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
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Abstract |
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Keywords: B cells, generation of diversity, H chain, Ig, somatic mutation
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Introduction |
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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. 1). 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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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.
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Methods |
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Oligonucleotides
5'L: 5'-CCGCTCGAGACACACTGACTCAAACCATG-3'
JH3: 5'-CCGCTCGAGACAGTGACCAGAGTCCCTTG-3'
LRI: 5'-CCGAATTCACACACTGACTCAAACCATG-3'
L3RI: 5'-AGAATTCCTCTTCCTCCTGTCAGTAAC-3'
CRI: 5'-GGAATTCCGGGGCCAGTGGATAGAC-3'
CH2: 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), CH2(R) and L3RI(F), C
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 TrisHCl, 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 1530 min, in 3xSSC, 10xDenhardt's solution at 42°C for 3060 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 (23°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.
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Results |
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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 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
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 3 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. 3
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. 3
, 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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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 VDJ 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 4 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. 4
(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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The sequence transfers found in RT-PCR clones from immunized VV5 splenocytes are highly correlated with additional somatic mutations (Fig. 4). 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 520% of anti-Ars IgG-producing hybridomas from immunized VV5 mice exhibited transgene conversion (20). The RT-PCR/blot analyses (Fig. 3) 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. 5, 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. 5
). These findings complicate the interpretation of the RT-PCR/blot analyses of InVVCµ transgene conversion.
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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 VDJ 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. 6) 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. 7
). 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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Discussion |
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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 538% 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 538%.
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. 4 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. 4). 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.
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Acknowledgments |
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Abbreviations |
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KLH keyhole limpet hemocyanin |
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Notes |
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Received 17 June 2001, accepted 8 October 2001.
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References |
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