| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
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
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
recombination in the plasmid.
Recombination of the plasmid in I.29µ cells does not require
treatment with inducers of switch recombination, suggesting that
recombinase activity is constitutive in I.29µ 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. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(reviewed in Refs. 1, 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, 4, 5, 6, 7, 8, 9, 10, 11, 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 double-strand breaks are rapidly induced in the S
3 region when
mouse 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 (DNA-PK), Ku70, and Ku80 are required
for switch recombination (16, 17, 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, 21, 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, 24, 25, 26). Transcription of germline RNA is regulated by
the cytokines IL-4, IFN-, and TGF-ß1, in concert with B cell
activators, and serves to direct recombination to specific S regions
(reviewed in Refs. 26, 27, 28). Transcription initiates at the
I exon located 5' 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, 30, 31, 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 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, 36, 37) found they could measure B cell-specific 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 sIg+ 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, 39, 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.29µ 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-ß1 (42, 43, 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-ß1 (45, 46, 47).
Our plasmid, p273, undergoes recombination between its two switch
region sequences (Sµ and S) in I.29µ 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.29µ cells
does not require treatment with inducers of switch recombination,
suggesting that recombinase activity is constitutive in I.29µ
cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The 1.8-kb HindIII mouse Sµ segment (BALB/c) from
pM2-20 (48) was inserted into the HindIII site
of the Bluescript SK- vector (Stratagene, La
Jolla CA). The 2.1-kb HaeIII S fragment cloned from the
I.29µ 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 Sµ fragment also includes about 330 bp 5' to
the SacI site where the tandem repeats begin. Both the Sµ
and S fragments have a binding site for the B cell-specific paired
domain transcription factor, BSAP (Pax-5), near their 5' ends (3, 8, 49). The herpes virus thymidine kinase (TK) gene (1.8 kb) was
excised from pZN(Sµ-S2b)tk.1 (38) with
BamHI, cloned into Bluescript KS-,
excised with EcoRI and BamHI, and inserted
between the Sµ and S 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 µ 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 promoter (I
promoter) segment from -489 to +46 relative to the first RNA
initiation site (51) was inserted in the forward direction
into the BamHI site between the TK and S 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 x 106 per transfection) were electroporated as previously described (52) with 1 µg 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.29µ 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 µg/ml), TGF-ß1 (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 µg/ml for all cells, except at 1.2 mg/ml for BW5147, and plated in 96-well plates at limiting dilution. For I.29µ, 2 x 104 cells per well were plated. For the M12.4.1, J558L, and EL-4 lines, 4 x 103 cells/well were plated, and for BW5147, 2 x 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.29µ cells
Class switching was assayed on day 2 by immunofluorescence microscopy as described (44).
PCRs and primers
The PCR to detect Sµ-S 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 µM primers, and 250 µM dNTPs were used. Primers
used were: µ-1A, 5'-CTCTACTGCCTACACTGGACTGTTC-3', identical with nt
5269–5293 of the germline BALB/c Sµ sequence (MUSIGCD07)
(15); µ-3, 5'-TGGCTTAACCGAGATGAGCC-3', identical with nt
5206–5226 of MUSIGCD07 (41); and R10,
5'-CTCTATCTAGGTCTGCCCCGTCTAGATAAG-3', complementary to nt 62–91
upstream of the 3' end of the 2.1-kb HaeIII S segment
(GenBank accession no. AF069385) in the switch plasmids. The sequences
of both µ primers differ from the sequence of the endogenous I.29
Sµ sequence (MUSIGHMY) at several nucleotides. The µ-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 µ-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: µ-2,
5'-CCTGGGGTGAGCTCAGCTATGCTACGC-3', identical with nt 5307–5333
of the BALB/c Sµ sequence (MUSIGCD07) (15); and R10B,
5'-GGAATTCATAAGCTCAGCCTTGTTCAGCCCATTCCATC-3', complementary to nt
87–117 upstream of the 3' end of the S segment, except for the
addition of an EcoRI site at the 5' end. The other PCR
products that were sequenced were amplified by a single round of PCR
using the µ-1 and R10 primers.
|
Primers for amplification of the TK-I 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): 5'-TGACTTACTGGCAGGTGCTG-3'
(corresponding to nt 769–789 of GenBank accession no. V00467) and
sequences complementary to the I segment at -33/-14 (GLR1):
5'-GGCTGCATGACTGTGTGTCT-3' (GenBank accession no. L04145).
RT-PCR to evaluate transcription of the TK-S and the
neor-Sµ segments
Two days after transfection of I.29µ 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 µg RNA using Moloney murine leukemia virus
(M-MLV)-Reverse-Transciptase (200 U; Promega, Madison, WI) with the 3'
primer R10 and with the 3' µ-dn2 primer,
5'-CTTGGTTCTTGGCCAGCCAGCTCTAC-3' (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 Sµ-S recombination, except
that for amplification of the TK-S segment a 5' primer specific to
the HSV-TK gene, TKF2, was used along with the 3' primer R10. For
amplification of the neor-Sµ segment,
PCR was performed using a 5' primer complementary to the
neor gene, Neo4,
5'-CGTCCAGATCATCCTGATC-3', and the µ-dn2 primer. An equal proportion
of the cDNA products were amplified for each sample and 3 µCi
[-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
5' primer IF, 5'-ACAGGCAATCACACACAGAG-3', corresponding to the
I region +13/+33 relative to the first RNA initiation site, along
with the 3' primer R10. As a positive control for the RT-PCR using the
µ-dn2 primer, an aliquot of the cDNA obtained from p273- and
p200-transfected cells was amplified using a 5' primer for the
VH sequence in the EP segment, 5'VHN,
5'-TGTTCTCTTTACAGTTACTGAGCACACAGGACC-3'. 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 5' Neo/µ-dn2
primers and also on p273 using the IF/R10 primers and on p273 and
p200 using 5'VHN/µ-dn2 primers. PCR products were separated on 1.0%
agarose gels, dried, and autoradiographed.
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 0.1x 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 Sµ, S, and TK-I probes were labeled
to a similar specific activity (50 x 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).
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 Sµ-S
fragments down to the smallest detectable Sµ-S 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 Sµ
and S 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 µ intron
enhancer and an Ig VH promoter (50),
inserted upstream of the Sµ segment. One plasmid (p273) also has a
535-bp fragment (I) containing the promoter and first RNA initiation
site for the germline transcripts inserted upstream of the S
segment (51). Fig. 1B diagrams an expected
switch recombination event in p273.
|
, 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. 1
B.
|
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 3' portion of the
neor gene, the 1-kb EP segment, and 0.3 kb
from the 5' portion of Sµ (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
5' portion of Sµ 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, left-most 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.
PCR and Southern blotting analyses detect Sµ-S fragments in
I.29µ 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.29µ
cells in the absence of LPS plus TGF-ß1, as cells expressing both IgM
and IgA are rarely observed in untreated cultures
(42, 43, 44). TGF-ß1 induces transcription from the promoter
for germline transcripts, but the role of LPS is unknown. In
addition, nicotinamide has been shown to stimulate class switch
recombination in I.29µ cells by inhibiting the nuclear enzyme,
poly(ADP-ribose) polymerase (44).
I.29µ cells were electroporated with p273 or p200, divided into aliquots that were treated with LPS, LPS plus TGF-ß1, LPS plus TGF-ß1 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 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 Sµ-S recombination. We developed a PCR assay
for recombination between the Sµ and S segments, using
oligonucleotide primers complementary to the 5' end of Sµ and to the
3' end of S (see Fig. 1B). This strategy was previously
used to amplify endogenous Sµ-S3 recombinants (56).
The µ primers are specific for the BALB/c Sµ sequence in the
plasmid, but the S primer is complementary to both the plasmid and
endogenous S sequences. The primers amplify Sµ-S 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, Sµ, S, and a probe
containing both the TK and I segments from p273. The probes
correspond to the segments indicated in the plasmid maps (Fig. 1). PCR
fragments that are due to direct Sµ-S recombination and therefore
lack the TK and I segments should be 
3.3 kb in size.
Southern blots of PCR products obtained from the genomic DNA of I.29µ
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 Sµ probe, the blots in the
middle panels are the identical blots hybridized with the S probe,
and the blots on the right are the identical blots hybridized with the
TK-I probe. The dots on the edge of the bands in the Sµ blots
indicate bands that also hybridize with the S probe. The great
majority of the amplified segments are detected by both the Sµ and
S probes, but not with the TK-I probe. However, from three of the
transfected clones that were not treated with inducers, 5.8-kb
fragments that hybridize with the Sµ, S, and TK-I 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-I probe, except for two
fragments from one clone induced with LPS without TGF-ß1. These data
suggest that the transfected switch plasmid has undergone numerous
direct Sµ-S recombination events in I.29µ cells. An average of
7.9 (±0.6) Sµ-S PCR products were amplified from each
p273-transfected I.29µ clone in two experiments, whether or not the
cells were treated with inducers of class switch recombination (Table I).
|
|
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.29µ 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 S and TK-I probes (perhaps due to
amplification of an endogenous I-S fragment), and a different
sized band (2.5 kb) was detected with the Sµ probe. Thus, the
primers used do not appear to amplify the endogenous Sµ-S
junctions in IgA+ cells derived from the I.29µ
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-ß1. This clone did not
yield Sµ-S PCR products, although it yielded a
neor PCR product (Fig. 2C) and
contains endogenous genomic Sµ and S fragments, as shown by
genomic Southern blot analysis (data not shown). Nucleotide sequencing
data described below further support the conclusion that the Sµ-S
fragments amplified from p273-transfected I.29µ cells are due to
recombination in the plasmid and are not due to recombination of
endogenous Sµ and S sequences.
|
segments from stably
transfected p273
To examine the nature of the recombination events that produced
the comigrating Sµ and S fragments, we determined the nucleotide
sequences of six Sµ-S segments amplified from I.29µ cells stably
transfected with p273. The PCR products were cloned from five
independent clones of p273-transfected I.29µ 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 Sµ to S junctions with 0, 1,
2, 6, or 8 nt of identity between the Sµ and S sequences at the
recombination sites (Fig. 5), quite
comparable to switch recombination junctions in genomic DNA
(2).
|
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
Sµ-S recombination. Fig. 5A presents the sequence
surrounding the Sµ-S junction of clone 153 aligned with the
plasmid Sµ and S 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 Sµ-S junction, we compared the entire Sµ and S
sequences of clone 153 with the p273 sequence (Fig. 6A and data not shown). The
Sµ 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 Sµ sequence. However, in the regions at the
5' and 3' ends of the sequence, the alignments obtained by three
programs using different algorithms are identical. In these regions the
153 Sµ sequence has 9-nt differences from the p273 sequence (in bold
type). The S sequence of 153 is identical with the S sequence in
p273 (data not shown).
|
B) The Sµ segments of all other
Sµ-S PCR products in Fig. 5 were also compared with the I.29 Sµ
sequence and found not to match it as well as they match the p273 Sµ
sequence (data not shown). Therefore, all of the PCR products analyzed
by nucleotide sequencing originated from Sµ-S 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.29µ
clone (EW 4) show no differences from the plasmid Sµ and S
sequences. The sequence of EW 4.1 surrounding the Sµ-S junction is
shown in Fig. 5B. The junction occurs within a 6-nt identity
(underlined) between the Sµ and S 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 Sµ-S recombination. Although they have
several nucleotide differences in comparison to the plasmid Sµ and
S sequences, we do not 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 Sµ and S sequences.
Switch recombination in the plasmid does not require high levels of transcription through the S regions
To determine whether transcription through the S 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 (I) promoter upstream of the S
segment, to undergo Sµ-S recombination. Twenty clones of I.29µ
cells stably transfected with p200, which were untreated or treated
with LPS plus TGF-ß1, were analyzed by PCR followed by Southern blot
analysis. An average of 3.6 (±0.36) Sµ-S fragments that were
devoid of TK-I signal were detected per clone, whether untreated or
treated with LPS plus TGF-ß1 (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).
|
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 Sµ-S
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-ß1 treatment, it is possible that it occurs earlier or its
frequency might be enhanced in induced cells.
We also examined Sµ-S recombination in a plasmid lacking
transcriptional activator elements upstream of either S region (p218,
see Fig. 1) and found an average of 5.6 (±0.54) Sµ-S PCR products
per clone (Table I). Therefore, it appears that Sµ-S recombination
within the plasmid does not require the germline I promoter
segment upstream of the S segment nor the EP segment located
upstream of the Sµ 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 I promoter due to run-through transcription
from the TK gene. We examined this possibility by examining RNA
transcripts from plasmids in I.29µ cells 2 days after transfection.
Total cell RNA was purified, transcribed with reverse transcriptase
using the same S primer (R10) that was used for the PCR experiments.
The reverse transcriptase products were amplified by PCR using the S
primer along with either a 5' primer that binds near the beginning of
the TK gene or a 5' primer that binds just downstream of the RNA
initiation site within the I segment (see Fig. 1A). The
I/R10 primers successfully amplified a 2.3-kb product from
p273-transfected cell cDNA, demonstrating the presence of the expected
I-S 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 I/S transcripts (0.5–1.0% of
the I-S RNA, as determined by phosphorimaging).
|
A. The cDNA preparation
was subjected to amplification using either a 5'
VH primer (5'VHN) or a 5' neo primer
(Neo4) and the µ-dn2 primer (Fig. 8B). The 5'VHN/µ-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/µ-dn2
primers (lanes 2, 5, and 8). The very low
level of transcription through the S 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.
Sµ-S 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
sIgG2a+ line (M12) that has not been reported to
undergo class switching in culture; 2) the plasmacytoma line J558L,
which secretes 
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 (I) segment in the presence
or absence of the Ig µ 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 (±0.7)
fragments per clone that hybridize with both Sµ and S probes, but
not with the TK-I probe, were detected. This is 6-fold fewer than in
I.29µ cells. Another method for comparing the frequency of Sµ-S
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 Sµ and S signals from
the entire lane of PCR products from the p273-transfected M12.4.1 clone
363 with those from all the I.29µ 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.29µ 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.29µ and M12.4.1
clones (Fig. 2C).
Altogether, these results indicate that Sµ-S 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.29µ clones. The low
frequency of Sµ-S 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 Sµ-S recombination.
This finding is consistent with a genomic Southern blot analysis
in which it was found that no S band 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
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 Sµ-TK-I-S
segment was detected in 7 of the 12 clones examined, and only four very
faint Sµ-S bands were detected (Fig. 10 and Table I). All of the clones
contain a neor gene (Fig. 2C).
Thus, the frequency of Sµ-S recombination of p273 in J558L cells
was 0.3 (±0.2) events per clone, which is 26-fold lower than in
I.29µ cells. Thus, like M12.4.1 cells, J558L cells appear to contain
only low levels of switch recombination activity.
|
To further explore the cell type specificity of Sµ-S
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 (±0.3) Sµ-S fragments that were devoid
of detectable TK-I signal per EL-4 clone and an average of 2.4
(±0.3) such fragments in the BW5147 clones (Fig. 11). These values are 2.5- and 3.3-fold
lower, respectively, than that obtained with I.29µ cells and are
significantly different (p < 0.001; Table I).
Unlike results with p273-transfected I.29µ 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.29µ 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.29µ 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.29µ cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The properties of the stably transfected plasmids described in
this report suggest that they are undergoing switch recombination. The
most compelling of these properties is that the frequency of Sµ-S
recombination in the optimal switch plasmid, p273, is greater in the B
cell line I.29µ, 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 Sµ-S fragments, three of the
four nonswitching cell lines produced Sµ-S fragments that gave
lower hybridization signals than did I.29µ 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 Sµ-S recombination of
the four nonswitching lines, although the difference from I.29µ is
still statistically highly significant. Furthermore there are
qualitative differences in the Sµ-S bands found in I.29µ 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.
Timing of Sµ-S 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 Sµ-S
bands. Thus, the difference in intensity of the Sµ-S bands in
I.29µ in comparison with M12.4.1, J558L, and BW5147 indicates that
Sµ-S recombination begins more rapidly and/or occurs more
frequently in I.29µ cells than in M12.4.1, J558L, and in BW5147
cells.
Because Sµ-S 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.29µ 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.29µ, recombination occurs frequently, thus resulting
in intense bands and probably saturation of the assay.
Evidence from transient transfection experiments with these plasmids
indicates that Sµ-S recombination begins before integration of the
plasmids in I.29µ 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 Sµ-S fragments. However, it is also possible that the
different Sµ-S 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-ß1. Very few gancyclovir-resistant cells were obtained,
suggesting that these plasmids were not accessible to recombinase.
The plasmids undergo rearrangements in addition to Sµ-S
recombination
Similarly to the previously reported plasmid switch substrates
(33, 34, 35, 36, 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 Sµ-S PCR
products. It was deleted from 50% of the M12.4.1 clones, 67% of the
EL-4 clones, 70% of I.29µ clones, but present in unrearranged form
in all of the J558L clones. The S segment of the plasmid is also
frequently deleted, except in J558L.
Deletion of the S segment would result in loss of the 3' primer
binding sites. Deletion of the S 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 Sµ-S 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 Sµ-S PCR
products from I.29µ 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 provide
a specific assay for Sµ-S 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
The fact that transcriptional promoters upstream of the Sµ or
S segments do not appear to stimulate recombination between switch
regions in the transfected plasmids is inconsistent with what is known
about switch recombination of endogenous genes. The requirement for
promoters upstream of S regions appears to be absolute in vivo. We
examined whether this was because transcription from the TK promoter
may continue through the S segment and found a very low level of
run-through transcripts (0.5–1.0% of the level of transcription
driven by the I promoter). Further support for a lack of requirement
for a high rate of transcription was obtained by the finding that p218,
which has no transcriptional activator segment upstream of either S
region segments, undergoes Sµ-S recombination in I.29µ cells
about as frequently as does p200.
The lack of requirement for specific transcriptional elements has several possible explanations. It is possible that low levels of run-through transcripts initiating in segments upstream of the S regions or initiating within the S regions themselves are sufficient for recombination. Alternatively, it is possible that whatever the role of germline transcripts, this role is not required for recombination of these plasmids. It is possible that the role for germline transcripts in vivo may be to contribute to synapsis of the switch regions and due to their close proximity in the plasmid, this is not required. Perhaps the chromatin structure of the transfected plasmid does not interfere with switch recombination, unlike the chromatin of endogenous switch regions and thus germline transcription is not required.
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 (S) which is mutated, and the
other daughter has a newly synthesized (mutated) donor switch region
(Sµ). 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 Sµ and S sequences at the
