Role of the Intronic Elements in the Endogenous Immunoglobulin
Heavy Chain Locus
EITHER THE MATRIX ATTACHMENT REGIONS OR THE CORE ENHANCER IS
SUFFICIENT TO MAINTAIN EXPRESSION*
Erik J.
Wiersma
,
Diana
Ronai
,
Maribel
Berru
,
Florence
W. L.
Tsui
§, and
Marc J.
Shulman
¶
From the Departments of
Immunology and
¶ Molecular and Medical Genetics, University of Toronto, and
§ The Toronto Hospital, Toronto, Ontario M5S 1A8, Canada
 |
ABSTRACT |
High level expression in mice of transgenes
derived from the immunoglobulin heavy chain (IgH) locus requires both
the core enhancer (Eµ) and the matrix attachment regions (MARs) that
flank Eµ. The need for both elements implies that they each perform a
different function in transcription. While it is generally assumed that
expression of the endogenous IgH locus has similar requirements, it has
been difficult to assess the role of these elements in expression of
the endogenous heavy chain gene, because B cell development and IgH
expression are strongly interdependent and also because the locus
contains other redundant activating elements. We have previously
described a gene-targeting approach in hybridoma cells that overcomes
the redundancy problem to yield a stable cell line in which expression
of the IgH locus depends strongly on elements in the MAR-Eµ-MAR
segment. Using this system, we have found that expression of the
endogenous µ gene persists at substantially (~50%) normal levels
in recombinants which retain either the MARs or Eµ. That is, despite
the dissimilar biochemical activities of these two elements, either one
is sufficient to maintain high level expression of the endogenous
locus. These findings suggest new models for how the enhancer and MARs
might collaborate in the initiation or maintenance of transcription.
 |
INTRODUCTION |
Expression activating elements in the immunoglobulin heavy chain
(IgH)1 locus (Fig. 1) have
been identified and characterized in diverse ways. An enhancer lying in
the VDJ-Cµ intron was originally detected by its capacity to
stimulate transcription from linked reporter genes that were
transfected into myeloma cell lines (1, 2). Experiments using
transgenic cell lines from which this enhancer could be deleted after
transcription had been initiated indicated that the enhancer was
required to maintain high level expression (3, 4). The enhancer-bearing
segment can be divided into two subregions on the basis of sequence
motifs and protein-binding characteristics. Thus, multiple
transcription factors that bind to characteristic motifs define a
central core (Eµ) region; this region is flanked by matrix attachment
regions (MARs) identified by their affinity for the nuclear matrix
(reviewed in Refs. 5 and 6). Both elements, Eµ and the MARs, are
required for high level expression of IgH-derived transgenes in mice,
albeit not in stably transfected cell lines (7). The switch (S) regions were first recognized as repetitive DNA segments that lie 5' of the
exons encoding heavy chain constant regions and which are the sites of
most of the breakage and rejoining events underlying the heavy chain
isotype switch. These switch regions also harbor elements that
stimulate expression of IgH-derived reporter genes in transgenic mice,
but again this activity has not been detected in assays of stably
transfected cell lines (8, 9). That all three types of elements are
required for high level expression in transgenic mice indicates that
each element performs a different and necessary function in
transcription. Evidence of the Eµ-MAR collaboration can also be seen
in assays of chromatin accessibility (10) and DNA demethylation
(11).
It is generally assumed that the expression-activating elements defined
in transgene expression assays function similarly in the transcription
of genes in their endogenous chromosomal loci. Thus, while the intronic
MARs and Eµ core enhancer are not needed to maintain expression of
the endogenous IgH locus in some B-cell lines (12-15), this difference
is generally ascribed to the presence of other, functionally redundant,
activating elements present in the IgH locus but absent from the
transgenes, viz. the MARs that are located 5' of the C
exons (16) and enhancers that are located 3' of the C
exons
(reviewed in Ref. 17). In fact, the capacity of the intronic elements
to contribute to IgH expression can be observed under conditions that
ablate this redundancy. Thus, targeted replacement of one of these 3'
E
enhancers by the neo gene extinguishes IgH expression
specifically in those myeloma cells that lack the intronic activating
elements (18).
We have previously described a related method of ablating redundancy in
which introduction of the gpt cassette 3' of the endogenous µ heavy chain gene of hybridoma cells apparently insulates the transcription unit from other activating elements and renders µ expression dependent on the intronic activating elements (6). That is,
in this configuration, deletion of the 5'-MAR-Eµ-3'-MAR segment
depresses µ expression to ~2% the normal level (6). Although the
mechanism by which the gpt cassette renders the µ gene
expression dependent on the intronic elements is unknown, this approach
offers the possibility of examining how these elements function in a
reproducible and nearly normal context. Thus, using this system we
showed that recombinants that retain a substantial segment of the MARs
maintain µ expression at a high (~60% normal) level. In the
context of models in which µ expression requires both MAR and Eµ
functions, this result implies that other enhancers in the recombinant
IgH locus can act on the µ gene, but that other MARs in the locus
cannot. We have now tested this interpretation by examining the
properties of the reciprocal recombinant that retained Eµ but lacked
the MARs. As reported here, we have found unexpectedly that this
recombinant also continued to express the µ gene at a high (~50%
the normal) level, a result that implies that other MARs in the
recombinant IgH locus, but not other enhancers, can act on the µ gene. These observations then pose the paradox that high level µ expression in the recombinant hybridoma cells requires a MAR or an
enhancer, even though the recombinant IgH locus can supply both of
these functions. We present two models that might account for this
puzzling combination of observations.
 |
MATERIALS AND METHODS |
Constuction of Targeting Vectors--
We have previously
described the Eµ+MAR+ recombinants (6). Fig.
1 illustrates the construction of the three targeting vectors, p0,
pEµ, and pMM, used to generate the
Eµ
MAR
,
Eµ+MAR
, and
Eµ
MAR+ recombinants, respectively. The
targeting vectors are composed from seven DNA segments (6). Thus, the
NcoI-NaeI segment A included the VDJ3 nucleotides
of GenBankTM/EMBL accession number X56936. Segment B was
specific for each recombinant, such that p0 lacked the entire
5'-MAR-Eµ-3'-MAR segment, while pEµ and pMM included the Eµ and
5'-MAR-3'-MAR segments, respectively, as illustrated in Fig.
1C. Segments D (bounded by restriction sites
SnaI-SphI) and F (bounded by restriction sites BglII-NdeI) are included in entries MUSIGCD07-09.
Segment C was derived from the multicloning site of two vectors
and has the sequence:
GCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCGGTACCCtaccagg, where the
lowercase letters denote the sequence beginning in the 3'-half of the
site denoted Sna1 (b) (Fig. 1A). Segment E
(bounded by restriction sites SphI-BamHI) was
derived from pSV2gpt (19). Segment G corresponds to pGEM5
(Promega Biotech). The notation B/B indicates that segments E and F
were joined by ligating the BamHI-generated end of E with
the BglII-generated end of F.
Segments A and C-G were obtained from the transfer vector "D" (6)
by digesting with NotI and either NaeI or its
isoschizomer NgoMI and purified by gel electrophoresis. The
insert segments denoted B were constructed by PCR using the primers
defined below in which restriction sites were fused to sequences that
would pair with specific regions of the intronic DNA. Thus, the Eµ
insert was generated from plasmid pRSp6 (20) by PCR using primers 5 and
6. Primers 7 and 8 were used to amplify the MARs-containing insert from
plasmid pµ
4, kindly provided by R. Grosschedl (7). The
NotI and NaeI/NgoMI sites in the
primers are italicized.
Cell Culture and Transfection--
Previous publications have
described the wild-type Sp6 (21) and mutant igm692 (22) and X10 (23)
cell lines and the methods for cell culture and transfection by
electroporation (24). As illustrated in Fig. 1, targeted recombinants
were identified by screening either for the correct 5' junction using
primers 1 and 2 (see below) or by testing culture fluid for the
presence of normal IgM using a Cµ1-specific ELISA. Transfectants
satisfying these criteria were further tested for both correct
junctions using primers 3 and 4 (see below). To ensure that the
recombinants had not acquired additional vector DNA, genomic DNA was
digested with BamHI and analyzed by Southern blot probing
with the Cµ1-2 (XbaI-BamHI) and Cµ3-4
(HindIII-HindIII) fragments. In most cases only
the predicted bands were seen, and only recombinants with the correct
structure were analyzed for µ mRNA production.
Analysis of DNA Structure and mRNA Production--
DNA
structure was analyzed by Southern blot and other methods, as described
(24). The primers used for PCR amplifications were as follows.
Primer 1, 5'-ACATGGATGTCCACAGCCTGAAAACA-3' (5' of VH homology
region); primer 2, 5'-GGGTACCGAGCTCTCCCATATGGT-3' (in
multicloning site); primer 3, 5'-CGATACGGTGATTGGCTACCG-3' (in
gpt gene); primer 4, 5'-GTGGCAAGAGGCTAATCTTGTCATGT-3' (3' of
homology region); primer 5, 5'-CGTCCTGCCGGCAATGTTGAGTTGGAGTCAAGAT-3'; primer 6, 5'-GCTGCAGCGGCCGCTCTCCAGTTTCGGCTGAATCCT-3'; primer 7, 5'-TGAGGGAGCCGGCTGAGAGAAGT-3'; primer 8, 5'-GCTGCAGCGGCCGCCGAAATAAGTCTAGATAAT-3'. RNA was
analyzed by Northern blot, as described (24).
 |
RESULTS |
Construction of Targeted Recombinants--
We have previously
described a gene-targeting system to remove various intronic elements
from the active IgH locus of a mouse hybridoma cell line, Sp6, which
produces IgM(
) specific for the hapten, trinitrophenyl (6, 24). This
system makes use of a mutant hybridoma, igm692, which encodes a
truncated immunoglobulin µ heavy chain lacking the Cµ1 and Cµ2
domains (Fig. 1). The targeting vector
bears the bacterially derived gpt gene for guanine-xanthine phosphoribosyltransferase, which allows cells to convert exogenous xanthine to XMP and to grow in medium containing xanthine and mycophenolic acid (MHX medium) (19). Homologous recombination between
the targeting vector and the endogenous µ gene can restore the
missing exons and render the recombinants gpt+,
so that they can then be selected in MHX medium. This feature makes it
possible to use an ELISA specific for the Cµ1 domain to detect
targeted recombinants, if the recombinants express the µ gene at
>0.1% the normal level. We have shown that recombinants bearing an
intact 5'-MAR-Eµ-3'-MAR segment express the µ gene at the same
level as the parental hybridoma (24).

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Fig. 1.
Structure of IgH loci and targeting
vectors. A, structure of the endogenous IgH locus of
Sp6 hybridoma cells. This diagram shows position of relevant
restriction sites and elements of interest: MARs, Eµ enhancer, and
Sµ switch region. B, generation of targeted recombinants.
As illustrated here, the targeted recombinants were produced by
recombination between the transfected vector and the endogenous IgH
locus of the mutant hybridoma igm692, which lacks the Cµ1 and Cµ2
exons and a segment of the switch region (22, 32). The construction of
the targeting vectors is described under "Materials and Methods."
The selection of targeted recombinants in MHX medium, screening for
normal IgM production and DNA junctions, and composition of the probes
and primers are described under "Materials and Methods."
C, structure of recombinant IgH loci. The recombinants bear
the indicated parts of the NaeI-SnaI (b) segment
of the JH-Cµ intron. Nucleotide positions are numbered from the first
nucleotide 3' of the indicated NaeI site and define segment
B represented in the targeting vector (see B, above). As
indicated, the Eµ and MAR segments were designed to overlap by 13-17
nucleotides in an effort to ensure that no enhancer-specific site was
interrupted. These short segments are expected to contribute no
significant MAR activity (28, 33).
|
|
As noted above, Forrester et al. (7) reported that high
level µ expression in transgenic mice requires both Eµ and MARs. Consistent with their findings, our analysis of nested deletions that
removed various segments of the JH-Cµ intron from the endogenous locus indicated that µ expression depends strongly on some component in the MAR-Eµ-MAR segment and that the level of µ expression was closely related to the extent of remaining MAR DNA (6). Because of the
importance of relating analyses of transgene and endogenous gene
expression, we have followed their definition of these elements very
closely. Accordingly we have constructed the pEµ, pMM, and p0
targeting vectors to generate the Eµ+MAR
,
Eµ
MAR+, and
Eµ
MAR
recombinants, respectively, such
that these recombinants bear very nearly the same DNA segments that
were used in the transgene expression study. Thus, the
Eµ+MAR+, Eµ+MAR
,
Eµ
MAR+ recombinants bear the complete
5'-MAR-Eµ-3'-MAR segment, the Eµ segment, and the 5'-MAR-3'-MAR
segment, respectively (Fig. 1). We have previously shown that the
deletion of the switch region does not alter the level of µ expression (25).
To generate the recombinants we transfected linearized vector DNA into
the igm692 recipient cells and plated these cells at limiting dilution
in MHX medium (Table I). The initial
MHXR recombinants which we generated with the p0 and pEµ
vectors were identified by PCR amplification of the 5'-junction
fragment (Fig. 1). These recombinants produced detectable levels
(>0.1%) of normal IgM, as measured using a Cµ-specific ELISA,
suggesting that these two screening methods might be used
interchangeably. For this reason, most subsequent recombinants were
identified by first testing culture fluid with the Cµ1-specific ELISA
and then testing positive colonies for proper junctions by PCR. The
recombinants were then subcloned at 0.1 cell/well. To assure that only
a single copy of the vector had inserted into the recombinant loci, we analyzed the µ-gpt DNA by Southern blots of genomic DNA
(results not shown). Multiple independent recombinants that yielded
only the predicted bands were then studied further.
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Table I
Generation of targeted recombinants
Vector DNA (defined in Fig. 1) was linearized and transferred to igm692
cells by electroporation. The cells were then plated at limiting
dilution (103-105 cells/well). Two days after
transfection, MHX medium was added to the wells, and 10 days
thereafter, the wells were scored for growth (MHXR
transformants). These colonies were tested either for the presence of
the correct recombinant 5'-junction or for production of full-length µ chain using a Cµl-specific ELISA. Transfectants satisfying these
criteria were further tested for proper targeting using a PCR assay for
the expected 3'-junction and Southern blotting. From these measurements
we calculated the frequency of properly targeted recombinants among the
cells that survived electroporation.
|
|
In principle, the structure of the intron in the targeted IgH locus
could affect expression of the linked gpt gene. However, an
effect that prevented growth in MHX medium would have been evident as a
marked decrease in the frequency of targeted MHX-resistant transfectants. As shown in Table I, we recovered all recombinants at
frequencies ranging from 2 × 10
7 to 5 × 10
6. We interpret this relatively consistent recovery
frequency to mean that the selected phenotype was typical of the
recombinant structure, i.e. the selected phenotype did not
require a (rare) mutation in addition to recombination.
Effects of Deletions on µ mRNA Production--
To assess the
importance of individual intronic elements in µ expression, we
measured the level of µ mRNA in recombinants of each structure,
as well as the level of actin mRNA, which is expected not to be
affected by the intronic deletions. As a negative control for
specificity we included RNA from the Sp6-derived mutant X10, which has
a deletion of the entire µ gene (denoted
µ) (23). Representative
results for particular recombinants are shown in Fig.
2. For each lane the indicated ratio of µ to actin mRNA levels was calculated and then expressed as the
percent of the value obtained for one of the
Eµ+MAR+ recombinants,
Eµ+MAR+-R1; this percentage is listed below
each lane of the blot.

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Fig. 2.
µ RNA production by recombinant
hybridomas. Cytoplasmic RNA was isolated from the indicated cell
lines and analyzed by Northern blotting with probes corresponding to
the Cµ3-4 exons and actin. The µ deleted cell line, X10, is
included as a negative control. The intensity of the µ- and
actin-specific bands were quantified by PhosphorImager analysis. The
ratio of µ to actin activity was calculated for each cell line. To
compensate for the low background of radioactivity due to nonspecific
binding, the value for a comparable region of the lane for the
µ-deleted cell line ( µ = X10) was subtracted from the value
obtained for the µ band of each recombinant. For the sake of
comparing independent blots the net µ/actin value for each
recombinant was normalized to the value obtained for the
Eµ+MAR+-R1 recombinant. The values
corresponding to this particular blot are listed below each lane. This
same analysis was applied to other independent recombinants as well as
the parental Sp6 hybridoma (Sp6/HL subclone), and the compilation of
these measurements is presented in Table II.
|
|
We have examined at least four independent recombinants of each
deletion type in this same way. Also RNA was prepared and analyzed
multiple times from most individual recombinants (blots not shown).
From these data we calculated the level of µ production compared with
the Eµ+MAR+ recombinant, and Table
II lists the mean (± S.E.) obtained from these measurements of multiple independent recombinants. As reported previously (6, 24), the Eµ+MAR+ recombinants
produced approximately the same level of µ mRNA as the Sp6
parental hybridoma, while the Eµ
MAR
recombinants produced only ~6% of the normal level of µ mRNA. The Eµ
MAR+ recombinant, which bears both
5'- and 3'-MARs produced ~60% of the normal level of µ mRNA,
thus comparable to what we found previously for a recombinant bearing
only the 3'-MAR (6). Interestingly, the
Eµ+MAR
recombinant produced a similarly
high level of µ mRNA (~50% normal). Our previous analysis of
the Eµ
MAR
recombinants by nuclear run-on
assays showed that their decreased µ mRNA content reflects
decreased transcription (6). The relatively high µ mRNA content
of the Eµ+MAR
and
Eµ
MAR+ recombinants therefore indicates
that these elements were each sufficient to maintain transcription of
the µ gene in the context of the recombinant IgH locus.
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Table II
µ mRNA content of recombinant hybridoma cell lines
Recombinants of the indicated structure were generated as described in
the text and Table 1. Each line represents an independently generated
recombinant. One or more RNA preparations were made from each
recombinant and analyzed by Northern blot, as illustrated in Fig. 2.
The ratio of µ to actin mRNA was measured for each preparation of
RNA, and the value obtained for the µ-deleted cell line ( µ = X10) was subtracted. This difference was then normalized to the value
obtained for the Eµ+MAR+ recombinant included in each
analysis. The values for each independent RNA preparation from each
recombinant were then averaged, as presented here with the standard
error in the second column. For each set of independent recombinants of
the same structure, e.g. the six recombinants with the
Eµ MAR structure, the average values were then
combined to obtain the average and standard error shown in the last
column.
|
|
 |
DISCUSSION |
As summarized in the Introduction, high level expression of
IgH-derived transgenes in mice requires both Eµ and MARs,
indicating that these elements each provide different and necessary
functions (7). The repeated observation that these elements are not
needed to maintain endogenous IgH expression in B cell lines has
suggested either that the locus contains other, functionally redundant
elements, or that continued expression of the heavy chain gene in these cell lines does not require enhancers or MARs of any type (12-15). The
finding that the 5'-MAR-Eµ-3'-MAR segment is in fact required for
continued expression of a transgene in a pre-B cell line then argues
that the normal IgH locus has activating elements that are redundant in
B cell lines (3, 4). It might, however, be inappropriate to meld these
experiments, as transgenes in mice and the endogenous IgH of cell lines
have been subjected to ontogenetic effects that might not apply to
transgenes introduced into cell lines.
To investigate the role of the intronic activating elements under more
uniform conditions, we have exploited our finding that insertion of the
gpt cassette into the IgH locus renders expression of the
endogenous µ gene dependent on at least one intronic element in the
5'-MAR-Eµ-3'-MAR segment (6). These conditions have allowed us to
analyze the role of the MARs and Eµ core enhancer in µ expression,
using closely related targeted recombinants that present the µ gene
in a constant chromosomal and cellular context. In contrast to the case
of µ expression from transgenes in mice where both Eµ
and MARs are required, our analysis of µ expression in the
recombinant IgH locus of hybridoma cells indicates that high level µ expression required either Eµ or the MARs.
Thus, for both the Eµ+MAR
and
Eµ
MAR+ targeted recombinant cell lines, µ expression was much higher than the case of the
Eµ
MAR
locus, and each of the single
element loci was roughly comparable with the complete
Eµ+MAR+ locus. These observations lead to an
interesting paradox, as follows. On the one hand, the large difference
in µ expression between the Eµ+MAR
and
Eµ
MAR
recombinants indicates that µ expression required the Eµ function, i.e. that Eµ
function could not be supplied to the
Eµ
MAR
locus by elements elsewhere in the
locus. Similarly, the large difference between the
Eµ
MAR+ and
Eµ
MAR
recombinants indicates that µ expression required the MAR function and that this function could not
be supplied by the locus. Thus, these results indicate that µ expression required both the MAR and Eµ functions, because the locus
could supply neither function. On the other hand, the relatively small
(~2-fold) difference between the Eµ+MAR+,
Eµ+MAR
, and
Eµ
MAR+ recombinants recombinants leads to
the contrary conclusion, namely that µ expression required neither
MAR nor Eµ function, because the locus could supply both functions.
We propose two models to account for this apparent paradox. One
possibility is that maintenance of µ expression does not in fact
require both Eµ and MAR functions, as it should be noted that the
experiments that tested for continued transgene expression in pre-B
cell lines examined the effects of deleting a segment which bore both
elements and did not test deletions of each element separately (3, 4).
That is, the role of Eµ and MARs in initiating expression might be
different from their role in maintaining expression. For example,
initiation might require demethylation of some special site in the IgH
locus, and both Eµ and a MAR might be required to render that site a
substrate for demethylation. Conversely, maintenance might be abrogated
by methylation, and either Eµ or a MAR might be sufficient to prevent
specific methylation.
A second possibility, suggested by the seemingly complementary actions
of Eµ and MARs, is that µ expression always requires both an
enhancer and MARs, but that one element in or near the transcription
unit can collaborate with another (complementary) element elsewhere in
the locus. Thus, either Eµ or a MAR would be sufficient to maintain
expression of the µ gene in the endogenous locus, because by
hypothesis Eµ can collaborate with a MAR, or, alternatively, a MAR in
the JH-Cµ intron can collaborate with an enhancer. The IgH locus is
known to include other enhancers 3' of C
; as well, other MARs lie
between C
and C
3 genes and 5' of the V region promoter (16, 26).
To explain why these other enhancers and MARs do not collaborate to
activate transcription in the Eµ
MAR
recombinants, we suppose that the functional unit must be properly situated, as noted above. Experiments using a MAR derived from the
interferon
gene have also led to the conclusion that the capacity
of a MAR to stimulate transcription depends strongly on its position
relative to the promoter (27), although the "distance," 1.5 kilobases between the µ promoter and the intronic MARs, is in the
range for which the interferon
MAR was inhibitory.
MARs are found both outside and within transcription units, and those
MARs within transcription units are often co-localized with enhancers
(28). It has been proposed that this association exists because the
functioning of these elements requires their collaboration (29). Thus,
MARs might facilitate promoter-enhancer contacts or sequester the
enhancer in the vicinity of transcription factors (30). Our results
indicate that Eµ and the MARs can function, although there are no
(recognizable) complementary elements in their immediate vicinity.
Initiation and maintenance of expression might each require both an
enhancer and MARs but differ in their dependence on linkage,
e.g. initiation might require close linkage between the
enhancer and the MARs, while more separated elements might suffice to
maintain expression. That is, the two models proposed above are not
mutually exclusive.
To judge from the well studied IgH and
-globin loci, transcriptional
activating elements might often be redundant in normal chromosomal
genes. As reviewed in the Introduction, the finding that IgH expression
was maintained at a high level in B-cell lines that had lost the
enhancer needed for transgene expression was an early suggestion that
the locus contained other, normally redundant activating elements.
Similarly, in the case of the
-globin locus, several DNase
hypersensitive sites that are required for high level transgene
expression can be deleted from the endogenous locus without greatly
altering
-globin gene expression (31). The redundant elements, which
permit transcription in the mutant globin and IgH loci, have not been
identified. It might be expected that functionally redundant elements
would resemble each other, and, indeed, the aforementioned 3' enhancers
in the IgH locus are good candidates for the elements that maintain
expression in the enhancerless mutant B-cell lines. However, our
finding that the MARs and Eµ, two elements that differ substantially
in their biochemical properties and activities, are functionally redundant in their capacity to maintain IgH expression underscores the
general possibility that redundant elements might appear dissimilar.
 |
FOOTNOTES |
*
This work was supported by grants from the Medical Research
Council, the Connaught Transformative Program of the University of
Toronto, and the Ciba-Geigy/Novartis Company.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Immunology Dept.,
Medical Science Bldg., University of Toronto, Toronto, Ontario M5S 1A8,
Canada. Tel.: 416-978-6730; Fax: 416-978-1938; E-mail: marc.shulman{at}utoronto.ca.
 |
ABBREVIATIONS |
The abbreviations used are:
Ig, immunoglobulin;
MAR, matrix attachment regions;
PCR, polymerase chain reaction;
ELISA, enzyme-linked immunosorbent assay.
 |
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