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Switch Recombination in a Transfected
Plasmid Occurs Preferentially in a B Cell
Line That Undergoes Switch Recombination
of Its Chromosomal Ig Heavy Chain Genes
Janet Stavnezer, Sean P. Bradley, Norman Rousseau, Todd
Pearson, Ananth Shanmugam, Debra J. Waite, Paul R.
Rogers and Amy L. Kenter
J Immunol 1999; 163:2028-2040; ;
http://www.jimmunol.org/content/163/4/2028
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Copyright © 1999 by The American Association of
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References
Switch Recombination in a Transfected Plasmid Occurs
Preferentially in a B Cell Line That Undergoes Switch
Recombination of Its Chromosomal Ig Heavy Chain Genes1
Janet Stavnezer,2* Sean P. Bradley,* Norman Rousseau,* Todd Pearson,*
Ananth Shanmugam,† Debra J. Waite,* Paul R. Rogers,* and Amy L. Kenter†
W
hen mature B lymphocytes, which express IgM and
IgD on their surface, are activated by Ag and receive
accessory signals, they switch to express downstream
Ig heavy chain constant region (CH) genes while maintaining the
same expressed variable region. Because the CH region determines
the Ab effector function, class switching allows an adaptive humoral immune response to a variety of different infectious organisms. Ab class switching is caused by an intrachromosomal DNA
recombination event called switch recombination, which occurs
between switch (S) regions consisting of G-rich tandem repeats,
located 59 of each CH gene, except Cd (reviewed in Refs. 1 and 2).
The means by which the DNA is cut, aligned, and rejoined is
unknown, as are the roles of several nuclear proteins that have
been shown to bind to S regions (3–12).
Base substitutions, duplications, and deletions found near switch
recombination junctions led to the proposal that an illegitimate
priming event, followed by error-prone DNA synthesis, creates the
recombination (2, 13, 14). Recently, it has been shown that doublestrand breaks are rapidly induced in the Sg3 region when mouse
*Department of Molecular Genetics and Microbiology and Program in Immunology
and Virology, University of Massachusetts Medical School, Worcester MA 01655;
and †Department of Microbiology and Immunology, University of Illinois College of
Medicine, Chicago IL 60612
Received for publication August 4, 1998. Accepted for publication June 9, 1999.
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 research was supported by grants from the National Institutes of Health, RO1
AI23283 to J.S., RO1 GM 57078 to A.L.K., and T32 AI07349 to S.P.B. Its contents
are solely the responsibility of the authors and do not necessarily represent the official
views of the National Institutes of Health.
2
Address correspondence and reprint requests to Dr. Janet Stavnezer, Department of
Molecular Genetics and Microbiology and Program in Immunology and Virology,
University of Massachusetts Medical School, Worcester MA 01655-0122. E-mail
address: [email protected]
Copyright © 1999 by The American Association of Immunologists
splenic B cells are treated with mitogens that induce class switch
recombination (CSR)3 to IgG3 (15). These data suggest that switch
recombination is initiated by a double-strand break. Consistent
with this is the finding that DNA-dependent protein kinase (DNAPK), Ku70, and Ku80 are required for switch recombination (16 –
18). The Ku complex is known to bind to DNA double-strand
breaks, nicks, gaps, and hairpins and is required for double-strand
break repair (19) and for V(D)J recombination (20 –22). However,
because Ku proteins and DNA-PK are required for viability of
mitogen-activated cells presumably due to induced double-strand
breaks, it is possible that the effects of these mutations on CSR are
indirect.
Numerous studies have established that transcription of unrearranged CH genes occurs before switch recombination, producing
what are termed germline, or switch, transcripts (23–26). Transcription of germline RNA is regulated by the cytokines IL-4,
IFN-g, and TGF-b1, in concert with B cell activators, and serves
to direct recombination to specific S regions (reviewed in Refs.
26 –28). Transcription initiates at the I exon located 59 to each S
region. Deletions of the I exons or segments of the I exons and
their upstream regulatory elements by gene targeting have demonstrated that germline transcripts are required for switch recombination (29 –32). Although cytokines induce germline transcripts,
cytokines themselves do not induce switch recombination.
To provide an assay for the enzymes and proteins involved in
cutting and recombining switch regions, several investigators have
reported attempts to develop plasmid and retroviral substrates for
assaying switch recombination. These substrates contain two
switch region segments separated by a DNA segment whose deletion allows selection for recombination events. One approach has
3
Abbreviations used in this paper: CSR, class switch recombination; Ia promoter,
segment of DNA from 2489/146 relative to the first start site of germline a RNA;
neor, G418 resistance; TK, thymidine kinase.
0022-1767/99/$02.00
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Ab class switching is induced upon B cell activation in vivo by immunization or infection or in vitro by treatment with mitogens,
e.g. LPS, and results in the expression of different heavy chain constant region (CH) genes without a change in the Ab variable
region. This DNA recombination event allows Abs to alter their biological activity while maintaining their antigenic specificity.
Little is known about the molecular mechanism of switch recombination. To attempt to develop an assay for enzymes, DNA
binding proteins, and DNA sequences that mediate switch recombination, we have constructed a plasmid DNA substrate that will
undergo switch recombination upon stable transfection into the surface IgM1 B cell line (I.29m), a cell line capable of undergoing
switch recombination of its endogenous genes. We demonstrate that recombination occurs between the two switch regions of the
plasmid, as assayed by PCRs across the integrated plasmid switch regions, followed by Southern blot hybridization. Nucleotide
sequence analysis of the PCR products confirmed the occurrence of Sm-Sa recombination in the plasmid. Recombination of the
plasmid in I.29m cells does not require treatment with inducers of switch recombination, suggesting that recombinase activity is
constitutive in I.29m cells. Recombination does not require high levels of transcription across the switch regions of the plasmid.
Fewer recombination events are detected in four different B and T cell lines that do not undergo switch recombination of their
endogenous genes. The Journal of Immunology, 1999, 163: 2028 –2040.
The Journal of Immunology
Materials and Methods
Construction of switch plasmids p273, p200, and p218
The 1.8-kb HindIII mouse Sm segment (BALB/c) from pM2-20 (48) was
inserted into the HindIII site of the Bluescript SK2 vector (Stratagene, La
Jolla CA). The 2.1-kb HaeIII Sa fragment cloned from the I.29m lymphoma (3, 13) was inserted into the BamHI site of the vector after ligation
of BamHI linkers. Both of these switch fragments are made up of tandem
consensus repeats, although the Sm fragment also includes about 330 bp 59
to the SacI site where the tandem repeats begin. Both the Sm and Sa
fragments have a binding site for the B cell-specific paired domain transcription factor, BSAP (Pax-5), near their 59 ends (3, 8, 49). The herpes
virus thymidine kinase (TK) gene (1.8 kb) was excised from pZN(SmSg2b)tk.1 (38) with BamHI, cloned into Bluescript KS2, excised with
EcoRI and BamHI, and inserted between the Sm and Sa segments at the
EcoRI and BamHI sites. The G418 resistance gene (neor) was excised from
pPGKneocbpA (received from Dr. Allan Bradley, Baylor College of Medicine, Houston TX) with XhoI, sticky ends filled in with T4 polymerase,
KpnI linkers ligated on, and the fragment inserted into the KpnI site. The
1.0-kb cassette (EP) which has the 0.7-kb XhoI/EcoRI fragment containing
most of the Ig m intron enhancer and a 0.3-kb fragment containing the VH
186.2 promoter was excised from pEP.B (50) (received from Dr. Fred Alt,
Harvard Medical School, Boston MA) with SphI and XhoI, sticky ends
filled in with Klenow and T4 polymerase, and cloned into the XhoI site
(filled-in). A 0.5-kb BglII fragment containing the germline a promoter (Ia
promoter) segment from 2489 to 146 relative to the first RNA initiation
site (51) was inserted in the forward direction into the BamHI site between
the TK and Sa segments. The plasmids were linearized at the NotI or SacII
site before transfection.
Stable transfection of plasmids, cell culture, and induction of
class switching
Cells (maximally 50 3 106 per transfection) were electroporated as previously described (52) with 1 mg plasmid DNA per 106 cells. Cells were
cultured at 37°C in bulk culture for 2 days in the absence or presence of
inducers, before drug selection. The components of the complete medium
and culture conditions for I.29m were described previously (43). All other
cell types were cultured in the same medium in a 5% CO2 incubator, using
10% FBS, without insulin. The concentrations and sources of LPS (50
mg/ml), TGF-b1 (2 ng/ml), and nicotinamide (10 mM) are as previously
reported (44). EL-4 cells were either untreated or treated with 10 mM PMA
(Sigma, St. Louis, MO) and ionomycin (1 ng/ml; Sigma)
To select for stably transfected cell lines, cells were pelleted and resuspended in medium (without inducers) containing G418 (Life Technologies,
Gaithersburg, MD) at 400 mg/ml for all cells, except at 1.2 mg/ml for
BW5147, and plated in 96-well plates at limiting dilution. For I.29m, 2 3
104 cells per well were plated. For the M12.4.1, J558L, and EL-4 lines, 4 3
103 cells/well were plated, and for BW5147, 2 3 102 cells/well were
plated. Cells were re-fed with medium supplemented with G418 subsequently about every 3 days. Transfected lines were identified and expanded. DNA was isolated as soon as the cultures contained 5–10 million
cells. This took 2–3 wk. The transfected lines obtained should be clonal
because they were generally obtained from experiments in which fewer
than 10% of the wells yielded G418-resistant cells. Since for the purposes
of this assay it is unimportant whether the transfected lines are clonal, they
have not been further subcloned.
Assay of class switching in I.29m cells
Class switching was assayed on day 2 by immunofluorescence microscopy
as described (44).
PCRs and primers
The PCR to detect Sm-Sa recombination was conducted for 30 cycles
using the Taq Expand High Fidelity System from Boehringer Mannheim
(Indianapolis, IN) in 2.0 mM MgCl2 under conditions recommended by the
manufacturer. The annealing temperature was 64°C. A total of 400 ng of
genomic DNA, 0.2 mM primers, and 250 mM dNTPs were used. Primers
used were: m-1A, 59-CTCTACTGCCTACACTGGACTGTTC-39, identical with nt 5269 –5293 of the germline BALB/c Sm sequence
(MUSIGCD07) (15); m-3, 59-TGGCTTAACCGAGATGAGCC-39, identical with nt 5206 –5226 of MUSIGCD07 (41); and R10, 59-CTC
TATCTAGGTCTGCCCCGTCTAGATAAG-39, complementary to nt
62–91 upstream of the 39 end of the 2.1-kb HaeIII Sa segment (GenBank
accession no. AF069385) in the switch plasmids. The sequences of both m
primers differ from the sequence of the endogenous I.29 Sm sequence
(MUSIGHMY) at several nucleotides. The m-3 and R10 primer combination was used for all PCR amplifications results presented, except for the
clones used for nucleotide sequencing that were amplified earlier using the
m-1A and R10 primers (see Fig. 4). For amplifying the PCR product from
clone 153 for sequencing, a second set of nested primers was used: m-2,
59-CCTGGGGTGAGCTCAGCTATGCTACGC-39, identical with nt 5307–
5333 of the BALB/c Sm sequence (MUSIGCD07) (15); and R10B, 59GGAATTCATAAGCTCAGCCTTGTTCAGCCCATTCCATC-39, complementary to nt 87–117 upstream of the 39 end of the Sa segment, except for
the addition of an EcoRI site at the 59 end. The other PCR products that
were sequenced were amplified by a single round of PCR using the m-1 and
R10 primers.
For amplification of a 550 bp segment of the neor gene from the plasmid
a primer at the T7 promoter of Bluescript: 59-GTAATACGACTCACTAT
AGGGC-39 and a primer for the neor coding region 59-ATGGCCGCTTT
TCTGGATTC-39 were used. Amplification was for 30 cycles using HotStar Taq polymerase (Qiagen, Chatsworth, CA). The annealing
temperature was 55°C and the concentration of MgCl2 was 2.5 mM.
Primers for amplification of the TK-Ia segment to produce an 1.8-kb
fragment for use as a probe on Southern blots of PCR products contain
sequences from the TK gene (TKF2): 59-TGACTTACTGGCAGGT
GCTG-39 (corresponding to nt 769 –789 of GenBank accession no.
V00467) and sequences complementary to the Ia segment at 233/214
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been to use transiently transfected plasmids that can replicate as
episomes by virtue of the presence of a fragment from the Polyoma
(Py) virus containing an origin of replication, early and late transcription initiation sites and encoding the T Ag. Using a drug selection assay, Leung and Maizels (33, 34) reported that such a
plasmid undergoes recombination in LPS-activated spleen cells at
a high frequency (21% of transfectants). Using a similar plasmid,
Lieber and coworkers (35–37) found they could measure B cellspecific recombination events in the plasmid by subtracting the
recombination that occurs immediately after transfection and only
considering recombination events occurring in the window between 20 to 50 h after transfection. B cells of the sIg1 stage continue to accumulate recombination events during this window,
whereas in most pre-B, plasmacytoma, and nonlymphoid lines no
further recombination events are detected after 20 h. However, the
high background levels of recombination occurring in non-B cells
before the 20-h time point suggested that these plasmid assays lack
some of the specificity expected for an assay of switch recombination enzymes. Furthermore, it was not determined whether these
plasmids recombine preferentially in B cells that undergo CSR of
their endogenous genes. Another approach was taken by Marcu
and coworkers (38 – 40), who demonstrated that integrated retroviral vectors containing two switch region fragments undergo recombination in pre-B and B cell lines within the tandem repeats,
and that the frequency of recombination is much lower in non-B
cell lines. Recently, they found that treatment of splenic B cells
with CD40 ligand induces recombination of the retroviral switch
substrate (41). We combined these two approaches, using plasmids
that lack the Py origin and assayed by the PCR for switch recombination after stable integration into the genome.
As a model system in which to study the mechanism of switch
recombination, we use the mouse I.29m B cell line, which can be
induced to switch from expression of surface IgM (sIgM) to expression of sIgA by treatment with LPS plus TGF-b1 (42– 44).
This model resembles splenic B cells in that switching to IgA in
mouse splenic B cells is also induced by a combination of LPS
plus TGF-b1 (45– 47).
Our plasmid, p273, undergoes recombination between its two
switch region sequences (Sm and Sa) in I.29m cells, but at a reduced level in two T cell lines and infrequently in the B lymphoma
M12.4.1 or in the plasmacytoma J558L, none of which undergo
class switching of their endogenous genes. Recombination of the
plasmid in I.29m cells does not require treatment with inducers of
switch recombination, suggesting that recombinase activity is constitutive in I.29m cells.
2029
2030
PLASMID ASSAY FOR SWITCH RECOMBINATION
(GLaR1): 59-GGCTGCATGACTGTGTGTCT-39 (GenBank accession
no. L04145).
RT-PCR to evaluate transcription of the TK-Sa and the neor-Sm
segments
Southern blotting analysis of PCR products
Southern blotting of PCR products was performed as previously described
for genomic Southern blotting (42, 44). After each hybridization and exposure to autoradiographic film, the probes were removed from the blots by
heating to nearly 100°C in 0.13 SSC and 0.1% SDS. Successful removal
of the probes was always verified by autoradiography. Probes were restriction enzyme fragments or PCR products that were labeled by random priming using hexamer primers (Boehringer Mannheim) and Klenow DNA
polymerase. The fragments used for plasmid construction were used as
hybridization probes in Southern blotting experiments. The Sm, Sa, and
TK-Ia probes were labeled to a similar specific activity (50 3 106 cpm per
25 ng), and equal amounts of cpm were used for each hybridization (with
one exception which is noted). The hybridized blots were all autoradiographed for equivalent times.
Nucleotide sequencing and data analysis
PCR products obtained after one or two rounds of amplification of DNA
from stably transfected clones were subcloned directly into pGEM-T (all
except clone 153) or digested with SacI, purified from gels, and subcloned
into Bluescript (clone 153). Nucleotide sequencing was performed using an
Applied Biosystems (Foster City, CA) model 373 Stretch DNA Sequencer
using Applied Biosystems Prism version 2.1.1 software. Sequences were
determined at least two times from every template, in both directions when
possible. Analysis and alignments of DNA sequences were performed using software from the GCG Wisconsin Package (Genetics Computer
Group, Madison WI) and/or the lfasta, lalign and SIMs programs placed on
the internet by the Human Genome Center at the Baylor College of Medicine and/or by the Clustal 1.4 program (53).
FIGURE 1. A, Maps of three switch plasmids. Plasmids consist of the
following fragments: EP, 1.0 kb Ig m intron enhancer/VH promoter casette;
Sm, 1.8-kb HindIII fragment containing tandem repeats; TK, a 1.8-kb segment containing an herpes virus TK gene and its promoter; Ia, 0.5-kb
segment containing nucleotides from 2489 to 146 relative to the first
RNA initiation site for germline a RNA; and Sa, 2.1-kb HaeIII fragment
containing tandem repeats. Arrows indicate transcriptional orientation of
fragments. The thin line indicates the pBluescript SK2 vector backbone.
The sizes of segments discussed in the text are indicated. The probes used
in the Southern analyses correspond to the labeled segments in the figure.
The primers used for the RT-PCR analysis of transcripts from the transfected plasmids are diagrammed beneath the p273 map. B, Diagram of
switch recombination in p273. Arrows indicate the location of the m-1A,
m-2 and m-3 Sm primers (indistinguishable on this map) and also the location of the R10 and R10B Sa primers used for assaying recombination
by PCR and for amplifying segments for sequence analysis. The segment
detected in unrearranged p273 (5.8 kb) and the segments detected in plasmids that have undergone Sm-Sa recombination (#3.3 kb) are indicated.
The location of the T7 and Neo primers for amplifying a 550-bp fragment
from the neor gene are indicated with arrows.
Densitometric quantitation
Quantitation of the PCR fragments detected by Southern blot analyses was
performed by measuring by densitometry the signal in the entire lane extending from the maximal size observed for Sm-Sa fragments down to the
smallest detectable Sm-Sa segments. The densitometer used was a Molecular Dynamics (Sunnyvale, CA) Personal Densitometer SI and analysis
was by Molecular Dynamics Image Quant 1.2. Quantitation of the RT-PCR
products was performed by a Bio-Rad (Richmond, CA) PhosphorImager.
Results
Construction and stable transfection of switch plasmids into
I.29m cells
The maps of the three switch plasmid substrates used in these
studies are shown in Fig. 1A. Each plasmid has a neor gene and the
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Two days after transfection of I.29m cells by electroporation, total cell
RNA was prepared using the Ultraspec RNA Isolation System (Biotecx
Laboratories, Houston TX) or by the hot-phenol method. cDNA was
synthesized from 3 mg RNA using Moloney murine leukemia virus (MMLV)-Reverse-Transciptase (200 U; Promega, Madison, WI) with the 39
primer R10 and with the 39 m-dn2 primer, 59-CTTGGTTCTTGGCCAGC
CAGCTCTAC-39 (positions 1037/1062 in MUSIGCD09), under reaction
conditions recommended by Promega. PCR was performed for 30 cycles
using the Taq Expand High Fidelity System, as described for analysis of
Sm-Sa recombination, except that for amplification of the TK-Sa segment
a 59 primer specific to the HSV-TK gene, TKF2, was used along with the
39 primer R10. For amplification of the neor-Sm segment, PCR was performed using a 59 primer complementary to the neor gene, Neo4, 59CGTCCAGATCATCCTGATC-39, and the m-dn2 primer. An equal proportion of the cDNA products were amplified for each sample and 3 mCi
[a-32P]dCTP was included in each reaction. As a positive control for the
RT-PCR using the R10 primer, an aliquot of the cDNA obtained from
p273-transfected cells was amplified using the 59 primer IaF, 59-ACAG
GCAATCACACACAGAG-39, corresponding to the Ia region 113/133
relative to the first RNA initiation site, along with the 39 primer R10. As a
positive control for the RT-PCR using the m-dn2 primer, an aliquot of the
cDNA obtained from p273- and p200-transfected cells was amplified using
a 59 primer for the VH sequence in the EP segment, 59VHN, 59-TGT
TCTCTTTACAGTTACTGAGCACACAGGACC-39. To control for DNA
contamination, PCR was performed on an equivalent amount of RNA
which had not been transcribed by reverse transcription. To control for the
PCR and to provide m.w. markers, PCRs were also performed on each
plasmid DNA (0.5 ng) with the TKF2/R10 primers, with the 59 Neo/m-dn2
primers and also on p273 using the IaF/R10 primers and on p273 and p200
using 59VHN/m-dn2 primers. PCR products were separated on 1.0% agarose gels, dried, and autoradiographed.
The Journal of Immunology
2031
PCR and Southern blotting analyses detect Sm-Sa fragments in
I.29m cells transfected with p273
A characteristic of switch recombination that differs from other
types of recombination is its inducibility by certain B cell mitogens. Switching from IgM to IgA expression occurs infrequently in
I.29m cells in the absence of LPS plus TGF-b1, as cells expressing
both IgM and IgA are rarely observed in untreated cultures (42–
44). TGF-b1 induces transcription from the promoter for germline
a transcripts, but the role of LPS is unknown. In addition, nicotinamide has been shown to stimulate class switch recombination
in I.29m cells by inhibiting the nuclear enzyme, poly(ADP-ribose)
polymerase (44).
I.29m cells were electroporated with p273 or p200, divided into
aliquots that were treated with LPS, LPS plus TGF-b1, LPS plus
TGF-b1 plus nicotinamide, or were left untreated and stable transfectants were selected and cloned by limiting dilution. We verified
that switching of the endogenous Ig genes had indeed been induced by using immunofluorescence microscopy to measure sIgM
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Sm and Sa segments described in Materials and Methods, separated by a herpes virus TK gene. Two of the plasmids, p273 and
p200, also have a 1.0-kb fragment (EP) containing the Ig m intron
enhancer and an Ig VH promoter (50), inserted upstream of the Sm
segment. One plasmid (p273) also has a 535-bp fragment (Ia)
containing the promoter and first RNA initiation site for the germline a transcripts inserted upstream of the Sa segment (51). Fig.
1B diagrams an expected switch recombination event in p273.
The switch plasmids p273 and p200 were transfected into the
22D or 22A10 clones of I.29m (54, 55), and stably transfected
clones were selected by limiting dilution in the presence of G418
(see Materials and Methods). To examine the copy number of the
transfected plasmids, we performed Southern blot analyses of
genomic DNA from the transfected clones, hybridizing with a
probe for the neor gene (Fig. 2, A and B), and/or used PCR to
amplify a segment extending from the vector backbone into the
neor gene segment (Fig. 2C). The location of the primers for the
PCR are indicated in Fig. 1B.
The data suggest that the copy number of integrated plasmids
does not differ more than 4-fold among most clones, although occasional clones have many copies (Fig. 2 and data not shown). As
a loading control, we show beneath Fig. 2B the signals obtained
from hybridization of the 5-kb cellular TK SacI fragment on the
same blot. Similar results were obtained upon analysis of the cellular TK signal for the DNA samples shown in Fig. 2A (data not
shown). There are no consistent differences in the endogenous TK
gene or neor gene copy numbers among the various cell lines and
treatments. In most p273- and p200-transfected clones, the neor
probe detects a 2.9-kb SacI fragment which contains the 39 portion
of the neor gene, the 1-kb EP segment, and 0.3 kb from the 59
portion of Sm (see Fig. 1A). As shown in Fig. 2A (clone 432) and
Fig. 2B (clone 6), rearrangements in this fragment are occasionally
observed. Such rearrangements may be due to recombinations in
the EP segment, in the 59 portion of Sm or within the neor gene, as
we have not mapped them. PCR amplification of a 550 bp segment
of the neor gene from stably transfected plasmids demonstrated
that all G418-resistant clones analyzed in this study contain the
neor segment, including M12.4.1 clone 1 in which the neo fragment was not detected on the genomic Southern blot (Fig. 2C). To
normalize between different PCR experiments, an identical sample
of p273 DNA was amplified in every experiment (Fig. 2C, leftmost lanes). To attempt to ensure that these neo gene amplifications can serve as internal controls for the analysis of S-S recombination, the same samples of diluted DNA prepared for the neo
amplifications are used in all the experiments described below.
FIGURE 2. Blots of genomic DNA from stable p200- and p273-transfected cell lines hybridized with a neo probe. A. Lanes from one blot of
SacI-digested genomic DNA from I.29m lines stably transfected with p200
or p273. Deleted lanes contained DNA that was only partially digested with
SacI. B. Lanes from one blot containing p273-transfected M12.4.1 cell
lines. Although clone 1 appears to have deleted the neor gene, in this blot,
the PCR analysis in C demonstrates a low amount of the fragment is
present. Beneath the panel showing the neo gene hybridization is a panel
from the identical blot hybridized with the TK probe, showing the 5-kb
endogenous TK gene as a DNA loading control. Additional TK bands due
to the plasmid (of approximately equal intensity to the genomic bands)
were present in 6 of the 11 stably transfected clones shown here. M12.4.1
clones analyzed here are the same as those analyzed in Fig. 9: clone 1 5
363, 2 5 364, etc.) C. Ethidium bromide stained gels showing PCR amplification of 550-bp neor gene fragments from stably transfected clones.
Lanes labeled p273 contain the product from 100-pg p273, N.T., product
from no added template, lanes J558L and I.29m are products from untransfected cellular DNA. Four percent of each reaction product was loaded
onto the agarose gel.
2032
PLASMID ASSAY FOR SWITCH RECOMBINATION
and sIgA expression on day 2 of induction (44), the day the cells
were distributed into wells for selection of clones (data not shown).
In the optimally induced cultures, an average of 6.5% of the cells
expressed IgA (data not shown).
Switch recombination occurs by direct recombination between
switch regions and thus we wanted to evaluate whether the switch
plasmids could undergo direct Sm-Sa recombination. We developed a PCR assay for recombination between the Sm and Sa segments, using oligonucleotide primers complementary to the 59 end
of Sm and to the 39 end of Sa (see Fig. 1B). This strategy was
previously used to amplify endogenous Sm-Sg3 recombinants
(56). The m primers are specific for the BALB/c Sm sequence in
the plasmid, but the Sa primer is complementary to both the plasmid and endogenous Sa sequences. The primers amplify Sm-Sa
recombinants from the transfected plasmid, but not from endogenous genes of the stably transfected clones (see below). After one
round of PCR amplification, the products were analyzed by Southern blotting, hybridizing sequentially with three probes, Sm, Sa,
and a probe containing both the TK and Ia segments from p273.
The probes correspond to the segments indicated in the plasmid
maps (Fig. 1). PCR fragments that are due to direct Sm-Sa recombination and therefore lack the TK and Ia segments should be
#3.3 kb in size.
Southern blots of PCR products obtained from the genomic
DNA of I.29m clones stably transfected with p273 that were cultured in medium alone (untreated) and from clones cultured with
the indicated inducers of switching are shown in Fig. 3. The blots
on the left were hybridized with the Sm probe, the blots in the
middle panels are the identical blots hybridized with the Sa probe,
and the blots on the right are the identical blots hybridized with the
TK-Ia probe. The dots on the edge of the bands in the Sm blots
indicate bands that also hybridize with the Sa probe. The great
majority of the amplified segments are detected by both the Sm and
Sa probes, but not with the TK-Ia probe. However, from three of
the transfected clones that were not treated with inducers, 5.8-kb
fragments that hybridize with the Sm, Sa, and TK-Ia probes and
which correspond to unrecombined plasmid were amplified (Fig.
3). (The 5.8-kb band in clone 438 can be seen on the original
autoradiograph.) None of the amplified fragments from clones induced to undergo switching hybridized with the TK-Ia probe, except for two fragments from one clone induced with LPS without
TGF-b1. These data suggest that the transfected switch plasmid
has undergone numerous direct Sm-Sa recombination events in
I.29m cells. An average of 7.9 (60.6) Sm-Sa PCR products were
amplified from each p273-transfected I.29m clone in two experiments, whether or not the cells were treated with inducers of class
switch recombination (Table I).
Because I.29m cells undergo switch recombination on their endogenous chromosomes, it was important to determine whether the
comigrating Sm-Sa bands detected in this experiment could be due
to endogenous switch recombination. To examine this possibility,
we performed the same PCR and Southern blot analysis on DNA
from a line of I.29m cells in which 50% of the cells express IgA
(see Fig. 9 below, left-most lanes in all three panels). A faint band
(;4 kb) was detected with both the Sa and TK-Ia probes (perhaps
due to amplification of an endogenous Ia-Sa fragment), and a
different sized band (;2.5 kb) was detected with the Sm probe.
Thus, the primers used do not appear to amplify the endogenous
Sm-Sa junctions in IgA1 cells derived from the I.29m cell line.
Further evidence for this comes from an examination of one of the
p273-transfected clones shown in Fig. 3 (no. 458), which was derived from cells induced with LPS plus TGF-b1. This clone did not
yield Sm-Sa PCR products, although it yielded a neor PCR product
(Fig. 2C) and contains endogenous genomic Sm and Sa fragments,
as shown by genomic Southern blot analysis (data not shown).
Nucleotide sequencing data described below further support the
conclusion that the Sm-Sa fragments amplified from p273-transfected I.29m cells are due to recombination in the plasmid and are
not due to recombination of endogenous Sm and Sa sequences.
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FIGURE 3. Southern blot of PCR products from
I.29m cells transfected with p273. The left-most two
lanes of the lower panels contain PCR products from
p273-transfected M12.4.1 clones. Blots of PCR products were hybridized with the TK-Ia probe first (right
panels), then with either the Sm probe (left panels) or
the Sa probe (middle panels), followed by the other S
region probe. All three probes were labeled to a similar specific activity (50 3 106 cpm per 25 ng), and
approximately equal amounts of cpm were used for
hybridization. Most of the fragments in these blots
were not visible by ethidium bromide staining (data
not shown). The sizes of Sm-Sa fragments lacking TK
and Ia segments should be #3.3 kb. The 5.8-kb band
is the product from intact unrecombined plasmid.
Dots next to bands on the left panels indicate fragments that were also detected with the Sa probe, but
not with the TK-Ia probe. These data are summarized
in Table I. The reason the DNA samples are not numbered sequentially is because some of the samples
were depleted during the many genomic Southern
blotting and PCR experiments performed during development of the assay. Results obtained from these
depleted DNA samples did not differ from the samples
shown.
The Journal of Immunology
2033
Table I. PCR Analyses of switch recombination in stably transfected plasmids
Plasmid and Cell Type
No. Clones
with Sm-Sa
Junctions
No. Clones with
Full-Length (5.8 kb)
PCR Band
Mean No. Sm-Sa
Junctions per Clone
10
1
7
4
10
1
6
4
3
0
0
0
9.0
11.0
8.6
7.5
10
10
0
6.2
10
10
10
10
2
6
3.6
3.5
3.6 6 0.36
12
12
0
5.6
5.6 6 0.54
10
4
0
1.3
1.3 6 0.7
,0.001
12
2
7
0.33
0.3 6 0.26
,0.001
12
11
10
9
11
10
9
9
3
2
0
4
1.5
2.3
4.1
4.3
3.1 6 0.31
,0.001
12
12
0
2.4
2.4 6 0.33
,0.001
Cloning and sequencing of recombined Sm-Sa segments from
stably transfected p273
To examine the nature of the recombination events that produced
the comigrating Sm and Sa fragments, we determined the nucleotide sequences of six Sm-Sa segments amplified from I.29m cells
stably transfected with p273. The PCR products were cloned from
five independent clones of p273-transfected I.29m cells, 153, EW4,
EW7, EW18, and JI. Southern blot analyses of the PCR products
from three of these clones are shown in Fig. 4. These DNA samples were chosen for analysis due to the presence of PCR products
that could be detected on an ethidium bromide stained gel (data not
shown). PCR products were excised from the gel, cloned, and sequenced. The sequences show direct Sm to Sa junctions with 0, 1,
2, 6, or 8 nt of identity between the Sm and Sa sequences at the
recombination sites (Fig. 5), quite comparable to switch recombination junctions in genomic DNA (2).
Significance of Difference
from p273(I.29m),
p Value (t test)
7.9 6 0.61
,0.001
0.03
three programs using different algorithms are identical. In these
regions the 153 Sm sequence has 9-nt differences from the p273
sequence (in bold type). The Sa sequence of 153 is identical with
the Sa sequence in p273 (data not shown).
Due to the finding of differences from the p273 Sm sequence,
we considered the possibility that the Sm sequence of clone 153
may have been derived by recombination with endogenous Sm
Recombined plasmid Sm-Sa junctions manifest mutations similar
to those found in endogenous switch recombination junctions
Clone 153. The nucleotide sequences of three products from three
independent nested PCRs from clone 153 were identical and demonstrate direct Sm-Sa recombination. Fig. 5A presents the sequence surrounding the Sm-Sa junction of clone 153 aligned with
the plasmid Sm and Sa sequences. To search for evidence of nucleotide substitutions, deletions, or insertions in comparison with
the parental p273 plasmid, as would be expected if switch recombination activities created the Sm-Sa junction, we compared the
entire Sm and Sa sequences of clone 153 with the p273 sequence
(Fig. 6A and data not shown). The Sm region of clone 153 has
undergone one or more deletion events during or subsequent to
switch recombination. The deletions were not generated during
PCR amplification because they are present in PCR products obtained from three independent reactions. Due to deletions in the
153 sequence, one cannot be completely confident of the entire
alignment with the p273 Sm sequence. However, in the regions at
the 59 and 39 ends of the sequence, the alignments obtained by
FIGURE 4. Southern blot analysis of PCR products from p273-transfected I.29m clones that were analyzed by nucleotide sequencing. Amplified products from these three cell lines were excised from ethidium bromide-stained gels, cloned, and sequenced. The sequences of the Sm-Sa
junctions are shown in Fig. 5. The EW 7.3 sequence was obtained from the
0.7-kb fragment amplified from EW 7, the EW 18.6 sequence was obtained
from the 0.9-kb fragment amplified from EW 18, and the 153 sequence was
obtained from the 0.5-kb fragment amplified from clone 153.
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p273 3 I.29m B cells
Expt. 1
Untreated
LPS
LPS 1 TGFb
LPS 1 TGFb 1 nic.
Expt. 2
Untreated
p200 3 I.29m B cells
Untreated
LPS 1 TGFb
p218 3 I.29m B cells
Untreated
p273 3 M12.4.1 B cells
Untreated
p273 3 J558L plasmacytoma
Untreated
p273 3 EL-4 T cells
Untreated
PMA 1 ionomycin
Untreated
PMA 1 ionomycin
p273 3 BW5147 T cells
Untreated
Mean No. 6 S.E.
Sm-Sa Junctions
per Cell Type
No.
Clones
2034
sequences, rather than due to recombination within the p273 plasmid. We compared the Sm sequence of clone 153 with the endogenous I.29 Sm sequence and with the p273 sequence and found that
the 153 sequence matches the p273 sequence better (Fig. 6B) The
Sm segments of all other Sm-Sa PCR products in Fig. 5 were also
compared with the I.29 Sm sequence and found not to match it as
well as they match the p273 Sm sequence (data not shown). Therefore, all of the PCR products analyzed by nucleotide sequencing
originated from Sm-Sa recombination events in the switch
plasmid.
Clone EW 4.1. Unlike the 153 PCR products, the nucleotide sequences of two independent PCR products (EW 4.1) from another
p273-transfected I.29m clone (EW 4) show no differences from the
plasmid Sm and Sa sequences. The sequence of EW 4.1 surround-
FIGURE 6. A. Nucleotide sequence of PCR products from clone 153
(p273-transfected I.29m cells treated with LPS and TGF-b1). Alignment of
the Sm sequence of the PCR products (GenBank accession no. AF069380)
with the p273 Sm sequence, beginning at the SacI site. There is no nucleotide identity between the Sm and Sa segments at the junction (see Fig. 4).
The computer program generated the alignments by minimizing the mutations and gaps; thus, the alignment may not represent the true events and
some of the differences in sequences may be due to choice of gap positions.
Nine nucleotides in the 153 sequence that differ from the p273 sequence
and are in regions where the alignments are convincing are in bold type.
Alignments were obtained using the BestFit program of GCG software
(Genetics Computer Group). Other alignments are possible and were obtained using the GCG program GapAlign or Clustal 1.4. B, Alignment of
the first 91 nt of 153 Sm with Sm sequences from the p273 and I.29
genomic DNA. Alignment of the first 91 nt 39 to the SacI site in 153 Sm
with the p273 (BALB/c) and I.29m Sm sequences (MUSIGHMY) by the
Clustal 1.4 program. p, Positions that are identical between all three sequences. By using two different GCG programs which weigh gaps differently, we found that the overall homology of the 153 Sm sequence with the
comparable region in the I.29 Sm sequence was lower than with the
BALB/c Sm segment present in p273 (89% or 90% vs 93% identity with
p273 Sm), and that wherever the 153 sequence differed from the p273 Sm
sequence, it never matched the I.29m endogenous Sm sequence. Although
the entire Sm segment of the 153 sequence was compared by the GCG
sequence alignment programs to derive the percent identity given above, it
is impossible to be confident of any one alignment 39 to position 91 due to
deletions in the 153 sequence and differences in the genomic sequences of
the two mouse strains. In all cases, however, the conclusions are the same,
that wherever the 153 sequence differs from the p273 sequence, it does not
match the I.29m genomic Sm sequence.
ing the Sm-Sa junction is shown in Fig. 5B. The junction occurs
within a 6-nt identity (underlined) between the Sm and Sa
sequences.
Clones EW 4.6, EW 7.3, EW 18.6, and JI-7. The nucleotide sequences of four other PCR products were also sequenced. These
products were each obtained from only one PCR. Fig. 5, C--F,
presents segments of the sequences surrounding the sites of Sm-Sa
recombination. Although they have several nucleotide differences
in comparison to the plasmid Sm and Sa sequences, we do not
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FIGURE 5. Compilation of nucleotide sequences of six Sm-Sa junctions in p273 from stably transfected I.29m cells. A, The 153 sequence near
the Sm-Sa junction in comparison with the p273 Sm and Sa sequences. The
sequence is from 3 independent PCRs. u, Sequence identities between the
compared sequences. The entire Sm sequence is presented in Fig. 6A. B,
EW 4.1 sequence (GenBank accession no. AF069381) is from two independent PCRs from clone EW 4 (A. Shanmugam and A. L. Kenter, data not
shown). The 6 nt that are identical between the Sm and Sa sequences are
underlined. C–F, Sequences surrounding Sm-Sa junctions (GenBank accession nos. AF069382 (EW4.6), AF069383 (EW7.3), AF069384
(EW18.6), and JI-7 (AF069379)) are of products from a single PCR performed on p273 transfected clones EW 4, EW 18, EW 7, and JI (A. Shanmugam and A. L. Kenter, data not shown). Nucleotides identical between
the Sm and Sa sequences at the junctions are underlined. The numbering
of Sm starts at 244 relative to the SacI site of the p273 Sm segment (5nt
5271 of MUSIGCD07). The numbering of Sa is according to the p273
sequence (GenBank accession no. AF069385).
PLASMID ASSAY FOR SWITCH RECOMBINATION
The Journal of Immunology
2035
FIGURE 7. Southern blot analysis of PCR products from I.29m transfected with p200 and left untreated or treated with LPS plus TGFb. Methods are
same as for Fig. 3. The 5.3-kb full-length Sm-TK-Sa segments are indicated and so is the position of the maximal size expected for direct Sm-Sa
recombinant (3.3 kb). Dots on the Sm blot mark bands that hybridize with the Sm and Sa probes, but not with the TK-Ia probe.
Switch recombination in the plasmid does not require high
levels of transcription through the S regions
To determine whether transcription through the Sa segment is required for switch recombination in the plasmid, as it is for endogenous class switch recombination, we tested the ability of p200, a
plasmid which lacks the germline (Ia) promoter upstream of the
Sa segment, to undergo Sm-Sa recombination. Twenty clones of
I.29m cells stably transfected with p200, which were untreated or
treated with LPS plus TGF-b1, were analyzed by PCR followed by
Southern blot analysis. An average of 3.6 (60.36) Sm-Sa fragments that were devoid of TK-Ia signal were detected per clone,
whether untreated or treated with LPS plus TGF-b1 (Fig. 7, Table
I). Although the difference between the numbers of S/S fragments
obtained from p273 and p200 is only about 2-fold, the difference
is statistically significant ( p , 0.001).
As true for p273, the numbers of Sm-Sa fragments obtained per
p200-transfected clone are similar in untreated and induced cells.
However, unlike p273, the intensities of the hybridization signals
of the amplified switch fragments from induced cells is greater
than from uninduced cells. This result suggests that the proportion
of cells in the clone which have each fragment is greater in the
induced cells than in the uninduced cells. The difference in intensities of the Sm-Sa fragments in untreated and induced cells is
unlikely to be due to different amounts of DNA used in the amplifications or due to different amounts of plasmids in these cells,
as attested to by the similar signals obtained from the neor gene
amplification of these same DNA samples (Fig. 2C). Thus, although S/S recombination occurs in p200 in the absence of LPS
plus TGF-b1 treatment, it is possible that it occurs earlier or its
frequency might be enhanced in induced cells.
We also examined Sm-Sa recombination in a plasmid lacking
transcriptional activator elements upstream of either S region
(p218, see Fig. 1) and found an average of 5.6 (60.54) Sm-Sa
PCR products per clone (Table I). Therefore, it appears that Sm-Sa
recombination within the plasmid does not require the germline Ia
promoter segment upstream of the Sa segment nor the EP segment
located upstream of the Sm segment. However, it appears to be
somewhat enhanced by these elements. The finding that transcriptional activator elements are not required appears to differ entirely
from chromosomal switch recombination, as it has been shown that
transcription of germline RNA is essential for CSR (29, 30, 32).
It was possible that recombination in the plasmid is not eliminated by deletion of the germline Ia promoter due to run-through
transcription from the TK gene. We examined this possibility by
examining RNA transcripts from plasmids in I.29m cells 2 days
after transfection. Total cell RNA was purified, transcribed with
reverse transcriptase using the same Sa primer (R10) that was used
for the PCR experiments. The reverse transcriptase products were
amplified by PCR using the Sa primer along with either a 59
primer that binds near the beginning of the TK gene or a 59 primer
that binds just downstream of the RNA initiation site within the Ia
segment (see Fig. 1A). The Ia/R10 primers successfully amplified
a 2.3-kb product from p273-transfected cell cDNA, demonstrating
the presence of the expected Ia-Sa transcript (Fig. 8A, lane 11).
The TK/R10 primers amplified very low amounts of 3.8- and
3.3-kb products from cDNA of cells transfected with p273, p200
or p218 (lanes 2, 5, and 8) corresponding to the sizes of comparable fragments amplified from the plasmids (lanes 1, 4, and 7).
These data suggest that run-through transcripts from the TK gene are
present in very low amounts relative to the Ia/Sa transcripts (0.5–
1.0% of the Ia-Sa RNA, as determined by phosphorimaging).
Although the neor segment of the plasmid is oriented in the
anti-sense direction relative to the Sm segment, it was possible that
transcription through the Sm segment was also occurring in the
plasmid lacking the EP segment (p218). To address this possibility, a similar RT-PCR experiment was performed, using cDNA
transcribed from RNA of the transfected cells with a 39 Sm primer,
m-dn2, indicated in Fig. 1A. The cDNA preparation was subjected
to amplification using either a 59 VH primer (59VHN) or a 59 neo
primer (Neo4) and the m-dn2 primer (Fig. 8B). The 59VHN/m-dn2
primers readily amplified cDNAs of the correct size from cells
transfected with either p273 or p200 (lanes 11 and 13), indicating
the presence of reverse transcriptase products in the reactions.
However, no RT-PCR products were detected from any transfected
cell RNA with the combination of the Neo4/m-dn2 primers (lanes
2, 5, and 8). The very low level of transcription through the Sa
segments of p218 and p200 may be sufficient to support recombination in the plasmid or, alternatively, transcription may not be
required for switch recombination on the plasmid.
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know whether these were generated during the PCR or occurred
during switch recombination. Altogether, the characteristics of the
sequences of the switch recombination junctions from transfected
p273 are typical of junctions from endogenous genes, including the
finding of deletions, both mutated and unmutated sequences and
the finding that recombination occurs at sites of no or short sequence identities between the Sm and Sa sequences.
2036
Sm-Sa recombination in p273 is infrequent in a B cell line that
does not undergo switch recombination
Because many B cell lines (and also non-B cell lines) do not undergo class switch recombination, we wanted to determine whether
switch recombination in p273 maintains this cell type specificity.
The mouse cell lines that we examined were 1) M12.4.1, a B
lymphoma line that does not express sIg and was derived from a
sIgG2a1 line (M12) that has not been reported to undergo class
switching in culture; 2) the plasmacytoma line J558L, which secretes l light chains; and 3) two T cell lines, EL-4 and BW5147.
M12.4.1 and EL-4 cells both express a transiently transfected reporter gene driven by the germline a (Ia) segment in the presence
or absence of the Ig m intron enhancer, whereas J558L and
BW5147 cells do not (A. Shanmugam, M.-J. Shi, J. Stavnezer, and
A. L. Kenter, manuscript in preparation) (M. J. Shi, laboratory
observations).
To determine whether p273 undergoes recombination in
M12.4.1 B lymphoma cells, stably transfected clones were derived
and analyzed by PCR (Fig. 9 and Table I). An average of 1.3
(60.7) fragments per clone that hybridize with both Sm and Sa
probes, but not with the TK-Ia probe, were detected. This is 6-fold
fewer than in I.29m cells. Another method for comparing the frequency of Sm-Sa recombination is to compare the intensity of the
hybridization signals obtained from the two cell types. We performed this analysis by comparative densitometric analysis of the
Sm and Sa signals from the entire lane of PCR products from the
p273-transfected M12.4.1 clone 363 with those from all the I.29m
lanes on the same gel (bottom panels in Fig. 3). The intensity of the
hybridization signals in the M12.4.1 lane were about 2.5% of those
obtained from p273-transfected I.29m cells. This difference in signal intensity does not appear to be due to a smaller amount of
plasmid in the M12.4.1 cells because the amplified neor bands are
of comparable intensities in the p273–transfected I.29m and
M12.4.1 clones (Fig. 2C).
Altogether, these results indicate that Sm-Sa recombination in
the plasmid occurs less frequently and in a much smaller proportion of the cells in each M12.4.1 clone than in the I.29m clones.
The low frequency of Sm-Sa recombination may be due to low
amounts of putative switch enzymes or other proteins required for
switch recombination. Surprisingly, the 5.8-kb band from unrecombined plasmid was not detected, suggesting that p273 undergoes frequent rearrangement events in M12.4.1 cells that do not
result in Sm-Sa recombination. This finding is consistent with a
genomic Southern blot analysis in which it was found that no Sa band
FIGURE 9. Southern blot of PCR products from M12.4.1 B lymphoma cells transfected with p273 and PCR products from untransfected I.29m cells.
The left-most lanes in each blot show PCR products from untransfected I.29 cells, 50% of which had switched to expression of IgA. The remainder of the
gel contains p273-transfected M12.4.1 cells, not treated with inducers of class switching. Fragments that were detected by both the Sm and Sa probes but
not with the TK-Ia probe are indicated with a dot on the Sm blot. Methods are the same as for Fig. 3, except the Sm blot was hybridized with 5 3 106
cpm. To achieve an autoradiographic exposure equivalent to or greater than those in all other figures in the manuscript, the duration of autoradiography
was increased to 70 h, which is 12- to 23-fold longer than that used for all other blots. See Fig. 3 for a direct comparison of the relative signal intensities
of the PCR products from p273-transfected M12.4.1 and I.29m clones. An irrelevant lane between the I.29m and p273-transfected M12.4.1 samples was
excised from the photos. Clones analyzed here are the same clones as shown in Fig. 2: (1 5 363, 2 5 364, etc.).
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FIGURE 8. RT-PCR products of transcripts from p273, p200, and p218
transiently transfected into I.29m cells. Autoradiographs of agarose gels of
RT-PCR products from plasmid p273, p200, and p218 transfected into
I.29m. A, PCR products of cDNA obtained from reverse transcriptase reactions primed with the R10 primer. Positive controls for the PCR amplification are the input plasmid DNAs amplified with TK/R10 primers (lanes
1, 4, and 7) or with the Ia/R10 primers (p273 only). Lanes 2, 5, and 8 are
the RT-PCR products of RNA from I.29m cells transfected with p273,
p200, or p218, respectively. Lane 11 shows the PCR products of the same
RT reaction of p273-transfected cell RNA as shown in lane 2, but amplified
with Ia/R10 primers. Lanes 3, 6, and 9 are the PCR products from mock
reactions performed without reverse transcriptase. Equal portions of all the
RT-PCRs and of the PCRs of input plasmid are loaded. B, PCR products
of cDNA obtained from reverse transcriptase reactions primed with the
m-dn2 primer. The lanes correspond to those in A, except the primers are
Neo4/m-dn2 for lanes 1–9 and 59VHN/m-dn2 for lanes 10 –13. Positive
controls for the PCR are input plasmid DNAs amplified with the Neo4/mdn2 primers (lanes 1, 4, and 7) or with the 59VHN/m-dn2 primers, p273 and
p200 (lanes 10 and 12).
PLASMID ASSAY FOR SWITCH RECOMBINATION
The Journal of Immunology
2037
FIGURE 10. Southern blots of PCR products
from J558L plasmacytoma cells transfected with
p273. The cells were not treated with inducers of
class switching. The 5.8-kb full-length Sm-TKIa-Sa segments are indicated with an arrow. The
bands that hybridize with both Sm and Sa probes
but not with the TK-Ia segment are indicated with
dots on the Sm blot.
was detectable in 7 of 12 and no plasmid TK segment was detected in
6 of 12 p273-transfected M12.4.1 clones (data not shown).
p273 undergoes infrequent S/S recombination in the
plasmacytoma line J558L
p273 recombines at a low frequency in EL-4 and BW5147 T cell
lines
To further explore the cell type specificity of Sm-Sa recombination
in p273, we assessed recombination between the plasmid switch
regions in the T cell lines, EL-4 and BW5147, which were stably
transfected and then treated with the T cell activators PMA and
ionomycin (EL-4), or left untreated (EL-4 and BW5147). The PCR
assay detected an average of 3.1 (60.3) Sm-Sa fragments that
were devoid of detectable TK-Ia signal per EL-4 clone and an
average of 2.4 (60.3) such fragments in the BW5147 clones (Fig.
11). These values are 2.5- and 3.3-fold lower, respectively, than
that obtained with I.29m cells and are significantly different ( p ,
0.001; Table I). Unlike results with p273-transfected I.29m cells,
more than half of the PCR products from the T cell lines were
#0.5 kb in length. These fragments were obtained in two sizes and
appeared identical in every clone and were obtained in two different EL-4 and two different BW5147 transfection experiments (data
not shown). These fragments were not obtained when DNA from
p273-transfected I.29m clone 472 was amplified in the same experiment with BW5147 (Fig. 11, left-most lanes). The #0.5-kb
fragments are not due to primer dimers because they were not
obtained in reactions performed without template DNA (lanes labeled N.T.). Although comparable #0.5-kb fragments are observed in some p273-transfected I.29m cells (Fig. 3), they are more
variable and contribute a much smaller portion of the total PCR
products. Thus, EL-4 and perhaps also BW5147 cells may have
some switch recombinase activity, although the activity appears to
differ qualitatively from that in I.29m cells.
Discussion
Cell type specificity of the switch plasmid
The properties of the stably transfected plasmids described in this
report suggest that they are undergoing switch recombination. The
Timing of Sm-Sa recombination in the plasmid
Recombination events which occur soon after transfection of the
plasmids will be present in a high proportion of the cells in a clone
and give intense hybridization signals. Cells in which the plasmid
recombines frequently would have a higher probability of recombining soon after transfection and therefore would have more intense Sm-S bands. Thus, the difference in intensity of the Sm-Sa
bands in I.29m in comparison with M12.4.1, J558L, and BW5147
indicates that Sm-Sa recombination begins more rapidly and/or
occurs more frequently in I.29m cells than in M12.4.1, J558L, and
in BW5147 cells.
Because Sm-Sa recombination can result in loss of targets for
further switch recombination, cells in which switch recombination
occurs frequently can saturate the assay. This is consistent with the
finding that when switch recombination of p273 is assayed in a
transient assay (A. Shanmugam, M.-J. Shi, J. Stavnezer, and A. L.
Kenter, manuscript in preparation) there is a greater difference in
the numbers of plasmid switch fragments obtained from I.29m and
M12.4.1 cells than when assayed in the stable assay described
here. It is possible that in cell lines in which the bands are faint,
e.g. M12.4.1 and BW5147, that recombinations occur infrequently
but continue to accumulate if the plasmid integrates at a permissive
site in the chromosome. In I.29m, recombination occurs frequently,
thus resulting in intense bands and probably saturation of the
assay.
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We also used the PCR assay to examine p273-transfected J558L
plasmacytoma clones. Plasmacytoma cells correspond to plasma
cells that are differentiated to secrete large amounts of Ab and that
do not undergo class switching (57). The 5.8-kb Sm-TK-Ia-Sa
segment was detected in 7 of the 12 clones examined, and only
four very faint Sm-Sa bands were detected (Fig. 10 and Table I).
All of the clones contain a neor gene (Fig. 2C). Thus, the frequency of Sm-Sa recombination of p273 in J558L cells was 0.3
(60.2) events per clone, which is 26-fold lower than in I.29m cells.
Thus, like M12.4.1 cells, J558L cells appear to contain only low
levels of switch recombination activity.
most compelling of these properties is that the frequency of Sm-Sa
recombination in the optimal switch plasmid, p273, is greater in
the B cell line I.29m, which undergoes switch recombination of its
endogenous heavy chain genes, than in four cell lines that do not
undergo switching of their endogenous genes. In addition to yielding a lower number of amplified plasmid Sm-Sa fragments, three
of the four nonswitching cell lines produced Sm-Sa fragments that
gave lower hybridization signals than did I.29m cells, indicating
they were present in a smaller proportion of the cells of the clone.
The T cell line, EL-4, gave the highest level of Sm-Sa recombination of the four nonswitching lines, although the difference from
I.29m is still statistically highly significant. Furthermore there are
qualitative differences in the Sm-Sa bands found in I.29m cells and
the two T cell lines, because the majority of the amplified fragments in the two T cell lines were small, #0.5 kb, and were identical among all clones. The finding of some switch activity in EL-4
cells is consistent with results obtained by Daniels and Lieber (35)
using their switch plasmid. We conclude that one reason T cells do
not undergo switch recombination in vivo may be because they
express low levels of switch recombinase activity, and it is also
likely that the switch regions are inaccessible to recombinase activity in the endogenous loci.
2038
PLASMID ASSAY FOR SWITCH RECOMBINATION
Evidence from transient transfection experiments with these
plasmids indicates that Sm-Sa recombination begins before integration of the plasmids in I.29m cells (A. Shanmugam, M.-J. Shi,
J. Stavnezer, and A. L. Kenter, manuscript in preparation).
Whether recombination continues after integration has not been
established. Data presented here suggest that it may continue after
integration, because the G418-resistant clones obtained by limiting
dilution cultures have different Sm-Sa fragments. However, it is
also possible that the different Sm-Sa fragments could be due to
integration of different recombined plasmids after cloning on day
2. To attempt to determine whether integrated plasmids can recombine, we subcloned a few clones that have integrated intact
plasmids in the presence of gancyclovir and LPS plus TGF-b1.
Very few gancyclovir-resistant cells were obtained, suggesting that
these plasmids were not accessible to recombinase.
The plasmids undergo rearrangements in addition to Sm-Sa
recombination
Similarly to the previously reported plasmid switch substrates (33–
37), the plasmids reported here undergo recombination events in
addition to switch recombination. These other recombinations occur in all cell lines we examined, except perhaps in J558L cells.
This was learned from extensive genomic Southern blot analyses
of stably transfected clones. For example, the TK gene of p273 is
undetectable in many clones that yield very few or no Sm-Sa PCR
products. It was deleted from 50% of the M12.4.1 clones, 67% of
the EL-4 clones, 70% of I.29m clones, but present in unrearranged
form in all of the J558L clones. The Sa segment of the plasmid is
also frequently deleted, except in J558L.
Deletion of the Sa segment would result in loss of the 39 primer
binding sites. Deletion of the Sa segment can explain why we do
not usually detect plasmid bands containing the TK gene in the
PCR assay, even in cells which retain the plasmid TK gene (identified by genomic Southern blotting). However, the loss of primer
binding sites cannot explain why the plasmid infrequently undergoes Sm-Sa recombination in M12.4.1 or the T cell lines, because
in experiments in which the plasmids were isolated 2 days after
transfection, at which time most of the plasmids in nuclei are
present in the intact form (A. Shanmugam, M.-J. Shi, J. Stavnezer,
and A. L. Kenter, manuscript in preparation), a greater difference
is found between the yield of Sm-Sa PCR products from I.29m and
the nonswitching cell lines than found here using the stable assay
(A. Shanmugam, M.-J. Shi, J. Stavnezer, and A. L. Kenter, manuscript in preparation). Further information about the other recombination events in the plasmids is reported by Shanmugam et al.
(A. Shanmugam, M.-J. Shi, J. Stavnezer, and A. L. Kenter, manuscript in preparation). Because of these other recombination
events, the use of drug selection against the TK gene does not
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FIGURE 11. Southern blots of PCR products from p273-transfected EL-4 and BW5‘47 T cell clones. The transfected cells were treated as indicated.
Fragments that were detected by both the Sm and Sa probes but not with the TK-Ia probe are indicated with dots on the Sm blots. The 0.5-kb bands are
indicated but the dots are not visible. The left-most lanes of the bottom panels contain PCR products from the I.29m clone 472, transfected with p273, to
be able to compare the intensity of the signals for BW5147 and I.29m-transfected cells.
The Journal of Immunology
provide a specific assay for Sm-Sa recombination on the plasmid.
However, the use of the PCR assay allows one to specifically assay
switch recombination on the plasmid.
Switch recombination does not require a high rate of
transcription of the plasmid
Nucleotide sequences of switch recombination junctions in the
plasmid
The presence of mutations and deletions on one side of the switch
recombination junction in the sequence from clone 153 is similar
to endogenous switch recombination events (2). These observations are predicted by a model for switch recombination involving
error-prone DNA synthesis in which one switch region (donor)
primes DNA synthesis on another (acceptor) switch region (2, 14).
Simultaneously, the other strand of the acceptor switch region
primes DNA synthesis on the donor switch region. According to
this model, after recombination and segregation of the products
into two different daughter cells, one of the two daughter cells has
a newly synthesized acceptor switch region (Sa) which is mutated,
and the other daughter has a newly synthesized (mutated) donor
switch region (Sm). The 153 sequence corresponds to the latter
type of product.
The illegitimate priming model might seem to predict the finding of sequence identities between the Sm and Sa sequences at the
sites of switch recombination. Although such identities are found
at switch recombination junctions which occur in endogenous
genes and are also observed in the PCR products from the switch
plasmid, there is no preference for junctions with long identities
(2). Thus, switch recombination differs from homologous recombination. The finding of short bits of identity at recombination
junctions is typical of illegitimate recombination, or end-joining,
in mammals (58). It is also typical of V(D)J recombination (59).
An observation that is consistent with the finding that error-prone
DNA synthesis occurs across switch recombination junctions (2,
13, 14) and also has similarity to recombination by end-joining is
the finding that end-joining in germinal vesicle extracts from Xe-
nopus oocytes requires deoxynucleotide triphosphates and thus appears to involve DNA synthesis (60).
Switch recombination of the plasmid occurs constitutively in
I.29m cells
Although induction of switch recombination of the endogenous
heavy chain genes in I.29m cells requires addition of LPS, recombination of the switch regions in transfected p273 occurs without
treatment of I.29m cells with LPS. The function of LPS in inducing
switch recombination is not understood, as it does not increase the
rate of transcription of germline a transcripts (43), nor does it
increase proliferation of I.29m cells (data not shown). One possibility was that it induces recombinase activity. However, the finding that switch recombination of the plasmid does not require LPS
treatment suggests that the switch recombinase activity is present
constitutively in I.29m cells, but the endogenous loci do not recombine due to a lack of accessibility. This result is reminiscent of
the finding that the V(D)J recombinase enzymes, Rag-1 and Rag-2,
are expressed in developing B lineage cells at stages in which
endogenous loci are inaccessible to their activity. Specific chromatin changes are required to induce accessibility of the Ig genes
to recombinase (61). The role of LPS may be to induce chromatin
remodeling, resulting in accessibility. It is possible that the endogenous switch regions are not accessible to the hypothetical constitutive recombinase activities due to proteins bound to switch regions that protect them from recombinase activities, and that the
role of LPS is to remove the repressor proteins.
Kinoshita et al. (62) recently demonstrated switch recombination in a plasmid substrate which was stably integrated into the B
cell line CH12F3-2, a cell line capable of undergoing switch recombination of its endogenous genes. They found that intact integrated plasmids did not undergo constitutive recombination, but
did recombine when cells were treated with reagents, e.g., cytokines and CD40 ligand, which induce switch recombination of the
endogenous genes. We postulate that the function of the inducers
is to stimulate accessibility of the integrated plasmid. In contrast,
our plasmid begins to recombine before integration and thus a
major difference between their experiments and ours is that our
plasmid does not have the constraints of endogenous chromatin
which may regulate the recombination. In conclusion, although
switch recombination of the endogenous genes in I.29m cells requires treatment with LPS, recombination of the plasmid does not.
This result suggests that the switch recombinase is constitutive in
I.29m cells and that LPS may induce chromatin changes at the
endogenous loci, allowing accessibility to the recombinase.
Acknowledgments
We thank Dr. Meng-Jiao Shi for examining the activity of reporter genes
driven by transcriptional elements from the switch plasmids; Drs. Ako
Jacintho, Gang Qiu, and Özlem Yazar and Mr. Curtis Barrett for performing some of the preliminary Southern blotting experiments; and Ms. Jennifer Nietupski for technical assistance. We thank Drs. Rachel Gerstein and
Carol Schrader for helpful comments on the manuscript and members of
our laboratories for helpful discussion. We thank Drs. Fred Alt for pEP.B,
Allan Bradley for the pPGK-neo plasmid, and Ken B. Marcu for
pZN(Sm-Sg2b)tk.1.
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PLASMID ASSAY FOR SWITCH RECOMBINATION