Sequence Transfers between Variable
Regions in a Mouse Antibody Transgene Can
Occur by Gene Conversion
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J Immunol 2005; 175:8133-8137; ;
doi: 10.4049/jimmunol.175.12.8133
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References
Nicole D'Avirro, David Truong, Bo Xu and Erik Selsing
The Journal of Immunology
Sequence Transfers between Variable Regions in a Mouse
Antibody Transgene Can Occur by Gene Conversion1
Nicole D’Avirro,* David Truong,‡ Bo Xu,† and Erik Selsing2*†‡
D
uring B cell development, V(D)J recombination of Ab
gene segments is a highly conserved mechanism for the
generation of Ab repertoire diversity in all jawed vertebrate species (1, 2). In warm-blooded vertebrates, other processes
are also involved in diversifying Ab repertoires. Somatic hypermutation is one mechanism for diversification that reflects the introduction of single point mutations into Ab V(D)J segments (reviewed in Ref. 3). Gene conversion is a second diversifying
mechanism that is characterized by transfers of homologous sequences from donor Ab V gene segments to an acceptor V gene
segment (4 –7). 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.
All warm-blooded vertebrate species appear to use somatic hypermutation to diversify their Ab repertoires. However, only some
species exhibit frequent gene conversion events during Ab diversification. Hypermutation and hyperconversion have been most extensively analyzed in mice and chickens. In chickens, both processes are observed (4, 8 –10) although hyperconversion appears to
dominate during early B cell development. In contrast, although
somatic hypermutation is common in mice during immune responses, definitive gene conversion events have not been detected
in developing or stimulated mouse B cells (11–16). The reasons
why mice (or other species such as sheep or humans) do not use
gene hyperconversion for Ab diversification are not known.
Hypermutation in mice and hyperconversion in chickens both
require the activation-induced cytidine deaminase (AID)3 enzyme
*Program in Genetics, †Program in Immunology, and ‡Department of Pathology,
Tufts University School of Medicine, Boston, MA 02111
Received for publication May 12, 2005. Accepted for publication October 6, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI24465 (to E.S.)
and CA65441 (to N.D.).
2
Address correspondence and reprint requests to Dr. Erik Selsing, Tufts University
School of Medicine, 150 Harrison Avenue, Boston, MA 02111. E-mail address:
[email protected]
3
Abbreviations used in this paper: AID, activation-induced cytidine deaminase; Ars,
p-azophenylarsonate; KLH, keyhole limpet hemocyanin; ES, embryonic stem.
Copyright © 2005 by The American Association of Immunologists, Inc.
that is also important for Ab gene class switching (17, 18). AID
deamination of cytidine residues in ssDNAs appears to lead to
DNA damage that can result in mutation, gene conversion, or
switch recombination events during DNA repair (18 –23). It has
been suggested that differences in available DNA repair pathways
might determine whether AID-induced DNA damage leads to hypermutation or hyperconversion. The strongest evidence supporting this notion comes from the DT40 chicken cell line that undergoes Ab gene hyperconversion during passage in culture; DT40
mutants lacking the XRCC2/3 DNA repair proteins have greatly
reduced hyperconversion activity but retain somatic hypermutation
activity (24).
The lack of Ab gene conversion events in some species could
indicate that hyperconversion pathways have only evolved in some
species or, instead, that hyperconversion pathways in some species
have become vestigial. Transgenic mice carrying a gene construct
(VVC␮), designed to optimize detection of sequence transfers,
provide the strongest evidence supporting a possible V-region conversion mechanism in mice (25). The VVC␮ transgene (depicted
in Fig. 1) contains two tandem VDJ segments; one functional VDJ
gene segment (R16.7 VDJ) that encodes a ␮ H chain capable, with
the correct L chain, of binding the hapten, p-azophenylarsonate
(Ars); and a second upstream promoterless VDJ segment (2B4
VDJ) that is identical to the R16.7 VDJ except for 17 single base
pair differences. Sequence transfers between the 2B4 and R16.7
VDJ segments are easily found in Ars-keyhole limpet hemocyanin
(KLH) immunized multicopy VVC␮ transgenic mice (25); however, these transfer events could represent either multiple (or reciprocal) recombinations mediated by a mechanism that is not related to the chicken V gene hyperconversion process, or they could
represent gene conversions (26). Differences in the importance of
the Rad54 repair protein in chicken hyperconversion (27) and in
mouse VVC␮ sequence transfers (28) might suggest that these two
processes are not closely related.
To determine whether mouse VVC␮ transgene sequence transfers are due to gene conversions or due to multiple/reciprocal recombinations, we have produced low-copy transgenic mice and
analyzed the relationships between donor and acceptor sequences
involved in the sequence transfers that occur in these low-copy
animals. We find that at least some VVC␮ sequence transfers are
0022-1767/05/$02.00
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Different vertebrate species show widely differing usage of somatic hyperconversion (SHC) as a mechanism for diversifying
expressed Ab V genes. The basis for the differing levels of SHC in different species is not known. Although no clear evidence for
SHC has been found in normal mouse B cells, transgenic mice carrying high-copy numbers of a gene construct designed to
optimize detection of SHC have previously been shown to exhibit sequence transfers that resemble gene conversion events.
However, these transgene sequence transfers could reflect multistep or reciprocal DNA recombination events rather than gene
conversions. We now find in low-copy number transgenic mice that transgene sequence transfers can exhibit the unidirectional
sequence information movement that is a hallmark of gene conversion. This indicates that gene conversion between V region
sequences can occur in mouse B cells; we propose that the lack of efficient SHC contributions to Ab diversification in normal mice
may be due, at least in part, to the particular pattern of V gene recombinational accessibility that occurs in differentiating mouse
B cells. The Journal of Immunology, 2005, 175: 8133– 8137.
8134
Ab GENE CONVERSION IN A MOUSE H-CHAIN TRANSGENE
unidirectional, indicating that gene conversion pathways are available in mouse B cells for the diversification of V regions.
Results
Materials and Methods
To assess whether sequence transfers in the VVC␮ transgene occur by gene conversion, we wanted to produce low-copy transgenic mice that would facilitate analyses of donor and recipient
sequences involved in the sequence transfer event. The VVC␮
construct was transfected into ES cells and transgene copy numbers were determined for individual clones by Southern blot analyses (see example in Fig. 1). Twenty-four ES cell clones were
analyzed and two clones were identified, each of which appeared
to carry one transgene copy. The two clones were separately microinjected into blastocysts to obtain chimeric mice and the chimeras were bred. Only one of these ES cell clones yielded chimeras that transmitted the transgene to offspring. However, extensive
efforts to obtain IgG-producing anti-Ars hybridomas after immunization of these offspring were unsuccessful, indicating that the
single VVC␮ transgene copy in the ES cell clone was not capable
of interchromosomal switch recombination. Because all sequence
transfers in multicopy VVC␮ transgenic mice have been found in
IgG-producing B cells (25, 28, 29), and because there is no information indicating whether such transfers can be found in IgMproducing B cells, these mice were not studied further. A third ES
clone carrying about two copies of the VVC␮ construct (Fig. 1)
was used to produce another transgenic line (VV29). VV29 mice
immunized with Ars-KLH exhibited serum levels of transgenederived anti-Ars IgG (data not shown) that were similar to previously analyzed VVC␮ transgenic mice (25, 30, 31). Thus, the
Animals
Transgenic mice were produced by cotransfecting the VVC␮ construct
(25) and a neoexpression construct into embryonic stem (ES) cells. ES cell
clones resistant to G418 were analyzed by Southern blotting to determine
approximate copy numbers of integrated VVC␮ transgenes. ES cell clones
having low VVC␮ transgene copy numbers were injected into blastocysts
to produce chimeric mice and these chimeras were bred to establish transgenic lines. All animal studies have been reviewed and approved by the
Tufts School of Medicine Institutional Animal Care Use Committee.
Immunization and hybridoma production
Analyses of transgene copy number and sequences
Transgene-derived ␥-chain mRNAs from the selected hybridomas were
analyzed for sequence transfers by RT-PCR amplification followed by
DNA sequencing as described previously (25). Transgene copy numbers
and sequences in genomic DNAs from germline cells and from hybridomas
were analyzed by Southern blotting and by sequencing of PCR clones
derived using primers specific for the VDJ regions in the VVC␮ transgene.
The transgene-specific primer pair consists of a leader sequence (5⬘CCGAATTCACACACTGACTCAAACCATG-3⬘) and a JH2 sequence
(5⬘-CCAGAATTCACCTGAGGAGACTGTGAGAG-3⬘) that are found in
both the R16.7 and 2B4 VDJ segments of the VVC␮ transgene.
Comparisons of gene sequences
Sequences of hybridoma RT-PCR products were compared with the VVC␮
transgene R16.7 and 2B4 VDJ sequences to assess whether sequence transfer events had occurred. As described previously (25), only the R16.7 VDJ
segments in the VVC␮ transgene have promoters and can be expressed as
mRNA. Sequence transfers are therefore indicated by mRNAs exhibiting a
pattern where the expressed R16.7 sequence contains a block of nucleotide
changes that match the 2B4 VDJ sequence rather than the R16.7
sequence (25).
Sequences of panels of cloned PCR-amplified genomic VDJ segments
from germline and hybridoma cells were compared with the VVC␮ transgene R16.7 and 2B4 VDJ sequences, as well as the expressed mRNA
sequence in the 591A6 hybridoma, to determine the origin of each PCR
clone. These clones were all found to correspond to one of the three sequences (either R16.7, 2B4, or 591A6) except for occasional PCR errors
that occurred at frequencies of about 1/700 nucleotides. This error frequency was higher than we expected but was similar to error frequencies
we have found in similar PCR amplifications of the endogenous C␮ gene.
Furthermore, the putative PCR errors were scattered throughout the sequences and, except for a few instances, did not occur in more than one
clone or at positions that differed between the R16.7, 2B4, and 591A6
sequences. Thus, these PCR errors were ignored in assigning the origin of
each PCR clone sequence. Most importantly, in all the clones that were
assigned as 2B4 sequences (the donors for sequence transfers) there were
no PCR errors that were found at positions that differed between the 2B4
and R16.7 VDJ sequences.
FIGURE 1. Diagram of the VVC␮ transgene and Southern blot analyses of VVC␮ copy number in the ESVV29 cell line and in VV29 transgenic mice. In A, the organization of the VVC␮ transgene is depicted.
Boxes represent various exons as labeled. The triangle indicates the promoter of the R16.7 VDJ exon; the 2B4 VDJ segment (black boxes) has no
promoter. The S␮ tandem repeat region is indicated by an oval. In B, DNAs
from the parental D3 ES cell line are compared with the ESVV29 clone
carrying a VVC␮ transgene. DNAs were digested with BamH1 and EcoR1
and hybridized with a probe containing the C␮1 exon. Bands at 8 kb correspond to the two germline C␮ loci present in the ES cells whereas the
band at 5 kb corresponds to the VVC␮ transgene. Quantitative phosphoimager analyses of the band intensities indicate that two copies of the
VVC␮ transgene are found in the ESVV29 cell line. In C, kidney DNAs
from VV29 transgenic mice and BALB/c nontransgenic mice are compared. DNAs were digested with BamH1 and EcoR1 and hybridized with
the pJ11 probe containing JH and E␮ enhancer sequences. Bands at 2 kb
correspond to the two germline JH-C␮ loci whereas the band at 6 kb corresponds to the VVC␮ transgene. Analyses of band intensities using densitometry of the autoradiogram indicated that about two copies of the transgene are found in VV29 mice.
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Transgenic lines were immunized with Ars-KLH to assess transgene sequence transfers as described previously (25). Mice between 2 and 3
months of age were immunized i.p. three times with 0.1 ml containing 100
␮g of Ars-KLH with IFA and then a fourth time with 100 ␮g of Ars-KLH
in saline. Three weeks separated each immunization. Splenocytes or blood
lymphocytes for RT-PCR analyses were collected after the third immunization. Four days after the fourth immunization, splenocytes were obtained
for hybridoma production. Sequence transfers in splenocytes and blood
lymphocytes from immunized transgenic mice were detected using RTPCR/Southern blot assays that have been described previously (28, 29).
Immunized transgenic mice exhibiting sequence transfers as indicated by
the RT-PCR/Southern blot assay were then used to produce hybridomas.
Hybridomas were initially screened for production of anti-Ars IgG using an
ELISA that has previously been described (30). Anti-Ars IgG-producing
hybrids were then screened for IgG reactive with the AD8 anti-idiotype
again using a previously described ELISA (30). Previous studies have
shown that many hybridomas from immunized VVC␮ transgenic mice that
exhibit transgene sequence transfers lack AD8 reactivity (25). All hybridomas producing anti-Ars IgG that lacked AD8 reactivity were selected to
be analyzed by sequencing for transgene sequence transfers.
Production of low-copy VVC␮ transgenic mice that exhibit
transgene sequence transfers
The Journal of Immunology
VV29 line provided low-copy VVC␮ transgenic mice that were
suitable for further analyses of the sequence transfer mechanism.
Transgene sequence transfers in immunized VV29 blood lymphocytes were assessed using an RT-PCR/Southern blot assay that
has been described previously (28, 29). In this assay, PCR products
that hybridize with the 2B4 probe are indicative of sequence transfers in the VVC␮ transgene. As shown in Fig. 2, sequence transfers were detected in two of three immunized VV29 animals, and
the levels of sequence transfers in the low-copy VV29 mice were
similar to levels in a representative multicopy VV5 mouse that has
been analyzed previously (29). In other experiments, splenocytes
from four of seven immunized VV29 mice exhibited sequence
transfer events either by the RT-PCR/Southern blot assay or by
sequencing VDJ segments in cloned splenocyte RT-PCR products
(data not shown). These findings showed that sequence transfer
events found in high-copy VVC␮ mice can also occur in
low-copy mice.
Three transgene VDJ regions in VV29 mice
FIGURE 2. RT-PCR/Southern blot assays for gene conversion in blood
lymphocytes from immunized VV29 mice. Transgene VDJ sequences in
␥-chain mRNAs were PCR amplified from the indicated samples and then
electrophoresed, transferred to nylon membranes, and hybridized with oligonucleotide probes specific for R16.7 and 2B4 CDR2 sequences. Specificity control PCR product samples are from the VV5-derived hybridomas
3A3 and 8B2; previous studies have shown that the 3A3 hybridoma exhibits transgene sequence transfers whereas the 8B2 hybridoma does not
(28). Samples from three immunized VV29 mice are also compared with
a splenocyte sample from an immunized VV5 mouse (VV5 no. 6) that has
been previously been found to exhibit levels of transgene sequence transfers that are average for VV5 mice (29).
Hybridoma cell lines from low-copyVV29 mice exhibit sequence
transfers
Hybridomas were produced from immunized VV29 mice and
these were screened for sequence transfer events using the AD8
anti-idiotypic Ab. The AD8 reagent reacts with H chains that carry
the R16.7 VDJ CDR2 sequence but not with H chains having the
2B4 VDJ CDR2 sequence (25, 28). Transgene-expressing B cells
from VVC␮ transgenic mice generally express the AD8-reactive
idiotope whereas those VVC␮ B cells that exhibit transgene sequence transfers often do not express the AD8-reactive idiotope
(25, 28, 29). Candidate hybridomas expressing anti-Ars IgG Abs
that were not reactive with AD8 were further screened by sequence
analyses of the expressed H chain VDJ from RT-PCR products.
Despite the results showing transgene conversion events in immunized splenocytes, it was difficult to find hybridomas that exhibited
sequence transfers. Among more than 600 hybridomas that were
screened, nine anti-Ars IgG and AD8-negative hybrids were found
but only one hybridoma (591A6) exhibiting sequence transfers
was obtained. Fig. 3 shows the 591A6 transgene-derived ␥-chain
mRNA VDJ sequence compared with the R16.7 and 2B4 VDJ
sequences present in the VVC␮ transgene. The block of nucleotide
changes in the expressed 591A6 VDJ that corresponds to the sequence of the 2B4 VDJ indicates a transgene sequence transfer
similar to those described previously (25, 28, 29).
Analyses of hybridoma VDJ regions show that sequence
transfers occur by gene conversion
The sequence transfer in the 591A6 hybridoma allowed further
analyses to determine the transfer mechanism. Sequence transfers
that occur by gene conversion are characterized by a unidirectional
movement of sequence information from a donor sequence to a
homologous acceptor sequence. The donor sequence in a gene conversion event is not altered. Such unidirectional sequence transfers
have been directly demonstrated for the chicken hyperconversion
mechanism (32). Sequencing of 591A6 transgene VDJ segments
was used to determine the status of donor sequences in the
hybridoma.
Transgenic VDJ gene sequences in genomic DNA from the
591A6 hybridoma were determined by sequencing cloned PCR
products that were generated using primers that did not distinguish
2B4 and R16.7 VDJ gene segments. A total of 69 VDJ sequences
were determined; 24 of these were found to come from transgene
R16.7 VDJ regions, 25 were from 2B4 VDJ regions, and 20 were
from the expressed 591A6 ␥-chain VDJ gene (criteria used to assign the sequences are described in Materials and Methods). Because the expressed 591A6 sequence must be derived from a single
H chain gene, the observed ratios of VDJ sequences indicate that
the 591A6 hybridoma genome contains three transgenic VDJ gene
segments: one R16.7 VDJ segment, one 2B4 VDJ segment, and
one R16.7-derived VDJ segment that has undergone a sequence
transfer event and that is expressed as ␥-chain mRNA. These results are consistent with the finding that germline VV29 cells contain three transgenic VDJ segments. The findings indicate that the
591A6 hybridoma retains all of the VDJ copies present in VV29
germline cells, and that one of the two 591A6 R16.7 VDJ segments has undergone both a switch recombination event and a
sequence transfer event as depicted in Fig. 3.
The expressed R16.7 VDJ transgene copy in the 591A6 hybridoma exhibits nucleotide changes that are clearly derived from a
donor 2B4 gene. Because there is only one 2B4 gene available in
VV29 B cells to serve as a donor for these nucleotide changes, then
the single 2B4 VDJ gene that is present in the 591A6 hybridoma
must have been the donor for the DNA sequence transfer that has
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Southern blot analyses had indicated that the VV29 mouse line
contained two copies of the VVC␮ transgene. However, these
analyses had used a hybridization probe located within the JH-C␮
intron region of the transgenes; this probe would not assess the
number of individual VDJ segments that were present within the
VV29 mouse line. To determine the number of VDJ segments in
the germline of VV29 mice, transgene VDJ gene segments were
amplified from VV29 germline (kidney) DNA and then characterized by sequencing. The PCR amplification of VDJ segments used
primers that did not distinguish the R16.7 and 2B4 VDJ gene segments. PCR products were cloned and clones were randomly chosen for sequencing. Analyses of the sequences from 14 of these
VDJ PCR clones showed that 9 were from the R16.7 VDJ gene
segment and 5 were from the 2B4 VDJ segment. Together with the
Southern blot analyses that indicated two JH-C␮ transgene segments, this 2:1 ratio of VDJ segments indicated that one of the
VVC␮ transgene copies in VV29 mice was truncated and lacked
an upstream 2B4 VDJ segment (Fig. 3B). These results indicate
that three transgene-derived VDJ segments are present in VV29
mice; this conclusion was further supported by analyses of VDJ
segments in a VV29 hybridoma as described below.
8135
8136
Ab GENE CONVERSION IN A MOUSE H-CHAIN TRANSGENE
altered the expressed ␥-chain 591A4 VDJ gene. The 25 PCR
clones from the single donor 2B4 VDJ gene in 591A6 show that
the sequence of this 2B4 gene is the same as found in the germline
and, therefore, has not been altered by the sequence transfer process. These results show that the sequence transfer event in 591A4
cells is unidirectional and has occurred by a gene conversion
mechanism.
Discussion
Analyses of the VV29 transgenic mouse line carrying only two
copies of the VVC␮ transgene show the same types of sequence
transfers between Ab V genes in stimulated B cells that have previously been reported for the high-copy VV1 and VV5 transgenic
lines (50 and 30 copies, respectively) (25). Thus, high transgene
copy numbers are not required for the sequence transfer mechanism. Furthermore, the levels of sequence transfer events in immunized VV29 mice appear to be similar to the levels in VV1 and
VV5 mice, indicating that copy number does not have a major
effect on sequence transfer efficiency. Characterizations of the donor and acceptor V gene sequences in a hybridoma derived from
an immunized VV29 mouse show the unidirectional sequence
transfers that are characteristic of a gene conversion mechanism
and that have been shown for hyperconversion events in chicken B
cells (32). These results also suggest that the numerous sequence
transfers found in mice carrying high copy numbers of the VVC␮
transgene (25, 28) also occur by gene conversion. The results indicate that mouse B cells can exhibit gene conversions between Ab
V genes, similar to the events that have been reported for chicken,
rabbit, and cow B cells.
Our results indicating a mechanism for gene conversion between V genes within a transgene construct in mouse B cells raises
questions about the apparent lack of such events between the endogenous V genes in nontransgenic mice. The frequency of transgene conversions in VVC␮ transgenic mice does appear to be low
because preferential Ag selection of cells that have undergone conversion events is required to easily detect the transfers in immunized animals (29). For endogenous mouse Ab genes, only a few
studies have found evidence to suggest possible V gene conversion
events (33, 34). Given the numerous studies of Ab responses to
immunization with various Ags that have been done in nontransgenic mice, it is surprising that no definitive examples of gene
conversion events have been detected.
Perhaps there are other features of mouse B cell differentiation
that limit gene conversions between germline and recombined V
genes during normal B cell maturation or during B cell responses
to Ag. Transgene conversion events appear to occur in Ag-stimulated B cells; all transgene sequence transfers in VVC␮ mice have
been found together with somatic mutations and in IgG-producing
cells. In normal mouse B cells, the germline V genes that would
provide donors in V gene conversion events are in “closed” chromosomal chromatin and might not be accessible to a hyperconversion mechanism. Furthermore, in those early normal pre-B cells
where germline and recombined V genes would presumably both
be in accessible chromatin, the AID protein does not appear to be
expressed (35, 36) and, therefore, hyperconversion is again not
likely to occur. Thus, the regulation of V gene accessibility in
mouse B cells might inhibit the conversion process. Nevertheless,
our results, showing that the VVC␮ transgene can exhibit conversion events between V genes, support a hyperconversion mechanism that has been conserved during evolution but that varies in
activity in different species due to distinct evolutions of Ab gene
organization and regulation in each individual species.
Acknowledgments
We thank Drs. Frederick W. Alt and Jianzhu Chen for help in the production of transgenic ES cell lines, Dr. Hwei-Fang Tsai for help in analyzing
the initial single-copy mice, and Dr. Naomi E. Rosenberg for critically
reading the manuscript.
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FIGURE 3. Sequences in the expressed ␥-chain transgene copy in the 591A6 hybridoma compared with the R16.7 and 2B4 VDJ sequences in the VV29
transgene. A, Sequence of the expressed 591A6 ␥-chain gene. The entire sequences of the R16.7 and 2B4 VDJ regions have been published previously (31).
Only those codons where differences are observed between R16.7, 2B4, and 591A6 are shown. In these codons the entire sequence is shown for R16.7
whereas, in the 2B4 and 591A6 sequences, only differences from R16.7 are shown and identities with R16.7 are indicated by dashes. In the 591A6 sequence,
differences from R16.7 that reflect a sequence transfer (similar to sequence transfers reported previously (25, 28, 29) are shown as capital letters whereas
differences that are due to somatic hypermutation are shown as lowercase. B, Transgene VDJ segments in the VV29 mouse line. Boxes indicate gene
segments as labeled. The diagram depicts the truncation of one VVC␮ copy in the VV29 line resulting in the loss of one 2B4 VDJ segment. The dotted
lines indicate that the relative locations and orientations of the two VVC␮ construct copies have not been determined. C, VDJ segments in the 591A6
hybridoma. The diagram is depicted to show the truncation of one VVC␮ copy in the 591A6 hybridoma, as also found in VV29 germline tissues in B. In
addition, the black portion of the expressed 591A6 ␥-chain VDJ gene segment depicts the transfer of sequence information from the 2B4 VDJ gene segment
into a R16.7 VDJ gene segment. This transgene copy has also undergone isotype switching as indicated by the heavier lines and shaded C␥ gene segment.
As in B, the dotted lines indicate that relative locations and orientations of the two construct copies have not been determined.
The Journal of Immunology
Disclosures
The authors have no financial conflict of interest.
References
19. Chaudhuri, J., M. Tian, C. Khuong, K. Chua, E. Pinaud, and F. W. Alt. 2003.
Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422: 726 –730.
20. Bransteitter, R., P. Pham, M. D. Scharff, and M. F. Goodman. 2003. Activationinduced cytidine deaminase deaminates deoxycytidine on single-stranded DNA
but requires the action of RNase. Proc. Natl. Acad. Sci. USA 100: 4102– 4107.
21. Pham, P., R. Bransteitter, J. Petruska, and M. F. Goodman. 2003. Processive
AID-catalysed cytosine deamination on single-stranded DNA simulates somatic
hypermutation. Nature 424: 103–107.
22. Dickerson, S. K., E. Market, E. Besmer, and F. N. Papavasiliou. 2003. AID
mediates hypermutation by deaminating single stranded DNA. J. Exp. Med. 197:
1291–1296.
23. Ramiro, A. R., P. Stavropoulos, M. Jankovic, and M. C. Nussenzweig. 2003.
Transcription enhances AID-mediated cytidine deamination by exposing singlestranded DNA on the nontemplate strand. Nat. Immunol. 4: 452– 456.
24. Sale, J. E., D. M. Calandrini, M. Takata, S. Takeda, and M. S. Neuberger. 2001.
Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. Nature 412: 921–926.
25. Xu, B., and E. Selsing. 1994. Analysis of sequence transfers resembling gene
conversion in a mouse antibody transgene. Science 265: 1590 –1593.
26. Selsing, E., B. Xu, and D. Sigurdardottir. 1996. Gene conversion and homologous
recombination in murine B cells. Semin. Immunol. 8: 151–158.
27. Bezzubova, O., A. Silbergleit, Y. Yamaguchi-Iwai, S. Takeda, and
J. M. Buerstedde. 1997. Reduced X-ray resistance and homologous recombination frequencies in a RAD54⫺/⫺ mutant of the chicken DT40 cell line. Cell 89:
185–193.
28. D’Avirro, N., D. Truong, M. Luong, R. Kanaar, and E. Selsing. 2002. Gene
conversion-like sequence transfers between transgenic antibody V genes are independent of RAD54. J. Immunol. 169: 3069 –3075.
29. Tsai, H. F., N. D’Avirro, and E. Selsing. 2002. Gene conversion-like sequence
transfers in a mouse antibody transgene: antigen selection allows sensitive detection of V region interactions based on homology. Int. Immunol. 14: 55– 64.
30. Durdik, J., R. M. Gerstein, S. Rath, P. F. Robbins, A. Nisonoff, and E. Selsing.
1989. Isotype switching by a microinjected ␮ immunoglobulin heavy chain gene
in transgenic mice. Proc. Natl. Acad. Sci. USA 86: 2346 –2350.
31. Gerstein, R. M., W. N. Frankel, C.-L. Hsieh, J. M. Durdik, S. Rath, J. M. Coffin,
A. Nisonoff, and E. Selsing. 1990. Isotype switching of an immunoglobulin heavy
chain transgene occurs by DNA recombination between different chromosomes.
Cell 63: 537–548.
32. Carlson, L. M., W. T. McCormack, C. E. Postema, E. H. Humphries, and
C. B. Thompson. 1990. Templated insertions in the rearranged chicken IgL V
gene segment arise by intrachromosomal gene conversion. Genes Dev. 4:
536 –547.
33. Cumano, A., and K. Rajewsky. 1986. Clonal recruitment and somatic mutation in
the generation of immunologic memory to the hapten NP. EMBO J. 5:
2459 –2468.
34. David, V., N. L. Folk, and N. Maizels. 1992. Germ line variable regions that
match hypermutated sequences in genes encoding murine anti-hapten antibodies.
Genetics 132: 799 – 811.
35. Ruckerl, F., and J. Bachl. 2004. Activation-induced cytidine deaminase fails to
induce a mutator phenotype in the human pre-B cell line Nalm-6. Eur. J. Immunol. 35: 290 –298.
36. Muramatsu, M., V. S. Sankaranand, S. Anant, M. Sugai, K. Kinoshita,
N. O. Davidson, and T. Honjo. 1999. Specific expression of activation-induced
cytidine deaminase (AID), a novel member of the RNA-editing deaminase family
in germinal center B cells. J. Biol. Chem. 274: 18470 –18476.
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1. Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302:
575–581.
2. Alt, F. W., T. K. Blackwell, and G. D. Yancopoulos. 1987. Development of the
primary antibody repertoire. Science 238: 1079 –1087.
3. Storb, U. 1998. Progress in understanding the mechanism and consequences of
somatic hypermutation. Immunol. Rev. 162: 5–11.
4. Reynaud, C. A., V. Anquez, H. Grimal, and J. C. Weill. 1987. A hyperconversion
mechanism generates the chicken light chain preimmune repertoire. Cell 48:
379 –388.
5. Thompson, C. B., and P. E. Neiman. 1987. Somatic diversification of the chicken
immunoglobulin light chain gene is limited to the rearranged variable gene segment. Cell 48: 369 –378.
6. Knight, K. L., and R. S. Becker. 1990. Molecular basis of the allelic inheritance
of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity. Cell 60: 963–970.
7. Parng, C. L., S. Hansal, R. A. Goldsby, and B. A. Osborne. 1996. Gene conversion contributes to Ig light chain diversity in cattle. J. Immunol. 157: 5478 –5486.
8. Arakawa, H., S. Furusawa, S. Ekino, and H. Yamagishi. 1996. Immunoglobulin
gene hyperconversion ongoing in chicken splenic germinal centers. EMBO J. 15:
2540 –2546.
9. Arakawa, H., K. Kuma, M. Yasuda, S. Ekino, A. Shimizu, and H. Yamagishi.
2002. Effect of environmental antigens on the Ig diversification and the selection
of productive V-J joints in the bursa. J. Immunol. 169: 818 – 828.
10. Arakawa, H., K. Kuma, M. Yasuda, S. Furusawa, S. Ekino, and H. Yamagishi.
1998. Oligoclonal development of B cells bearing discrete Ig chains in chicken
single germinal centers. J. Immunol. 160: 4232– 4241.
11. Kong, Q., L. Zhao, S. Subbaiah, and N. Maizels. 1998. A ␭ 3⬘ enhancer drives
active and untemplated somatic hypermutation of a ␭ 1 transgene. J. Immunol.
161: 294 –301.
12. Chien, N. C., R. R. Pollock, C. Desaymard, and M. D. Scharff. 1988. Point
mutations cause the somatic diversification of IgM and IgG2a antiphosphorylcholine antibodies. J. Exp. Med. 167: 954 –973.
13. Crews, S., J. Griffin, H. Huang, K. Calame, and L. Hood. 1981. A single VH gene
segment encodes the immune response to phosphrylcholine: somatic mutation is
correlated with the class of the antibody. Cell 25: 59 – 66.
14. Ford, J. E., M. G. McHeyzer-Williams, and M. R. Lieber. 1994. Analysis of
individual immunoglobulin ␭ 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 –1822.
15. Bernard, O., N. Hozumi, and S. Tonegawa. 1978. Sequences of mouse immunoglobulin light chain genes before and after somatic changes. Cell 15:
1133–1144.
16. Wysocki, L. J., and M. L. Gefter. 1989. Gene conversion and the generation of
antibody diversity. Annu. Rev. Biochem. 58: 509 –531.
17. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and
T. Honjo. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell
102: 553–563.
18. Arakawa, H., J. Hauschild, and J. M. Buerstedde. 2002. Requirement of the
activation-induced deaminase (AID) gene for immunoglobulin gene conversion.
Science 295: 1301–1306.
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