From the Instituto de Investigaciones Bioquímicas, Fundación Campomar and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Patricias Argentinas 435, (1405) Buenos Aires, Argentina
Received for publication, January 11, 2001
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ABSTRACT |
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By taking advantage of the extreme stability of a
protein-DNA complex, we have obtained two highly specific monoclonal
antibodies against a predetermined palindromic DNA sequence
corresponding to the binding site of the E2 transcriptional regulator
of the human papillomavirus (HPV-16). The purified univalent antibody fragments bind to a double-stranded DNA oligonucleotide corresponding to the E2 binding site in solution with dissociation constants in the
low and subnanomolar range. This affinity matches that of the natural
DNA binding domain and is severalfold higher than the affinity of a
homologous bovine E2 C-terminal domain (BPV-1) for the same DNA. These
antibodies discriminate effectively among a number of double- and
single-stranded synthetic DNAs with factors ranging from 125- to
20,000-fold the dissociation constant of the specific DNA sequence used
in the immunogenic protein-DNA complex. Moreover, they are capable of
fine specificity tuning, since they both bind less tightly to another
HPV-16 E2 binding site, differing in only 1 base pair in a noncontact
flexible region. Beyond the relevance of obtaining a specific anti-DNA
response, these results provide a first glance at how DNA as an antigen is recognized specifically by an antibody. The accuracy of the spectroscopic method used for the binding analysis suggests that a
detailed mechanistic analysis is attainable.
Unveiling the molecular rules for protein-DNA recognition is a
necessary step for the understanding of gene function and regulation. A
large number of proteins and cognate DNA sequences displaying a large
variety of natural structures and recognition modes have been and are
being identified as involved in physiological and pathological
mechanisms (1, 2). In addition, the ability to design new DNA binding
activities constitutes a major scientific challenge with technological
applications such as control of gene function, gene therapy, genome
research, and diagnostics.
Antibodies that bind to DNA are a hallmark of the autoimmune disease in
systemic lupus erythematosus (3), but these are not specific to
particular sequences of single- or double-stranded DNA, or at least the
putative specific sequences that elicit them have not been yet
identified. Although a number of natural anti-DNA antibodies have been
described, DNA is known to be a poor immunogen (4), and it has been
virtually impossible to generate antibodies against a specific DNA
sequence to date. DNA binding antibodies were obtained using phage
display technology, but these bound to repetitive, nonspecific
sequences (5). A chimeric sequence-specific DNA binding antibody was
engineered by incorporating the DNA binding domain of a transcription
factor into the CDR3 of the heavy chain (HCDR3) from a recombinant Fab
molecule (6). This elegant engineering approach can be further
exploited through antibody display in phage, but it cannot take
advantage of the natural diversity of antibody repertoires. Other
approaches for obtaining novel DNA binding activities arise from the
combination of phage display technology in a zinc finger framework (7);
some of these chimeras bind DNA in the low nanomolar range as judged by
electrophoretic methods (8). A major goal for generating sequence
specific anti-DNA activities is the intracellular expression of the
resulting protein for control of gene function (9).
Can antibodies be raised against specific and predetermined DNA
sequences? If so, how might such antibodies interact with the DNA
compared with natural DNA binders? In an attempt to answer these
questions, we used a natural protein-DNA complex, the DNA binding
domain of the human papillomavirus E2 protein (E2C) bound to its target
DNA (10), as immunogen. This domain shows a particular dimeric
Anti-DNA Monoclonal Antibodies--
The C-terminal DNA binding
domain of the E2 protein from human papillomavirus strain-16 was
expressed in BL21-DE3 Escherichia coli cells as described
previously (13). The sequence of the A chain of the synthetic
oligonucleotide is 5'-GTAACCGAAATCGGTTGA-3', corresponding to E2 site 35 in the HPV-16 genome, where the underlined sequence indicates the consensus E2 binding site. A protein-DNA complex
was formed by mixing E2C and DNA at a 1:1 ratio. Details of the
immunization will be described elsewhere. Briefly, BALB/c mice were
immunized intraperitoneally with 25 µg of protein-DNA 1:1 complex
emulsified in MPL® + TDM Adjuvant System (Sigma). Second and
third boosters were administered intraperitoneally at 20-day intervals
using similar doses. The immune response elicited was monitored by
indirect ELISA using the 18-bp oligonucleotide and the protein-DNA
complexes as antigens, as described below. Final boosting involved an
intraperitoneal injection of 20 µg of protein-DNA 1:1 complex
dissolved in TBS (25 mM Tris, 150 mM NaCl, pH
7.4) 4 days prior to the somatic cell hybridization. Spleenocytes
obtained from the immunized mouse with the highest anti-DNA titer were
fused with a NSO mouse plasmacytoma cell line following
established techniques (14). For the selection of anti-DNA antibodies
we used the site 35 double-stranded oligonucleotide as antigen (1 µg/well in TBS). Of 92 culture supernatants screened after the
fusion, eight displayed anti-site 35 DNA reactivity. Two hybridomas
producing anti-DNA monoclonal antibodies (ED-10 and ED-84) were
expanded and isotyped as IgG1 by ELISA. Ascites fluids were produced
and IgGs and their derived Fab fragments prepared following standard
procedures (15). An additional gel chromatography step was included to
ensure that only univalent and highly purified Fab fragments were
present. The variable regions of both antibodies were cloned (16) and
according to their VH sequences, they belong to the J558
superfamily, shared by several pathogenic autoimmune anti-DNA
antibodies (17) (Fig. 1B).
Electrophoretic Mobility Assays--
A site 35 DNA solution of 3 µM was incubated with the different proteins at different
ratios, and the concentrations were determined from the respective
molar extinction coefficients. A 6% acrylamide native gel was run in
0.1 M MOPS/Imidazol, pH 6.5, at 5 V
cm Quantitative ELISA Binding--
A quantitative ELISA assay for
the determination of the KD was carried out as
described previously (19). Briefly, ELISA plates were coated with
streptavidin (1 µg/well in TBS) for 60 min. After blocking with 1%
BSA in TBS (BSA/TBS), 5 ng/well of a 5'-biotinylated site 35 DNA in
BSA/TBS was added to the plate and further incubated for 15 min. ED-10
or ED-84 IgGs were incubated at 0.8 nM concentration with
different concentrations of site 35 oligonucleotide solution in
BSA/TBS. The 300-µl sample was incubated in microcentrifuge tubes for
30 min at room temperature. The entire mixture was transferred to the
biotinylated DNA-coated ELISA plates for 60 min and subsequently washed
three times with TBS. The retention of a-DNAb IgGs in the solid phase,
representing the degree of binding to the biotinylated specific DNA,
was developed using an anti-IgG peroxidase-conjugated polyclonal
antibody. For each separate tube, the same mixture was incubated in the
absence of biotinylated oligonucleotide as a blank. The resulting
A at 492 nm value was used to calculate the antibody
bound fraction, Fluorescence Binding Experiments--
Fluorescence spectra were
carried out using an Aminco Bowman Series 2 instrument. Excitation
wavelength was fixed at 295 nm and the emission registered from 310 to
410 nm. Buffer base lines were subtracted. Tryptophan fluorescence
titrations were carried out with excitation at 295 nm, monitoring the
emission at 340 nm. The proteins were incubated in TBS in 3.0 ml
volumes at 25 ± 0.1 °C, with protein concentration ranging
from 5 to 100 nM, depending on the sequence used (see Table
I and Fig. 4). DNA oligonucleotides were gradually added, and the
tryptophan fluorescence was measured after 5 min equilibration at each
point. The maximum dilution was 10%, and the fluorescence was
corrected accordingly. The data were fitted to a simple binding model
where both protein and DNA concentrations are considered (20).
Generation of High Affinity Anti-DNA Antibodies--
Using the
HPV16-E2C-site 35 DNA complex as immunogen, we obtained a set of
monoclonal antibodies against the E2C protein as well as antibodies
that reacted against the free or E2C bound DNA but not with the protein
(Fig. 1 and see "Materials and
Methods"). We focused our studies on two selected a-DNAbs,
ED-10 and ED-84, which showed reactivity in ELISA assays toward the
HPV-16 site 35 double-stranded DNA oligonucleotide either free or bound
to the HPV-16 E2C domain (Fig. 1A). These antibodies also
recognize the DNA when bound to the bovine (BPV-1) E2C domain.
We cloned the variable regions of the two a-DNAbs antibodies, and the
amino acid sequence analysis indicates hypermutation in the
VH domains. In addition, the surprisingly identical CDR3s of the VH domain suggest a strong antigen-driven selection
(Fig. 1B). These CDR3s show no sequence homology to any
antibody described in data banks. Contrary to previously reported for
germ line gene J558-derived anti-DNA antibodies, there is not an
evident predominance of positively charged residues in the a-DNAbs as
the main source for binding affinity as discussed elsewhere (21). Both
a-DNAbs have different VL chains, explaining at least in
part the fine differences in specificity.
Analysis of DNA Binding by the Antibodies--
The interaction of
the a-DNAbs with the E2 site 35 oligonucleotide was preliminary
analyzed by an EMSA. No shift, and therefore no detectable binding,
appeared when the EMSA was performed using a radioactively
phosphorylated oligonucleotide (not shown). Thus, we used an unmodified
oligonucleotide and developed the bands using ethidium bromide
staining. Fig. 2A shows the
result of such a binding experiment where the two Fabs, ED-10 and
ED-84, produced a band shift as expected from the initial ELISA assays.
The absence of a band shift using radioactively phosphorylated
oligonucleotide strongly suggest that DNA binding by both antibodies is
sensitive to the phosphorylation at the 5' terminus of the
oligonucleotide (see below). Incremental additions of a-DNAb to the
free DNA produce a gradual band shift to the antibody-bound form as
shown in Fig. 2B. However, no reliable quantitative binding
analysis would be possible (see "Materials and Methods").
The binding was quantitatively analyzed by competition ELISA in
solution (19) and yield dissociation constants of 1.85 ± 0.05 and
1.69 ± 0.18 nM for ED-10 and ED-84, respectively
(Fig. 2C). This is the first strong indication that the
affinity of both antibodies for the E2 site 35 DNA is very high.
Nevertheless, we wanted to test a spectroscopic method that would
confirm the tight binding and provide the highest accuracy in solution.
The tryptophan fluorescence of the purified Fabs is quenched by
30-40% upon binding to the DNA oligonucleotide, depending on the
antibody (Fig. 3A). The DNA
does not quench the spectrum of a control Fab that recognizes the E2C
protein (Fig. 3A). We carried out equilibrium titration
experiments in solution monitored by tryptophan fluorescence. Titration
of site 35 double-stranded DNA with ED-10 and ED-84 Fabs confirms high
affinity-saturable binding and a clear 1:1 stoichiometry, as expected
from univalent Fabs (Fig. 3B).
For an accurate determination of the dissociation constants
(KD) for DNA binding, we carried out tryptophan
fluorescence binding experiments at near-dissociation conditions for
both antibodies. A typical DNA binding experiment for ED-10 is shown in
Fig. 3C. Such analysis yielded dissociation constants of
0.73 ± 0.07 and 2.5 ± 0.4 nM for ED-10 and
ED-84, respectively (Table I). The affinity of the same site 35 DNA oligonucleotide for the E2C domain, the natural partner, is 0.2 nM (18).
Sequence Discrimination--
The untranslated regulatory region of
the HPV16 genome contains three similar, but not identical, high
affinity E2 binding sites. To test whether our anti-site 35 DNA
antibodies were capable of fine specificity tuning as the E2C domain,
we analyzed the binding of the a-DNAbs to another natural E2 binding
site, site 7450 (Fig. 4A).
Both antibodies are able to discriminate HPV-16 E2 site 7450 from site
35 with 2-3-fold lower affinity (Table I).
In fluorescence binding experiments, phosphorylation of site 35-18 bp
produced an increase in KD of 20- and 50-fold for
ED-10 and ED-84 Fabs, respectively (Table I), in agreement with
preliminary EMSA experiments using radioactively phosphorylated site
35. Since the phosphorylation at the 5'-OH affects the binding of the
a-DNAbs to the 18-bp site, we tested a longer double-stranded oligonucleotide containing the E2 site 35. Table I shows that a 26-mer
(site 35-26 bp) still binds with high affinity, albeit lower than the
18-bp site by 1.3 and 0.4 kcal mol
We determined the binding properties of purified Fab fragments
corresponding to both a-DNAbs to a set of different palindromic and
nonpalindromic double-stranded DNA oligonucleotides of similar length
(Fig. 4A). The affinities for most sequences tested were markedly lower than the target sequence, with discrimination factors (KD, nonspecific/KD, site 35) ranging from 125 to 20,000 (Fig. 4B and Table I). ED-84
binds with 3-fold less affinity for specific DNA, but binds to most nonspecific double- and single-stranded DNAs with higher affinity than
ED-10. This suggests that the slightly lower affinity of ED-84 for the
specific DNA could eventually be linked to a lower capacity of sequence
discrimination ranging from 2- to 7-fold depending on the DNA sequence,
evidenced in the ratio of discrimination factors
(DED-10/DED-84, see
legend for Table I).
The discrimination factor for single-stranded site 35 DNA was 20,500 and 3,000 for ED-10 and ED-84, respectively. Binding affinity for two
nonspecific single-stranded DNAs was noticeably higher, but since it is
essentially a nonspecific interaction, we shall await detailed
structural information to fully understand the molecular basis of this
phenomenon. In any case, it should be pointed out that discrimination
between single- and double-stranded DNA in natural antibodies found in
autoimmune disease appears to be at the level of backbone recognition
(22), i.e. conformational, while the type of interaction we
are now describing is sequence-specific.
We have shown that it is possible to generate antibodies against a
desired and predetermined double-stranded DNA sequence. The strong
anti-DNA response generated could be the result of the high stability
of the protein-DNA complex conferring a longer lifetime to the DNA,
possibly enhanced by the high immunogenicity of the E2C protein.
Further analysis of the immune response to the E2C-DNA complex will be
required to fully understand the process.
The fluorescence spectroscopic method developed allowed us to evaluate
the binding affinities with high accuracy and is therefore promising
when aiming at a full characterization of the binding mechanism.
Although the structural basis for this change cannot be unequivocally
assigned, the HCDR3s contain a high proportion of aromatic residues as
potential targets for fluorescence quenching. We have shown that a
classical phosphorylation-based EMSA missed the binding event, as it
was described for some protein-DNA interactions using filter assays
(23). The binding was physically confirmed by ELISA and ethidium
bromide-stained EMSA, which underestimate the binding by at least 1 order of magnitude in our simplified assay conditions (Fig.
2B and see "Materials and Methods").
The monoclonal antibodies obtained display high specificity and
affinity approaching that of the natural DNA binding domain E2C from
the human infecting high risk strain, HPV-16, determined in our
laboratory by spectroscopic methods to be 0.2 nM (18). The
affinity of the highly related bovine (BPV-1) E2C domain for the site
35 DNA oligonucleotide was recently determined in our laboratory in
similar buffer conditions and was shown to be over 300-fold lower than
the specific anti-site 35 DNA antibodies we now describe (18). On the
other hand, the a-DNAbs discriminate the HPV16 E2 binding site, 7450, from site 35, used to raise the antibodies. The two sites are almost
identical, except for a single base change in a flexible
noncontact region. These two results are a clear indication of the high
affinity and sequence discrimination of the specific a-DNAbs we
describe, even for a minimal change in sequence.
The free 5'-OH in the specific oligonucleotide appears to participate
in high affinity binding, but the difference in binding energies of
phosphorylated species is well below the 3-5 kcal mol The fact that the a-DNAbs specifically bind to the E2 site 35 DNA
oligonucleotide does not imply that they recognize the E2 consensus
sequence, i.e. ACCGN4CGGT, in the same manner as
the viral domains. The a-DNAbs could well recognize part of the
conserved consensus sequence and part of the flexible noncontact
region, as the lower binding affinity for a related site suggests.
Alternatively, they may recognize bases outside the consensus sequence
of the synthetic site 35 DNA oligonucleotide. The precise sequence
binding requirements will be mapped using a variety of synthetic
oligonucleotides or hopefully uncovered by detailed structural
analysis. The palindromic nature of this oligonucleotide suggests a
possible duplex-hairpin equilibrium in solution, but more evidence is
needed to ascertain the precise recognition motif.
It is hardly possible that the DNA recognition mechanism by the a-DNAbs
is similar to the natural DNA binding domain. The latter binds the DNA
at the surface, conferring a substantial bent to it (11), while
antibody binding sites frequently display large flat and extended
surfaces or deep cavities (24). Furthermore, given the evident lack of
positive charges at the combining site of the antibodies, an
electrostatic component as high as that found in natural
DNA-binding proteins is also unlikely. Detailed atomic structures and
thermodynamic binding analysis will shed light into this puzzling novel
protein-DNA recognition interface.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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-barrel topology consisting of an eight-stranded (four per subunit)
-barrel, with major DNA binding and minor
-helices, packed
against opposite sides of the barrel (11). Based on the large stability
of the E2C-DNA complex (12), we used it as the antigen to produce a
specific anti-DNA response against this key viral regulatory DNA
binding site. We characterize two newly obtained sequence-specific
anti-DNA antibodies
(a-DNAbs)1 using an accurate
spectroscopic method in solution.
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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1 and the bands visualized by standard
ethidium bromide staining. Overall, the EMSA experiments in our
simplified buffer conditions (0.2 M NaCl, 25 mM
Bis-Tris HCl, pH 7.0, 1 mM dithiothreitol, and 10%
glycerol for loading) appear to be dissociating, and relative affinities cannot be inferred from these type of experiments (18). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA).
. The data were linearized according to Klotz: 1/
versus 1/Ag concentration (19). KD values
were obtained from the slope of the plot.
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Fig. 1.
Characterization of two anti-DNA monoclonal
antibodies. A, ELISA reactivity of ED-10 (open
bars) and ED-84 (filled bars) anti-DNA IgGs
toward the different antigens, where DNA refers to E2 binding site 35 in HPV-16 genome (see above). Serum from a nonimmunized mouse was used
as a negative control (shaded bars). The anti-DNA
IgGs did not react toward a panel of control protein antigens, and none
of the specific free or E2C-bound DNA antigens were recognized by a
control antibody. B, comparison of the amino acid sequences
of the VH and VL CDR regions of the newly
obtained a-DNAbs with their corresponding germ line sequences (17, 25,
26). VH germ line sequences analysis indicates they belong
to the J558 family, used by several pathogenic autoimmune anti-DNA
antibodies (17). They present a total HCDR3 homology and hypermutation,
suggesting a marked antigen-driven selection.
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Fig. 2.
Binding of ED-10 and ED-84 to site 35 DNA. A, electrophoretic mobility shift assay. Site 35 DNA at 3 µM (Free, first lane) was
incubated with 4-fold either ED-10 (second lane) or ED-84
Fabs (third lane) for 30 min at room temperature as
described under `Materials and Methods,` prior to gel loading.
B, same as A. Site 35 DNA at 3 µM
was incubated with increasing amounts of a-DNAbs (0-12
µM). ED-10 was shown as an example. Running conditions
and gel staining are explained under "Materials and Methods."
C, quantitative ELISA binding experiment. The binding of the
a-DNAb IgGs (1.6 nM per site) to the DNA-coated plate was
prevented by incubation with increasing amounts of free site 35 DNA,
and the resulting optical density decrease is a measure of the binding
(inset). The data were transformed and plotted as 1/
against 1/Ag (Klotz), and the KD was obtained from
the slope, after linear regression analysis (see "Materials and
Methods").
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Fig. 3.
DNA binding by fluorescence
spectroscopy. A, fluorescence spectral change upon
binding of DNA. Proteins were incubated at 2 µM
concentration in TBS buffer, pH 7.4, and the spectra were recorded
prior and after addition of 2.5 µM site 35 DNA. The
spectra of ED-84 Fab were shown as an example, no addition (open
squares), plus DNA (closed squares). As a control, we
used an anti-E2C Fab, ED-5: open circles, no addition;
closed circles, plus DNA. B, a stoichiometric
titration was carried out for both a-DNAbs (ED-10 shown in the figure),
at 200 nM protein concentration, monitoring fluorescence
upon addition of site 35 DNA, as described under "Materials and
Methods." C, the dissociation constants,
KD, were determined in titration experiments at
near-dissociation conditions. The figure shows the titration of ED-10
Fab, at 5 nM concentration. The data were fitted as
described under "Materials and Methods," and the residuals are
shown (inset). The binding data of both antibodies to all
the sequences tested are shown in Table I.
Sequence discrimination and affinity of a-DNAbs ED-10 and ED-84
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Fig. 4.
Sequence discrimination by the a-DNAbs.
A, DNA sequences of various oligonucleotides tested for
binding. Site 35, HPV-16 E2 site; site 7450,
HPV-16 E2 site; ARC, the specific operator sequence for the
arc repressor; CRE, cyclic AMP-responsive element;
EBNA, Epstein-Barr nuclear antigen binding site;
VH mouse, a randomly selected mouse VH
sequence; HPV-16 2300, an internal DNA sequence of the
HPV-16 genome. B, logarithmic plot of the binding of ED-84
Fab to site 35 (open circles), single strand site 35 (closed circles), cyclic AMP-responsive element
(CRE) (closed triangles), VH mouse
(open squares).
1,
respectively, for each a-DNAb. The binding appears to be weakened to a
lesser extent when compared with phosphorylated site 35-18 bp. In
addition, we analyzed the binding of the a-DNAbs to a 36-bp site
containing the other natural E2 site (7450), with both 5'-OH ends
modified with fluorescein (FITC). Using tryptophan fluorescence quenching, we determined that the binding affinities are marginally modified with respect to site 7450, marking a difference with the site
35 DNA, which was used as immunogen. FITC fluorescence intensity and
anisotropy of site 7450-36 bp-FITC remain completely unchanged after
addition of a-DNAbs (not shown), suggesting that in longer DNA
stretches, the 5'-OH is not involved in recognition.
DISCUSSION
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ABSTRACT
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RESULTS
DISCUSSION
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1 difference observed for nonspecific
sequences. Moreover, the a-DNAbs bind tightly to the specific sequence
contained within longer DNA oligonucleotides and even to
oligonucleotides with 5'-OH of both strands modified with FITC. Binding
of a site 7450 oligonucleotide of 36 bp is as tight as the 18-bp site
7450, suggesting that modifications in the 5'-OH of shorter
oligonucleotides would interfere with the binding. Without structural
data, at this stage we can only speculate about the origin of the
effect of 5' phosphorylation, which could in part be that it is
recognized by the a-DNAb, but possible effects on the DNA conformation
or stability, particularly in an 18-bp palindrome, cannot be ruled out.
This could also lead to slow conformational changes that might affect
the binding at equilibrium. All the evidence accumulated so far
strongly suggests that the major determinant for binding is still the
DNA base sequence.
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ACKNOWLEDGEMENTS |
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We thank A. Day and R. Giraldo for helpful criticisms.
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FOOTNOTES |
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* This work was supported by the Fundación Bunge y Born and Fundación Antorchas.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.
Holds a fellowship from Consejo Nacional de Investigaciones
Científicas y Tecnológicas.
§ To whom correspondence should be addressed. E-mail: pratgay@ iib.uba.ar.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M100260200
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ABBREVIATIONS |
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The abbreviations used are: a-DNAb(s), anti-DNA antibodie(s); bp, base pair(s); ELISA, enzyme-linked immunosorbent assay; MOPS, morpholinepropanesulfonic acid; EMSA, electrophoretic mobility shift assay; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate.
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