From the George Whipple Laboratory for Cancer
Research and Departments of Pathology, Urology, and Biochemistry,
University of Rochester, Rochester, New York 14642 and the
¶ Department of Medical Pharmacology, Utrecht University, 3584 CG
Utrecht, The Netherlands
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ABSTRACT |
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Amino acid sequence analysis indicates that the
human TR4 orphan receptor (TR4) is a member of the estrogen/thyroid
receptor subfamily of the steroid/thyroid receptor superfamily and
recognizes the AGGTCA direct repeat (DR) of the hormone response
element. Here we demonstrate using the electrophoretic mobility shift
assay that TR4 binds specifically to DR with a spacing of 1 and 5 base pairs (DR1 and DR5), which are the response elements for retinoic acid
receptor (RAR) and retinoid X receptor (RXR), respectively. A reporter
gene assay using chloramphenicol acetyltransferase demonstrated that
TR4 repressed RA-induced transactivation in a TR4
dose-dependent manner. Inhibition of the retinoid signal pathway also occurs through natural response elements found in CRBPII
and RAR genes. Our data suggest that the mechanism of repression may
not involve the formation of functionally inactive heterodimers between
TR4 and RAR or RXR. Instead, we show that TR4 may compete for hormone
response elements with RAR and RXR due to its higher binding affinity.
Furthermore, treatment of F9 murine teratocarcinoma (F9) cells with
10
6 M all-trans-retinoic
acid increased TR4 mRNA levels, and this change was accompanied by
an increased amount of endogenous TR4 protein that can bind to RXRE in
electrophoretic mobility shift assay. Our data therefore strongly
suggest that the retinoid signal pathway can be regulated by TR4 in a
negative feedback control mechanism, which may restrict retinoic acid
signaling to certain elements in a cell-specific fashion.
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INTRODUCTION |
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The steroid/thyroid hormone receptor superfamily is a large group of related transcriptional factors that control cellular differentiation, development, and homeostasis by direct interaction with distinct cis-elements in target genes (1). This superfamily includes receptors for steroids, thyroid, vitamin D3, retinoids, and a large number of orphan receptors whose cognate ligands are still unknown (2). Regardless of whether transcriptional activity is controlled by ligand binding, each of these proteins is able to bind to specific DNA sequences called hormone response elements (HREs)1 in their target genes. The sequence-specific DNA binding properties of nuclear receptors are determined by their highly conserved DNA binding domains (DBD). The P box of the DBD formed by three amino acids at the C-terminal base of the first zinc finger is responsible for the recognition of response elements. Based on the sequence within the P box and the generic recognition sequence of the P box for the HRE, the steroid/thyroid hormone receptor superfamily can be divided into the GR and ER/TR subfamilies (3). The GR group, which includes GR, MR, PR, and AR, prefers to bind to the glucocorticoid response element (5'-AGAACAnnnTGTTCT-3') palindromic consensus sequence. Other receptors that have glutamic acid and glycine at the first two positions of the P box are assigned to the ER/TR subfamily. This subfamily, which includes ER, T3R, VD3R, RARs, RXRs, and most of the orphan receptors, recognize the AGGTCA direct repeat or palindromic motif. However, some members prefer to bind to the single half-site of AGGTCA as a monomer, such as the steroidogenic factor 1 (SF-1)(4), TR3 orphan receptor/NGFI-B/nur77 (5), and the thyroid receptor (T3R) (6).
The human and rat TR4 were originally isolated from human prostate, testis, and hypothalamus cDNA libraries (7). The open reading frame of human TR4 encodes a protein of 615 amino acid residues with a calculated molecular mass of 67.3 kDa. Based on the modular structure and presence of a conserved DBD, which includes two zinc fingers that have a high degree of nucleotide sequence homology (65%) with the TR2 orphan receptor (7-9), TR4 belongs to the subfamily of TR2 orphan receptors within the steroid/thyroid receptor superfamily. Northern blotting and in situ hybridization studies reveal that TR4 is widely expressed in the adult rat brain (10), and most intense labelings for TR4 transcripts are detected within the granule cells of the hippocampus and cerebellum. On the basis of the sequence in the P box, TR4 has been speculated to belong to the ER/TR subfamily because of its ability to bind to AGGTCA direct repeats. In our previous studies, we found that TR4 may repress the expression of SV40 major late promoter, which contained an imperfect AGGTCA motif with a spacing of 2 bp between the half-sites (DR2) (11). By contrast, TR4 may also induce transcription of a thyroid receptor-regulated gene with a DR4 motif (12).
The retinoid signaling pathway is mediated by retinoid X receptors (RXRs) and retinoic acid receptors (RARs) through interaction with their HREs. This happens by formation of either a RXR-RAR heterodimer, a RXR-RXR homodimer, or heterodimers with other orphan receptors, such as LXR or NGFI-B. The RXR-RAR heterodimer mediates the effects of atRA and 9-cis-retinoic acid (9cRA) through interaction with DR5. In this complex, RXR functions as a silent partner that occupies the 5' half-site of DR5-RE (13). Alternatively, RXR may become an active ligand-binding heterodimer partner with LXR or NGFI-B and mediate the response of target genes to 9cRA (14, 15). Finally, RXR can mediate 9cRA action by binding through a DR1 element under the form of an RXR-RXR homodimer (16).
Here we present evidence that TR4 can bind strongly to response elements for RXR (RXRE-DR1) and RAR (RARE-DR5). The consequence of TR4 interaction with DR1-RXRE and DR5-RARE in the promoter context was then determined by using a transfection gene assay. The molecular mechanism for this regulation was further examined by Northern blotting analysis and EMSA. Together these results strongly suggest that TR4 is a central regulator in the retinoic acid signal transduction pathway.
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EXPERIMENTAL PROCEDURES |
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Construction of Plasmids-- The chimera receptor TR4-AR-TR4 was constructed by PCR site-directed mutagenesis to create two restriction enzyme sites, one is in front of DBD (BglII), the other is right after the DBD (XhoI), in both TR4 and AR. The DBD of AR was then removed by cutting pSG5AR with BglII and XhoI and ligated into pCMX-TR4, which had also been digested with these two enzymes.
Production of Monoclonal and Polyclonal Antibodies against Human TR4-- To obtain a large amount of TR4 for use as an antigen in raising specific monoclonal antibodies, the Escherichia coli pET expression system was used. The expression of TR4 from the pET system was performed according to the manufacturer's instruction (Novagen) with the addition of six consecutive histidine residues at the N-terminal TR4. The lysates were centrifuged at 40,000 rpm for 20 min at 4 °C. The cellular extraction was then either analyzed on SDS-PAGE followed by Coomassie Blue staining or purified by a one-step metal chelating chromatography (Novagen). For the production of monoclonal and polyclonal antibodies, the antigen was prepared by directly cutting from the SDS-PAGE and emulsified with Freund's complete adjuvant.
In Vitro Transcription/Translation of Nuclear
Receptors--
Four expression vectors, pCMX-TR4, pCMX-4A4,
pSG5-RAR, and pCMX-RXR
, were utilized to produce in
vitro transcribed and translated proteins in a rabbit
reticulocyte-based transcription/translation kit (TNT
coupled reticulocyte lysate system; Promega, Madison, WI).
Nuclear Extracts-- Nuclear extracts were prepared following the mini-extract procedure (17). In short, the cells, either with or without treatment of RA for 24 h, were harvested and lysed by pushing through a 25-gauge hypodermic needle. The nuclear pellet was resuspended in buffer C (500 mM NaCl, 20 mM Hepes, 25% glycerol, 1.5 mM MgCl, 0.2 mM EDTA, 0.5 mM dithiothreitol) and incubated on a rotating wheel for 30 min at 4 °C. The nuclear debris was pelleted by centrifugation for 30 min, and supernatant was dialyzed for 2 h against buffer D (20 mM Hepes, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol). Protein concentration was determined by Bradford reagent (Bio-Rad). Two µg of nuclear extracts were used in each 20-µl DNA-protein interaction.
Cell Culture and Transient Transfection-- Chinese hamster ovary (CHO) cells were routinely maintained in DMEM with 5% heat-inactivated fetal bovine serum (FBS). CHO cells (3 × 105) were seeded in 6-cm culture dishes 24 h before transfection. The medium was changed to DMEM with 5% charcoal dextran-treated FBS at least 1 h before transfection. The cells were transfected using a modified calcium phosphate precipitation method previously described (18).
Northern Blotting Analysis--
F9 (106) cells were
seeded in DMEM containing 5% charcoal dextran-treated FBS. After
24 h, the cells were treated with 106 M
atRA and harvested at 0.5, 1, 2, 4, 8, 24, 48 h, and even longer to 6 days after atRA treatment. Total RNA from the RA-treated F9 cells
was prepared by the ultracentrifugation method as described previously
(19). A probe covering the N-terminal of TR4 was released by the
digestion of EcoRI and AatII and labeled with [
-32P]dCTP by using a random primer DNA labeling
system (Life Technologies Inc.).
In Situ Hybridization Analysis-- Embryo collection, section preparation, and in situ hybridization were performed as described previously (20).
Other Methods-- EMSA, DNA-protein binding assay, and Western blot analysis were performed as described previously (21, 22).
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RESULTS |
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Production of Anti-TR4 Monoclonal Antibodies-- Using an E. coli expression system, we were able to generate large quantities of the N-terminal domain of TR4 to use as an antigen for the production of monoclonal antibodies. The reason we used the N-terminal domain of TR4 as an antigen is that this domain is the least conserved domain compared with other members of steroid/thyroid receptors. As shown in Fig. 1A, the N-terminal domain of TR4 encoded a 21-kDa IPTG-inducible protein (Fig. 1A, lane 1 versus 2). This induced protein, which was tagged with six N-terminal His residues, was further purified by one-step affinity chromatography and analyzed on 12.5% SDS-PAGE. As shown in Fig. 1A, lane 3, a major band of the right size was detected. For the production of the monoclonal antibody, the purified N-terminal of the TR4 peptide was cut from the gel and used for monoclonal antibody production. After screening, at least 40 monoclonal antibodies showed positive in enzyme-linked immunosorbent assay, and most of them were IgG or IgM subtypes. Western blotting analysis demonstrated that monoclonal antibody G232-151.4 can specifically recognize endogenous TR4 in CHO cell extract with a much lower intensity (Fig. 1B, lane 3, co-transfection of the expression vector only) than the cell extract from the co-transfection of TR4 (lane 1). There are no further enhanced TR4 bands that can be detected in CHO cell extract, which was transfected with TR2, indicating that the antibody does not cross-react with TR2 (lane 2).
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TR4 but Not Chimera Receptor 4A4 Binds to RXRE-DR1
Specifically--
Previously we were able to identify a DNA response
element for TR4 (TR4RE-SV40) containing an imperfect direct repeat of
the AGGTCA consensus motif with a 2-base pair spacing (DR2) in the transcriptional initiation site of the SV40 major late promoter (11).
Because the RXRE and RARE in the 5' promoter region of CRBPII and
RAR were similar to TR4RE-SV40, we were interested in determining if
TR4 might also bind specifically to these HREs and if it plays a role
in the regulation of the retinoid signal pathway. Wild-type TR4 and
chimera 4A4 were in vitro translated to produce proteins of
the expected molecular mass of 67.3 kDa as shown in Fig.
2A. To characterize the
binding specificity for TR4 and 4A4 to RXRE-CRBPII-DR1, EMSA was
performed. As shown in Fig. 2B, a specific DNA-protein
complex was revealed when 1 µl of in vitro translated TR4
was incubated with 0.1 ng of 32P end-labeled CRBPII
oligonucleotides (lane 2, arrow) which
was different from mock-translated control protein (lane
1). This complex could be eliminated in the presence of a
10- and 100-fold molar excess of unlabeled RXRE-CRBPII oligonucleotides
(lanes 3 and 4). Furthermore, the
monoclonal anti-TR4 antibody (G232-151.4) could supershift this
DNA-protein complex (lane 5,
arrowhead). In contrast, there is no specific interaction
between the probe and the chimera receptor 4A4 protein
(lanes 6-9). Similar results were detected when we replaced the 32P-CRBPII-DR1
with 32P-RAR
-DR5 oligonucleotide (data not shown). These
data demonstrated that TR4 can specifically bind to and form a single
complex with RXRE-CRBPII-DR1, and also the DNA binding domain of TR4 is
essential for the DNA binding.
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Suppression of RAR and CRBPII Promoter Activities by
TR4--
Transient transfection in CHO cells of CAT genes driven by
promoters with retinoid response elements were then used to study the
potential roles of TR4 in regulating RXR/RAR transcriptional activation. atRA (10
6 M) induced the
expression of both pRARE-CAT (a CAT expression vector with insertion of
RARE oligonucleotides) and pRXRE-CAT (a CAT expression vector with
insertion of RXRE oligonucleotides) nearly 7-fold (Fig.
3, lanes 4 and
10). This induction was repressed to basal levels when the
TR4 expression vector (pCMX-TR4) was co-transfected into the CHO cells
(Fig. 3, lanes 5 and 11). However, the
TR4-mediated repression could not be detected when we replaced TR4 with
the chimera receptor TR4-AR-TR4 (4A4), in which the DNA binding domain
of TR4 is exchanged with that of androgen receptor (Fig. 3,
lanes 6 and 12). The effects of TR4
and 4A4 expression on the basal promoter activity levels were also
tested, and there is no significant change (Fig. 3, lanes
1, 2, 7, and 8). Moreover, our data suggested that TR4-mediated suppression of pRARE-CAT and
pRXRE-CAT was TR4 dose-dependent both in the presence and absence of exogenous RAR or RXR (by co-transfection with pSG5-RAR
or
pCMX-RXR
(Fig. 4)).
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Interaction between TR4, RAR/RXR, and RARE/RXRE--
To explore
the direct interaction between TR4 or RAR/RXR upon binding to the RARE
or RXRE, we performed a series of studies using an EMSA. As shown in
Fig. 6, in vitro translated
TR4 binds specifically to RXRE with one clear band shift
(lane 3). As expected, no visible band shifts
were obtained when in vitro translated TR4 was replaced by
either in vitro translated RAR (lane
1) or RXR
(lane 2). However, a
clear band appeared (lane 4) when both in
vitro translated RAR
and RXR
were added. This band migrated differently from that of the TR4, which migrated more slowly
(lane 3 versus 4). When
in vitro translated TR4 was added together with in
vitro translated RAR
/RXR
, two distinct bands could be
observed, one for TR4 and another one for RAR
/RXR
. There was
clearly no intermediate band between TR4 and the RAR
/RXR
heterodimer (lane 5), even when the amount of
in vitro translated RXR
in the assay was increased
(lanes 6 and 8). Similarly, no
intermediate band could be visualized between TR4 and the RAR
/RXR
heterodimer when RXRE-CRBPII was replaced with RARE-RAR
(data not
shown). Together, these data indicate that there is no significant
heterodimer formation between TR4 and RAR or RXR. Clearly, these
results suggest that TR4 functions as a competitor for RARE/RXRE
binding.
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RXRE-CRBPII Binds to TR4 with Higher Affinity Than to the RAR/RXR
Heterodimer--
To explore the affinity between these receptors and
HREs, we performed EMSA to determine the dissociation constant by
Scatchard plot analysis. Scatchard plot analysis of the DNA-protein
complexes in the EMSA demonstrated that the dissociation constants
(Kd) for TR4 to RXRE-CRBPII and RAR/RXR
for
RXRE-CRBPII were 0.5 nM and 10.0 nM,
respectively (Fig. 7). Therefore, TR4 has
an affinity for RXRE-CRBPII that is 20-fold greater than that of the
RAR
/RXR
heterodimer. Our data suggest that a simple competition
between TR4 and RXR
/RAR
for the same HRE may be the potential
mechanism for the TR4-mediated suppression of the retinoid-RAR/RXR
pathway.
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Negative Feedback Control of the Retinoid Pathway by TR4--
To
explore the mechanism for TR4-mediated repression of the retinoid
signal pathway, we checked to see whether retinoids can regulate TR4 in
F9 cells that are sensitive to the RA treatment. To investigate
expression of TR4 during differentiation of F9 cells, 25 µg total RNA
from untreated F9 cells, or after treatment with 106
M atRA at different times, was analyzed by Northern
blotting. As shown in Fig. 8A,
an inducible band, which corresponds to the size of TR4 mRNA (9 kilobases), was visible in atRA-treated F9 cells. The induction
increased gradually from 30 min to 2 h after RA treatment (Fig.
8A, lanes 1-4) and reached a plateau
2 h after treatment (Fig. 8A, lanes
5-8).
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TR4 Expression Domains Overlapped with Those of RA Receptors-- It is well accepted that retinoic acids affect differentiation in various developmental systems via nuclear RA receptors. To analyze whether the interaction between TR4 and RA receptors could occur in vivo, we examined the TR4 expression pattern by in situ hybridization and compared it to that of RA receptors. As shown in Fig. 9, TR4 transcripts were intensively accumulated in the paraventricular brain area, retina, vestibular epithelium, perichondrium, kidney, and hair follicles of an embryo at gestation day 14 (Fig. 9A). The TR4 labeling was also detectable in tooth bud and whisker follicles at gestation day 16 (Fig. 9, B and C). It is worth noting that sense TR4 riboprobe did not detect any specific signal. Thus, this TR4 distribution pattern appears to be specific. This pattern does not directly follow expression patterns reported for any RA receptors.
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DISCUSSION |
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The data reported here suggest an important biological role of TR4 as a negative feedback regulator for the retinoid signaling pathway. Because the level of RA-induced transactivation can be repressed by co-transfection of TR4, but not the chimera receptor, TR4-AR-TR4 (Fig. 3), the DBD of TR4 is essential for repression. This result also argued against the possibility that TR4 may compete with RAR or RXR for the co-factors needed for the RA activation. Moreover, our data (Fig. 5) suggested that the repression of RA-induced transactivation by TR4 is RARE- and RXRE-dependent. Together, these results indicate that both the DNA sequence in HRE and the promoter context may contribute to the repression of RA-induced transactivation by TR4.
There are two possible mechanisms to explain how nuclear receptors can
repress the RA-induced transactivation pathway. The first one is
through protein-protein interaction by formation of heterodimer with
RXR. For example, COUP-TF has been demonstrated to repress RA-induced
transactivation by forming a heterodimer with RXR (22). However, our
results from the EMSA demonstrated that the mechanism of repression by
TR4 may be different from that displayed by COUP-TF. Our data showed
that both TR4 and RXR/RAR heterodimer can bind to RARE or RXRE probes
specifically but failed to show any unique bands formed between TR4 and
RXR/RAR when these three receptors were combined in EMSA analysis. This
strongly suggests that TR4 may repress RAR/RXR without
heterodimerization with either RAR or RXR
. The second mechanism
for repression is competition with these receptors for binding to a
common response element. In this regard, we compared the binding
affinity between TR4 and RXR/RAR to the RXRE-CRBPII DNA fragment in
EMSA. TR4 binds to RXRE-CRBPII with a 20-fold higher affinity than
RXR/RAR. The strong correlation observed between effective binding of
TR4 to a response element and its repressing effect provides convincing evidence that TR4 represses the retinoid signaling pathway by a simple
competition mechanism. Therefore, TR4 can compete with and displace the
RXR/RAR heterodimer from the DNA binding site to achieve effective
repression. Our data further confirmed that this repression of
RA-mediated transactivation is TR4-dose dependent, as increasing levels
of transfected TR4 led to a stronger inhibition of RA-mediated
transactivation. Such a finding is similar to our previous results for
the TR2 orphan receptor, a closely related subclass member (21, 27,
28). Both TR2 and TR4 can potentially function as repressors for the
retinoid-RAR/RXR signal transduction pathway by competition with the
HRE. A similar example of the dose-dependent regulation of
gene expression in this steroid/thyroid superfamily is the repression
of OCT3/4 gene by COUP-TFs. It has been demonstrated that
COUP-TFs can repress the OCT3/4 gene by binding to the
RAREoct site with much higher affinity than the RXR/RAR heterodimer
(29).
Based on the above conclusions, it is possible that gene regulation can be controlled by different ratios and relative affinities of a gene's inducers and repressors. In F9 cells that differentiated upon treatment of RA, we noted that TR4 mRNA was increased nearly 4-fold by adding RA. This inducibility was also supported by EMSA results. Although many nuclear hormone receptors may bind to RXRE-CRBPII in differentiating F9 cells (Fig. 8B, lanes 2 and 3), TR4 may represent one of the very few proteins that can also be induced during the RA treatment. It may indicate that retinoid pathways involve a very complicated and balanced control between TR4 expression and RA concentration.
Taken together, the repressive effects of TR4 on RA-induced transactivation suggest that TR4 may play a role in a negative feedback system, which controls RA-mediated modulation of gene expression. Moreover, TR4 may represent a master regulator in the retinoid signal pathway due to the dominant effects it exerts with higher affinity to the HREs and the potential amplification of this repression effect by its increasing expression during RA treatment.
In summary, the discovery of negative feedback control regulation of RA-mediated gene induction by TR4 provides evidence that TR4 is a regulator of cell proliferation and differentiation. Further studies of TR4 in retinoid systems may provide us with clues as to their physiological role in humans and other systems.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. M. Evans for
pRAR-CAT, p
RAR
-CAT, pCMX-RXR
, pCRBPII-CAT, and
p
CRBPII-CAT plasmids, Drs. T.-M. Lin and H.-J. Lee for their
valuable discussions. We also thank Dr. A. Saltzman for his helpful
comments and editing of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK47258 and CA68568.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 first two authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
716-273-4500; Fax: 716-756-4133.
1
The abbreviations used are: HRE, hormone
response element; TR4, TR4 orphan receptor; AR, androgen receptor; ER,
estrogen receptor; GR, glucocorticoid receptor; PR, progesterone
receptor; MR, mineralocorticoid receptor; T3R, thyroid
receptor; VD3R, 1,25-dihydroxyvitamin D3 receptor; COUP-TF, chicken ovalbumin upstream promoter transcription factor; atRA, all-trans-retinoic acid; 9cRA,
9-cis-retinoic acid; RARE, retinoic acid receptor response
element; RXRE, retinoid X receptor response element; EMSA,
electrophoretic mobility shift assay; CAT, chloramphenicol
acetyltransferase; CHO cell, Chinese hamster ovary cell; DBD, DNA
binding domains; PCR, polymerase chain reaction; PAGE, polyacrylamide
gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; FBS,
fetal bovine serum;
IPTG,isopropyl-1-thio--D-galactopyranoside.
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REFERENCES |
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