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*
Basel Institute for Immunology, Basel, Switzerland; and
GSF-National Research Center for Environment and Health, Institute of Clinical Molecular Biology and Tumor Genetics, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-kb stretch of DNA that includes the rearranged V(D)J segment and
thus defines Ag receptor specificity (4, 5, 6). Hypermutation
increases the diversity of the Ab repertoire, allowing the organism to
cope with the immense variability of pathogenic agents and organism.
Despite an elaborate effort by many research groups over more than two
decades, the molecular mechanism of somatic hypermutation remains
elusive. However, mounting evidence suggests that transcription is critical for the hypermutation process. This is supported by the observation that the transcription initiation site defines the 5' boundary of the hypermutated area (7, 8, 9). Many studies have demonstrated that the presence of Ig enhancers is required to target a transgene for hypermutation (10, 11, 12). The Ig enhancers probably serve a dual role for the hypermutation process. They presumably contain cis-element(s) to target the mutator system and provide transcriptional activation of the Ig loci (13, 14). A correlation between the mutation frequency and the transcription level in the Ig loci has been reported by Fukita et al. (15, 16). We used here a previously described green fluorescent protein (GFP)3-based assay (17, 18) to directly investigate the linkage of transcription levels and mutation rate in the hypermutating pre-B cell line 18-81 (19). A GFP transgene containing a premature stop codon, which renders the GFP gene nonfunctional, was stably transfected into cell line 18-81. The GFP gene only becomes functionally expressed in revertant cells when the stop codon is reverted, for example, by a point mutation. GFP-expressing revertant cells can be easily and reliably detected by flow cytometry or fluorescence microscopy (17). In this study the GFP transgene is driven by the inducible tet-on promoter (20, 21). By titration of doxycyclin (Dox) the transcription level of the GFP transgene can be modulated. Thus, the reversion rate at the premature stop codon can be assayed at different transcription levels. Here we demonstrate that the reversion rate of the GFP transgene is linked to its transcription level. Increased transcription levels correlate to higher reversion rates. We further show that trichostatin A (TSA), a histone-acetylating drug, caused an additional 2- fold increase in the mutation rate. This is the first demonstration of a drug to affect the mutator system of the cell line 18-81.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the plasmid pI, the AseI-SalI fragment of plasmid pEGFP-N1 (Clontech, Palo Alto, CA) was replaced by the XhoI-SalI fragment from plasmid pUHC 13-3 (22) containing the tet operon. To generate the plasmid pI-Enh, the 1-kb XbaI fragment from plasmid phyp#2 (23) containing the Ig heavy chain large intron enhancer (ELi) was blunt-ended and cloned into the AflII site of plasmid pI. The premature stop codon was introduced at aa position 107 of the EGFP gene by oligonucleotide-directed in vitro mutagenesis using the oligo 5'-GGA CGA CGG CAA CTA GTA TAC CCG CGC CGA GG-3'. To verify expression levels, all constructs were generated with and without the premature stop codon. Constructs pI and pI-Enh were linearized before transfection with Alw44I and than cotransfected with plasmid pRT-TA (20) by electroporation in a Bio-Rad electroporator (Hercules, CA). The copy number of the stably integrated plasmids ranged from one to four copies.
Cell cultures
The hypermutation-active pre-B cell line 18-81 was cultured in RPMI medium containing 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 IU/ml penicillin-streptomycin, and 50 mM 2-ME.
Flow cytometry
Before expansion the cell cultures were purged of pre-existing revertant cells by sorting on a MoFlo high speed sorter (Cytomation, Fort Collins, CO) using the sort mode: single one drop. PKH-26 (Sigma, St. Louis, MO) staining was performed according to the recommendation of the manufacturer. On day 0, 3–5 million nonfluorescent cells were seeded into 100 ml of fresh medium. The volume of the culture was slowly expanded up to 500 ml over the course of 4 days. Each day (every 24 h) a sample was removed, and the numbers of fluorescent and nonfluorescent cells among a million viable cells were analyzed by flow cytometry. The mutation rate was determined by applying the curve fit function from program CA-Cricket Graph III 5.2 (Computer Associates International, Islandia, NY). For phenotypic analysis, cells were washed once in PBS and resuspended with PBS and 1% BSA. Flow cytometric analysis was performed on a FACSCalibur (BD Sciences, Mountain View, CA).
Preparation of mRNA samples and Northern blot analysis
Northern blot analysis was performed essentially as previously
described (24). mRNA was extracted from expanding cultures
with Oligotex Direct mRNA Midi Kit (Qiagen, Chatsworth, CA). For each
Northern blot, 1 µg of mRNA was loaded per lane. The probes used were
fragments from the GFP gene, the GAPDH gene and the activation-induced
cytidine deaminase (AID) gene. The probes were radiolabeled using a
random primer labeling kit (Roche, Indianapolis, IN) and
[
-32P]dCTP (Amersham Pharmacia Biotech,
Arlington Heights, IL).
RT-PCR and nucleotide sequence analysis
Total RNA was extracted from sorted revertant cells with TRIzol (Life Technologies, Gaithersburg, MD), and cDNA was prepared by RT with Superscript II reverse transcriptase (Life Technologies) using a oligo(dT)11 primer. PCR amplification was performed with Taq polymerase using the buffer supplied by the manufacturer (Roche). The PCR primers for the GFP gene were: sense, 5'-GTG CCC ATC CTG GTC GAG CTG GAC-3'; and antisense, 5'-GTG ATC GCG CTT CTC GTT GGG GTC-3'. The purified PCR product of 617-nt length was directly sequenced with the GFP nested primer (5'-GGCGAGGGCGATGAC-3') using the dideoxy chain termination method with T7 polymerase (Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the influence of transcription levels on the
mutability of a reporter gene, we generated construct pI (Fig. 1
A) and stably transfected it
into cell line 18-81. In construct pI the GFP gene is driven by the
inducible tet-on promoter, Ptet. Transcription at Ptet is induced by
supplementing Dox to the growth medium and can be modulated by altering
the Dox concentration. The GFP gene contains a premature TAG stop codon
and is therefore nonfunctional. In stably transfected clones revertant
cells express the functional GFP. Flow cytometric analysis enables the
enumeration of revertant cells and the measurement of GFP expression
levels within individual revertant cells, shown as the relative
fluorescence intensity (RFI). The RFI value of revertant cells can be
used as an approximate and indirect measurement for the transcription
level of the GFP transgene (17).
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Mutation rate is coupled to transcription level
In a screen of roughly 100 pI transfection clones, three
independent transfection clones were identified that were Dox
inducible, had a very low basal transcription level in the absence of
Dox, and showed a significant number of revertant cells. Fig. 2 shows the 5-day flow analysis
for one of the transfection clones, pI#1, at different Dox
concentrations. Transfection clone pI#1 harbors three copies of the
transgene, as determined by Southern blot analysis (data not shown). In
Fig. 3 the flow cytometry data of Fig. 2
are plotted graphically as a function of revertant frequency vs cell
divisions. Very few revertant cells were detected in the absence of Dox
(culture A). This is not surprising, because transcription of the
transgene is required to express GFP in revertant cells. With
increasing Dox concentrations, higher numbers of revertant cells were
generated. The mutation rate in culture B is 0.8 x
10-5 mutations/base pair/generation, in culture
C it is 2.1 x 10-5 mutations/base
pair/generation, and in culture D it is 5.0 x
10-5 mutations/base pair/generation. The mean
GFP expression level of the revertant cells (shown as RFI values)
increased with higher Dox concentrations. The RFI values were 650 for
culture B, 1023 for culture C, and 1681 for culture D. The increase in
transcription levels reached a threshold at a Dox concentration of 0.8
µg/ml, beyond which no further effect was seen. To verify the
correlation between RFI values and the transcription level of the GFP
transgene, Northern blot analysis was performed on the steady state
mRNA levels of the GFP transgene in cultures A, B, C, and D (Fig. 4). The RFI values of the revertant cells
corresponded to the mRNA level of the GFP transgene and can be used as
an indirect measurement of the transcription activity of the GFP
transgene.
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The higher mutation rates at increased Dox concentrations could be due to a potential mutagenic effect of Dox. To address this possibility, a constitutively transcribed GFP transgene, driven by the thymidine kinase promoter and the large intron enhancer (18), was stably transfected into cell line 18-81. The reversion frequency of three independent transfection clones was then analyzed in the presence or the absence of Dox. The transfection clones displayed no significant differences in their mutation frequencies when expanded at 1.0 µg/ml Dox or in the absence of Dox. The RFI values of the revertant cells were also unaffected by the addition of Dox (data not shown).
Mutation rate responds directly to changes in transcription level
It is also possible that the decreased mutation rate at lower transcription levels (cultures B and C) is due to undetected, low GFP-expressing revertants, which are hidden in the GFP-negative cell population. These putative, GFPlow revertants should become detectable by raising the Dox concentration and thus increasing the GFP expression level. To address this possibility, culture B was split on day 2. Half of the culture continued to expand at 0.1 µg/ml Dox (culture B), while the Dox concentration for the other half of the culture was raised to 1.0 µg/ml (culture E). Similarly, the Dox concentration for half of culture C was raised to 1.0 µg/ml Dox (culture F). If the same number of revertants had accumulated over the first 2 days in cultures B, C, and D, then a similar frequency of revertants would be expected on day 3 in cultures D, E, and F upon full transcription induction with 1.0 µg/ml Dox. Alternatively, if cultures B and C generated fewer revertants than culture D during the first 2 days, a lower frequency of revertants would be expected upon full transcription induction.
Raising the Dox concentration to 1.0 µg/ml caused a rapid increase in the mutation rate in cultures E and F to the level of culture D. The mutation rates (4.0 x 10-5 mutations/base pair/generation for culture E and 4.7 x 10-5 mutations/base pair/generation for culture F) were comparable to that for culture D (5.0 x 10-5 mutations/base pair/generation). The RFI values for revertant cells also reached comparable levels in culture D (1681 RFI), E (1619 RFI), and F (1769 RFI), indicating the enhanced transcriptional activity in cultures E and F after raising the Dox concentration. Thus, alterations of the transgenic transcription level caused rapid changes in the mutation rate at the premature stop codon. However, the absolute number of revertants remained lower in both cultures E and F than in culture D. This confirms that at lower transcription levels of the GFP transgene, fewer revertant cells were generated.
TSA treatment boosts the mutation rate
TSA treatment of cells can increase the transcription level of
endogenous genes and transgenes (25, 26, 27, 28). TSA blocks
histone deacetylases and causes nonspecific opening of the chromatin
structure by hyperacetylation of histones (29, 30). To
determine the effect of TSA on the mutability of the GFP transgene,
cells from culture D were further expanded at different TSA
concentrations (17 and 34 nM). Culture D was consequently split on day
2 into three equal cultures (D, G, and H), all of which were maintained
at 1.0 µg/ml Dox. Culture G was supplemented with 17 nM TSA, culture
H was supplemented with 34 nM TSA, and culture D contained no TSA.
Addition of TSA caused several dose-dependent effects on the cells
(Fig. 5). First, the generation time was
prolonged for cultures G and H. Second, the RFI values for revertant
cells increased to 1941 for culture G and 2917 for culture H,
indicating higher transcription levels of the GFP transgene compared
with 1681 in culture D. Northern blot analysis confirmed increased
steady state mRNA levels of the GFP transgene in the TSA-treated
cultures (data not shown). Third, the frequency of revertant cells was
higher in the TSA-treated cultures than in culture D. The prolongation
of the generation time and the higher revertant frequency both
translate into increased mutation rates for culture G (9.4 x
10-5 mutations/base pair/generation) and culture
H (1.4 x 10-4 mutations/base
pair/generation). Therefore, TSA treatment caused a 2-fold increase in
the mutation rate.
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The presence of the Ig enhancers is required for an efficient
targeting of the Ig mutator system (12, 23). In
particular, the ELi appears to play an essential role in the
hypermutation mechanism, although by itself the ELi is not sufficient
to confer maximum levels of hypermutation (31). Construct
pI-Enh (Fig. 1
B) was generated and stably transfected into
18-81 to address whether targeting of the inducible GFP transgene can
be improved. Construct pI-Enh is identical with construct pI, except
that it includes the ELi downstream of the GFP gene. In contrast to
construct pI, 80% of the pI-Enh transfection clones had a significant
number of revertant cells (data not shown). Thus, the presence of the
large intron enhancer greatly increases targeting of the GFP transgene
by the 18-81 mutator system. The pI-Enh transfection clones showed an
elevated level of background transcription for the GFP transgene even
in the absence of Dox. The ELi presumably activates the Ptet promoter
independently of Dox. Yet, addition of 1.0 µg/ml Dox increased the
transcription level 2- to 10-fold for most of the pI-Enh transfection
clones. Fig. 6 shows the reversion
analysis for four independent pI-Enh-transfected clones grown in either
the presence or the absence of Dox. The mutation rates for all four
pI-Enh transfection clones ranged from 2.5 to 4.6 x
10-6 mutations/base pair/generation without Dox
and from 1.6 to 2.2 x 10-5 mutations/base
pair/generation at 1.0 µg/ml Dox. Thus, the mutability of the GFP
transgene increased when 1.0 µg/ml Dox was added to the medium. The
RFI levels of the four pI-Enh clones ranged from 251 to 311 without Dox
and from 1237 to 2033 at 1.0 µg/ml Dox, and increased mutation rates
again coincided with higher RFI values and thus consequently with
higher transcription levels. Yet, the transfectant clone with the
highest transcription level (pI-Enh#2) did not display the highest
mutation rate. The differences in the mutation rate among the four
transfection clones were also not related to the copy number of the
transgene. Transfection clones 1 and 3 contained one copy of the
transgene each, transfection clone 2 contained three copies, and
transfection clone 4 contained four copies (Southern blot analysis not
shown). This indicates that other parameters besides the transcription
levels and the copy number of the transgene may influence the
mutability of the GFP transgene (32).
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A recent study by Muramatsu et al. (33) implicated a
crucial role of AID in both somatic hypermutation and Ig class
switching. Somatic hypermutation was drastically reduced in AID gene
knockout mice, indicating that the expression of the AID gene is
essential for the hypermutation mechanism (33, 34). To
investigate the expression of the AID gene in the hypermutating cell
line 18-81, Northern blot analysis of total RNA was performed (Fig. 7). Two AID transcripts, 1.9 and 1.3 kb
in length, were detectable in the hypermutating cell line 18-81. In
contrast, various control cell lines (28-C-9, 70Z/3, and Raw 8.1) that
failed to hypermutate the GFP transgene were devoid of detectable AID
transcripts. Both AID transcripts in the cell line 18-81 were shorter
than the predicted length of the murine AID transcript at 2.3 kb and
will require further characterization. Yet, the presence of AID
transcripts in the hypermutating cell line 18-81 compared with
nonhypermutating cell lines supports the assumption that its mutator
mechanism is indeed closely related to the in vivo somatic
hypermutation process.
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The genetic event that caused the reversion of the GFP transgene
was determined for eight pI-Enh revertants (obtained from eight
independent pI-Enh transfection clones) and three pI revertants
(obtained from three independent pI transfection clones). All 11
revertants were caused by point mutation in the TAG stop codon. Nine
revertants had a G to C transversion, and two revertants had a G to T
transversion. The strong GC bias is characteristic of mutation hot
spots in the Ig genes (35, 36, 37, 38). Due to the limited pool of
our sequencing data (4 kb), obtained by sequencing 11 revertant
cells, only one additional point mutation, a G to T transversion, was
detected. This additional point mutation also occurred in an Ig hot
spot motif, yet on the opposite strand (data not shown). This indicates
that the mutator system of cell line 18-81 may recognize Ig hot spots
on both DNA strands.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have adapted our previously published GFP-based reversion assay (17) to investigate the influence of transcription levels on the mutator mechanism of the cell line 18-81. The inducible tet-on promoter enables us to modulate the transcription levels in individual transfection clones and directly measure the mutation rate of the GFP reporter gene.
The importance of transcription for the Ig hypermutation mechanism has been demonstrated in vivo by several groups (8, 9, 15, 47, 48). In particular, Fukita et al. (15) demonstrated a direct correlation between the pre-mRNA transcription levels and the mutation frequency at the Ig heavy chain locus. Our studies, using the inducible GFP reversion assay, provide direct evidence that this correlation also exists in the hypermutating cell line 18-81. Increasing the transcription level of the GFP transgene by modulating the Dox concentration resulted in higher mutation rates at a premature stop codon. The mutator of cell line 18-81 is not an all-or-nothing system, because intermediate transcription levels at 0.1 and 0.2 µg/ml Dox resulted in a corresponding gradual increase in mutation rates. The mutator system responds rapidly to changes in the transgenic transcription level. The model proposed by Peters and Storb (9) accounts for such a correlation between transcription level and mutator activity. This model predicts that a specific mutator factor binds to the initiating RNA polymerase complex. Binding of this putative mutator factor is thought to cause premature pausing of the elongating RNA polymerase. Stalling of the RNA polymerase acts as a signal to attract the nucleotide excision repair (NER) system (49, 50, 51). The NER system introduces a single-strand gap in the DNA adjacent to the stalled RNA polymerase. A putative, error-prone DNA polymerase, which is part of the B cell-specific NER system, would then introduce point mutations in the repaired region during the fill-in reaction (52, 53). However, the hypermutation rate in NER knockout mice is not decreased (54, 55), suggesting that the mechanism of somatic hypermutation may be more complex than predicted by that model. Alternatively, the transcription machinery may not directly interact with the mutator factor(s). Instead, transcription could be required to alter the chromatin structure and thus provide increased accessibility to mutator factor binding sites. For V(D)J recombination in T lymphocytes, a linkage of histone H3 acetylation status at a transgenic V(D)J recombination reporter gene and its accessibility for V(D)J recombination machinery were recently demonstrated (56).
TSA, an inhibitor of histone deacetylases, has been shown to change the chromatin structure by hyperacetylation of histones (57, 58). Adding TSA to the fully induced pI transfection clone caused a further dose-dependent increase in the mutation rate. TSA treatment also increased the transcription level beyond that of the fully Dox-induced (i.e., 1.0 µg/ml) GFP transgene. This elevation of the GFP transcription level would be the most straightforward explanation for the hypermutation-boosting effect of TSA. Alternatively, treating the cells with TSA could up-regulate the expression of the mutator factor(s) in the cell line 18-81, because the effect of TSA is nonspecific and therefore not restricted to the GFP transgenic locus (59).
Does TSA treatment influence the mutability of the GFP transgene in the absence of Dox-induced transcription? Our attempts to answer this question have been unsuccessful to date, because we found that TSA treatment before Dox induction abolishes the inducibility of the GFP transgene by Dox. We are presently determining whether we can overcome this problem.
The ELi plays probably a dual role for the somatic hypermutation mechanism: it contains a putative cis-element(s) to target the mutator system, and it provides a transcriptional activation site(s) for the heavy chain gene. The presence of the ELi in plasmid pI-Enh increased the targeting of the GFP transgene by the mutator system of the cell line 18-81. The search for the cis-element(s) has been hindered particularly by the lack of a suitable model system. Deletions within the ELi (including the matrix attachment region) regularly resulted in a marked reduction of both the mutability and the transcription levels (13). Yet, to elucidate the putative cis-element(s) in the ELi it will be necessary to ensure that manipulations of the enhancer do not also affect the transcription level of the reporter gene. The inducible GFP system described here provides transcription independently of the enhancer and may therefore be a useful tool to search for the mutator targeting element(s).
Depending on its integration site, a transgene lacking the ELi can nevertheless become hypermutable, as seen for the rare hypermutable pI transfection clones. The GFP gene may have integrated into one of the Ig loci or, alternatively, the mutator mechanism is not exclusively restricted to the Ig loci. In vivo data indicated that the Bcl-6 gene in humans, but not that in mice, can be hypermutated (60, 61, 62, 63). It remains to be shown whether the Bcl-6 locus shares mutator targeting elements with the Ig loci or whether the mutations in the Bcl-6 gene are somewhat the "collateral damage" of the mutator system.
In summary, the inducible GFP reversion assay is a powerful tool to study the influence of transcription levels on mutability. This system is easily applicable and may prove useful to test other cell lines. Our results using TSA show that the complex interaction of several parameters, such as generation time, expression level, and mutability, can be assessed simultaneously. In addition, the GFP-based system can be exploited for a systematic screening of drugs, to find inhibitors and stimulators of the mutator system.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jürgen Bachl, GSF-National Research Center for Environment and Health, Institute of Clinical Molecular Biology and Tumor Genetics, Marchioninistrasse 25, 81377 Munich, Germany.
3 Abbreviations used in this paper: GFP, green fluorescent protein; Dox, doxycyclin; AID, activation-induced cytidine deaminase; ELi, Ig heavy chain large intron enhancer; RFI, relative fluorescence intensity; TSA, trichostatin A; NER, nucleotide excision repair.
Received for publication September 6, 2000. Accepted for publication February 1, 2001.
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References |
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H. Nagaoka, S. Ito, M. Muramatsu, M. Nakata, and T. Honjo DNA cleavage in immunoglobulin somatic hypermutation depends on de novo protein synthesis but not on uracil DNA glycosylase PNAS, February 8, 2005; 102(6): 2022 - 2027. [Abstract] [Full Text] [PDF]
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X. Wu, P. Geraldes, J. L. Platt, and M. Cascalho The Double-Edged Sword of Activation-Induced Cytidine Deaminase J. Immunol., January 15, 2005; 174(2): 934 - 941. [Abstract] [Full Text] [PDF]
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L. Wang, W. C. Jackson, P. A. Steinbach, and R. Y. Tsien Evolution of new nonantibody proteins via iterative somatic hypermutation PNAS, November 30, 2004; 101(48): 16745 - 16749. [Abstract] [Full Text] [PDF]
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 C. L. Wang, D. C. Yang, and M. Wabl Directed molecular evolution by somatic hypermutation Protein Eng. Des. Sel., September 1, 2004; 17(9): 659 - 664. [Abstract] [Full Text] [PDF]
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L. Delpy, C. Sirac, C. Le Morvan, and M. Cogne Transcription-Dependent Somatic Hypermutation Occurs at Similar Levels on Functional and Nonfunctional Rearranged IgH Alleles J. Immunol., August 1, 2004; 173(3): 1842 - 1848. [Abstract] [Full Text] [PDF]
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F. J. X. Spillmann and M. Wabl Endogenous Expression of Activation-Induced Cytidine Deaminase in Cell Line WEHI-231 J. Immunol., August 1, 2004; 173(3): 1858 - 1867. [Abstract] [Full Text] [PDF]
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M. Cascalho Advantages and Disadvantages of Cytidine Deamination J. Immunol., June 1, 2004; 172(11): 6513 - 6518. [Abstract] [Full Text] [PDF]
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K. M. McBride, V. Barreto, A. R. Ramiro, P. Stavropoulos, and M. C. Nussenzweig Somatic Hypermutation Is Limited by CRM1-dependent Nuclear Export of Activation-induced Deaminase J. Exp. Med., May 3, 2004; 199(9): 1235 - 1244. [Abstract] [Full Text] [PDF]
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K. Machida, K. T.-N. Cheng, V. M.-H. Sung, S. Shimodaira, K. L. Lindsay, A. M. Levine, M.-Y. Lai, and M. M. C. Lai Hepatitis C virus induces a mutator phenotype: Enhanced mutations of immunoglobulin and protooncogenes PNAS, March 23, 2004; 101(12): 4262 - 4267. [Abstract] [Full Text] [PDF]
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 Z. Li, C. J. Woo, M. D. Iglesias-Ussel, D. Ronai, and M. D. Scharff The generation of antibody diversity through somatic hypermutation and class switch recombination Genes & Dev., January 1, 2004; 18(1): 1 - 11. [Full Text] [PDF]
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T. Eto, K. Kinoshita, K. Yoshikawa, M. Muramatsu, and T. Honjo RNA-editing cytidine deaminase Apobec-1 is unable to induce somatic hypermutation in mammalian cells PNAS, October 28, 2003; 100(22): 12895 - 12898. [Abstract] [Full Text] [PDF]
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C. L. Morvan, E. Pinaud, C. Decourt, A. Cuvillier, and M. Cogne The immunoglobulin heavy-chain locus hs3b and hs4 3' enhancers are dispensable for VDJ assembly and somatic hypermutation Blood, August 15, 2003; 102(4): 1421 - 1427. [Abstract] [Full Text] [PDF]
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B. Reina-San-Martin, S. Difilippantonio, L. Hanitsch, R. F. Masilamani, A. Nussenzweig, and M. C. Nussenzweig H2AX Is Required for Recombination Between Immunoglobulin Switch Regions but Not for Intra-Switch Region Recombination or Somatic Hypermutation J. Exp. Med., June 16, 2003; 197(12): 1767 - 1778. [Abstract] [Full Text] [PDF]
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A. Sohail, J. Klapacz, M. Samaranayake, A. Ullah, and A. S. Bhagwat Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations Nucleic Acids Res., June 15, 2003; 31(12): 2990 - 2994. [Abstract] [Full Text] [PDF]
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M. S. Gordon, C. M. Kanegai, J. R. Doerr, and R. Wall Somatic hypermutation of the B cell receptor genes B29 (Igbeta , CD79b) and mb1 (Igalpha , CD79a) PNAS, April 1, 2003; 100(7): 4126 - 4131. [Abstract] [Full Text] [PDF]
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A. Martin and M. D. Scharff Somatic hypermutation of the AID transgene in B and non-B cells PNAS, September 17, 2002; 99(19): 12304 - 12308. [Abstract] [Full Text] [PDF]
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D. D. Dudley, J. P. Manis, A. A. Zarrin, L. Kaylor, M. Tian, and F. W. Alt Internal IgH class switch region deletions are position-independent and enhanced by AID expression PNAS, July 23, 2002; 99(15): 9984 - 9989. [Abstract] [Full Text] [PDF]
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 K. Yoshikawa, I.-m. Okazaki, T. Eto, K. Kinoshita, M. Muramatsu, H. Nagaoka, and T. Honjo AID Enzyme-Induced Hypermutation in an Actively Transcribed Gene in Fibroblasts Science, June 14, 2002; 296(5575): 2033 - 2036. [Abstract] [Full Text] [PDF]
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X. Chen, K. Kinoshita, and T. Honjo Variable deletion and duplication at recombination junction ends: Implication for staggered double-strand cleavage in class-switch recombination PNAS, November 20, 2001; 98(24): 13860 - 13865. [Abstract] [Full Text] [PDF]
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C.-G. Lee, K. Kinoshita, A. Arudchandran, S. M. Cerritelli, R. J. Crouch, and T. Honjo Quantitative Regulation of Class Switch Recombination by Switch Region Transcription J. Exp. Med., August 6, 2001; 194(3): 365 - 374. [Abstract] [Full Text] [PDF]
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