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*
Department of Pediatrics, Division of Basic Sciences, and
Division of Biostatistics, National Jewish Medical and Research Center, Denver, CO 80206; and
Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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clusters and by human VH genes,
suggesting that a common mutation mechanism exists for all Ig V genes
of both species. Using di- and trinucleotide target preferences, we
performed a comprehensive analysis of human and murine germline V genes
to predict regional mutabilities. Heavy chain genes of both species
exhibit indistinguishable patterns in which complementarity-determining
region 1 (CDR1), CDR2, and framework region 3 (FR3) are predicted to be
more mutable than FR1 and FR2. This prediction is borne out by
empirical mutation data from nonproductively rearranged human
VH genes. Analysis of light chain genes in both species
also revealed a common, but unexpected, pattern in which FR2 is
predicted to be highly mutable. While our analyses of nonfunctional Ig
genes accurately predicts regional mutation preferences in
VH genes, observed relative mutability differences between
regions are more extreme than expected. This cannot be readily
accounted for by nascent mRNA secondary structure or by a supplemental
gene conversion mechanism that might favor nucleotide replacements in
CDR. Collectively, our data support the concept of a common mutation
mechanism for heavy and light chain genes of mice and humans with
regional bias that is qualitatively, but not quantitatively, accounted
for by short nucleotide sequence composition. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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regulatory elements
(17). On the basis of these data, Storb et al. (17) proposed a model in
which a mutator travels with the transcription machinery and stalls at
points of nascent RNA secondary structure, thereby leading to higher
frequencies of mutagenesis in adjacent DNA. intron and 3' enhancers
play an essential role in the mutation process as revealed in
transgenic studies (15, 18, 19, 20), yet the analogous heavy chain gene
enhancers appear to be insufficient for full mutagenesis (21, 22, 23, 24).
These differences raise the possibility that different mutation
mechanisms may operate on heavy and light chain V region genes. Somatic mutations are not distributed randomly throughout V region genes. In part this is due to selection pressures that favor alteration of the complementarity-determing region (CDR).3 However, an intrinsic target bias of the mutation mechanism has been revealed by the nonrandom nature of mutations within sequences that are not influenced by cellular selection processes (8, 25, 26). Lebecque and Gearhart (27) observed that particular types of mutations occurred more frequently than others, leading them to deduce that the mechanism possesses a strand bias. Rogozin and Kolchanov (28) observed a higher than average frequency of mutations in RGYW and TAA nucleotide sequences. Similarly, Betz et al. (29) observed that AGY serine codons in a passenger transgene mutated at an inordinately high frequency. This led them to propose that CDR are more intrinsically mutable than FR because of differences in AGY vs TCN serine codon use by segments encoding the two types of regions (30). However, somatic mutations often occur in nucleotide sequences that are not included in these motifs, and this led us to examine the relative mutability of all di- and trinucleotides. Our analysis was confined to mutations within Ig intronic sequences that presumably are not subject to the indirect, but substantial, influences of selection (31). The results of this work revealed a hierarchy of mutability among all di- and trinucleotide sequences. In addition, the results helped to refine previously proposed motifs, identifying AGC, for example, as the mutable component of the AGY motif. Defining intrinsic mutability provides clues to the mechanism of mutation and is important to interpretations concerning Ag-driven selection in immune and autoimmune processes that are often drawn on the basis of mutational distribution within V genes.
In this manuscript we compare relative intrinsic mutabilities of di- and trinucleotides in heavy and light chains and V genes of mice and humans in search of evidence for common or distinct mutation mechanisms. In addition, we used di- and trinucleotide mutability preferences in a comprehensive analysis of germline V region gene sequences to predict intrinsic regional mutabilities of CDR and FR. We compared these predictions with empirical mutation data from nonproductively rearranged human VH genes to determine whether regional mutability indexes based upon di- and trinucleotides composition alone could predict the pattern and relative extent of mutational accumulation in different segments of Ab V genes. Finally, we examined the relationship between predicted RNA secondary structure and observed regional mutability. Our results support the idea that all Ig genes in mice and humans mutate by a common mechanism and that di- and trinucleotide sequence composition alone can predict regional mutation patterns, but in a nonquantitative manner. The quantitative discrepancy is not obviously resolved by taking predicted nascent secondary RNA structure into consideration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For regional mutability predictions of human V genes, sequences
were extracted from the V BASE index (32) which contains all known
human germline heavy and light chain V genes. When sequence
discrepancies due to potential mistakes or polymorphisms occurred, the
Tomlinson et al. (33) sequence was used. Murine Ig V gene sequences
were extracted from the ABG germline directory of mouse Ig sequences,
organized by the Ab Group of Instituto de Biotecnologia, Universidad
Nacional Autonoma de Mexico (34). In those few cases where identical
amino acid sequences were specified by different nucleotide sequences,
only one sequence was selected for analysis. Because complete sequence
information was not available for all V genes of any strain, the known
sequences from all strains were combined in our analyses. Altogether,
47, 40, and 29 human heavy, , and 
genes as well as 72 and 42
murine heavy and genes were included in the analyses.
Empirical mutation frequencies were determined using somatically
mutated, but unselected, Ig sequences derived from several sources.
Mutations in murine intronic sequences for the JH
and J clusters that flank assembled V gene exons were analyzed using
raw data (A/J + literature + autoimmune) obtained by Smith et al. (31).
Mutations in human VH genes were analyzed from
sequences of nonproductively rearranged VH genes
obtained by Dorner et al. (35) and Dunn-Walters and Spencer (36). In
all cases insertions and deletions were not included in the analyses,
and nonproductive rearrangements were defined by out-of-frame J
segments or junctional stop codons. We also analyzed somatic mutations
in a transgenic construct containing a 100-nt insertion with
repeated EcoRV and PvuII restriction sites under
the control of regulatory elements (EPS insert) (17). Forty-six
such mutated transgene sequences (27,278 nt) containing 135 mutations
were obtained from data reported by Storb et al. (17).
Sequence manipulation
All unmutated germline sequences were manipulated and analyzed with MacVector version 5.0 software (Oxford Molecular Group, Beaverton, OR). Sequences were divided into FR and CDR according to both the Kabat (37) and Chothia (38, 39, 40) definitions; the former is based on sequence variability, and the latter is based on location of structural loop regions. In each sequence file used in the dinucleotide analysis, one extra nucleotide was included from adjacent sequence at each end of the region under consideration. Similarly, sequence files for trinucleotide analyses included two extra nucleotides at each end. Mutated nonproductive human VH sequences were treated identically, except that 10 nt were deleted from the 3' end of FR3 to insure that discrepancies with germline sequence were due to somatic mutagenesis as opposed to junctional diversification during V segment assembly (41, 42). The transgene of Storb et al. (17) was divided into segments of 100 nt in a manner that left the artificial EPS insertion intact in one file. Collectively, 6507 files containing regional sequence data for V genes were generated for analysis.
Oligonucleotide mutability indexes
Di- and trinucleotide observed/expected (obs/exp) mutability indexes were calculated as previously described (31). Briefly, the number of times a given oligonucleotide within a segment of DNA contained a mutation was divided by the number of times the oligonucleotide was expected to be mutated for a mechanism with no bias. Mutability indexes are normalized for the di- and trinucleotide compositions of unmutated templates covering the precise regions for which mutational data were analyzed.
Predicting regional mutability
Regional mutability based solely on DNA sequence was predicted using di- and trinucleotide obs/exp mutability indexes (A/J and literature) defined by Smith et al. (31). The mutability index of a di- or trinucleotide is a normalized measure of its tendency to mutate, where a value of 1 indicates the average mutability for di- or trinucleotides. The predicted mutability index for a region was calculated by determining the number of times each di- or trinucleotide occurred within each region (file) of each gene (regardless of frame of reference) and multiplying by its mutability index. The resulting products for the 16 dinucleotides or 64 trinucleotides were summed and then divided by the total number of di- or trinucleotides in the region (file) under consideration. That is, di- and trinucleotide mutabilities were summed in all frames of reference. The composite mutability index predicted for a type of region, for example nucleotide sequences encoding human VH FR1, was determined by summing all di- or trinucleotide products (occurrences x mutability index) and dividing this number by the sum of all di- or trinucleotide occurrences in the region for all such sequences in the database. When predicting regional mutabilities for human VH genes within the databases of nonproductively rearranged VH genes, calculations were weighted proportionally to the number of times a mutated version of a given gene appeared in the database. Microsoft Excel 98 and version 4.0 (Redmond, WA) were used for database management and calculations.
Observed mutability
The empirical relative mutabilities (observed mutability index)
for each region of the nonproductively rearranged human
VH genes and the murine transgene were
determined by dividing the number of mutations per nucleotide for a
region by the number of mutations per nucleotide for the entire gene.
In essence, this gives the obs/exp mutability ratio for a region, where
the expected frequency is the average frequency of mutations for the
whole gene. For a given subregion, such as nucleotide sequences
encoding VH FR1, the observed composite
mutability index was calculated as the number of mutations per
nucleotide in all VH FR1 of the database divided
by the number of mutations per nucleotide for the entire length (FR1,
CDR1, FR2, CDR2, FR3) of all V regions within the database.
Relationship of mutation distribution to mRNA secondary structure stability
Six-base intervals of the nonproductive human VH genes were defined by aligning all the sequences while adjusting for CDR length variability. Predicted folding energy of nascent mRNA was used as a measure of secondary structure stability. Members of the mutated nonproductive human VH gene databases from Dorner et al. and Dunn-Walters and Spencer (35, 36) were divided into 51-nt intervals in steps of 6 nt. The mRNA folding energy for each interval was calculated by the online version of Mfold (43) and reported in kilocalories per mole, where stability increases as the number becomes more negative. For each 6-nt segment, we determined the mRNA folding energy by averaging the folding energies of the 51-nt interval whose 3' end coincided with the 6-base segment under consideration and the two immediately flanking 51-nt intervals. This analysis correlates a 6-nt segment with upstream RNA secondary structure that has the potential to influence the polymerase complex. Calculations for some 6-nt segments in FR1 could not be performed due to lack of leader sequence information.
Statistics
Continuous data are summarized as the mean and SEM. Mean ratios
were compared between regions using one-way ANOVA. The percentages of
nucleotides with mutations were compared with the expected percentages
under the assumption of uniform mutation rates by
2 tests for comparing observed to expected
proportions. Pearson correlation coefficients were used to evaluate
associations between continuous variables. Simple linear regression
with confidence intervals and tests of hypotheses on the intercepts and
slopes were used to evaluate linear relationships between continuous
variables. All tests of hypotheses were considered significant at an
level of 0.05, except that tests for significance of mutation
indexes were performed at the 0.01 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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If a common mechanism generates somatic mutations within Ig heavy
and light chain V genes, then the two should reveal a common di- and
trinucleotide target bias. To test this idea, we calculated normalized
obs/exp mutability indexes for all di- and trinucleotides within
mutated JH and J intronic DNA from a panel of
murine hybridomas (31). The index is a measure of the tendency to
mutate, where a value of 1 is the average. As shown in Fig. 1
A and Table I, a comparison of dinucleotide
mutability indexes in the two regions revealed a close agreement. This
is most clearly seen in a simple regression analysis, which yielded a
slope and y-intercept of 1 and 0, respectively, within a
95% confidence interval. Similar results and statistical significance
were obtained upon comparative analyses of trinucleotide mutability
indexes for the two regions of DNA (Fig. 1B and Table II).
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DNA (Fig. 2A and Table I
). This was
demonstrated by the slope of their linear regression equaling 1 and
their y-intercept falling within a 95% confidence interval
of 0. In all cases GC and TA were the preferred targets. The
trinucleotide mutability indexes were also in close agreement as shown
in Fig. 2B and Table II. This consistent hierarchy of di-
and trinucleotide mutability in all analyzed Ig sequences supports the
idea that a common mutation mechanism acts on heavy and light chain
genes of both species regardless of whether DNA is located within an
intron or an exon encoding a V region domain.
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To further investigate the consistency of sequence-specific
mutability, we calculated the mutability index for each position of all
di- and trinucleotides in the murine JH and J
and human VH sequences. As shown in Table III, the JH and
J as well as the murine and human Ig sequences possessed a striking
consistency of mutational bias at the level of individual positions.
Besides providing further support for a common mutation mechanism,
these data indicate that positions within di- and trinucleotides are
targeted to differing degrees (29, 44).
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To address the importance of DNA sequence in directing somatic
mutation, we first comprehensively analyzed di- and trinucleotide
sequence compositions of murine and human germline V region genes.
Human and murine germline-encoded genes were divided into regions
encoding FR and CDR based on the definitions of Kabat (37) and Chothia
(38, 39, 40). For each region in every category, we calculated a composite
obs/exp mutability index. Our analysis includes all available unmutated
human and murine Ig sequences: 47, 40, and 29 human heavy, , and
genes and 72 and 42 murine heavy and Ig genes, respectively,
organized into 4780 regional sequence files.
The composite regional mutability indexes predicted for murine and
human VH genes are shown in Fig. 3. There is an excellent agreement
between results obtained with VH genes of the two
species. With obs/exp mutability indexes >1, CDR1 and CDR2 in both
species are predicted to mutate the most when normalized for length. In
contrast, FR1 and FR2 are predicted to mutate the least, while FR3 is
predicted to mutate at an intermediate level. The regional patterns
calculated using the Kabat and Chothia CDR and FR definitions are
similar. Although modest in extent, the predicted regional mutability
differences are remarkably consistent among individual
VH genes (data not shown) and regardless of
whether they are calculated using di- or trinucleotide mutability
indexes. This finding together with the large number of regions
examined and consistency between species suggest that the predicted
regional mutability differences are not due to chance alone.
Statistical analysis of the data support this interpretation (two-way
ANOVA for comparing means with p < 0.0001).
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).
Nearly identical regional trends were seen for human V,
human V, and murine V genes, with a remarkable consistency among
individual light chain genes (data not shown). Murine V genes were
excluded because their numbers were insufficient to permit statistical
analysis. As with the heavy chain genes, there was good agreement
between results obtained using di- and trinucleotide mutability
indexes according to either the Kabat or Chothia regional definition.
Predicted regional differences were statistically tested as before
and were significant (p < 0.0001).
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Our predictions of regional mutabilities are based solely on di-
and trinucleotide composition. To assess the importance of these short
sequences in directing mutation into a longer segment of DNA, we
compared the regional predictions to empirical data from somatically
mutated V genes. To exclude potential bias due to cellular selection,
only somatic mutations within nonproductively rearranged V genes were
analyzed. Only two available sets of human VH
sequences satisfied this important criterion, and no comparable data
were available for light chain genes. Empirical regional mutability
indexes were determined by dividing the actual number of mutations in
each region by the expected quantity, assuming a nondiscriminatory
mechanism would randomly distribute mutations throughout the
VH genes. Observed regional mutabilities in these
VH genes were compared with predicted regional
mutabilities for germline-encoded correlates, such that the germline
database was weighted according to the frequency with which each gene
was found in the database of mutated genes. As shown in Fig. 5, the results of these comparisons
revealed a generally good agreement between predicted and observed
mutability patterns. This was true for results obtained using both di-
and trinucleotide mutability indexes and according to the Kabat and
Chothia regional definitions. However, the observed differences in
mutability among regions were more extreme than predicted. For example,
among the VH genes divided by the Kabat
definition, CDR1 mutated 2.8 times more frequently than FR1, while it
was predicted to mutate only 1.5 and 1.2 more frequently on the basis
of di- and trinucleotide compositions, respectively. Thus, our
predictions were qualitatively, but not quantitatively, accurate.
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transgene construct (10) to mutational
distribution predicted on the basis of di- and trinucleotide
composition. The transgene includes a 100-nt segment of DNA that
contains repeated EcoRV/PvuII restriction sites
and that is unrelated to V region gene DNA. Work by Storb et al. (17)
had indicated that this segment of DNA mutated disproportionally to its
trinucleotide composition. The results in Fig. 6 confirm this and reveal that, as with
the human VH genes, we were able to qualitatively
predict regional mutability preferences from di- and trinucleotide
composition, but as before, the observed differences were more extreme
than predicted. These quantitative differences between predicted and
observed mutations in an artificial sequence are therefore unlikely to
be due to mutagenesis by gene conversion.
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To address the possibility that mRNA secondary structure amplifies
mutagenesis in CDR, we adopted an approach used by Storb et al. (17) to
predict the stability of mRNA secondary structure within 51-nt-long
regions at six-nt steps for germline correlates of the mutated
VH genes described above. Fig. 7A shows a comparison of
average predicted mRNA secondary structure and mutation distribution
for 60 nonproductively rearranged human VH genes
encompassing four families. Segments encoding CDR1 and CDR2 were most
mutable, and substantial mRNA secondary structure occurred 5' of each.
However, for CDR1, mRNA stability peaked about 30 bases before the peak
of mutation, whereas for CDR2, peaks of mRNA stability and mutation
nearly coincided. Furthermore, while FR3 was substantially mutated, a
distinct decrease in the predicted mRNA stability occurred within this
region. Finally, FR1 possessed considerable mRNA secondary structure
yet contained few somatic mutations. A correlation coefficient of 0.09
and a linear regression with a p value of 0.55 indicated no
significant linear correlation between observed mutability and the
position of predicted stability of mRNA secondary structure (data not
shown). To further investigate the relationship between mutational
distribution and mRNA secondary structure, we analyzed two
VH genes for which the most mutational data were
available (235 and 172 mutations in 18 and 12 sequences for 4–34 and
5–51, respectively). As shown in Fig. 7, C and D, there was
no obvious linear correlation between predicted mRNA secondary
structure and mutation frequency. However, it is intriguing that the
pattern of mRNA secondary structure stability was somewhat conserved
even among VH genes belonging to different
families (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
JH intronic DNA. While not entirely surprising,
the consistency in the mutation bias is an important result because
distinct regulatory elements differentially control developmental
expression of H and L chain genes and because the intron and 3' L chain
enhancers are essential for full mutation, while analogous H chain
enhancers are not sufficient (15, 18, 19, 20, 21, 22, 23, 24). We cannot entirely exclude
the possibility that distinct mutation mechanisms operate on H and L
chain genes and that the two only share elements that generate
sequence-specific bias. For example, observed somatic mutations are
ones that have been fixed and propagated by B cells. Accordingly,
sequence-specific mutation bias could be due to a bias in nucleotide
repair rather than to a bias in the initial mutagenic event.
Nevertheless, our interpretation is the most straightforward given the
nature and consistency of the results. Predicted and observed higher mutability of DNA encoding VH CDR vs FR suggests that evolution has carved the VH sequence to optimize beneficial effects of somatic mutation while minimizing potential detrimental effects during Ag-driven somatic diversification. This view is consistent with the observations of Chang and Casali (45) that codon use by CDR is such that random mutations will produce amino acid changes at an inordinately high frequency and with a similar finding by Kepler (46), who also took into consideration intrinsic codon mutability. Kepler’s results show the same general mutability trend as ours, except in FR2 of light chains, where he found no differential localization of synonymous codons that preferentially mutate to produce amino acid changes, while we predicted this region to be highly mutable (46). These previous studies have been limited to in-frame codon analysis without evidence that the mutation mechanism has a frame of reference. Our analysis is thus unique in that all di- and trinucleotides were considered regardless of reading frame.
On the basis of di- and trinucleotide composition, the sequences
encoding light chain FR2 were predicted to mutate more frequently than
those for CDR2. This was true for humans and mice and for and
light chain genes. At present, there are insufficient sequence data
from nonproductively rearranged light chain genes to permit a test of
this unexpected result. However, quantitative differences between
predicted and actual regional mutabilities in VH
genes suggest that other undefined factors may influence mutational
accumulation. Thus, it will be interesting to determine whether
regional trends in light chain mutability follow predictions based only
on short sequence composition.
We considered several possible explanations for the quantitative
discrepancy between predicted and observed regional mutabilities. The
mutation mechanism might recognize oligonucleotide motifs longer than
trinucleotides. However, this is difficult to reconcile with the
consistent hierarchy of di- and trinucleotide mutation indexes observed
for different types of V genes in both coding and noncoding regions for
two species as well as the consistent positional mutation preferences
among the different sequence types. We have conducted a more limited
regional predictive analysis using tetranucleotide sequences that have
been proposed to be most mutable by other investigators (28, 47),
including RGYW, as well as sequences composed of overlapping
mutation-prone trinucleotides (AGCT, GTAC, CTAC, GCTA, TAGC, GTAG). The
results of this analysis failed to resolve the quantitative discrepancy
between predicted and observed regional mutabilities (data not shown).
Furthermore, using alignment algorithms on the entire human database of
germline V genes, we have been unable to identify a new motif that is
preferentially located in regions of high or low mutability. Again
without success, we tested the possibility that only some triplets were
targets for mutagenesis by performing the predictive analysis with
selected trinucleotides of highest mutability with or without regard to
reading frame (data not shown). Finally, the quantitative discrepancy
between predicted and observed regional mutation in the transgene
containing the artificial insert argues against a supplemental
mutagenesis mechanism involving a recombinational process with
unrearranged donor V genes.
Storb et al. (17) found a region of secondary structure stability in
RNA located 40 nt downstream of a highly mutable segment in their
transgene. From this observation they proposed that mutations are
directed into a region by pausing of the RNA polymerase due to
secondary structures within the nascent RNA, and the DNA sequence is
responsible for the fine specificity of the mutation mechanism.
However, our observations are not consistent with this model, in that
we found no linear correlation between areas of observed high
mutability in human VH genes and positions of
predicted RNA secondary structure stability. Yet, we cannot rule out
the possibility that RNA secondary structure plays a more elusive and
complex role than we were able to detect. We note, for example, that
there appears to be a somewhat recurrent pattern of predicted secondary
structure in mRNA for VH genes, even among
members of different families (Fig. 7B). If very short or
long segments of RNA secondary structure were influencing mutagenesis,
these would not necessarily be detected by our analysis, which was
predicated on stability within 51-base-long segments. Our analysis does
not take into consideration the possibility that secondary
substructures within this length interval or superstructures formed by
spatially separated regions of RNA could be involved in mutagenesis. As
suggested by other investigators, other components, such as palindromes
and repeats (28, 48, 49), could be acting together with oligonucleotide
motifs to direct mutations. Palindromes and repeats are abundant in the
most mutable region of the murine artificial transgene, but are
virtually absent from adjacent regions. This distribution might account
for the more extreme discrepancies between observed and expected
regional mutabilities found for the artificial transgene compared with
those found for human VH genes.
Our findings are perhaps most consistent with the accumulation model of
Gearhart and Bogenhagen (50) and observations by O’Brien et al. (9),
who found clustered somatic mutations within Ig transgenes.
Collectively, the results suggest that once a region of DNA is
somatically mutated, it is more likely to be targeted for mutation
again. If duplex melting were required for mutagenesis and if
heteroduplex DNA remained unrepaired for a significant period of time,
mutations might enhance further localized mutagenesis. This could
explain why di- and trinucleotide motifs can accurately predict the
pattern, but not the number, of mutations within a region. Despite
quantitative issues, our results indicate that short nucleotide
sequences play an important role in distributing somatic mutations
within V genes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lawrence J. Wysocki, Department of Pediatrics K902, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address:
3 Abbreviations used in this paper: CDR, complementarity-determining region; FR, framework; obs/exp, observed/expected.
Received for publication December 28, 1998. Accepted for publication April 9, 1999.
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References |
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G. D. Wiens, A. Lekkerkerker, I. Veltman, and M. B. Rittenberg Mutation of a Single Conserved Residue in VH Complementarity-Determining Region 2 Results in a Severe Ig Secretion Defect J. Immunol., August 15, 2001; 167(4): 2179 - 2186. [Abstract] [Full Text] [PDF]
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X. Zhang, D. S. Smith, A. Guth, and L. J. Wysocki A Receptor Presentation Hypothesis for T Cell Help That Recruits Autoreactive B Cells J. Immunol., February 1, 2001; 166(3): 1562 - 1571. [Abstract] [Full Text] [PDF]
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 H. M. Shen, N. Michael, N. Kim, and U. Storb The TATA binding protein, c-Myc and survivin genes are not somatically hypermutated, while Ig and BCL6 genes are hypermutated in human memory B cells Int. Immunol., July 1, 2000; 12(7): 1085 - 1093. [Abstract] [Full Text] [PDF]
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M. Brown, M. A. Schumacher, G. D. Wiens, R. G. Brennan, and M. B. Rittenberg The Structural Basis of Repertoire Shift in an Immune Response to Phosphocholine J. Exp. Med., June 19, 2000; 191(12): 2101 - 2112. [Abstract] [Full Text] [PDF]
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