Gene conversion–like sequence transfers in a mouse antibody transgene: antigen selection allows sensitive detection of V region interactions based on homology

Hwei-Fang Tsai1, Nicole D'Avirro2 and Erik Selsing1,2,3

1 Immunology and
2 Genetics Programs, and
3 Department of Pathology, Tufts University School of Medicine, Boston, MA 02111, USA

Correspondence to: E. Selsing, Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Gene conversion is important for antibody diversification in chickens, rabbits and cows. In mice, however, conversion events appear to be infrequent among endogenous antibody genes. DNA sequence transfer events that resemble gene conversions have been reported for a mouse H chain transgene (VVCµ) that contains two closely spaced homologous VDJ segments. Surprisingly, these reported VVCµ sequence transfers were found frequently among mouse B cells responding to immunization. Transgene sequence transfers could be occurring at high frequency in responding VVCµ B cells or could be occurring at lower frequency with subsequent amplification by preferential antigen selection. To distinguish these possibilities, we have analyzed a second transgene (InVVCµ) that is identical to VVCµ except that the two VDJ regions have been exchanged in position. We find that transgene sequence transfers are much less frequent among responding B cells in InVVCµ mice, demonstrating the importance of selection in the frequent transgene conversions observed in VVCµ mice. These results suggest that mice, like other species, can use gene conversion to diversify antibodies. Such diversification events are apparently infrequent, however, and might only be detected among endogenous Ig genes with a favorable arrangement of V genes and an antigenic stimulation that selects cells with conversions. For both VVCµ and InVVCµ mice, conversion-like sequence transfers are strongly correlated with somatic hypermutation. Based on these results, we hypothesize that, in mice, gene conversions represent infrequent alternative reactions of a homology-based DNA repair process that is central in the somatic hypermutational mechanism.

Keywords: B cells, generation of diversity, H chain, Ig, somatic mutation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The V(D)J recombination process that joins antibody gene segments during B cell development is an important mechanism in the generation of a diverse repertoire of antibodies in all vertebrate species (1,2). In higher vertebrates, other processes are also involved in diversifying antibody V regions. Somatic hypermutation is one diversifying mechanism typified by the introduction of single point mutations into antibody V(D)J segments (reviewed in 3). Gene conversion is a distinct diversifying mechanism characterized by transfers of homologous sequences from a donor antibody V gene segment to an acceptor V gene segment (47). If donor and acceptor segments have numerous sequence differences then gene conversion can introduce a set of sequence changes into a V region by a single event.

Somatic hypermutation appears to operate to diversify antibody genes in all higher vertebrate species. For the most part, hypermutation appears to be activated in B cells by antigenic stimulation (reviewed in 8). On the other hand, only some species have been found to exhibit frequent gene conversion events during antibody diversification. Depending on the species, gene conversion events can occur before and/or after antigen exposure during B cell differentiation. Chickens have been definitively shown to exhibit gene conversion in maturing B cells (4,5), whereas rabbits and cows also show DNA sequence transfers between V genes that resemble gene conversion (6,7). A major hallmark of gene conversion is that sequence transfers occur in a non-reciprocal manner from donor to acceptor; this feature has not yet been definitively demonstrated in rabbits or in cows.

In mice, several published analyses have investigated a possible role for gene conversion in diversifying mouse antibodies. All of these studies have examined particular V genes to look for evidence of conversion events. Although some studies have observed V gene alterations that could be due to conversion events (913), many analyses have not detected any evidence of conversion (1419).

Despite the fact that definitive gene conversion events have not been detected among endogenous mouse Ig genes, studies in our laboratory have shown that the mouse VVCµ H chain transgene, which contains two homologous VDJ segments separated by 1.5 kb, can undergo DNA sequence transfers that resemble gene conversion events in immunized mice (20). Surprisingly, the frequency of sequence transfers observed among responding B cells from immunized VVCµ transgenic mice is quite high; up to 20% of antigen-specific IgG-producing hybridomas derived from immunized animals display conversion-like sequence transfers.

Sequence transfers in the VVCµ transgene are closely linked with somatic hypermutation (20). We have suggested that this observation could indicate that somatic hypermutation of Ig genes occurs by error-prone DNA repair during homology-based sequence transfers (21). The apparently low frequency of detectable gene conversions among mouse Ig genes could indicate that this hypothesized somatic hypermutational mechanism operates more frequently during sequence transfers between chromatids rather than between similar, but non-identical, sequences (21). Recent studies of DNA cleavages associated with somatic hypermutation also suggest that homology-based DNA repair might be important in the hypermutational mechanism (22).

The large numbers of responding B cells that display sequence transfers between the tandem transgene VDJ segments in VVCµ mice appear to be at odds with the notion that gene conversion between non-identical sequences is low among mouse Ig genes. This raises questions whether the VVCµ sequence transfer mechanism is unique to transgenes. The frequent sequence transfers observed in VVCµ mice could reflect a high frequency of sequence transfer events occurring in responding B cell populations or, alternatively, a low frequency of sequence transfers giving rise to a small number of cells that are then preferentially amplified due to affinity selection. In the VVCµ transgene, the upstream VDJ segment is derived from the 2B4 hybridoma and the downstream VDJ is derived from the R16.7 hybridoma; these two VDJ regions differ by only 17 nucleotides. Binding measurements indicate that the affinity of the 2B4 antibody for the Ars hapten is only 2-fold higher than the R16.7 antibody affinity (20). In addition, sequence comparisons of VVCµ transgene H chains from hybridomas exhibiting sequence transfers did not appear to show selection for particular amino acid sequences (21). These two observations suggested that sequence transfers in the VVCµ transgene might be occurring with an unusual high frequency in B cells responding to immunization.

On the other hand, if antigen selection does preferentially amplify B cells that have undergone sequence transfers within the VVCµ transgene, then this could explain why the activity of such a transfer mechanism could be easily detected in an appropriate transgene but much more difficult to detect among endogenous antibody responses. To more directly investigate the possible role of antigen selection in preferential amplification of B cells exhibiting VVCµ sequence transfers, we have produced a second transgene construct which is identical to the VVCµ transgene except that the 2B4 and R16.7 VDJ regions are exchanged in position (Fig. 1Go). In this transgene, designated as InVVCµ, the upstream VDJ segment is derived from the R16.7 hybridoma and the downstream VDJ is from 2B4.



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Fig. 1. VVCµ and InVVCµ transgene constructs. Exons are shown as open boxes, the VH promoter is shown as a solid triangle, the tandemly repeated switch region is shown as a hatched oval and plasmid sequences are indicated by the hatched boxes.

 
The design of the InVVCµ transgene is based on the notion that, if preferential antigenic amplification plays a dominant role in the high frequency of B cells displaying sequence transfers in immunized VVCµ mice, then this selection process must reflect a favorable group of H chain V region amino acid changes that are transferred into the expressed R16.7 VDJ from the silent upstream 2B4 VDJ segment. Because the InVVCµ transgene already has these favorable 2B4 amino acid sequences in the expressed VDJ segment, most sequence transfers from the silent upstream R16.7 VDJ segment would result in the introduction of unfavorable groups of amino acid changes into the expressed VDJ. Thus, InVVCµ B cells that have undergone sequence transfers would presumably not be preferentially selected during the immune response due to these unfavorable amino acid sequences in the Ig BCR and, therefore, cells showing sequence transfers in immunized InVVCµ mice should be found at much lower levels due to the reverse order of the two VDJ segments. On the other hand, if preferential selection does not play a significant role in preferentially amplifying VVCµ B cells that have undergone sequence transfers then we would expect similar frequencies of B cells exhibiting transgene conversion in immunized VVCµ and InVVCµ mice.

The analyses of InVVCµ transgenic mice described here show that the numbers of B cells which display transgene sequence transfers are much lower in immunized InVVCµ mice and indicate that antigen selection plays a major role in the high frequency of transgene conversion events detected in immunized VVCµ mice. These results provide a possible explanation for the absence of detectable sequence transfers in studies of gene conversion in specific endogenous mouse Ig genes. Our findings suggest that mice, like other species, can use gene conversion to diversify antibodies. Such diversification events are apparently infrequent, however, and might represent low-level side reactions of a somatic hypermutational mechanism that involves homology-based DNA repair processes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The InVVCµ transgene
The InVVCµ construct (Fig. 1Go) was produced using the same approach employed in constructing the previously described VVCµ transgene (20). The R16.7 VDJ sequence was PCR amplified using 5'L and JH3 primer pairs together with a pK18 (23) plasmid template. The PCR-amplified R16.7 VDJ was inserted into p5.4 (24) to produce the pInVV clone and sequence analyses confirmed the sequence of the inserted R16.7 VDJ segment. The InVV fragment was isolated from this construct and inserted into a vector containing the BALB/c Cµ exons (23) to produce the InVVCµ transgene.

Oligonucleotides
5'L: 5'-CCGCTCGAGACACACTGACTCAAACCATG-3'

JH3: 5'-CCGCTCGAGACAGTGACCAGAGTCCCTTG-3'

LRI: 5'-CCGAATTCACACACTGACTCAAACCATG-3'

L3RI: 5'-AGAATTCCTCTTCCTCCTGTCAGTAAC-3'

C{gamma}RI: 5'-GGAATTCCGGGGCCAGTGGATAGAC-3'

C{gamma}H2: 5'-CCGAATTCTTTGGGGGGAAGATGAAGAC-3'

TgND: 5'-AGATCGAATTACTATGGTGGT-3'

2B4CDR2: 5'-CCGAATTCTATAAAAGCTTCCAGTACTTT-3'

R16.7CDR2: 5'-GTACTTAGTATAAACATTTCCAGGATTAA-3'

pUC18F: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'

Transgenic mice, immunization and serology
The VV5 transgenic line carrying the VVCµ transgene has been described previously (20). InVVCµ transgenic mice were produced as described previously using fertilized C57BL/6 eggs (23). Founder mice were identified by Southern blot analysis of tail DNAs. Mice were immunized and the levels of various antibodies were determined by ELISA assays as described earlier (25).

RT-PCR Amplification of Ig messages and RT-PCR/Southern blot `conversion assay'
Total RNAs were isolated from splenocytes using either Trizol reagent (Gibco/BRL, Gaithersburg, MD) or as described in (26). Approximately 500 ng of RNA and oligo dT (Promega, Madison, WI) were used to make cDNA with reverse transcriptase (Gibco/BRL).

For the amplification of transgene specific IgG messages, two nested rounds of amplifications were performed on cDNA templates using LRI(F), C{gamma}H2(R) and L3RI(F), C{gamma}RI(R) as primers and Taq polymerase (Gibco/BRL). The cycling program consisted of 25 cycles at 95°C for 1 min 20 s, 55°C for 2 min and 72°C for 2 min with 1-s increases in the time of extension in each cycle and was performed in a thermocycler (Perkin-Elmer, Shelton, CA). Amplified IgG messages were subjected to 1% agarose gel electrophoresis, Southern transfer, and hybridization with the 2B4CDR2 and R16.7CDR2 5'-labeled CDR2-specific oligonucleotides.

Dot-blots, hybridization and DNA sequencing
Cloning of RT-PCR products was done using the TA and TOPO TA cloning kits (Invitrogen, Carlsbad, CA). PCR clones with inserts were identified by the EcoRI restriction pattern. Positive clones were subjected to dot-blot analysis using the 2B4CDR2, R16.7CDR2 and TgND oligonucleotide probes. Samples of plasmid miniprep DNAs were boiled for 10 min, adjusted to 5xSSC and placed on ice. Nylon membranes were soaked in 6xSSC for 10 min and aliquots of the DNA preparations were applied. Membranes were denatured (1.5 M NaCl, 0.5 M NaOH for 10 min), neutralized (1 M NaCl, 0.5 M Tris–HCl, pH 7.0 for 5 min), air-dried and UV cross-linked (1200 J, UV Stratalinker).

Oligonucleotides were labeled using polynucleotide kinase (New England Biolabs, Beverly, MA). Blots were incubated in 3xSSC at 42°C for 15–30 min, in 3xSSC, 10xDenhardt's solution at 42°C for 30–60 min, in prehybridization solution (3xSSC, 10xDenhardt's solution, 0.1% SDS, 50 µg/ml salmon sperm DNA boiled for 5 min) at 42°C for 2 h. Hybridization reactions were done with hybridization solution (labeled oligonucleotides in prehybridization solution) at different temperatures according to the probe used. The hybridization reactions were incubated for >18 h. The hybridization temperature for the TgND oligonucleotide was 44°C. Hybridization temperatures for 2B4 CDR2 and R16.7 CDR2 oligonucleotides were 42°C. After hybridization, blots were rinsed in 3xSSC solution at room temperature. The blots were washed at washing temperature (2–3°C lower than hybridization temperature) twice for 15 min each in 750 ml 3xSSC, 5mM EDTA, 0.1% SDS, 5xDenhardt's solution, 50 µg/ml sonicated salmon sperm DNA) and washed for 15 min in 750 ml 1xSSC, 5mM EDTA, 0.1% SDS. The blots were air dried and analyzed by autoradiography.

Miniprep DNAs were prepared for sequencing using Qiagen (Valencia, CA) mini-columns. Sequence analyses were performed by the Tufts Sequencing Facility using the pUC18F or LRI oligonucleotides as sequencing primers.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
InVVCµ transgenic mice
The InVVCµ transgene construct (Fig. 1Go) was microinjected into C57BL/6 one-cell embryos and two founder mice carrying the transgene were identified by Southern blot analysis. Analyses of preimmune serum from the lines showed titers of transgene-derived IgMa antibodies (18–32 µg/ml) that are comparable to the preimmune titers of transgenic IgM in the previously described ARSµ and VVCµ mice that carry similar transgene constructs (20,2325). After immunization, titers of anti-Ars IgG serum antibodies in the InVVCµ lines (total average of 390 µg/ml for eight InVV4 mice and eight InVV5 mice) were slightly lower than but similar to titers (average of 710 µg/ml) in nine mice of the VV5 strain that carries the VVCµ transgene (Fig. 2Go). Thus, the InVVCµ and VVCµ constructs appear to be expressed similarly in transgenic mice. VV5 mice have previously been reported to carry ~20–30 copies of the VVCµ transgene (20). The InVV4 line carries about six integrated copies of the transgene, whereas the InVV5 line carries ~20 copies (data not shown).



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Fig. 2. Serum titers of immunized VV5 and InVVCµ mice. Titers of serum anti-Ars IgG for individual immunized mice (solid circles) are shown. All mice were immunized with three injections of Ars–KLH.

 
PCR assay for transgene conversion
Transgene conversion events were previously found in VVCµ transgenic mice by sequence analyses of hybridomas (20). We designed an RT-PCR/Southern blot approach for assessing transgene conversion that was more rapid than hybridoma production and that could be used to analyze a number of animals concurrently. In the VVCµ transgene, only the downstream R16.7 VDJ has a promoter element (Fig. 1Go) so that transcribed transgenic mRNAs are derived from this VDJ sequence. Transgene conversion events act to transfer sections of 2B4 VDJ sequences into this transcribed downstream R16.7 VDJ region. Therefore, transgene-derived mRNAs in cells that have undergone transgene conversion events would be expected to exhibit segments of 2B4 sequences. Because there are 17 nucleotide differences between the R16.7 and 2B4 VDJ segments, oligonucleotide hybridization probes specific for either R16.7 or 2B4 sequences can be used to assess whether or not sequence transfers had occurred.

Based on this design, RT-PCR reactions were used to amplify DNA fragments from transgene mRNA molecules present in splenocyte populations. Transgene-derived VDJ sequences were amplified using L and C{gamma} region primers that did not distinguish the R16.7 and 2B4 VDJ sequences. In these RT-PCR amplifications, mRNAs that have transgene sequence transfers and mRNAs that do not have sequence transfers should be equally amplified. The PCR products from the amplification were then transferred to nylon filters and probed with R16.7 and 2B4 oligonucleotides. Because the greatest number of nucleotide differences between the R16.7 and 2B4 VDJ sequences are clustered in the CDR2 region and because most VVCµ transgene conversions include the CDR2 region (20), we used CDR2 oligonucleotides for specificity in the hybridizations. In our assays, PCR products derived from VVCµ transgenes that have undergone transgene conversion would hybridize with the 2B4 CDR2 probe but not with the R16.7 CDR2 probe. C{gamma} primers are used in the RT-PCR/blot assay because sequence transfers are frequent among Ars-specific IgG-producing cells in immunized VVCµ mice (20), but have not yet been detected among the many transgene-expressing, IgM-producing B cells in VVCµ splenocytes (N. D'Avirro and E. Selsing, unpublished).

Figure 3Go shows results from this RT-PCR/blot assay for transgene conversion in immunized transgenic mice carrying the VVCµ transgene. The specificity of the probe hybridization is indicated by the control samples from 61E, a hybridoma that exhibits transgene conversion, and from VV5pre, a splenocyte sample from an unimmunized VV5 mouse. PCR products from 61E hybridize with the 2B4 CDR2 probe and not with the R16.7 CDR2 probe, whereas the PCR products from VV5pre show the inverse pattern. Also analyzed in Fig. 3Go are PCR products from splenocytes from six immunized VV5 transgenic mice. In all six of these samples, some PCR products hybridize with the R16.7 CDR2 oligonucleotide probe; this is expected because, in these mice, the majority of splenic IgG-producing B cells that express transgene-derived mRNAs will have no transgene conversion. As also seen in Fig. 3Go, three of the six immunized VVCµ mice clearly exhibit PCR products that hybridize with the 2B4 probe. These results indicate that, within the splenocytes in these three mice, there are transgene-expressing cells that have undergone transgene conversion events. In addition, there clearly appears to be variability in the levels of transgene conversion found in different immunized animals.



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Fig. 3. RT-PCR/blot assay for transgene conversion in splenocytes from immunized VV5 mice. PCR products from the indicated samples were electrophoresed, transferred to nylon membranes, and hybridized with the R16.7 and 2B4 CDR2 oligonucleotide probes.

 
To confirm that RT-PCR products hybridizing with the 2B4 CDR2 probe in Fig. 3Go actually represent VVCµ mRNAs that exhibit transgene conversion, amplified DNA fragments from the PCR reaction for mouse #6 were cloned for further analysis. A panel of 113 clones was assayed for hybridization to the 2B4 and R16.7 CDR2 oligonucleotide probes by dot-blot analyses. In this panel, 61 clones hybridized only to the R16.7 CDR2 probe, seven clones hybridized only to the 2B4 CDR2 probe and 45 clones did not hybridize to either probe. This analysis confirms that the hybridization signals in Fig. 3Go represent individual PCR products with different hybridization patterns.

A random set of 36 PCR clones from mouse #6 was also chosen for sequencing prior to the dot-blot analyses. Among these 36 clones, 18 clones hybridized only to the R16.7 probe, three hybridized only to the 2B4 probe and 15 did not hybridize to either probe. This indicates that this set of clones fairly represents the entire clone panel. Sequence analyses show that seven of the 36 clones were not derived the transgene; these sequences were either not VDJ segments or were VDJ segments produced by recombination of endogenous C57BL/6 VH, D and JH regions as indicated by V sequences and V–D–J junctions that were clearly different from the transgene VDJ. None of these seven clones hybridized with either the R16.7 or 2B4 probes.

Sequences of the remaining 29 PCR clones indicated that all were derived from the VVCµ transgene. Figure 4Go shows that 11 of these clones exhibit patterns of shared nucleotide substitutions that are consistent with being derived from the upstream 2B4 VDJ segment in the VVCµ transgene. These patterns in the PCR clones are identical to the patterns found previously in hybridomas derived from VVCµ transgenic mice (20). A number of the PCR clones show 2B4-like sequences throughout the entire VDJ region, similar to genes found among hybridomas (20). For hybridomas, a PCR primer specific for the 5'-untranslated region of the R16.7 VDJ region (and which is not present upstream of the 2B4 VDJ region) has previously been used to show that VDJ transcripts exhibiting sequence transfers have 5'-untranslated regions derived from the R16.7 VDJ gene segment (20). Using this specific primer, we have also generated a smaller number of splenocyte RT-PCR clones; sequence transfer patterns are found in some of these and are similar to those in Fig. 4Go (not shown). All of these results clearly indicate sequence transfers from the 2B4 VDJ to the R16.7 VDJ within the transgene. Thus, the splenocyte RT-PCR/blot assay can detect the transgene conversion events that have previously been demonstrated by analyses of hybridomas in VVCµ mice.



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Fig. 4. Sequences of VV5 RT-PCR clones that exhibit transgene conversion. Transgene conversion is indicated by a pattern of nucleotide substitutions derived from sequence transfers between the 2B4 and R16.7 VDJ segments present in the VVCµ transgene. The R16.7 and 2B4 sequences are shown at the top. All nucleotides in the codons are shown for R16.7; for other clones only the differences from the R16.7 sequence are shown and identities are indicated by dashes. Only those codons that exhibit differences from the R16.7 sequence within the set of VV5 RT-PCR clones are shown. Clones 158, 61, 36, 62 and 45 show sequence patterns indicating that these may be derived from sister cells originating from a single precursor B cell.

 
All of the splenocyte PCR clones that displayed sequence transfers also exhibited numerous somatic mutations, consistent with the correlation between the sequence transfer and hypermutational processes that has been reported previously (20). Among the 11 clones with transfers, eight hybridized only very weakly or not at all with the 2B4 oligonucleotide probe. All of these eight clones showed somatic mutations within the CDR2 site of hybridization for the 2B4 probe. These results indicate that a majority of the splenocyte B cells in immunized VVCµ mice that have undergone transgene conversion events will be missed by the RT-PCR/Southern blot or PCR clone dot-blot assays due to somatic mutations in the VDJ CDR2 region that disrupt hybridization with the 2B4 oligonucleotide probe.

The sequence transfers found in RT-PCR clones from immunized VV5 splenocytes are highly correlated with additional somatic mutations (Fig. 4Go). Discounting nucleotide changes introduced from the upstream 2B4 sequence, the RT-PCR clones that exhibit sequence transfer exhibit an average of twice as many somatic mutations (~10/clone) as found in RT-PCR clones that do not show sequence transfers (~5 mutations/clone). In addition, among RT-PCR clones without sequence transfers, five of 18 show no hypermutation even though the RT-PCR clones with sequence transfers all exhibit five or more additional somatic mutations. Thus, as was observed in VV5 hybridomas (20), there is a strong linkage between sequence transfers and somatic hypermutation in VV5 splenocytes.

Our analyses showed that 11 of the 29 sequenced PCR clones (38%) from VVCµ mouse #6 exhibit transgene conversion events. The level of conversion-like sequence transfers found in this analysis is somewhat higher than, but similar to, previous work where 5–20% of anti-Ars IgG-producing hybridomas from immunized VV5 mice exhibited transgene conversion (20). The RT-PCR/blot analyses (Fig. 3Go) also confirm the results from hybridomas indicating that different immunized VV5 mice exhibit different levels of transgene conversion. Thus, despite that fact that somatic mutations interfere with the detection of many B cells exhibiting transgene conversion, the RT-PCR/blot assay appears to be a rapid and simple approach for the qualitative detection of sequence transfer events in heterogeneous splenocyte populations.

Southern blot analysis of transgene conversion in InVVCµ mice
Immunized InVV4 and InVV5 transgenic lines were analyzed by the RT-PCR/Southern blot assay to assess gene conversion. RT-PCR reactions using splenocyte RNAs from a panel of InVV4 and InVV5 mice were analyzed by hybridization with the 2B4 and R16.7 CDR2 oligonucleotide probes. In these mice carrying the InVVCµ transgene, conversion-like sequence transfers should be indicated by hybridization with the R16.7 CDR2 probe, whereas PCR products having no conversion should hybridize with the 2B4 probe. As shown in Fig. 5Go, results from these analyses show that, although some of the immunized InVVCµ mice show increased levels of hybridization to the R16.7 probe, one of the immunized non-transgenic control mice also shows increased R16.7 probe hybridization levels. Thus, these results suggest that C57BL/6 mice may express an endogenous H chain V gene that can hybridize with the R16.7 CDR2 probe. In contrast, we have never observed a detectable signal from immunized non-transgenic C57BL/6 mice using the 2B4 CDR2 oligonucleotide probe, suggesting that C57BL/6 mice do not express any endogenous VH genes that can hybridize with the 2B4 CDR2 probe. The expression of an endogenous C57BL/6 VH gene that can hybridize with the R16.7 CDR2 oligonucleotide indicates that the R16.7 probe hybridization bands observed in some InVVCµ splenocyte samples might also represent expression of endogenous VH genes that have undergone V(D)J recombination rather than expression of transgenes that have undergone conversion. Although we did not find evidence for expression of such recombined endogenous VH genes in our sequence analyses of PCR clones from VV5 mouse #6 (see above), this could reflect the fact that individual immunized C57BL/6 mice appear to exhibit quite different levels of this VH gene expression (Fig. 5Go). These findings complicate the interpretation of the RT-PCR/blot analyses of InVVCµ transgene conversion.



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Fig. 5. RT-PCR/blot assay for transgene conversion in splenocytes from immunized InVVCµ mice. PCR products from the indicated samples were electrophoresed, transferred to nylon membranes, and hybridized with R16.7 and C{gamma} oligonucleotide probes. EtBr staining of the electrophoresed PCR product bands is also shown.

 
Sequence analyses of transgene conversion in InVVCµ mice
Because some of the immunized InVV mice in Fig. 5Go had R16.7 probe hybridization bands that were somewhat more intense than those in the control non-transgenic mice, we cloned RT-PCR products from the samples and analyzed these by dot-blots and sequencing to determine whether transgene conversions could be found. Samples from the InVV5.3 and InVV4.2 mice were analyzed by these approaches. A total of 521 clones from the InVV5.3 mouse and 119 clones from the InVV4.2 mouse were analyzed. Among these clones, 19 InVV5.3 clones and nine InVV4.2 clones hybridized with the R16.7 CDR2 probe. These clones were all sequenced and found to represent VDJ segments produced by recombination of endogenous C57BL/6 VH, D and JH genes as indicated by VH sequences and V–D–J junction sequences (including N regions) that were clearly distinct from the VDJ segments present in the InVVCµ transgene. All of these clones had CDR2 regions that were more closely related to the R16.7 sequence rather than the 2B4 sequence, although several nucleotide differences from the R16.7 sequence were found in each clone. These results indicate that C57BL/6 mice do express one or more VH genes that can hybridize to the R16.7 CDR2 probe. This finding provides an explanation for the elevated levels of hybridization to the R16.7 probe that were observed in the RT-PCR/blot analyses of some immunized non-transgenic C57BL/6 mice in Fig. 5Go.

Because our analyses of VV5 PCR clones indicated that somatic mutations could disrupt hybridization with the CDR2 oligonucleotides, we decided to screen the InVV5.3 and InVV4.2 dot-blots with an additional oligonucleotide probe, TgND, which corresponded to the V–D–J junction of the R16.7 and 2B4 VDJ segments present in the InVVCµ transgene. A total of 190 clones hybridized with the TgND probe. Of these, 158 also hybridized with the 2B4 CDR2 probe, suggesting that transgene sequence transfers were less likely in this group. The remaining 32 clones did not hybridize with either the 2B4 or R16.7 CDR2 probes and were sequenced. All of these were found to have somatic mutations in the CDR2 region that apparently disrupted detection with the 2B4 CDR2 probe.

Inspection of the entire VDJ sequence for all but one of these 32 clones did not show any evidence of transgene sequence transfers. However, one clone obtained from InVV5.3 (clone 4.5.8) did show nucleotide changes at codons 75 and 89 (Fig. 6Go) that could be derived from a sequence transfer in the InVVCµ transgene from the upstream R16.7 VDJ region to the expressed downstream 2B4 VDJ region. It is also possible that the changes at codons 75 and 89 in clone 4.5.8 represent somatic mutations. However, both of these changes are silent with respect to the amino acid sequence of the expressed VDJ. Furthermore, there is little evidence to suggest that these sites are hotspots for somatic hypermutation as indicated by the pattern of mutations seen in the FW3 region for all the transgene-derived PCR clones (except 4.5.8) that were sequenced in our analyses (Fig. 7Go). This supports the notion that this single clone may represent a transgene sequence transfer event in the InVVCµ transgene. Our dot-blot and sequencing analyses indicated that, of the 521 InVV5.3 PCR clones, ~310 clones were transgene-derived and 211 were either non-VDJ or were derived from various endogenous genes. Thus, the percentage of transgene conversion among transgene-derived clones in the InVV5.3 mouse was maximally 1/310 or ~0.3%.



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Fig. 6. Sequence of the InVVCµ RT-PCR clone 4.5.8 that shows a potential transgene conversion event. The full sequence of the 2B4 VDJ region is shown; only differences are shown for other sequences, whereas identities are indicated by dashes.

 


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Fig. 7. Mutation frequencies in the FW3 regions of transgene-derived RT-PCR clones from the InVV5.3 transgenic mouse. The sum of observed mutations at each codon position among the PCR clones is indicated for each FW3 codon in the sequence. The arrows indicate codon positions that, in the 4.5.8 PCR clone, show sequences suggesting a transgene conversion event.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
VVCµ transgenic mice display a high frequency of gene-conversion-like sequence transfers among cells responding the Ars–keyhole limpet hemocyanin (KLH) immunization (20). However, these findings from VVCµ transgenic mice contrasted with numerous studies of endogenous mouse Ig genes, which had not detected any gene conversion events contributing to antibody diversity (1419). The presence of multiple transgene copies and the close spacing (1.5 kb) of the VVCµ VDJ segments involved in the sequence transfers were suggested to be possible features that might account for the disparity between the findings in transgenic and wild-type mice (21).

Our studies of InVVCµ transgenic mice now show that transgene sequence transfers are observed at a much lower frequency in these mice than in VVCµ mice. In InVVCµ mice <0.3% of the responding B cells exhibit transgene conversion, compared to the 5–38% found in VVCµ mice. Because the only difference between the InVVCµ and VVCµ transgenes is the location of the 17 nucleotide differences that distinguish the two VDJ segments in the transgenes, the intrinsic frequency of sequence transfers for the two transgenes is almost certainly the same. Furthermore, transgene copy number is not likely to affect the sequence transfer frequency because VVCµ mice with about three, 20 and 100 copies all exhibit high levels of sequence transfers (20), whereas InVVCµ mice with about six or 20 copies both exhibit low levels. In addition, transgene expression levels also do not appear to affect the transfer frequency because all VVCµ and InVVCµ mice exhibit similar transgene expression as indicated by levels of transgene-derived IgM on the surface of splenic B cells (not shown). These considerations indicate that the large differences in the number of sequence transfers found after immunization of the two types of mice are due to differential antigenic selection of those B cells that have undergone transgene conversion events. Thus, although tandem transgene arrays and the close transgene VDJ spacing might affect the intrinsic frequencies of sequence transfers, these transgene features (which are present in InVVCµ) are not sufficient to lead to an easily detectable level of sequence transfers in immunized mice. Preferential selection clearly plays a major role in the level of conversion-like events found in responding transgenic B cells.

It is notable that the single InVV5 PCR clone that displayed a potential transgene sequence transfer exhibited this transfer in the FW3 region of the H chain sequence rather than in the CDR2 region, which was the location that was always included within the sequence transfers in VV5 mice (21). Perhaps selection for the 2B4 CDR2 sequence preferentially amplifies those transgene conversions that span the CDR2 region in VVCµ mice and selects against the equivalent conversions in InVVCµ mice. There is an ~15- to 65-fold difference in the frequency of observed transgene sequence transfers in the VVCµ and InVVCµ mice. Because it seems likely that sequence transfers are selected against in InVVCµ mice as much as they are positively selected in VVCµ mice, perhaps the intrinsic frequency of transgene conversion among responding cells is roughly midway between 0.3 and 5–38%.

The affinities of the R16.7 and 2B4 hybridoma antibodies for the Ars antigen have been measured to be 1.6x106 and 3.2x106 M-1 respectively (20). Our results suggest, therefore, that a 2-fold difference in antibody affinity can have a strong effect on selecting a small number of B cells that have undergone transgene conversion for preferential amplification during the immune response. Similar effects of antigen selection have been observed in other situations although the affinity differences have been somewhat larger (27).

Comparison of the amino acid sequences in expressed VVCµ transgenes that have undergone sequence transfers should indicate those amino acid residues that might contribute most strongly to the preferential antigen selection of B cells having transgene conversions. Based on previous data from hybridomas (20) together with the PCR clone sequencing data in this study (Fig. 4Go and additional data not shown), the 2B4 VDJ amino acid sequences found at codons 31, 51, 52 and 53 appear to be most strongly selected (present in >85% of the genes with sequence transfers). It is noteworthy that the amino acids present at each of these codon positions in 2B4 are not individually frequent among anti-Ars, CRI-A+ antibodies (2830). Perhaps this provides a possible explanation for the selection for sequence transfers in VVCµ mice; the 2B4 residues at codons 31, 51, 52 and 53 might collectively provide a significant increase in affinity whereas each residue alone might well have little effect on affinity. Sequence transfers, which would introduce all the critical 2B4 residues in a single event, might provide a strong affinity advantage during the expansion of anti-Ars B cell clones when compared to cells that might only be marginally increasing affinity by hypermutation.

The low level of gene conversion events detected in immunized InVVCµ mice may explain why few V region gene conversion events have been found among endogenous Ig genes in the mouse. In VVCµ mice, where frequent sequence transfers are observed, the nearby upstream donor VDJ segment is highly homologous to the downstream acceptor VDJ segment and can provide nucleotide changes that appear to increase the antibody affinity for the Ars antigenic determinant. Such a situation is likely to be infrequent among normal expressed Ig gene loci. Even in the occasional situation where a nearby, upstream germline V gene segment is highly homologous to a downstream H or L chain V(D)J segment expressed in an individual B cell, it would seem unlikely that the B cell would both be stimulated by the appropriate antigen and undergo a gene conversion event that provided the increased affinity required for experimental detection. This suggests that transfers of nucleotide sequence differences by gene conversion are likely to be only infrequently observed in mouse antibodies.

Although mouse VDJ gene conversion events may only play a minor role in the development of stronger antigen binding during immune responses, even infrequent VDJ sequence transfers might have a significant role in the receptor editing processes that are involved in the disruption of self-antigen binding. Autoreactive mouse B cells can undergo H chain receptor editing events that generally result from VH replacement mechanisms (31,32). Some VH replacement events certainly appear to involve V(D)J recombinase activity (31,32). However, other VH replacement events show features consistent with gene conversion/homologous recombination (33,34) and might reflect the same B cell activities involved in transgene sequence transfers.

Gene-conversion-like sequence transfers in VVCµ mice are invariably accompanied by numerous untemplated somatic mutations (20) (Fig. 4Go). The one sequence transfer that we have detected in InVVCµ mice also displays numerous additional somatic mutations. These results are entirely consistent with our previous suggestion that conversion and hypermutation might both reflect the operation of an error-prone gene conversion mechanism (21). Such a mechanism could also account for hypermutation and conversion events found in other species and others have suggested similar models to account for these results (3537).

It is important to note that the reduced levels of transgene conversion observed in the InVVCµ mice do not suggest that error-prone conversion models for somatic hypermutation are incorrect. It seems likely that gene conversions can take place between identical sequences and, thus, might give rise to untemplated mutations due to an error-prone DNA synthesis. In the mouse, it may be that conversions between identical sequences are much more common than between homologous, but non-identical sequences. This could explain a low frequency of detectable conversion events even among cells that exhibit a high frequency of hypermutation. Recently, DNA breaks within VDJ regions have been found to correlate with somatic hypermutation (22,3840) and these breaks appear to occur during the cell cycle G2 phase where DNA repair mechanisms generally involve homology-based repair (22). The lack of some homology-based DNA repair proteins such as Msh2, Mlh1 and Pms2 appear to reduce somatic hypermutation (4147); this could also suggest a role of homology-based DNA repair in the introduction of some somatic mutations. The availability of VVCµ mice, which amplify conversion events due to antigenic selection, may facilitate analyses of the enzymatic requirements for homology-based sequence transfers by the use of genetic crosses with mice lacking specific repair proteins.


    Acknowledgments
 
We thank Kevin Wright and Sharon Lo for help with analyses of the VVCµ RT-PCR clones and Naomi Rosenberg for helpful comments on the manuscript. This work was supported by NIH grants AI24465 and AI42569 to E. S., and CA65441 to N. D.


    Abbreviations
 
KLH keyhole limpet hemocyanin

    Notes
 
Transmitting editor: E. A. Clark

Received 17 June 2001, accepted 8 October 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302:575.[ISI][Medline]
  2. Alt, F. W., Blackwell, T. K. and Yancopoulos, G. D. 1987. Development of the primary antibody repertoire. Science 238:1079.[ISI][Medline]
  3. Storb, U. 1998. Progress in understanding the mechanism and consequences of somatic hypermutation. Immunol. Rev. 162:5.[ISI][Medline]
  4. Reynaud, C. A., Anquez, V., Grimal, H. and Weill, J. C. 1987. A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell 48:379.[ISI][Medline]
  5. Thompson, C. B. and Neiman, P. E. 1987. Somatic diversification of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment. Cell 48:369.[ISI][Medline]
  6. Knight, K. L. and Becker, R. S. 1990. Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity. Cell 60:963.[ISI][Medline]
  7. Parng, C. L., Hansal, S., Goldsby, R. A. and Osborne, B. A. 1996. Gene conversion contributes to Ig light chain diversity in cattle. J. Immunol. 157:5478.[Abstract]
  8. Kelsoe, G. 1999. V(D)J hypermutation and receptor revision: coloring outside the lines. Curr. Opin. Immunol. 11:70.[ISI][Medline]
  9. Cumano, A. and Rajewsky, K. 1986. Clonal recruitment and somatic mutation in the generation of immunologic memory to the hapten NP. EMBO J. 5:2459.[Abstract]
  10. Linton, P. J., Decker, D. J. and Klinman, N. R. 1989. Primary antibody-forming cells and secondary B cells are generated from separate precursor cell subpopulations. Cell 59:1049.[ISI][Medline]
  11. Krawinkel, U., Zoebelein, G., Bruggemann, M., Radbruch, A. and Rajewsky, K. 1983. Recombination between antibody heavy chain variable region genes: evidence for gene conversion. Proc. Natl Acad. Sci. USA 80:4997.[Abstract]
  12. David, V., Folk, N. L. and Maizels, N. 1992. Germ line variable regions that match hypermutated sequences in genes encoding murine anti-hapten antibodies. Genetics 132:799.[Abstract/Free Full Text]
  13. Maizels, N. 1993. Preimmune diversification creates a repertoire while somatic hypermutation fine-tunes affinity—implications for the processes of mutation. Res. Immunol. 144:459.[ISI][Medline]
  14. Wysocki, L. J. and Gefter, M. L. 1989. Gene conversion and the generation of antibody diversity. Annu. Rev. Biochem. 58:509.[ISI][Medline]
  15. Chien, N. C., Pollock, R. R., Desaymard, C. and Scharff, M. D. 1988. Point mutations cause the somatic diversification of IgM and IgG2a antiphosphoryl-choline antibodies. J. Exp. Med. 167:954.[Abstract]
  16. Crews, S., Griffin, J., Huang, H., Calame, K. and Hood, L. 1981. A single VH gene segment encodes the immune response to phosphorylcholine: somatic mutation is correlated with the class of the antibody. Cell 25:59.[ISI][Medline]
  17. Bernard, O., Hozumi, N. and Tonegawa, S. 1978. Sequences of mouse immunoglobulin light chain genes before and after somatic changes. Cell 15:1133.[ISI][Medline]
  18. Ford, J. E., McHeyzer-Williams, M. G. and Lieber, M. R. 1994. Analysis of individual immunoglobulin lambda light chain genes amplified from single cells is inconsistent with variable region gene conversion in germinal-center B cell somatic mutation. Eur. J. Immunol. 24:1816.[ISI][Medline]
  19. Kong, Q., Zhao, L., Subbaiah, S. and Maizels, N. 1998. A lambda 3' enhancer drives active and untemplated somatic hypermutation of a lambda 1 transgene. J. Immunol. 161:294.[Abstract/Free Full Text]
  20. Xu, B. and Selsing, E. 1994. Analysis of sequence transfers resembling gene conversion in a mouse antibody transgene. Science 265:1590.[ISI][Medline]
  21. Selsing, E., Xu, B. and Sigurdardottir, D. 1996. Gene conversion and homologous recombination in murine B cells. Semin. Immunol. 8:151.[Medline]
  22. Papavasiliou, F. N. and Schatz, D. G. 2000. Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes. Nature 408:216.[ISI][Medline]
  23. Durdik, J., Gerstein, R. M., Rath, S., Robbins, P. F., Nisonoff, A. and Selsing, E. 1989. Isotype switching by a microinjected µ immunoglobulin heavy chain gene in transgenic mice. Proc. Natl Acad. Sci. USA 86:2346.[Abstract]
  24. Gerstein, R. M., Frankel, W. N., Hsieh, C.-L., Durdik, J. M., Rath, S., Coffin, J. M., Nisonoff, A. and Selsing, E. 1990. Isotype switching of an immunoglobulin heavy chain transgene occurs by DNA recombination between different chromosomes. Cell 63:537.[ISI][Medline]
  25. Sohn, J., Gerstein, R. M., Hsieh, C. L., Lemer, M. and Selsing, E. 1993. Somatic hypermutation of an immunoglobulin µ heavy chain transgene. J. Exp. Med. 177:493.[Abstract]
  26. Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162:156.[ISI][Medline]
  27. Giusti, A. M., Coffee, R. and Manser, T. 1992. Somatic recombination of heavy chain variable–diversity–joining transgenes with the endogenous immunoglobulin heavy chain locus in mice. Proc. Natl Acad. Sci. USA 89:10321.[Abstract]
  28. Wysocki, L. J., Gefter, M. L. and Margolies, M. N. 1990. Parallel evolution of antibody variable regions by somatic processes: consecutive shared somatic alterations in VH genes expressed by independently generated hybridomas apparently acquired by point mutation and selection rather than by gene conversion. J. Exp. Med. 172:315.[Abstract]
  29. Manser, T. 1989. Evolution of antibody structure during the immune response. The differentiative potential of a single B lymphocyte. J. Exp. Med. 170:1211.[Abstract]
  30. Fish, S., Zenowich, E., Fleming, M. and Manser, T. 1989. Molecular analysis of original antigenic sin. I. Clonal selection, somatic mutation, and isotype switching during a memory B cell response. J. Exp. Med. 170:1191.[Abstract]
  31. Chen, C., Nagy, Z., Prak, E. L. and Weigert, M. 1995. Immunoglobulin heavy chain gene replacement: a mechanism of receptor editing. Immunity 3:747.[ISI][Medline]
  32. Chen, C., Prak, E. L. and Weigert, M. 1997. Editing disease-associated autoantibodies. Immunity 6:97.[ISI][Medline]
  33. Dildrop, R., Bruggemann, M., Radbruch, A., Rajewsky, K. and Beyreuther, K. 1982. Immunoglobulin V region variants in hybridoma cells. II. Recombination between V genes. EMBO J. 1:635.[ISI][Medline]
  34. Kleinfield, R., Hardy, R. R., Tarlinton, D., Dangl, J., Herzenberg, L. A. and Weigert, M. 1986. Recombination between an expressed immunoglobulin heavy-chain gene and a germline variable gene segment in a Ly 1+ B-cell lymphoma. Nature 322:843.[ISI][Medline]
  35. Weill, J. C. and Reynaud, C. A. 1996. Rearrangement/hypermutation/gene conversion: when, where and why? Immunol. Today 17:92.[ISI][Medline]
  36. Kong, Q., Harris, R. S. and Maizels, N. 1998. Recombination-based mechanisms for somatic hypermutation. Immunol. Rev. 162:67.[ISI][Medline]
  37. Diaz, M. and Flajnik, M. F. 1998. Evolution of somatic hypermutation and gene conversion in adaptive immunity. Immunol. Rev. 162:13.[ISI][Medline]
  38. Bross, L., Fukita, Y., McBlane, F., Demolliere, C., Rajewsky, K. and Jacobs, H. 2000. DNA double-strand breaks in immunoglobulin genes undergoing somatic hypermutation. Immunity 13:589.[ISI][Medline]
  39. Sale, J. E. and Neuberger, M. S. 1998. TdT-accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line. Immunity 9:859.[ISI][Medline]
  40. Kong, Q. and Maizels, N. 2001. DNA breaks in hypermutating immunoglobulin genes: evidence for a break-and-repair pathway of somatic hypermutation. Genetics 158:369.[Abstract/Free Full Text]
  41. Wiesendanger, M., Kneitz, B., Edelmann, W. and Scharff, M. D. 2000. Somatic hypermutation in MutS homologue (MSH)3-, MSH6-, and MSH3/MSH6-deficient mice reveals a role for the MSH2–MSH6 heterodimer in modulating the base substitution pattern. J. Exp. Med. 191:579.[Abstract/Free Full Text]
  42. Rada, C., Ehrenstein, M. R., Neuberger, M. S. and Milstein, C. 1998. Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting. Immunity 9:135.[ISI][Medline]
  43. Cascalho, M., Wong, J., Steinberg, C. and Wabl, M. 1998. Mismatch repair co-opted by hypermutation. Science 279:1207.[Abstract/Free Full Text]
  44. Winter, D. B., Phung, Q. H., Umar, A., Baker, S. M., Tarone, R. E., Tanaka, K., Liskay, R. M., Kunkel, T. A., Bohr, V. A. and Gearhart, P. J. 1998. Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2. Proc. Natl Acad. Sci. USA 95:6953.[Abstract/Free Full Text]
  45. Phung, Q. H., Winter, D. B., Cranston, A., Tarone, R. E., Bohr, V. A., Fishel, R. and Gearhart, P. J. 1998. Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein. J. Exp. Med. 187:1745.[Abstract/Free Full Text]
  46. Phung, Q. H., Winter, D. B., Alrefai, R. and Gearhart, P. J. 1999. Hypermutation in Ig V genes from mice deficient in the MLH1 mismatch repair protein. J. Immunol. 162:3121.[Abstract/Free Full Text]
  47. Kim, N., Bozek, G., Lo, J. C. and Storb, U. 1999. Different mismatch repair deficiencies all have the same effects on somatic hypermutation: intact primary mechanism accompanied by secondary modifications. J. Exp. Med. 190:21.[Abstract/Free Full Text]