From the Departments of Pediatrics,
§ Pathology, and ¶ Biochemistry and Molecular Biology,
the University of Chicago, Chicago, Illinois 60637
Received for publication, November 1, 2000, and in revised form, February 2, 2001
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ABSTRACT |
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Aggrecan is a large chondroitin sulfate
proteoglycan whose expression is both cell-specific and developmentally
regulated. Cloning and sequencing of the 1.8-kilobase genomic
5'-flanking sequence of the chick aggrecan gene revealed the presence
of potential tissue-specific control elements including a consensus
sequence found in the cartilage-associated silencers, CSIIS1 and
CSIIS2, that were first characterized in the type II collagen promoter sequences, as well as numerous other cis elements.
Transient transfections of chick sternal chondrocytes and fibroblasts
with reporter plasmids bearing progressively deleted portions of the
chick aggrecan promoter and enhancer region demonstrated cell
type-specific promoter activity and identified a 420-base pair region
in the genomic 5-flanking region responsible for negative regulation of
the aggrecan gene. In this report, three complementary methods, DNase I
footprinting assays, transient transfections, and electrophoretic
mobility shift assays (EMSA), provided an integral approach to better
understand the regulation of the aggrecan gene. DNase I footprinting
revealed that six regions of this genomic sequence bind to nuclear
proteins in a tissue-specific manner. Transient transfection of
reporter constructs bearing ablations of these protected sequences
showed that four of the six protected sequences, which contain the
sequence TCCTCC or TCCCCT, had repressor activities in transfected
chick chondrocytes. Cross-competition EMSA using nuclear protein
extracted from chondrocytes or fibroblasts explored the contributions
of the different sequence elements in formation of DNA-protein
complexes specific to cell type. This is the first parallel examination of the EMSA patterns for six functionally defined cis
elements with highly similar sequences, using protein from primary
cultured cells.
Aggrecan is a large chondroitin sulfate proteoglycan found
predominantly in cartilage that is essential for maintaining the integrity of this tissue. Because of its ability to bind to hyaluronate and link protein, aggrecan forms large space-filling aggregates in the
extracellular matrix produced by chondrocytes, providing the resilient
and compressible properties of cartilage (1, 2). Aggrecan consists of a
225-250-kDa core protein with three globular domains and two linear
domains to which about 100 chondroitin sulfate chains
(CS)1 and 25-30 keratan
sulfate chains covalently bind (3). The genomic structure of the chick
aggrecan gene consists of 18 exons; multiple exons encode the three
globular domains, G1, G2, and G3, the latter being composed of
epidermal growth factor, lectin, and complement regulatory
protein-like domains (4, 5).
A mutation in the CS exon of the chick gene that produces a premature
stop codon results in a shortened and nonfunctional protein that leads
to the lethal chondrodystrophy nanomelia, the phenotype of which is
most notably characterized by shortened limbs (4, 6). A mutation in the
mouse aggrecan gene (cmd, cartilage matrix-deficient)
produces shortened limbs in utero, cleft palate, and death
shortly after birth (6-8). Furthermore, aggrecan is important for
maintenance of the cartilage phenotype later in life. Aggrecan
degradation, concomitant with matrix destruction, is the hallmark of
osteoarthritis and rheumatoid arthritis, in which aggrecan catabolism
is elevated compared with normal articular cartilage (9). Therefore, a
precise understanding of the regulation of aggrecan expression is
critical to investigating the mechanisms of normal development and of
diseases that involve abnormalities of the extracellular matrix.
The dynamics of transcriptional control of gene expression are complex
and intriguing, such that it is often the context of transcription
factors and DNA elements that determines the functionality of a
regulatory region. It has been proposed that some transcription factors
may have multiple domains, some specific for repression and others for
activation (10). Protein-protein interactions can influence the basal
DNA-protein interactions observed in a regulatory region. Thus
formation of protein complexes and how they interact with DNA may
influence the transcriptional machinery that enables specific
expression of genes at a precise time and in response to appropriate
stimuli (11). An emerging paradigm is that there are a variety of
mechanisms by which individual cis elements interact with
trans factors. The initial interaction with a cis
element may be a site of nucleation to which a series of other proteins
are recruited, and it may then be this complex, rather than a single
factor, that interacts with the RNA polymerase machinery. In fact, the
DNA may act as a tether to enable localized and distal interactions
between transcription factors.
Recently, we reported (12) the cloning and sequencing of a 1.8-kilobase
genomic fragment containing the 5'-flanking sequence of the chick
aggrecan gene. By using transient transfections of chick sternal
chondrocytes and fibroblasts with reporter plasmids bearing
progressively deleted portions of the chick aggrecan promoter and
enhancer region, we demonstrated tissue-specific promoter activity and
identified a 420-base pair region in the genomic 5'-flanking sequence responsible for negative regulation of the aggrecan gene. Analysis of this nucleotide sequence revealed the presence of potential tissue-specific control elements including a
consensus sequence found in the cartilage-associated silencers, CSIIS1
and CSIIS2, that were first characterized in the type II collagen
promoter sequences, as well as numerous other potential cis
elements (13).
To continue our analysis of the chick aggrecan promoter and enhancer
region, we have conducted DNase I footprinting assays, transient
transfections, and electrophoretic mobility shift assays (EMSA) with
the 420-base pair region previously found to confer repressor activity
and with native and mutated sub-sequences drawn from that region. These
three complementary methodologies provide an integrated approach to
understand better the regulation of the aggrecan gene, allowing us to
determine the nature and extent of some actual protein-DNA interactions
and clarify which cis elements, of the many predicted, were
responsible for our previous findings. By using sequences characterized
in these studies, we later assayed for the binding of previously
defined transcription factors and characterized novel tissue-specific
binding sites that eventually led to the identification of a novel
factor that binds to the aggrecan promoter and enhancer
region.2
Materials--
Oligonucleotides were made with an
Applied Biosystems 3808 DNA Synthesizer. Reagents for biochemical and
molecular cloning experiments were of the highest quality available
from commercial vendors. Restriction endonucleases were from New
England Biolabs unless otherwise stated. T4 DNA ligase, T4 kinase,
DNase I, and Klenow polymerase were from Promega. Taq
polymerase was from PerkinElmer Life Sciences.
Mutagenesis--
The Altered Sites II kit from Promega was
employed for oligonucleotide-mediated mutagenesis strategies, following
the manufacturer's protocol. The Ag-1(+) genomic clone was used as a
template sequence for the oligonucleotide-mediated alterations of
cis elements (12). Briefly, the Ag-1(+) clone was denatured
and annealed with a mutant oligonucleotide, an ampicillin-repair
oligonucleotide, and a tetracycline-knockout oligonucleotide.
Subsequent synthesis, ligation, and selection for ampicillin resistance
were used to identify mutant constructs. Positive mutants were
sequenced to ensure that the correct mutation was made. The
oligonucleotides used to make the mutant constructs are displayed in
Table I.
Cell Cultures--
Cultures of day-14 chick sternal chondrocytes
were established according to the procedures described by Cahn et
al. (14) and as modified by Campbell and Schwartz (15). Cultures
of fibroblasts were established from skin of day-14 chick embryos
following trypsinization (15). Cells were plated at an initial density
of 1.5 × 106/100-mm tissue culture dish (Falcon) in
either F-12 medium (chondrocytes) or Dulbecco's modified Eagle's
medium (fibroblasts) and supplemented with 10% fetal calf serum and 10 µg/ml ascorbic acid (chondrocytes). The cells were permitted to
attach to the dishes, and subsequent growth (2-3 days) was maintained
by a complete change of the medium every 2 days (16). On the day of
transfection chondrocyte cultures were trypsinized, and single cells
were suspended in F-12 medium, replated, and allowed to attach to the
dishes for 3-4 h before treatment as described below.
Purification of Nuclear Proteins--
Standard protocols were
used to purify nuclear proteins from either chick chondrocytes or
fibroblasts. Briefly, cells from confluent monolayer or suspension
cultures were scraped and collected into 50-ml conical tubes (Falcon).
The cells were pelleted for 10 min in a JS-4.2 rotor at 3000 rpm,
resuspended in five times the packed cell volume of phosphate-buffered
saline (usually 15 ml), and then pelleted for another 5 min at 3000 rpm. The phosphate-buffered saline was poured off, and the pellets were
resuspended in a hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.2 mM PMSF, 0.5 mM DTT) and allowed to swell for
10 min. The cells were lysed by Dounce homogenization, and the lysate
was centrifuged at 3300 × g for 15 min. The
cytoplasmic supernatant was discarded, and the nuclear pellet was
suspended in a low salt buffer (20 mM HEPES, pH
7.9, 0.02 M KCl, 1.5 mM MgCl2, 25%
glycerol, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT); nuclear proteins were then extracted in high salt
buffer (20 mM HEPES, pH 7.9, 1.2 M KCl, 1.5 mM MgCl2, 25% glycerol, 0.2 mM
EDTA, 0.2 mM PMSF, 0.5 mM DTT) for 30 min. The
solution was centrifuged at 25,000 × g, and the
supernatant was dialyzed at 4 °C against dialysis buffer (20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl,
0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT). The nuclear extracts were used immediately for DNase I
footprinting experiments, gel shift assays, or further purification procedures.
Transfection--
Standard methods were followed for calcium
phosphate transient transfections (17). Duplicate plates containing
~5 × 106 cells (either chondrocytes or fibroblasts)
received 20 pmol of a given plasmid construct to be assayed, and
incubation continued for 36 h. Five µg of a Cell Recovery and Assays--
Reagents for the luciferase and
Preparation of DNA Probes for DNase I Footprinting--
Probes
used for DNase I footprinting experiments were generated via PCR using
the previously described genomic clone Ag-1(+) as template DNA, Fig. 1
(12). For probes 1 and 2, XhoI sites were introduced at the
5'-end of the amplified fragment, whereas BglII sites were
introduced via the primers at the 3'-end. The defined 5'-3'
orientation of the cloned PCR fragments corresponds to the native
5'-3' orientation of the Ag-1(+) clone sequence relative to the
aggrecan gene. PCR fragments were purified using Qiaquick PCR Preps
(Qiagen). Samples of purified and unlabeled PCR fragments were
electrophoresed on agarose gels to determine sizes, and sequencing of
the PCR products was done to exclude PCR artifacts in the probes.
Approximately 0.3 µg of a given probe was digested overnight at
37 °C with the restriction enzyme BglII and then treated
with 0.5 units of alkaline phosphatase. The digested PCR products were
again purified using a Qiaquick Nucleotide Removal Kit (Qiagen). The
purified double-stranded end-digested probes were end-labeled using
[ DNase I Footprinting--
For each footprinting experiment
~0.015 µg of end-labeled probe was added to 25 µl of binding
buffer A (10 mM Tris, pH 8, 5 mM
MgCl2, 1 mM CaCl2, 2 mM
DTT, 50 µg/ml bovine serum albumin, 2 µg/ml calf thymus DNA, 100 mM KCl) with the addition of 25 µg of nuclear protein
from either chick chondrocytes or fibroblasts in 25 µl of dialysis
buffer. Control samples that had no protein added included only 25 µl
of dialysis buffer. Binding mixtures were kept at room temperature for
90 min. Prior to digestion with DNase I, 50 µl of 5 mM
CaCl2, 10 mM MgCl2 was added, and
the mixture was incubated for 60 s. Control lanes that contained
no nuclear protein were digested with 0.06 units of DNase I, and lanes
with nuclear protein were digested with 0.12 units of DNase I for
90 s at room temperature. The reaction was stopped with 90 µl of 200 mM NaCl, 30 mM EDTA, 1% SDS, and 250 µg/ml yeast RNA. Reactions were extracted with equal volumes of
phenol/chloroform and precipitated with 2 volumes of 100% EtOH.
Precipitated DNA was washed once with 70% EtOH, and the pellets were
resuspended in gel loading buffer. The products were electrophoresed on
6% polyacrylamide sequencing gels. Dideoxynucleotide termination
sequencing reactions were done simultaneously for each DNA fragment and
run next to the control footprinting reactions to match the observed
footprints with the published sequence.
EMSA--
100 pmol of each single-stranded oligonucleotide
complementary pair was incubated in annealing buffer (100 mM Tris, pH 7.5, 500 mM NaCl, and 250 mM MgCl2) at 90 °C for 5 min and then
allowed to cool slowly for ~1 h. Standard methods were employed to
end label the resulting double-stranded oligonucleotides. Briefly, 10 pmol of probe was incubated for 30 min at 37 °C in kinase buffer (Promega) containing 20 units of T4 kinase, and
[ Sequence Search for Transcription Factor Binding Sites--
The
Wisconsin Package (version 9, Genetics Computer Group, Madison, WI)
program FINDPATTERNS was used to scan the protected footprint sequences
for cis elements listed in the Transcription Factor Database.
DNase I Footprinting with Probes Spanning the Aggrecan Repressor
Region--
Fig. 1 shows a
representation of a portion of the 1.8-kb aggrecan gene genomic
5'-flanking sequence. The 420-bp region from
Probe 1 yielded three protected segments with chondrocyte extracts,
designated footprints G-I, which spanned the sequences
Two major segments of probe 2 were protected by fibroblast extracts,
spanning the regions
Comparison of the protected sequences with the Transcription Factor
Database search results determined that a number of previously defined
cis element consensus sequences were contained within the
observed footprint regions (Fig. 2C). Footprint G included MalT_CS, H2A, P5, Aldosterone_CAP_box, D4(rev), and D1 sequences. Footprint H had an SP1_CS4 sequence in the reverse orientation. Footprint I contained a Zeste sequence, also in the reverse
orientation. The large footprint J is composed of multiple sites, most
importantly a CIIS2- (CACCTCC) containing sequence in addition to a
MalT_malPp (TCCTCC) and a CK-8-mer sequence. Finally, footprint
K contained a second MalT-malPp sequence.
Sequence comparison among the protected sequences revealed several
common motifs largely composed of various combinations of the bases
thymine and cytosine. Both footprints J and K contain the sequence
CTCCTCC (which includes the above-mentioned MalT-malPp site), and
footprint J had two repeats of the sequence TCCCC, which occurs once in
the footprint L sequence. Furthermore, J contained a CTTCAC sequence,
whereas L contained the very similar sequence, CTTCAG, and both
contained a CACCTCC sequence.
Functional Analysis via Mutagenesis of the Aggrecan Promoter
Repressor Region--
The extent to which these protected sequences
contribute to the previously reported repressor activity of this
promoter segment was addressed by introducing mutations into the native
1.8-kb Ag-1(+) sequence, which preserved the nucleotide spacing but
altered the bases putatively involved in nuclear protein binding.
Alternating series of either As and Ts (poly(dA)·poly(dT)) or
Cs and Gs (poly(dC)·poly(dG)) were substituted for the normal
footprint sequences (Table I). Sternal
chondrocytes and fibroblasts were transfected with these mutant
sequences in reporter plasmid constructs; initial experiments suggested
that the simple introduction of either a series of As and Ts or a
series of Cs and Gs did not alter activity in itself nor did it matter
which substitution sequence was used (data not shown). These
experiments included use of either poly(A-T) or poly(G-C) at a given
protected sequence mutation site with highly similar results. Use of
these sequence blocs to mutate other locations in Ag-1(+) produced
either the opposite or no effect on reporter expression. Finally, in
the results described below, it was found that a given mutational
sequence produced different effects depending on which protected
sequence was mutated. In sum, effects of mutations correlated with
their location and not with the type of sequence bloc used.
Since the single, large protected sequence observed for footprint J
with fibroblast extract appeared as two discrete regions when protected
by chick chondrocyte protein, two mutations were made in the J
sequence, mJ.2 that altered the nucleotides
Transient transfections of chondrocytes or fibroblasts with the
construct Ag-1(+) (the forward orientation of the 1.8-kb insert in the
promoter/enhancer-free pGL-2-Basic reporter vector) were compared with
Ag-1(+) constructs bearing the various footprint mutations. Mutations
of the sequence described as footprint G had little effect on reporter
expression in transfected chondrocytes when compared with the normal
Ag-1(+) construct (Fig. 3A);
however, construct mG produced a 30% increase in luciferase activity
compared with that for Ag-1(+) in transfected chick fibroblasts (Fig.
3B). Similarly, mutation of protected sequence H, which had
an Sp1_CS4 site in the reverse orientation, had little effect in
transfected chondrocytes, whereas reporter activity in transfected
fibroblasts dropped nearly 25% when construct mH was compared with the
normal Ag-1(+) construct. Disruption of the protected sequence I, which contains a Zeste-like sequence, caused a significant increase in
reporter activity in both chondrocytes and fibroblasts, to nearly 400 and 175%, respectively. Alteration of the large protected sequence J
caused substantial increases in luciferase activity in both transfected
chondrocytes and fibroblasts. The construct mJ.2, which mutated the
overlapping CIIS2 and MalT-maLPp sites, exhibited the highest
increases in relative reporter activity, i.e. transfected
chondrocytes showed an increase in luciferase activity of nearly 600%,
and luciferase activity in transfected fibroblasts peaked at 230%.
Removal of the CK-8-mer site, in construct mJ.1, increased relative
reporter activity by nearly 400% in transfected chondrocytes but only
35% in transfected fibroblasts. Interestingly, both constructs mK and
mL, which ablated separate MalT-malPp and CIIS2 sites, respectively,
produced large reporter activity increases in both cell types, but
neither of these equaled the activity increases for the mutant
construct mJ.2 which eliminated overlapping sequences of each
cis element. Construct mL exhibited greater activity than mK
in transfected fibroblasts; this reporter activity was similar to that
observed for the mutant construct mJ.2 in fibroblasts. Whereas mL
exhibited less activity than mK in transfected chondrocytes, in both
cell types alteration of the sequence CACCTCC (mL) caused a significant
increase in luciferase activity, suggesting the presence of repressor,
silencer, or modulation complexes acting at that site alone.
Examination of DNA-Protein Interactions by EMSA--
To explore
further the nature and extent of the DNA-protein interactions at the
observed protected sequences in chick chondrocytes, double-stranded
probes corresponding to these sequences were made for use in EMSA
experiments. Table II shows the
sequences, names, and sizes of the eight oligonucleotide pairs used in
combination with their respective complements to form double-stranded
32P-labeled probes that correspond to the repressor region
footprint sequences. Fig. 4A
shows the results of incubating five of those probes with nuclear
protein extracts derived from day-14 sternal chondrocytes seeking to
determine if there are any similarities in the DNA-protein complexes
formed. The 48-base pair probe J, which contains CIIS2,
MalT_malPp, CTTCAC, and two TTCCCC sequences, yielded three
specific DNA-protein complexes, labeled B-D, although production of
complex B appeared variable for the full-length probe J in repeated
experiments. The 30-base pair probe J.1 produced the same DNA-protein
band patterns as the full-length probe J, although lacking the CIIS2
and MalT_malPp sequences. It did, however, contain the sequences
CTTCAC and TTCCCC, in addition to a CK-8 site. Probe J.2 contains three
of the shared sequences, CIIS2, TTCCCC, and a CTCCTCC, yet it only
forms two complexes, A and C, when incubated with chick sternal
chondrocyte extract. EMSA with probe K, which contains a CTCCTCC
sequence, produced only the prominent band A, which co-migrates with
the slower migrating complex observed with probe J.2. The only
similarity between probes K and probe J.2 is the presence of a CTCCTCC
sequence, implying band A can be attributed to nuclear proteins binding
to that sequence. Probe L produces two bands, B and C. Sequence
comparison between L and J.1, which produces those same EMSA bands,
reveals the presence of a CTTCAG and a TCCCC sequence in both. Band C
is also present in reactions incubated with probe J.2, which contains a
TCCCC but not a CTTCAG sequence, suggesting that the TCCCC motif
produces the DNA-protein complex C. Surprisingly, probes J.2 and L,
which both contain the CIIS2 site, do not generate a unique band that can be definitively associated with this sequence alone. Indeed they
both produce band C but that is also present for probe J.1, which does
not contain the CIIS2 site. In sum, both probe K and J.2 contain the
sequence CTCCTCC and produce band A. Probes J, J.1, J.2, and L produce
band C, and all contain the sequence TCCCC. Probes J, J.1, and L share
the sequence CTTCA(G/C), and each produces the band B. The D complex
was only seen for probes J and J.1 suggesting that it is unique to this
larger sequence that is not included in the other probes.
Probes that corresponded to the protected sequences G-I were also
synthesized; Fig. 4B represents the EMSA patterns that are observed when these probes bind with day-14 chick sternal chondrocyte nuclear proteins. Incubation with probe G produced three specific complexes (labeled 1-3) that do not align with any other observed bands, suggesting that these DNA-protein complexes are unique. Probe H
produced one band that migrated between the complexes 1 and 2 observed
for probe G. Probe I produced one specific band that similarly migrated
between the complexes 1 and 2 observed with probe G. None of the 5 complexes from probes G-I aligned with bands B-D observed for probe
J, nor did they co-migrate with band A produced by probes J.2 and K
(data not shown). Sites H and I contain only one predicted
cis element each, a Sp1(rev) site and a Zeste (rev) site,
respectively. Thus, it is not surprising that each produces only one
specific complex. Footprint G contained 6 potential cis
elements yet generated only 3 specific complexes.
Because all of the EMSA experiments described above were done with one
concentration of nuclear protein, and protein-DNA interactions can be
affected by relative concentrations of both the DNA and protein,
varying amounts of nuclear protein were added to each of the probes to
determine if any other complex formation could occur. Indeed, nuclear
extracts may contain a limiting amount of cofactors needed to allow
proper protein-protein interactions to form complexes that then jointly
bind to the DNA elements. The footprint sequences, H, I, J, J.1, J.2,
and K, did not exhibit significant differences in band formation upon
addition of excess nuclear protein, except for the expected increase of
band intensities (data not shown). However, addition of varying amounts
of nuclear protein to probes G and L had significant effects on complex
formation by these two probes.
For probe G, 3 bands were observed that increased in intensity in a
dose-dependent manner and that could be competed out with excess unlabeled double-stranded probe G (Fig.
5A). Bands 1 and 3 appear with
chondrocyte extract at protein concentrations of 1 µg per reaction.
Band 2 did not appear distinctly until higher concentrations of nuclear
protein were added. Band 3 was of lower intensity (or more diffuse)
than band 1 at low concentrations, but the converse was observed at
higher concentrations of proteins (compare lanes 1 and
3). Band 3 was the most difficult to compete out with excess
unlabeled probe (persisting with a 100-fold excess), whereas bands 1 and 2 competed out at a 25-fold excess. A faster migrating band was
present in all of the samples and was not dependent on protein
concentration or amount of unlabeled probe, suggesting that it
represents nonspecific binding. A protein dose-dependent change in complex formation was also seen for probe L (Fig.
5B). With increasing concentrations of chick chondrocyte
nuclear protein, a complex appeared that migrated slower than band B
(labeled with an asterisk). Concomitant with the appearance
of this upper band, there is a visible decrease in the amounts of
complexes B and C. The new band was distinct from band A, seen in the
probe K lane.
Different cis Elements with Similar Sequences May Interact with the
Same Trans Factor(s) to Cause Repression--
Additional
competition-EMSA studies were carried out with chondrocyte extracts and
radiolabeled probes J.1, J.2, K, and L (20 fmol), using as unlabeled
competitor the same probes at either 10- or 50-fold (200 fmol or 1 pmol) molar excess. Radiolabeled probe J.1 (Fig.
6A) incubated with 5 µg of
chondrocyte nuclear protein yielded three complexes. As expected, the
formation of complexes B-D was competed against by unlabeled probe J.1
at 50-fold molar excess. Complex B could not be competed for by
unlabeled probes J.2 and K at 10-fold molar excess. Surprisingly, with
50-fold molar excess of unlabeled probes J.2 or K, which contain the
MalT-malPp site, complex B increased in intensity concomitant with a
reduction of complex C. Probe L was the only oligonucleotide sequence
that competed for complex B and C formation at both 10- and 50-fold. No
competition was observed with a 50-fold molar excess of the TA
oligonucleotide.
The competition EMSA results suggest that probe L has a greater
affinity for complexes B and C than any of the other probes, as it
competed for these complexes more strongly than the native J.1
sequence. Probe J.2 competed only for complex C; the sole similarity it
has with probe J.1 is the presence of a TCCCC sequence. This finding
supports the previous EMSA outcome that the TCCCC sequence is required
to form the DNA-protein interactions resulting in band C. Since band B
increased in intensity with the addition of probes J.2 and K, whereas
band C weakened in intensity, it may be that, when in excess, these
probes disrupted the dynamics of the normal J.1 DNA-protein
interactions. Both probes J.2 and K contain the sequence TCCTCC, which
bears similarity to TCCCC. Perhaps TCCTCC has a stronger affinity for
some component of complex C than does the TCCCC sequence, hence the
loss of band C with the addition of probes J.2 and K.
Probe J.2 (Fig. 6B) yielded two bands, complexes A and C. As
expected, complex C formation was competed for by the addition of 10- and 50-fold excess probe J.1, but complex A was more difficult to
compete out at either concentration than complex C. Addition of J.2 at
10-fold molar excess diminished band C, whereas 50-fold excess seemed
to somewhat diminish the intensity of A and further reduce band C. Probe K at 10-fold molar excess slightly competed for band C, and a
higher molar excess (50-fold) nearly ablated it. Complex A, however,
was only slightly reduced by the addition of 50-fold unlabeled probe K. Thus in two separate instances, for labeled probes J.1 and J.2,
addition of K competitor reduces the intensity of complex C permitting
formation of the slower migrating complexes A (probe J.2, Fig.
6B) or B (probe J.1, Fig. 6A). These results
further support the notion that the DNA sequence TCCTCC competes for a
complex C component.
Excess unlabeled probe L competed with J.2 for formation of both
DNA-protein complexes A and C, which suggests that complex A contains a
protein that can bind to the TCCCC sequence or another consensus
sequence embedded in probe L. However, probe L does not form complex A
by itself so probe L may not be acting directly on complex A, rather it
may compete away a protein common to both complexes A and C making the
formation of complex A impossible. Addition of the TA probe did not
change the intensity of the complexes when compared with control
lane C, Fig. 6B.
Probe K incubated with 10 µg of nuclear protein (Fig. 6C)
yielded one major complex, A. Unlabeled J.1, J.2, or L probes at 50-fold molar excess competed for the formation of this complex. Again,
probe L competed for this complex more strongly than the other probes,
exhibiting competition at 10-fold molar excess. In fact, probe L
competed for band A at both excess levels more strongly than did the
unlabeled probe K. The TA probe did not compete with probe K formation
of band A.
Radiolabeled probe L (Fig. 6D) produced two complexes, bands
B and C. Even at high molar excess, probes J.1, J.2, and K could not
compete for either complex formed by probe L. The bands were competed
for by unlabeled native sequence L at 10- and 50-fold excess.
Interestingly, the upper band B was competed before the lower band C by
a 10-fold excess of unlabeled competitor and continued to be reduced
with a 50-fold excess. Again, the TA probe had no competitive effect;
thus it is unlikely that any of the observed competition resulted
simply from nonspecific interactions with the excess competitor probes.
Nuclear protein from primary cultures of day-14 chick fibroblasts, a
cell type of mesenchymal origin that does not express aggrecan,
produced some differences in DNA-protein interactions when compared
with chick chondrocyte nuclear extracts but, perhaps more
significantly, some similarities (Fig.
7). Band C, which is associated with the
sequence (T)TCCCC(T), was also present for chick fibroblast extracts
only with probe L; the appearance of complex C was not observed with
fibroblast extract and probes J.1, J.2, or J, even though incubation of
chick chondrocyte nuclear protein results in the formation of complex C
for all of these probes. Thus the sequence (T)TCCCC(T) in probes J.1,
J.2, and J is not sufficient to form complex C with chick fibroblast
extract, but it is capable of forming a similar band in the context of probe L. Another similarity is that the band labeled D appears to be
identical for probes J and J.1 with both fibroblast and chondrocyte
extracts. Interestingly, the faster migrating complexes observed with
probes J, J.1, and L are seemingly identical in both chondrocytes and
fibroblasts, suggesting that these bands may represent a primary
DNA-protein interaction, perhaps with the same transcription factor(s).
The higher mobility bands seen for each of the probes exhibit
differences that are unique to each cell type, suggesting that other
proteins, specific to each type, are also binding to these
cis elements or to the aforementioned primary complexes,
causing slower migrating DNA-protein(s) complexes. The fundamental
basis of aggrecan promoter DNA-protein interactions could be identical
in these two mesenchyme-derived cell types, but the multiple
protein-protein interactions appear unique to each.
DNA-Nuclear Protein Interactions in the Aggrecan Promoter--
The
approaches used to survey trans factor-cis
element interactions in the
Alternatively, perhaps the complexes seen with chick fibroblast
extracts are more tightly bound, thus preventing aggrecan expression.
These complexes could individually inhibit transcription, or the four
binding sites could act synergistically to inhibit more strongly
aggrecan expression. In chick chondrocytes this region, as shown in
earlier work (12), can act as a modulator of high expression. If the
cells are exposed to injury or are in the developmental context where
greater amounts of aggrecan need to be expressed rapidly, removal of
the trans complexes from these conserved cis
sites could allow rapid up-regulation of the gene. It is tempting to
speculate that such removal could be at least a two-stage process
because both probes J.2 and K produce complex A, whereas probes L and
J.1 produce complex B. Removal of a functional site by repression,
squelching, or quenching (10) could increase aggrecan expression. Thus,
it is conceivable that an individual cis element could
interact with both negative and positive transcription factors.
Footprints J.1, J.2, and L exclusively contain the sequence TCCCC and
are the only probes to produce band C in EMSAs using chondrocyte
protein, but when fibroblast extract is used only probe L forms band C. If TCCCC is in fact the cis element directing complex C
formation, as seems likely, then it is clear that the sequences
adjoining a cis element can influence its behavior in different cell types. Similarly, probe L out-competed J.1 in formation of bands B and C, J.2 for bands A and C, and K for band A. Probe L
shares various sequence elements with the other three, so the difference in affinity for particular complex components likely stems
from the exact arrangement of elements. Also, since L can compete
against band A formation even though it does not form that complex
itself, it is possible that bands A and B, although never observed
together, share some protein components.
Examination of the Defined Repressor cis Elements in the Context of
Other Genes and Exogenous Stimuli--
Footprint J contained the
sequence (CACCTCC), a motif present in the 80- and 100-base pair
silencers CIIS1 and CIIS2 defined in the rat type II collagen promoter
(13). Studies on the collagen
Footprint K contained the similar MalT-malPp consensus sequence
(TCCTCC), a site important in the positive regulation of the malT gene (18). The malT gene is regulated by
Mlc, which has homology to the protein NagC, a gene regulator
functioning as a repressor of enzymes that control the uptake and
release of N-acetylglucosamine (19). Regulation of the
malT gene can be modulated by the amount of internal free
glucose or glucose derived from disaccharides (20-22). It is tempting
to speculate that these aggrecan repressor sites are in some way linked
to the availability of sugar precursors since aggrecan is extensively
glycosylated. Furthermore, the TCCTCC motif has also been found in the
human COL1A2 promoter (23) and the decay-accelerating factor gene (DAF) (24). When the TCCTCC site was mutated in the COL1A2
promoter, there was a significant reduction of basal promoter activity
in transfection experiments using human fibroblasts (23); deletion of a
region containing this motif in the DAF gene resulted in complete loss of promoter activity as determined by chloramphenicol acetyltransferase assays in transfected COS cells (24). In these promoters then, the TCCTCC motif appears to bind an activator of
transcription. Interestingly, when the same group put the
TCC-containing motif in the opposite direction to drive the expression
of a thymidine kinase promoter system, basal promoter activity
was significantly increased, suggesting that in a different context and
system this motif has the capacity to act as a repressor. The authors
(23) suggest that these phenomena could be due to conformational
changes induced by the trans factors that bind to this site,
indicating that this sequence has a dynamic role that probably involves
precise positioning relative to adjacent sequences.
The human COL1A2 promoter also has a TCCCCC motif, mutation of which
increased promoter activity by nearly 6-fold (23). Interestingly, the
TCCCCC motif was located at
In sum, we have dissected a functionally significant 420-bp region of
the aggrecan promoter that was previously shown to repress chick
aggrecan gene expression (12). We have identified cis elements mediating the observed repressor activity and have
characterized the propensity of these sequences to form cell
type-specific DNA-protein interactions with nuclear proteins derived
from day-14 chick chondrocytes or fibroblasts. DNase I footprinting
protected several forward-strand sequences primarily composed of
combinations of the bases thymine and cytosine; three of these were
found by EMSA to participate in specific DNA-protein interactions as
follows: the sequence CTCCTCC produced band A, CTTCA(G/C) produced band
B, and TCCCC produced band C in chondrocytes. Band C was observed with
chick fibroblast nuclear proteins only in the context of probe L, which was shown to have the strongest affinity for nuclear proteins in
cross-competition EMSA experiments; clearly the location of a TCCCC
sequence greatly influences its binding behavior. Three of the four
demonstrated repressor areas have a CCTCC motif, alluded to in other
reports in a variety of capacities as a silencer or even an enhancer,
and thus important in binding to transcription factors in a number of
genes. To our knowledge this is the first parallel examination of the
EMSA patterns for six functionally defined cis elements with
highly similar sequences, using protein from normal primary cells. By
purifying and characterizing transcription factors that bind to these
sequences, we are now able to investigate how these elements function
to regulate aggrecan expression during development and in response to
either chemical stimuli by cytokines or physical stresses on cartilage
tissues, both in normal and disease states.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase
reporter plasmid were co-transfected with each experimental construct
to correct for cell loss. Duplicate transfection sets were prepared
three times, each time yielding similar results.
-galactosidase assays were purchased from Promega. Since both
luciferase assays and
-galactosidase assays were performed, Reporter
Lysis Buffer from Promega was used (RBL, E3971) to prevent the
inhibition of
-galactosidase activity that occurs in buffers
containing detergents such as Triton X-100. No deviations were made
from the manufacturer's protocols for preparation of extracts from
tissue culture cells and the enzyme activity assays. Three sample
aliquots from each of the duplicate transfection plates were assayed.
The enzymatic activity of luciferase was measured with a luminometer
(Analytical Luminescence Laboratory, Monolight 1500). The enzymatic
activity for
-galactosidase was measured with a microplate reader
(Dynatech) at 409 nm. Average luminescence values and standard
deviations were determined for the set of six assays performed within
each experimental group.
32P]ATP and T4 DNA kinase according to standard
protocols. The previously undigested end of each probe was digested for
2 h at 37 °C using the appropriate restriction enzyme
(XhoI) to ensure that only one end of the probe contained a
radiolabeled phosphate. The probes were then extracted twice with
phenol/chloroform, precipitated using standard protocols (12), and then
stored at 4 °C in TE8 buffer (10 mM Tris, 1 mM EDTA, pH 8).
32P]ATP (PerkinElmer Life Sciences). To stop the
labeling reaction, 2 µl of 0.5 M EDTA was added to the
reaction, and the labeled probes were purified with a Nucleotide
Removal Kit (Qiagen) to remove the excess unincorporated
[
32P]ATP. For each binding reaction, 20 fmol of the
probe were incubated with varying amounts of nuclear protein (between
0.1 and 10 µg) for 30 min at 4 °C in a 15-µl reaction mixture
containing 1.5 µl of binding buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 5 mM DTT, 375 mM KCl), 2 µg of dI-dC (Amersham Pharmacia Biotech), 3.75 µl of 20% Ficoll,
and water. For competition experiments, excess unlabeled
double-stranded probes were added to the binding reaction (amounts
ranging from 0.2 to 1.0 pmol, or as defined in the figure legend). The DNA-protein complexes were electrophoresed on 5% polyacrylamide gels in TBE buffer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1200 to
780 relative
to the most 5' transcription start site was previously shown to play a
negative regulatory role (12). This sequence was searched against the
Transcription Factor Database using the program FINDPATTERNS, and some
of the resulting predicted cis elements embedded in that
sequence are represented in the diagram. DNase I footprinting
experiments were conducted using two PCR-generated, single-end
32P-labeled probes (Fig. 1), which span 430 bp of sequence
and overlap with each other by 18 bp. Each of these probes was
incubated with nuclear proteins extracted from primary cultures of
either day-14 chick sternal chondrocytes or day-14 chick fibroblasts.
Fig. 2, A and B,
shows the results of these experiments using probe 1 and probe 2, respectively.
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Fig. 1.
Schematic representation of the 1.8-kb chick
aggrecan promoter and enhancer region and DNase I footprinting
strategy. The 1.8-kb genomic fragment Ag-1 is shown in relation to
a 420-bp promoter sequence involved in the negative regulation of the
chick aggrecan gene. Within the box are predicted
cis motifs in order as defined using the program
FINDPATTERNS or from published papers. The bars below the
box represent two of the DNase I footprinting probes used
with indication of which end of the probe contained the radiolabeled
phosphate. The names and positions of the resulting protected sequences
(Fig. 2) are indicated below each probe. Note the diagram is
not drawn to scale. An additional protected sequence, L ( 730 to
703), was identified using a probe taken from sequence adjacent to
probe 2.
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Fig. 2.
Footprinting analysis, sequences, and
embedded cis elements. Probes 1 and 2 were used
for footprinting analysis in A and B,
respectively. Lanes 1 and 3 contained DNA not
incubated with nuclear protein. Lanes 2 and 4 contained DNA protected by nuclear protein extracted from either day-14
chick chondrocytes or fibroblasts (lanes 1 and 2 and lanes 3 and 4, respectively). DNase I
digestion was performed on all as described under "Experimental
Procedures," and the products were electrophoresed on 6%
polyacrylamide sequencing gels. C lists the nucleotide
sequences protected by day-14 chick sternal chondrocyte or fibroblast
extracts. The 1st column shows the protected sequence; the
2nd column lists the designated footprint name; and the
3rd column lists the consensus sequences for potential
transcription factor binding sites that were identified using the
program FINDPATTERNS or from published reports. The L protected
sequence resulting from footprinting with a third probe (not shown) is
included.
1054 to
1020,
1015 to
996, and
960 to
937, respectively (Fig.
2A, lanes 1 and 2). Footprint G was observed with
fibroblast extracts (Fig. 2A, lanes 3 and 4), but
the resultant protection was less than in parallel experiments
utilizing chondrocyte protein (Fig. 2A, lanes 1 and
2). Experiments using fibroblast extracts yielded no
observable footprint in the chondrocyte-protein-protected sequence H
under four different buffer conditions, as well as at other DNase I
concentrations (data not shown). Finally, fibroblast extracts produced
footprint I, with protection comparable to that obtained with
chondrocyte extract.
878 to
831 and
808 to
791 (Fig. 2B,
lanes 3 and 4); these protected sequences were
designated as footprints J and K, respectively. One protected segment,
footprint K, had greater DNase I protection in its 5' portion using
fibroblast versus chondrocyte extracts under otherwise
identical conditions (Fig. 2B, lanes 1 and 2).
The upper region of footprint J varied slightly; the protected region
designated as J observed for fibroblast extracts was replaced by two
smaller non-overlapping footprints that spanned the same overall region
when chondrocyte nuclear protein was used. Similarly, another
footprint, L (Fig. 2C), was obtained using a third probe
from sequence just 3' of the probe 2, from
730 to
703 (data not
shown). This segment was protected by both chondrocyte and fibroblast
nuclear protein.
Oligonucleotides used to form mutant constructs for transfection
experiments
878 to
86, and mJ.1
that altered the sequence spanning
860 to
830. The mutant construct
mJ.2 ablated the CIIS2-containing sequence and the MalT_malPp
consensus sequence, whereas the mutant construct mJ.1 removed only the
CK-8-mer site. There is a high degree of similarity, and a 3-base
overlap, between the CIIS2 and MalT_malPp sequences in footprint
J. To evaluate these elements separately, we mutated the sequence that
corresponds to footprint K, which also contained a consensus sequence
for a MalT_malPp site but not the very similar CACCTCC sequence.
A second observed CIIS2 sequence, located in footprint L, was mutated
to determine to what extent that sequence alone was affecting activity,
thus mK ablated a MalT_malPp site, mL removed a CIIS2 sequence,
and mJ.2 mutated the composite sequence CACCTCCTCC.
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Fig. 3.
Effects of footprint sequence mutations on
Ag-1(+) promoter activity. This figure shows the relative
luciferase activities resulting from transfections with the Ag-1(+)
construct and mutated forms of Ag-1(+) produced with the
oligonucleotides described under "Experimental Procedures." Ag-1(+)
is the 1.8-kilobase promoter/enhancer region from the aggrecan gene
placed in the reporter vector pGL2-Basic. Duplicate plates were
transfected, and each plate was assayed for luciferase activity three
times. An average value and standard deviation were determined for all
six assays at each data point. Results were normalized by
co-transfection of 5 µg of a -galactosidase reporter gene
(Promega). Both day-14 chick sternal chondrocytes (A) and
day-14 chick fibroblasts (B) were transfected. At the time
of transfection cell density per dish was ~5 million. The
transfection was allowed to proceed for 36 h.
Nucleotide sequences used to form double-stranded EMSA probes
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Fig. 4.
EMSA using probes J, J.1, J.2, K, L, G, H,
and I. A shows the results for probes J, J.1, J.2, K,
and L (Table II) incubated with nuclear extracts from day-14 chick
chondrocytes. Bands that appear to be affected by protein concentration
and addition of excess of cold probe in other experiments are labeled
A D, and the free probe position is marked. B
shows the bands resulting when probes G, H, and I (Table II) were
incubated with nuclear extracts from day-14 chick chondrocytes. The
major DNA-protein complexes are labeled 1-3.
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Fig. 5.
Dose dependence of probe G and L EMSA
bands. A shows EMSA results for probe G (Table II)
incubated with various amounts of chick chondrocyte nuclear protein.
Reactions for lanes 1-3 contained 1, 5, and 10 µg of
nuclear protein, whereas the lane 4 and 5 reactions contained 5 µg of nuclear protein with the addition of
increasing concentrations of excess unlabeled probe G. Bands that
appear to be affected by protein concentration are labeled, and the
free probe location is indicated. B shows the EMSA results
for probe L (Table II) incubated with various amounts of chick
chondrocyte nuclear extract. Lane K is a reference lane with
probe K to denote the position of complex A. The lanes L5,
L10, and L20 reactions contained 5, 10, and 20 µg of
protein, respectively. Bands that appear to be affected by protein
concentration are labeled.
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Fig. 6.
Competition EMSA analyses with probes J.1,
J.2, K, and L. A-D display the results of incubating
20 fmol of 32P-labeled probes J.1-L (Table II) with 5 µg
of day-14 chick chondrocyte nuclear extract, either alone or with
excess unlabeled competitor probes. For each panel, the control
(C) lane reaction contained only the labeled probe for that
panel. Pairs of lanes designated with the same probe name were from
reactions that contained 200 fmol (left) or 1 pmol
(right) of that probe as unlabeled competitor.
Lanes marked TA contained reactions with 1 pmol
of unlabeled TA probe (Table II) as nonspecific DNA competitor.
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Fig. 7.
EMSA analyses with chick chondrocyte and
fibroblast extracts. A and B show the bands
resulting when probes G-L were incubated with nuclear extracts from
day-14 chick chondrocyte and fibroblast cultures. The lowercase
letters c and f, appended to the names of the probes
and the major complex bands, refer to the source of nuclear protein
(chondrocytes or fibroblasts, respectively) in the binding
reactions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1200 to
780 region of the chick
aggrecan promoter yielded much mutually supportive information. Five of
the six DNase I footprint sequences observed (excepting H) were
protected to varying degrees by both chondrocyte and fibroblast nuclear proteins. Four of those five sequences (I-L) have negative roles in
the transcriptional regulation of aggrecan, as evidenced by the
increases in reporter activity observed for mutated Ag-1(+) constructs
that preserved the native spacing while ablating individual footprint
regions. All of the protected sequences were found to contain known
cis elements, and the J-L regions were found to share,
pairwise, a number of identical or similar sequence elements. Comparison of EMSA results for the various footprint sequence probes
produced correlations of certain sequence elements to specific DNA-protein complex bands. Additionally, comparison of EMSA band patterns obtained with chondrocyte and fibroblast proteins revealed differences for all footprint probes, implying that cell type-specific transcription factor complexes were binding. For all four of the repression-associated footprint sequences, the chondrocyte/fibroblast EMSA difference is a reduction in mobility for the chondrocyte extract
band compared with the slowest band observed with fibroblast extract
(Fig. 7). Since chondrocytes express aggrecan (at various levels)
throughout life, whereas fibroblasts do not express aggrecan at any
time (1), a plausible interpretation of the EMSA data is that
up-regulation of aggrecan expression in chondrocytes is accomplished in
part by substitution or addition of specific proteins in/to the
complexes that act at the repressor region cis elements in
fibroblasts. Other bands that occur for both cell types may represent
core complexes with which the cell type-specific factors interact.
1(II) promoter defined this site in
the context of a 100-bp sequence abolishing transcription in
fibroblasts and HeLa cells but not in chondrocytes (13). Our studies
demonstrate that this sequence is acting as a repressor of the aggrecan
gene in both mesenchyme-derived cell types. Most likely, the sequence
(CACCTCC) that is present in footprints J and L and the rat collagen
1(II) CIIS1 and CIIS2 regions is sufficient to produce silencer
activity in both chondrocytes and fibroblasts, and the CACCTCC sequence
is a minimal core or half-site element responsible for the binding of a
general mesenchyme-specific repressor. EMSA results for both
chondrocytes and fibroblasts suggest that elements flanking this
sequence are important in the cell type-specific interactions observed
in our study and can produce cell type-specific DNA-protein complexes.
159 in the human genomic clone, only 31 base pairs from the TCCTCC motif at
128. A similar pattern is seen in
the chick aggrecan promoter with the TCCTCC sequence contained in
protected regions J.2 and K, with very close proximity to the TCCCC
sequences found in sequences J.1 and L.
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ACKNOWLEDGEMENTS |
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We thank Dr. Miriam Domowicz for helpful discussion, Glenn Burrell for manuscript preparation, and James Mensch for advice and manuscript review.
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FOOTNOTES |
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* This work was supported by United State Public Health Service Grants AR-19622 and HD-09402 (to N. B. S.), Training Grant HL-07237, and a Fellowship from the Markey Program in Molecular Medicine (to E. W. P.).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 the correspondence should be addressed: The University
of Chicago, 5841 S. Maryland Ave., MC 5058, Chicago, IL 60637. Tel.:
773-702-6426; Fax: 773-702-9234.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M009944200
2 E. W. Pirok III and N. B. Schwartz, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CS, chondroitin sulfate; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction; kb, kilobase pair; bp, base pair.
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