega) and labeled pGL3-en. Digested, purified V10AS and V10CS promoter fragments were ligated into pGL3-en linearized with HindIII. Orientation and promoter sequence confirmation were determined by sequencing. Plasmids, including control vector pCMV-ß (Clontech, Palo Alto, CA), were purified by double banding in CsCl.

Transfections and lysate production

All electroporations were performed using a Bio-Rad (Richmond, CA) gene pulser (0.22 kV, 960 µFd) and 0.2-cm path-length cuvettes containing 6 x 10⁶ cells in 250 µl electroporation media (RPMI 1640, 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, nonessential amino acids 1x final concentration, 50 µM 2-ME, and 2 mM glutamine). pCMVß (0.32 µg) was cotransfected with either 5 pmol of pGL3-en/V10AS, pGL3-en/V10CS, or pGL3en-only into the pre-B cell lines 18-81 and NFS-467, the immature B cell line Wehi 231.4, and the plasmacytoma cell line Sp2/0. The pre-B cell lines NFS-5 and 70Z/3 were each cotransfected with 10 pmol of the promoter plasmids and pGL3-en plasmids and 0.32 µg of pCMV-ß as a control. The mature B cell line A20 was cotransfected with 0.5 pmol pGL3-en/V10AS, pGL3-en/V10CS, or pGL3-en-only, and 1.7 µg pCMV-ß. Following transfection, cells were transferred to T75 flasks containing 15 ml complete RPMI and incubated for 24 h at 37°C in 5% CO₂. After 24 h, cells were harvested by centrifugation, washed once in PBS, and lysed for 15 min in 70 µl 1x reporter lysis buffer (Promega). Cellular debris was pelleted by centrifugation at 11,000 rpm for 2 min at 4°C. Supernatants were transferred to new tubes and stored at -70°C.

Luciferase and ß-galactosidase assays

For luciferase assays, 20 µl of lysate was combined with 100 µl of luciferase assay substrate (Promega) in the wells of a microlite I 96-well tray (Dynatech, Chantilly, VA), and light production was measured in a luminometer. For ß-galactosidase assays, 5 µl of lysate was added to the wells of a microlite I 96-well tray and combined with 50 µl of galacton plus (Tropix, Bedford, MA) diluted 1/100 in 0.1 M sodium phosphate, pH 7, and incubated for 15 min at room temperature. A total of 50 µl of emerald enhancer (Tropix) diluted 1/10 in 0.2 N NaOH was added to each well, and light production was measured in a luminometer. Luciferase activity was calculated by dividing the luciferase luminometer value by the ß-galactosidase value for each well. Statistical differences in V10 promoter efficiencies were analyzed using one-way ANOVA.

V10 DNA recombination

BALB/c genomic DNAs (100 ng) from spleen and ThB-enriched bone marrow B cells (see below) were amplified with the Gen7 and J 5–3 primer pair to amplify all V10 rearrangements. PCRs were performed in duplicate 100-µl reactions (pooled before gel loading) with 1.5 mM MgCl₂, 0.05 mM dNTPs, and 50 pmol each primer under the following conditions: 95°C, 5 min; 60°C, 1 min/72°C, 2 min/95°C, 1 min (30 cycles); and 72°C, 10 min, 4°C hold. Thirty-seven microliters of each sample were electrophoresed on triplicate 2.3% agarose gels for 3 h at 150 V. Included on each gel as specificity controls were genomic clones for V10A (2.1-kb EcoRI/BamHI fragment from pC13-13 (22)) and V10C (2.4-kb EcoRI fragment from the pBluescipt 91-3), and an RT-PCR clone for V10B (4B16 EcoRI digest). EcoRI digests of RT-PCR clones A5 and 4C17 were included on the V10A and V10C gels, respectively, as additional positive controls. EcoRI-digested 4B16 was used as both the specificity and positive control on the V10B gel, since a genomic clone of V10B was not available. DNA was transferred to positively charged nylon membranes in 10x SSC overnight by capillary action, and UV cross-linked to filters in a Bio-Rad gene linker (150 mJ). Oligonucleotide labeling and washing methods were described by Pennycook et al. (41). Membranes were prehybridized in separate roller tubes with 20 ml 5x SSC, 2.5% skim milk powder, 0.1% N-laurylsarcosine, and 0.02% SDS at 42°C for 4 h. Oligonucleotides V10A3, V10B, and V10C were end labeled with [-³²P]dATP and purified on Select-D G25 spin columns (5 prime3 prime, Boulder, CO). A quantity amounting to 3 x 10⁷ total cpm of either the V10A3, V10B, or V10C probe was added to the tubes containing the prehybridization solution and hybridized overnight at 42°C. Blots were washed twice for 5 min with 2x SSC, 0.1% SDS at room temperature and twice for 15 min at 42°C (V10A3), 51°C (V10B), or 57°C (V10C). The V10C primer differs from V10A and V10B template by 2 and 4 bases, respectively, V10B primer differs from V10A and V10C template by 2 and 3 bases, respectively, and V10A3 primer differs from V10B and V10C template by 7 bases. The specificity of each probe was determined experimentally by washing nylon strips containing the V10A and V10C genomic clones and the V10B RT-PCR clone 4B16 at increasing temperatures until each probe hybridized to its own V10 family member and not the other two. Membranes were placed in Molecular Dynamics (Sunnyvale, CA) PhosphorImager cassettes overnight. Screens were developed using a Molecular Dynamics PhosphorImager and Image Quant software.

ThB enrichment of bone marrow B cells

Bone marrow cells collected from both femurs and tibiae were resuspended at 1 to 4 x 10⁷ cells/ml PBS and incubated with biotin-labeled anti-ThB at 4°C with gentle shaking for 30 min. Cells were washed twice in HBSS, pH 7.4, and mixed with Dynabeads M-280 streptavidin at a ratio of 4:1 beads/cell, with the beads at 1 to 2 x 10⁷ beads/ml. Cells and beads were incubated at 4°C with rotation for 30 min. The cells bound to the beads were collected with a Dynal (Great Neck, NY) MPC-1 or MPC-2 magnet. Genomic DNA was prepared from the ThB⁺-selected population. Flow-cytometric analysis of cells before enrichment showed ThB⁺ cells ranging from 12 to 24% of total bone marrow cells. After enrichment, 4 to 5% of unselected cells were ThB⁺ (data not shown).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation and sequencing of V10C germline gene

The V10 family contains three members (37). Southern blots of BALB/c BamHI-digested or EcoRI-digested kidney genomic DNA with a 0.9-kb PC3386 probe labeled with ³²P revealed two and three strongly hybridizing bands, respectively (data not shown and (37)). The smaller (5.2-kb) BamHI band is a doublet that is known to contain both V10A and V10B. The V10A gene resides on a 5.2-kb EcoRI fragment, while the V10B gene resides on a 3.7-kb EcoRI fragment (42). Although unconfirmed, the 7.4-kb BamHI and the 2.6-kb EcoRI bands most likely contain the V10C gene.

The V10C germline gene was isolated from a BALB/c kidney genomic library and sequenced beginning from 600 bp 5' of the transcription start site to 1700 bp 3' of the heptamer/nonamer. Analysis of the V10C sequence (Fig. 1) shows it is structurally functional. There are no obvious defects such as frameshifts or missense mutations to easily explain the lack of detection of V10C in functional Abs. Comparison of the germline sequence of V10C with germline V10A and V10B sequences reveals that it is most closely related to V10B, sharing 97% homology in the coding region and 94% homology with V10A. The majority of base substitutions among the V10 family members occur in the CDRs. The promoter region, splice site, and RSS of V10C are also intact. V10C differs from V10A in the last position of the nonamer; however, this difference is shared with V10B, which is known to be expressed, and most likely does not account for a decrease in recombination efficiency.

The V10C amino acid sequence (Fig. 2) is 91 and 94% homologous to V10A and V10B, respectively, differing primarily in the CDRs. The V10C sequence contains two unusual substitutions: a Cys to Tyr substitution at position -1 of the leader peptide, and a Thr to Ala substitution at position 69 in framework 3. Neither of these substitutions is thought to interfere with light chain processing, folding, or the ability to pair with heavy chain (see Discussion).

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Translation of V10 germline genes. V10A and V10B sequences were obtained from GenBank, V10C was translated from the germline sequence.

Semiquantitation of spleen V10 mRNA

A search of DNA databases revealed that both V10A and V10B light chains are utilized in response to a wide variety of T-dependent and T-independent Ags in different inbred mouse strains, while V10C has not yet been detected in a functional Ab. We wanted to determine whether this was due to the lack of appropriate Ags selecting for V10C or if V10C is underexpressed in the spleen. It has been estimated in murine spleen that individual V exons represent approximately 0.6% of total mRNA (43).

RT-PCR experiments to detect the presence of V10A, B, and C message in an adult BALB/c mouse were performed with the 5' V10A, B, and C primers shown in Figure 1 and Table I and a 3' primer (3'KC) from the C region (Table I). Specific conditions were determined in cross-priming experiments in which cDNA from both V10A (226.1)- and V10B (H24C2)-producing hybridomas was amplified with the three gene-specific V10 primers and the C region primer 3'KC (data not shown). Using specific conditions established in the hybridoma cross-priming experiments, we were able to detect V10A, B, and C mRNA in the spleen of an adult mouse (Fig. 3). V10A and V10B appeared as intense bands, while V10C was barely visible. This was the only mouse tested that had visible V10C bands after a single PCR (see below). The RNA from this mouse was consumed during assay development and was not quantitated. Nineteen clones each of V10A, V10B, and V10C RT-PCR products were sequenced to establish the specificity of each reaction (data not shown). Mispriming events were not observed.

View larger version (79K): [in this window] [in a new window]   FIGURE 3. V10 RT-PCR of BALB/c spleen. A total of 5 µg of splenic RNA was reverse transcribed, and 2 µl of cDNA was used as template for PCR with the V10-specific primers and the C region primer 3'KC under conditions specific for each gene (see Materials and Methods).

View larger version (53K): [in this window] [in a new window]   FIGURE 4. Semiquantitative V10A RT-PCR. A, representative semiquantitative RT-PCR of V10A message from BALB/c spleens. A total of 1 µl of cDNA and 1 µl of diluted A5 standards was amplified with the V10A and 3'KS primer, as described in Materials and Methods. Thirty-five microliters of each sample and standard were electrophoresed in a 3% gel and stained with ethidium bromide. B, Band intensities from the A5 standards in the gel shown in A were measured in a fluorimeter and plotted versus the number of standard molecules calculated at each dilution. Band intensities for the four samples were interpolated into the A5 standard curve to calculate the number of V10A target structures in the cDNA. Similar experiments were performed to measure V10B. V10C was not detectable by this method, and was thus deemed to be present at or below the detection limit of the assay (approximately 1100 target structures).

View larger version (24K): [in this window] [in a new window]   FIGURE 5. V10 and total semiquantitative RT-PCRs from BALB/c spleens. Summary graph of V10 and total semiquantitative RT-PCRs from BALB/c spleens. For each mouse, the V10A, V10B, and total quantitations were determined from the same cDNA preparation in multiple assays. The V10C data shown represent experiments from all four mice.

V10 expression in bone marrow and fetal liver

It has been shown that V10 genes contribute substantially to the repertoire in early B cell development and that the V10 sequences detected in fetal liver, fetal omentum, and bone marrow all derived from the V10A or V10B genes (23, 24). Our examination of the expression of V10 genes in bone marrow and fetal liver revealed that V10A mRNA was readily detectable using a single RT-PCR reaction in both adult bone marrow and fetal livers. In fetal liver, V10A was detectable as a faint band at day 16 and increased in intensity through day 19 (data not shown). Neither V10B nor V10C was detectable using a single RT-PCR reaction in either tissue. Using a nested PCR reaction, both V10B and V10C mRNAs could be detected in the bone marrow, and by day 18 in fetal livers (data not shown).

Comparison of V10A and V10C promoter efficiency

Since V10C is poorly expressed in splenic B cells, we next compared the V10C promoter efficiency with that of V10A. The major elements of a V gene promoter are the octamer (44, 45) and a TATA box. These elements are contained within a 70- to 100-bp region that has been defined as the minimal promoter necessary for driving transcription (44, 46, 47). A comparison of the V10A and V10C gene promoters is shown in Figure 6A. Both promoters contain identical octamers that differ from the consensus octamer by 2 bases. Neither V10A nor V10C contain a consensus TATA box, but both genes have the sequence TAATT at position -27. V10A and V10C also contain the pentadecamer and Y sites, which have also been implicated in activation of transcription (44, 48). The pentadecamers for both genes are identical, lie upstream of the octamer, and differ from the consensus pentadecamer by a single base (TGCAGCTGTGCTCAG). The Y site, unlike those reported by Atchison (48), is downstream of the octamer and differs from the consensus by 2 bases (CTTCCTAT). Overall, the 142-bp V10A promoter differs from that of V10C at three positions, an A to C substitution at position -6, a C to G substitution at position -42, and an additional A nucleotide at position -97 just upstream of the octamer. Two of these substitutions, the C to G at position -42 and the A to C at position -6, lie within a potential transcription-regulatory element (CANNTG), the E-box (49, 50). At position -42, the V10A E-box sequence (CAGATT) differs from the V10C sequence by a single base (CACATT). In the downstream E-box beginning at position -10, both V10A (CAGCCTG) and V10C (CAGCATG) contain unique insertions into an E-box sequence.

View larger version (34K): [in this window] [in a new window]   FIGURE 6. V10A and V10C promoter alignments and efficiencies in B cells of different lineages. A, V10C and V10A promoters were made by PCR (see Materials and Methods) and cloned into the PGL3 luciferase reporter vector containing the intronic enhancer. Known regulatory sequences are in bold. PGL3enAS, PGL3enCS, and PGL3en without insert were cotransfected into B, immature B cells (Wehi 231.4), mature B cells (A20), and the plasmacytoma line Sp2/0, and C, pre-B cells with the control vector pCMV-ß. Each transfection was repeated six times. Luciferase expression was assayed after 24 h, and differences in V10A and V10C promoter-driven expression of luciferase were compared by one-way ANOVA.

V10-J recombination

The RSS of V10A and V10C differ at the last position of the nonamer, a T to G substitution. The V10B and V10C RSS, however, are identical, and V10B mRNA is present at similar levels as V10A in adult spleen, suggesting that the RSS difference between V10A and V10B/C does not influence the efficiency of V10C recombination. While the efficiency of the V10C RSS is likely to be equivalent to that of V10A and V10B, we do not know whether the frequency of V10C recombination is also equivalent. To examine this question, PCR was performed on genomic DNA using a 5' probe and an annealing temperature that would amplify all three family members, and a 3' probe lying downstream of J5. Thus, all amplified rearrangements would be in the context of the C region and not as a part of a reciprocal joint to a VJ recombination. We examined DNA isolated from both spleen and ThB-enriched bone marrow cells. ThB is a differentiation Ag expressed on thymocytes and on B cells, with its expression first seen on pre-B cells, the differentiation stage at which light chain rearrangement begins (55, 56). Thus, enriching for ThB-expressing cells would capture all B-lineage cells in the bone marrow undergoing the transition to or already having made the transition to an immature B cell.

V10 recombination products were hybridized with ³²P-labeled V10A3, V10B, and V10C oligonucleotide probes. Recombinations to all four J genes were detectable for all V10 family members in both ThB-enriched bone marrow and spleen (Fig. 7). This assay is not quantitative, and the less intense V10C bands most likely result from the higher wash temperature needed for specificity of the V10C probe. In preliminary experiments done for probe specificity, specific band intensities (for all probes) were shown to decrease as wash temperatures were elevated (data not shown). Additionally, although the V10 PCR annealing temperature was adjusted to amplify all V10 products, the Gen7 primer differs from V10C by a single base and may influence yield of V10C products. Because V10C recombinations are detected in both tissues, these data suggest that most V10C-rearranged cells do not undergo negative selection and elimination in the bone marrow. It is not known whether these V10C rearrangements are productive or nonproductive.

View larger version (57K): [in this window] [in a new window]   FIGURE 7. V10 recombination products. A, V10-J recombination products from BALB/c spleen and ThB-enriched bone marrow genomic DNAs were PCR amplified and blotted with 32P-labeled V10A3, V10B, or V10C oligonucleotides, as described in Materials and Methods. Shown are representative results from one of three mice. Blots were washed at the indicated temperatures 2 x 15 min in 2times] SSC/0.1% SDS. B, Positive and specificity controls for V10A, B, and C were included on each PCR gel and blotted and washed, as described above.

Sequence analysis of V10A and V10C PCR clones

We analyzed the translated sequences of cloned V10A and V10C RT-PCR products derived from the initial control assays. Nineteen sequences for each gene were analyzed. Of these 19 sequences, 10 of 19 V10A and 13 of 19 V10C sequences were established as unique based on their nucleotide sequences.

Of the 10 V10A rearrangements, 7 recombined with J1, and 3 with J2 (Table II). Three of these rearrangements were nonproductive due to out-of-frame rearrangements (2 involving J1, and 1 with J2). Of the 13 V10C sequences examined, 9 were to J1, 2 to J4, and 1 each to J2 and J5 (Table II). As with V10A, three of the V10C rearrangements were also nonproductive due to out-of-frame rearrangements (2 to J 1, and 1 to J4 rearrangement).

View this table: [in this window] [in a new window]   Table II. Cloned V10 junctions1

V10A and V10C harbor LINE elements downstream of the coding region

V10 gene segments, as well as up to 40% of all V genes, lie in opposite transcriptional orientation relative to the J locus and rearrange by inversion (22). Recombination by inversion results in the formation of reciprocal products that are retained on the chromosome. The V10 family and other families that rearrange by inversion, such as V4, V8, and V12,13, contain a conserved BamHI site approximately 1 kb downstream of the coding region (22, 57, 58). Rearrangement of one of these genes with J1 results in an 8-kb BamHI reciprocal product. An analysis of splenic DNA for 8-kb BamHI reciprocal products resulted in an estimate that approximately 25% of V alleles retain this BamHI site. Since this site is conserved downstream of many different V genes, it has been postulated that this site may play a role in regulation of V rearrangement (57). Sequences 1 kb downstream from the germline V10A and V10C genes and a germline V8 gene were determined. Both V10A and V8 germline sequences contain the 1-kb BamHI site, whereas the V10C sequence does not. It is known from the restriction maps of other investigators (42) that the V10B germline gene also has a 3' BamHI site. The BamHI sites for V10A and V8 are contributed by LINE elements (59) that lie in an inverted orientation 600 and 850 bp downstream of the V10A and V8 coding regions, respectively. The LINEs are truncated at different points in their respective 3' regions, but both contain the BamHI site at position 6989 of the LINE sequence. Like V10A, V10C harbors a LINE element 600 bp downstream of the coding region, but the region containing the BamHI site at position 6989 is absent due to a truncation at position 5690 of the LINE sequence.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies examining the generation of Ab diversity have compared the expression of different V_H or V_L families (10, 11, 12, 14, 15, 16, 17, 18). Others have looked at promoters or RSS of individual genes and identified differences that lead to more or less efficient transcription (4, 6, 35) or recombination (60). Recently, Buchanan et al. compared the promoters of V_H families and showed that there may be differential transcription and regulation of V_H promoters in vivo (35). In this study, we have examined and compared both the promoters and RSS of individual members of one V family. This study demonstrates that a structurally functional V gene, with no apparent defect in its promoter sequence or RSS, is underutilized in both the emerging and adult repertoires.

The V10 family contains three members, V10A and V10B, whose sequences have been previously published (42, 61), and V10C, whose sequence is reported in this work. It was of interest to study this family because V10A and V10B are utilized by a variety of inbred strains in response to a diverse array of Ags, including T dependent, T independent, and autoantigens, while V10C has not been detected in a functional Ab. All of the V10 sequences in the GenBank database could be assigned to either the V10A or V10B genes. Indeed, a V10C sequence has only been reported once, as part of a reciprocal element to a VJ join that has undergone a V10C to J1 rearrangement (22).

The V10C gene is structurally intact and does not contain stop codons, deletions, or insertions that might explain its underrepresentation in splenic B cells. It is 94% homologous at the nucleic acid level and 90% at the amino acid level with V10A and V10B. The V10C promoter, splice sites, and RSS are intact. V10C contains a rare Thr to Ala substitution at position 69 in framework 3. Threonine is highly conserved at this position, but an Ala residue has been detected at this position in a V10A sequence (62), and is therefore not believed to impede light chain folding or pairing with heavy chain. Another rare substitution in V10C is Cys to Tyr at position -1 of the leader peptide. Leader peptides are variable at this position (63), and one human light chain signal peptide is known to contain a Tyr at position -1 (64). Therefore, the Cys to Tyr substitution at position -1 of the leader peptide is unlikely to interfere with light chain processing.

We have shown that V10A and V10B are transcribed at equivalent levels in adult spleen, while V10C is present at levels at least 1000-fold lower. V10A mRNA was readily detectable in day 16 to 19 fetal livers and in bone marrow. V10B and V10C were only detectable in day 18 to 19 fetal livers and bone marrow using a nested PCR, indicating that both V10B and V10C mRNAs are present at significantly lower levels than V10A in early development. Clones expressing V10B may be expanded in the spleen in response to environmental Ags. Low levels of V10C mRNA in spleen may be due to the lack of a suitable environmental stimulus to expand V10C-expressing clones. We observed no increase, however, in V10C mRNA levels after stimulation of splenic B cells with LPS.

To further examine the low levels of V10C mRNA expression, the sequences of the V10A and C promoters and their ability to drive expression of reporter constructs were compared. V promoters contain several motifs that have been demonstrated to play a role in transcription. The most important of these sequences is the octamer (44, 45) element to which bind the Oct-1 and Oct-2 proteins. Other regulatory elements of the promoter include the Y site, which has been shown to lie upstream of the octamer and can compensate for a mutated octamer in a V19 gene (48), and the pentadecamer (44), which is highly conserved among V promoters. E-boxes, originally described by Ephrussi (49), are contained within the pentadecamer sequence (44) and function as promoter elements. Evidence that these sites can function as promoter elements was provided by Hogbom et al. (50), who demonstrated that a range of nuclear proteins bound to the SP6 promoter pentadecamer. Interestingly, these sites had variable efficiency as promoter elements depending on the cells/cell lines used in the experiments.

A comparison of the V10A and C promoter sequences shows there are no differences between V10A and C in the major promoter elements, the octamer, pentadecamer, Y site, and TATA box (Fig. 6A). V10A and V10C promoters each contain one E-box motif within the pentadecamer and two potential E-boxes downstream of the octamer. There are three nucleotide differences between the V10A and C promoters. At position -97, the V10C promoter contains a string of four As immediately upstream of the octamer, while the V10A promoter contains five A nucleotides. Baumruker et al. (65) have shown that octamer-flanking sequences can alter the affinity of Oct-1 binding to the V_H octamer. Likewise, Sigvardsson et al. (66) have shown that 3' flanking sequences of the SP6 promoter can affect the affinity of Oct-2A binding to the octamer. The other differences are in two potential E-box sites at positions -42 and -6. At position -42, the V10A (CAGATT) and V10C (CACATT) E-box sequences differ by a single base. At position -6, V10A and V10C each have a unique insertion into the E-box sequence.

To determine whether differences in the V10A and V10C promoters could explain the difference in mRNA levels between these genes, we examined the V10A and V10C promoter efficiencies in transiently transfected B cell lines representing different developmental stages. While the V10C promoters worked as efficiently as V10A promoters in immature B cells, mature B cells, and plasma cells, a statistically significant difference was observed in pre-B cells (Fig. 6C). This phenomenon was seen in all four pre-B cell lines tested. The biologic significance of this difference is unclear. It is possible that a complex of pre-B cell nuclear transcription factors binds with less affinity to the V10C promoter due to one or all of the three nucleotide changes in this promoter, resulting in lower levels of V10C transcripts. Experiments are in progress to address this question. The late pre-B cell stage in development is when light chain rearrangement begins. If the V10C promoter is inefficient in pre-B cells, one possibility might be that insufficient amounts of V10C protein are produced at this critical point in the B cell developmental pathway to express surface IgM. In such a scenario, this cell could undergo further light chain recombination, receptor editing, or be eliminated by apoptosis.

We next examined the ability of the V10C gene to rearrange. The V10C RSS differs from that of V10A by one nucleotide, the terminal base in the nonamer. This position has not been shown to affect recombination efficiency. Since V10C and V10B have identical RSS, this change is not likely to be responsible for the underutilization of the V10C gene. Indeed, we demonstrated that V10C is capable of recombination, as rearrangements are detected in both spleen and bone marrow of adult mice. The J 5–3 primer used in the PCRs is located downstream of J5; thus, the recombination products are in the context of the C region and are not the result of secondary recombinations with displaced J resulting from inversional recombinations of other V genes. Since small amounts of V10C mRNA are detectable in spleen, bone marrow, and fetal liver, some of the rearrangements may result in productive V-J joins, although it has been shown that nonproductive rearrangements can be transcribed at levels comparable with or even greater than productive rearrangements (38).

Analysis of 13 unique V10C and 10 unique V10A junctions revealed some interesting differences. Seven of the nine in-frame V10C-J1 junctions were missing the proline residue at position 95. This was not seen for any in-frame V10A junctions. Proline 95 is invariant in murine -chains and is present in all of the V10A or V10B sequences obtained from GenBank. It is possible that the loss of proline 95 results in light chains that cannot fold properly or pair effectively with heavy chains.

Finally, it is not known how overall chromatin configuration affects the expression of V genes. LINE elements are commonly found in the V and V_H loci of mice (67, 68, 69). In some cases, LINEs have been involved in aberrant rearrangements of these genes (70). LINEs are transposable elements that contain two open reading frames, one of which codes for a reverse transcriptase (59), and recently, LINE proteins have been identified (71, 72). LINE elements are present at 10⁴ copies per haploid genome, and many of these are truncated, usually at the 5' end (59). It has been shown that at least 25% of V genes contain a common BamHI site 3' of the coding region (57). We have shown that this BamHI site is contributed by LINE elements. Both V10A and V10C contain LINE elements in this position, but only V10A has the conserved BamHI site. We have not sequenced a V10B gene to see whether there is a LINE element present, but, from published restriction maps (42), it does contain the conserved BamHI site. It is not known whether the truncation of the V10C LINE affects the overall expression of this gene or interferes with proper recombination, resulting in a high frequency of V10C rearrangements that lack the invariant proline 95.

Because the V10C gene is structurally functional, the lack of hybridomas and myelomas utilizing V10C suggests that V10C-expressing cells are negatively selected. Alternatively, V10C-rearranged cells may not be actively selected, either negatively or positively. Positive selection has been demonstrated for a specific VHCDR3 sequence to make the transition from a pre-BI cell to a pre-BII cell (73) and for certain V_H-VL pairs to move immature cells from the bone marrow into the immunocompetent B cell pool in the periphery (8). Because V10C rearrangements are detected in both the spleen and bone marrow and the V10C promoter is inefficient in pre-B cells when rearrangement begins, V10C-rearranged cells may be unable to undergo selection. There may be a threshold of surface Ig expression for selection to occur. Inefficient V10C transcription may not produce enough V10C protein to reach this threshold. Similarly, an intrinsic defect in recombination of V10C, resulting in proteins unable to pair efficiently with heavy chain, would have the same outcome. Because such cells are not selected, they can rearrange the other allele, the locus, or undergo receptor editing. Such cells would then have a chance to undergo positive or negative selection, but this selection would not be due to V10C expression. Regardless of the reason for the underutilization of V10C in the Ab repertoire, it is clear that not all structurally functional genes contribute to Ab diversity.

	Acknowledgments

 
We thank Drs. Fred Mushinski and Walter Gerhard for gifts of cell lines, Dr. Kevin Holmes and Larry Lantz for biotin-labeled ThB, and Drs. Stuart Rudikoff, Steve Bauer, and Mark Brunswick for critical review of the manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Marjorie Shapiro, Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Bldg. 29B, Room 5E12, Rockville, MD 20852. E-mail address:

² Abbreviations used in this paper: RSS, recombination signal sequence; CDR, complementarity-determining region; TAE, 40 mM Tris-Cl, pH 7.8, 20 mM sodium acetate, 1 mM EDTA.

³ The V10A and V10B germline genes are the V10.1^b (AJ1) and V10.2^b (AJ2) genes characterized by Kim et al. (42) from A/J mice and the V10 ars-a gene and V10b gene characterized by Victor-Kobrin et al. (61) from BALB/c mice.

Received for publication October 16, 1997. Accepted for publication May 4, 1998.

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