Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel and § National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland 20850
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ABSTRACT |
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Xenopus oocytes respond to trypsin
with a characteristic chloride current, virtually indistinguishable
from responses mediated by a large number of native and expressed G
protein-coupled receptors. We studied the involvement of G proteins of
the Gq family as possible mediators of this and other G
protein-coupled receptor-mediated responses in Xenopus
oocytes. We have cloned the third member of the G
q
family, Xenopus G
14, in addition to the
previously cloned Xenopus G
q and
G
11 (Shapira, H., Way, J., Lipinsky, D., Oron, Y., and
Battey, J. F. (1994) FEBS Lett. 348, 89-92).
Amphibian G
14 is 354 amino acids long and is 93%
identical to its mammalian counterpart. Based on the G
14
cDNA sequence, we designed a specific antisense DNA oligonucleotide
(antiG
14) that, together with antiG
q and
antiG
11, was used in antisense depletion experiments.
24 h after injection into oocytes, either antiG
q or
antiG
14 reduced the response to 1 µg/ml trypsin by
70%, whereas antiG
11 had no effect. A mixture of
antiG
q and antiG
14 virtually abolished the response. These data strongly suggest that G
q and
G
14 are the exclusive mediators of the trypsin-evoked
response in Xenopus oocytes. Similar experiments with the
expressed gastrin-releasing peptide receptor and muscarinic m1 receptor
revealed the coupling of G
q and G
11, but
not G
14, to these receptors in oocytes. These results
confirm the hypothesis that endogenous members of the G
q family
discriminate among different native receptors in vivo.
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INTRODUCTION |
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Heterotrimeric GTP-binding proteins (G proteins) of the
Gq family activate the
phosphatidylinositol-bisphosphate-inositol-trisphosphate-calcium pathway in a pertussis toxin-insensitive manner. Among
G
q family members, G
q and
G
11 share 88% homology and are expressed ubiquitously (2-4). G
14 is 81% identical to G
q and
is restricted in its tissue distribution mainly to spleen, lung,
kidney, and testes (5). G
15 and its human counterpart,
G
16, show only 58 and 57% amino acid identity,
respectively, to mouse G
q and are restricted to a subset
of hematopoietic cells (5, 6). Upon activation, all G proteins of the
G
q family activate
isomers of phospholipase C.
The coexistence of closely related G proteins in the same cell suggests
different functions or different interactions between individual G
proteins and either receptors or phospholipases. However, in
reconstitution systems and transfected cells, Gq and
G
11 have been shown to couple indiscriminately to a wide range of receptors, e.g. muscarinic m1 receptor
(m1-R)1 (7, 8) and m3-R (9),
thyrotropin-releasing hormone (10, 11), parathyroid hormone (12),
gastrin-releasing peptide (GRP) and vasopressin (13),
gonadotrophin-releasing hormone (14), angiotensin II and bradykinin
(15), histamine (16), and
1-adrenergic receptors (17).
G
15/16, despite restricted distribution, are capable of
coupling many serpentine receptors tested, including those natively
coupled to G
s and G
i (for review, see
Ref. 18). Similarly, an attempt to assign different roles to
G
q and G
11 using agonist-induced
down-regulation failed to distinguish between the two G proteins
(19-21).
This lack of discrimination may reflect real interchangeability
in vivo among the G proteins of the Gq family
or merely be a result of the assays used. To address this question, we
have used the Xenopus oocytes system to study receptor-G
protein specificity in vivo. Coexpression of
thyrotropin-releasing hormone receptors with either mouse
G
q or G
11 demonstrated different
modulation of the response to thyrotropin-releasing hormone by each one
of these G proteins (22). Applying the antisense approach, we
demonstrated for the first time different coupling preferences of
neuromedin B receptor for G
q and G
11
(1).
Although some information is available for other Gq
family members, little is known of G
14-receptor coupling
specificity. Wu et al. (17, 23) demonstrated in transfected
COS-7 cells that G
14 mediates responses evoked by the
1-adrenergic and the interleukin-8 receptors. Kuhn
et al. (16) showed that histamine receptors couple to all
G
q family members.
In this study, we have used the Xenopus oocyte system to
investigate the coupling of native or expressed receptors to
G14. Xenopus oocytes express an endogenous
protease receptor that is activated by trypsin (24). The response,
typical for the activation of the phosphatidylinositol-specific
phospholipase C signal transduction pathway, is manifested as
Ca2+-dependent Cl
current.
Trypsin, along with several other proteases, stimulates fertilization-like responses in starfish eggs (25). In
Xenopus oocytes, there is an elevation of G proteins of the
G
q family during maturation and early development (26).
These data suggest that protease receptors coupling via G proteins of
the G
q family may be of importance in fertilization and
early development. Using the antisense approach, we show here that
G
q and G
14 are the main mediators of the
response elicited by trypsin, in contrast to the response evoked by the
activation of GRP-Rs, which utilize G
q and
G
11, and the response to activation of m1-Rs, which
utilize G
q and G
o.
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EXPERIMENTAL PROCEDURES |
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Cloning of Xenopus G14--
A cDNA library of
Xenopus oocytes (constructed in
gt10) was screened at low
stringency with a mouse G
q probe, as described previously (1). Positive plaques were isolated and sequenced with
universal and gene-specific primers using fMOLTM sequencing kit
(Promega), according to the manufacturer's instructions. A partial
cDNA clone that displayed high homology to mouse G
14 was used as a probe in a second round of screening at high stringency: overnight hybridization at 37 °C in NT hybridization buffer (27) followed by a 4 × 10-min wash at room temperature in 1 × SSC (0.15 M NaCl and 0.015 M sodium citrate), 0.1% SDS and 20 min wash
at 42 °C in 0.1 × SSC, 0.1% SDS. Positive clones were
isolated and either subcloned into the EcoRI site of pGEM-4
plasmid (Promega) or PCR-amplified with pfu DNA polymerase
(Stratagene) and sequenced on both strands with universal and
gene-specific primers, as described above.
Antisense Oligonucleotides Sequence--
antiGq:
5'-ATTCTCAAAAGAGGCGACC-3'; antiG
11:
5'-CTGTTCAAAGGTACATACT-3'; antiG
14:
5'-GTTTCCTTTCAAGACTGGAT-3'; antiG
o:
5'-GCGCTCAGTCTGCAGCCCAT-3'.
Handling of Oocytes--
Oocytes were excised from
Xenopus females (South African Xenopus Facility,
South Africa), defolliculated with collagenase, and kept in NDE
solution at 20 °C, essentially as described previously (28). Stage V
oocytes were injected with the desired RNAs (0.5-2 ng/oocyte) and/or
phosphorothioate DNA antisense oligonucleotides (S-oligos, 50 ng/oocyte). The injections resulted in deterioration of some oocytes,
particularly those injected with antiG11. Viable oocytes
were used in three assays: functional assay and Northern and Western
analyses.
Functional Assay in Oocytes--
Measurements of
electrophysiological responses to agonists were performed essentially
as described previously (28). Briefly, individual oocytes were
voltage-clamped at 70 mV. Agonists (trypsin, GRP, or acetylcholine
(ACh)) were added directly to the bath, and membrane currents were
continuously recorded. The mean amplitude of the control group in each
experiment was set as 100%, and the effect of each treatment was
calculated as the percent of that control response. N
denotes the number of different donors, and n denotes the
number of oocytes tested. Results are represented as means ±S.E.
Paired Student's t test was used to estimate the statistical significance of the data.
Northern Analysis--
Groups of 80 oocytes were homogenized in
8 ml of cold guanidynium isothiocynate buffer, centrifuged for 5 min at
3000 × g, 4 °C, and the supernatant was loaded onto
4 ml of CsCl (5.7 M) cushion for standard total RNA
isolation (27). The precipitated RNA was dissolved in 40 µl of
H20. 6 µg of total RNA were loaded and resolved on RNA
gel (agarose/formaldehyde) and blotted onto a nitrocellulose filter.
Filters were air-dried and then baked at 80 °C in a vacuum for
1 h. Full-length clones of Xenopus Gq, G
11, and G
14 and partial clone of rat
glycerol aldehyde phosphate dehydrogenase (GAPDH) were randomly labeled
with 32P (Random Primer labeling kit, Life Technologies,
Inc.) and hybridized to the filters at high stringency (see cloning
section above). Filters were autoradiographed for 3-7 days on XAR 5 Kodak films. Quantitation of the relevant bands was done by
densitometry and normalization with the 28S and the 18S bands on the
RNA gels and the 1.2-kilobase GAPDH bands on the autoradiograms.
Western Analysis--
Groups of 60 oocytes were homogenized in 1 ml of cold hypotonic membrane buffer (5 mM Hepes, pH 7.4, 5 mM NaCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 µg/ml soybean trypsin inhibitor) and centrifuged twice in a
microcentrifuge for 5 s at 5,000 rpm to pellet the yolk. The
supernatant was centrifuged for 20 min at 14,000 rpm. The pelleted
membranes were resuspended in 60 µl of hypotonic membrane buffer and
kept at 70 °C. Before use, loading buffer was added (1:1 v/v), and
samples were heated to 90 °C for 5 min. 20 µl (equivalent to
membranes of 10 oocytes) were resolved on 10% SDS-polyacrylamide gels
and electrotransferred onto nitrocellulose filters. Filters were
blocked for 3 h in blocking solution (TBS-T, Tris-buffered saline + 0.05% Tween 20, with 5% nonfat milk powder) and then exposed to the
first antibody (1:500 dilution in blocking solution) overnight, with
gentle shaking at 4 °C. Filters were then washed 5 times in washing
solution (TBS-T) and exposed to the second antibody (1:1500 dilution in
blocking solution of goat anti-rabbit IgG conjugated to horseradish
peroxidase). Enhanced chemiluminescence system (ECL kit, Amersham
Pharmacia Biotech) was used for developing the blots.
Materials-- Collagenase (Type 1A), trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated), Hepes, aprotinin, leupeptin, soybean trypsin inhibitor, goat anti-rabbit IgG, and ACh were purchased from Sigma; GRP14-27 from Bachem, S-oligos from Oligos Etc.; primers from Genemed Biotechnologies; and radiodeoxynucleotides from NEN Life Science Products. All molecular biology reagents were of molecular biology grade. All other chemicals were of analytical grade. WO82, B825, and Z808 antibodies were a gift of Dr. P. Sternweis, and QL antibody was a gift of Dr. T. Jones.
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RESULTS |
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Cloning of Xenopus Oocyte G14--
Initial
screening of a Xenopus oocyte cDNA library with mouse
G
q probe at low stringency resulted in the cloning of
the amphibian counterparts of G
q, G
11,
and an additional partial clone that most resembled mouse
G
14 (see "Experimental Procedures"). This partial
clone was used as a probe in a second round of screening at high
stringency to isolate a full-length clone of Xenopus
G
14. The cloned Xenopus G
14
cDNA (accession number AF059182) had an open reading frame of 1065 bases, predicting a 354-amino acid protein. The cDNA sequence was
78% identical to mouse G
14 (GenBankTM accession number
M80631). The predicted protein was 90% identical to its mammalian
counterpart, with most differing residues representing conservative
substitutions, except for a cysteine missing at position 4 (Fig.
1). The consensus sequences for the GTP
binding site and the carboxyl terminus were identical to the mammalian
protein. Northern analysis of the native RNA with the cloned
G
14 cDNA as a probe detected a major 3.8-kilobase
band and an additional 3.1-kilobase band (Fig.
2).
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Antisense-induced RNA Degradation--
To test the antisense
S-oligos selectivity for the corresponding mRNAs, we injected
S-oligos into oocytes (50 ng/oocyte) and isolated RNA 48 h after
injection. Northern analysis of three independent experiments after
normalization with internal GAPDH transcript levels (see
"Experimental Procedures") showed that all the three S-oligos were
selective in promoting the degradation of their target mRNAs.
Although antiG11 and antiG
14 exhibited absolute selectivity, antiG
q was less effective and
caused some degradation of G
14 (Fig. 2). 48 h
post-S-oligos injection, antiG
14 degraded
G
14 RNA by 86% and had no effect on G
q
and G
11 RNA levels. antiG
11 completely
abolished its cognate RNA and had no effect on the other two RNAs.
antiG
q caused degradation of both G
q and
G
14 transcripts by 62 and 42%, respectively, and had no
effect on G
11 mRNA. The kinetics of RNA degradation
show that most of the RNA is already degraded 3 h after antisense
injection. Although a 3-h treatment with S-oligos did not always result
in a complete disappearance of the G
11 or
G
14 mRNA bands, low molecular mass products
indicated substantial partial degradation (Fig. 3A). In a representative
experiment designed to test early effects of antisense S-oligos (3 h),
densitometry of the autoradiograms after normalization with endogenous
GAPDH resulted in the reduction of G
q by 67%,
G
11 by 89%, and G
14 by 77%. 24 h
after the injection of S-oligos, the G
11 and
G
14 mRNAs were fully degraded. Since some reports
demonstrated recovery of the response 4-7 days post-antisense oligonucleotides injections (29, 30), we checked for possible reappearance of RNAs encoding these G proteins within the time frame of
our experiments. Oocytes were injected with antisense oligonucleotide
and subjected to RNA isolation 1, 2, 3, and 4 days post-injection and
then to Northern analysis with the respective probes. No
G
14-encoding RNA recovery was detected during this period (Fig. 3B). Similar results were obtained for the
other two mRNAs (not shown).
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Antisense-induced Depletion of Oocyte G Proteins--
To assess
the effect of S-oligos-induced mRNA depletion at the protein level,
we used Western analysis with antisera developed against the mammalian
G proteins of the Gq family. Antibodies that
specifically discriminate between mouse G
q and
G
11 (WO82, B825; Refs. 15 and 31) interacted with a
large number of proteins in the 30-50-kDa range and could not,
therefore, detect amphibian G proteins unambiguously. Antibodies raised
against the common carboxyl terminus of the G
q family
(Z808 or QL, see Ref. 32) were more specific and, despite their
interaction with a number of other proteins, we detected a faint band
migrating at a position corresponding to approximately 42 kDa (Fig.
4). These data confirm the findings of
Gallo et al., (26), showing only very small amounts of
proteins of the G
q family in oocytes. The small amount of native G proteins made the quantitation of turnover rates difficult. To circumvent this problem, we overexpressed Xenopus
G
q, G
11, or G
14 in oocytes
by injecting (1 ng/oocyte) in vitro transcripts encoding
their open reading frames. 48 h later, an increase in the
intensity of the 42-kDa band was observed. The results indicated that
G
11 and G
q were overexpressed, albeit to
a different extent. G
14 did not seem to overexpress;
however, injection of antiG
14 markedly decreased the
signal. This could be interpreted either as the oocyte natively
expressing mainly G
14 or that the expression of
G
14 repressed the expression of other G proteins of the
family. We have used oocytes injected with the transcripts of each of the three G
q family members to estimate their turnover
rates. 24 h post-injection of the cognate S-oligos, membranes were
subjected to Western analysis with QL and Z808 antisera. S-oligos
treatments caused substantial degradation of each protein (Fig.
4A). Densitometric analysis of the overexpressed 42-kDa
bands showed that G
q, G
11, and
G
14 were degraded by 75, 50, and 50%, respectively
(Fig. 4B). Since RNA degradation was very rapid, these
numbers approximate the degradation rates of the overexpressed
proteins. Mitchell et al. (33) found that the turnover of
G
q/11 in CHO cells was best described by monoexponential
curve with t1/2 = 18 h. Our data indicate
similar G protein turnover rates in oocytes. In control oocytes,
24 h post-injection of antiG
q, the endogenous
42-kDa proteins were degraded by 30%, suggesting that native G
proteins of the G
q family include approximately 40%
G
q. These results taken together demonstrate that
antisense S-oligos treatment caused rapid and significant depletion of
oocyte G proteins.
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The Effects of Antisenses on the Endogenous Responses to
Trypsin--
To study the involvement of Gq,
G
11, and G
14 in the response evoked by
trypsin, oocytes were injected with each of the S-oligos (50 ng/oocyte)
and challenged with 0.1-5 µg/ml (1-50 benzoyl-L-arginine ethyl ester units/ml) trypsin at
different time intervals after the injections. 24 h after
antiG
q injection, the response was reduced by 69 ± 4% (n = 57, N = 7, Fig.
5). antiG
14 reduced the
response by 68 ± 7% (n = 58, N = 7). antiG
11 decreased oocytes viability, but the
response in the surviving cells was not affected. Injection of only
half of the amount of antiG
11 (25 ng/oocyte) did not
compromise oocyte viability and had no effect on the response (81 ± 15% of control, n = 30, N = 3, not significant). In oocytes injected with a mixture of
antiG
q and antiG
14, the response to
trypsin was reduced to 7 ± 3% that of control (n = 57, N = 7). Despite the relatively high amounts of S-oligos injected in the mixture (50 ng/oocyte, each), oocyte deterioration was very limited and did not exceed that of oocytes injected with each one of the S-oligos alone. Similarly, membrane potentials and holding currents did not indicate any nonspecific functional damage due to the co-injection of the two S-oligos. These
results suggest that G
q and G
14, but not
G
11, are the main mediators of the endogenous response
to trypsin in Xenopus oocytes.
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DISCUSSION |
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All G proteins of the Gq family have been shown to
couple to GPCRs in the pertussis-toxin-insensitive
phosphatidylinositol-specific phospholipase C signal transduction
pathway. With the exception of our previous report in oocytes (1),
transfection and reconstitution experiments failed to functionally
discriminate between G
q and G
11. However,
sequence and distribution differences (e.g. Ref. 4) between
the two G proteins as well as among all the family members in general
suggest individual physiological roles in regulating signaling in
target tissues and organs. The antisense approach has the advantage of
relying on the natural milieu of each component of the signal
transduction pathway. By applying this approach in Xenopus
oocytes, we have previously demonstrated functional in vivo
preferences of neuromedin B receptor coupling via G
q (1).
To broaden the scope of the study, we have cloned Xenopus
G14 in order to develop an antisense oligonucleotide
that could be used to deplete cells of G
14 protein.
Similarly to the previously cloned Xenopus G
q
and G
11, Xenopus G
14 showed a
high degree of homology to its mammalian counterpart, demonstrating the
conservation of these protein sequences during vertebrate evolution.
Northern analysis in oocytes demonstrated the intrafamily selectivity
of the designed antisenses (antiG
q being less selective,
degrading mainly G
q but also G
14) and the
rapid elimination of the cognate mRNAs after antisense injection.
Thus, proteins turnover rates were the limiting factor in the depletion
process. The turnover rate of G
q/11 has been
shown in Chinese hamster ovary cells to fit a first-order reaction with
a t1/2 of 18 h (33). Our data in oocytes,
obtained from both Western analysis and functional assays, exhibit
similar kinetics of degradation, but distinguish between
G
q and G
11. The turnover rate of
G
14 was similar to that of G
11. Our
functional assays were executed 24-80 h after antisense injection, a
time window in which most of the G
q and more than 50%
of G
11 and G
14 were degraded. Therefore,
a 40% reduction in the m1 muscarinic response by either
antiG
q or antiG
11 and the fact that this
response was not further reduced after 48 and 80 h support the
existence of an additional mediator for this receptor. Indeed,
antiG
o caused a 20% reduction in the response to ACh
24 h after its injection. Mixtures of
antiG
q/antiG
11 caused pronounced oocytes
deterioration, preventing a more conclusive interpretation. We cannot
exclude the possibility that even residual amounts of any G protein
(undetectable by Western analysis) can mediate a full response,
provided there is a large excess of G protein over the receptor and
high affinity between the two proteins.
The antisense oligonucleotides designed against the different G
proteins could have achieved their effects by interfering with the
synthesis of different phospholipase C isoforms. This possibility,
however, appears unlikely since there is no evidence for multiple
phospholipase C
species in oocytes.
Since there is little information about the G14 ability
to couple GPCRs, we tested a number of receptors (mouse m1, GRP and neuromedin B receptors) and the native trypsin response to investigate its specificity. Only the endogenous protease receptor exhibited sensitivity to G
14 depletion. Two antisense
oligonucleotides, antiG
q and antiG
14,
each diminished the responses to about 30%, and a mixture of the two
reduced it to 7% that of control, whereas antiG
11 had
no effect. These results strongly suggest that the response to trypsin
is mediated exclusively by G
q and G
14.
Although it appears that the contribution of these two G proteins to
the trypsin response is similar, the limited degree of degradation of
G
14 mRNA caused by anti G
q precludes
a firm conclusion regarding this issue. Thus, G
14
mediates the response to protease in addition to its ability to couple
1-adrenergic, interleukin-8 receptors (17, 23), and
histamine receptors (16). Wu et al. (37) have shown by
altering selected domains within the third intracellular loop of the
1B-adrenergic receptor, sequence-related specificity for
either G
q/11 or G
14. It would
have been interesting to compare the sequence of the yet uncloned
Xenopus protease receptor, which appears to couple to
G
q and G
14, but not to
G
11, with that of the
1B-adrenergic
receptor.
The finding that antiG11 did not affect the response
confirms our previous report that in vivo G
q
and G
11 have distinct receptor preferences. It is
interesting to note that so far in reconstitution, transfection, and
in vivo models, every GPCR tested (with the possible
exception of neuromedin B receptor expressed in oocytes, Ref. 1) was
able to couple to more than one G protein of the G
q
family. Since different GPCRs appear to be able to discriminate among
the G proteins, the interaction of a single receptor with more than a
single G protein implies biological significance. In this respect, the
recent report by Macrez-Lepretre et al. (38) points to
discrimination between G
q and G
11 for specific effector molecule in the signaling pathway. The G proteins specificity for the receptors on the one hand and for the distal parts
of the signal transduction pathway on the other may serve to fine tune
receptor-mediated biological responses.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. P. Sternweis for his generous donation of anti-G protein antibodies and to Dr. T. Jones for her gift of the QL antiserum.
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FOOTNOTES |
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* This work was supported in part by a grant of BiNational Science Foundation (BSF) (to H. S. and J. F. B.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF059182.
To whom correspondence should be addressed: Dept. of Physiology
and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel. Tel.: 972-3-640-9862; Fax: 972-3-640-9113; E-mail: hshapira{at}post.tau.ac.il.
1 The abbreviations used are: m1-R, m1 muscarinic receptor; GPCRs, G protein-coupled receptors; GRP, gastrin-releasing peptide; GRP-R, GRP receptor; ACh, acetylcholine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; S-oligos, phosphorothioate oligodeoxynucleotides; PCR, polymerase chain reaction.
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REFERENCES |
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