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J Biol Chem, Vol. 273, Issue 23, 14516-14522, June 5, 1998
From the Department of Biochemistry and Molecular Biology,
Faculty of Medicine, University of Manitoba, Winnipeg,
Manitoba R3E OW3, Canada
Nucleosomes associated with transcribing
chromatin of mammalian cells have an unfolded structure in which the
normally buried cysteinyl-thiol group of histone H3 is exposed. In this
study we analyzed transcriptionally active/competent DNA-enriched
chromatin fractions from chicken mature and immature erythrocytes for
the presence of thiol-reactive nucleosomes using organomercury-agarose column chromatography and hydroxylapatite dissociation chromatography of chromatin fractions labeled with
[3H]iodoacetate. In mature and immature
erythrocytes, the active DNA-enriched chromatin fractions are
associated with histones that are rapidly highly acetylated and rapidly
deacetylated. When histone deacetylation was prevented by incubating
cells with histone deacetylase inhibitors, sodium butyrate or
trichostatin A, thiol-reactive H3 of unfolded nucleosomes was detected
in the soluble chromatin and nuclear skeleton-associated chromatin of
immature, but not mature, erythrocytes. We did not find thiol-reactive
nucleosomes in active DNA-enriched chromatin fractions of untreated
immature erythrocytes that had low levels of highly acetylated histones H3 and H4 or in chromatin of immature cells incubated with inhibitors of transcription elongation. This study shows that transcription elongation is required to form, and histone acetylation is needed to
maintain, the unfolded structure of transcribing nucleosomes.
Acetylation of the core histones (H2A, H2B, H3, and H4) is a
dynamic process catalyzed by histone acetyltransferases and histone deacetylases (1, 2). In chicken immature erythrocytes, 4% of the
modifiable lysine sites participate in dynamic histone acetylation.
These core histones are rapidly acetylated (t1/2 = 12 min for monoacetylated H4) and rapidly deacetylated
(t1/2 = 5 min for the tetraacetylated isoform of H4)
(3, 4). Histones undergoing rapid acetylation and deacetylation are
associated with transcriptionally active chromatin (5-7). The recent
findings that histone acetyltransferases and deacetylases are
transcriptional coactivators and corepressors have increased our
understanding of how the process of dynamic histone acetylation is
established on transcriptionally active chromatin (2, 8).
Transcriptionally active chromatin has a soluble and insoluble nature
(9). Transcribed DNA is found in chromatin fragments that are soluble
in 0.15 M NaCl and/or 2 mM MgCl2
and in chromatin fragments associated with the low salt-insoluble
residual nuclear material (nuclear skeletons) (for review, see Davie
(10)). Chromatin engaged in transcription is thought to be retained by
the nuclear skeleton by multiple dynamic attachments between the
nuclear matrix and transcribed chromatin; hence rendering the
transcribing chromatin insoluble (11, 12). As histone acetyltransferase
and deacetylase activities are associated with the nuclear matrix (7,
13), we proposed that these nuclear matrix-bound enzymes may mediate some of the dynamic attachments between active chromatin and nuclear matrix (13, 14).
Most information on the structure and composition of transcriptionally
active nucleosomes is from studies that analyze soluble transcriptionally active chromatin. However, most of the transcribed chromatin fragments partition with the low salt-insoluble nuclear material (nuclear skeleton) (7, 15, 16). We presented evidence that
dynamically acetylated histones are associated with the nuclear matrix-bound transcriptionally active chromatin (7). Otherwise, little
is known about the structure and composition of transcribing nucleosomes attached to the nuclear skeleton.
Allfrey and co-workers demonstrated that nucleosomes in the transcribed
regions of soluble chromatin of mammalian cells unfold exposing the
cysteinyl-thiol groups of histone H3 (17, 18). The unfolding of the
nucleosome was dependent upon ongoing transcription. Exploiting this
feature of transcribing nucleosomes, a procedure to isolate soluble
transcriptionally active chromatin by organomercury-agarose affinity
chromatography was developed. The transcribing chromatin was associated
with highly acetylated histones (18-20). However, current evidence
argues that histone acetylation is not involved in the generation of
the unfolded nucleosome. Reconstitution of nucleosomes with highly
acetylated histones did not result in the formation of a thiol-reactive
nucleosome (21). Further, treating mammalian cells with the histone
deacetylase inhibitor, sodium butyrate, did not increase the level of
thiol-reactive nucleosomes (6).
Analysis of chicken mature erythrocyte salt-soluble polynucleosomes
highly enriched in transcriptionally competent DNA and highly
acetylated histones (22, 23) showed that this chromatin fraction lacked
thiol-reactive nucleosomes.1
To address the question of whether unfolded nucleosomes exist in
chicken erythrocytes, we investigated the H3 thiol reactivity of
salt-soluble and low salt-insoluble (nuclear skeleton-associated) chromatin from mature (transcriptionally silent) and immature (transcriptionally active) chicken erythrocytes. We report that the
thiol-reactive, unfolded nucleosome exists in immature, but not mature,
erythrocyte salt-soluble chromatin fragments and chromatin fragments
associated with the nuclear skeleton. However, histone deacetylase
activity had to be suppressed to detect thiol-reactive nucleosomes in
immature erythrocyte chromatin. These studies show that highly
acetylated histones maintain the unfolded nucleosome structure formed
by transcriptional elongation.
Isolation and Treatments of Immature and Mature Chicken
Erythrocytes--
Mature and immature erythrocytes were isolated from
normal and anemic young adult White Leghorn chickens, respectively, as described previously (24). Immature and mature erythrocytes were
collected in 75 mM NaCl and 25 mM EDTA, while
cells to be incubated with sodium butyrate or trichostatin A were
collected in 75 mM NaCl, 25 mM EDTA, and 30 mM sodium butyrate. Cells were incubated at 37 °C in
Swims S-77 medium (Sigma) with 30 mM sodium butyrate or 100 ng/ml trichostatin A for 90 min to prevent the deacetylation of highly
acetylated histones. To inhibit transcription elongation, cells were
incubated at 37 °C for 90 min with transcription inhibitors
actinomycin D (0.04 µg/ml) (24),
5,6-dichloro-1- Erythrocyte Chromatin Fractionation--
The fractionation of
chromatin was done as described previously (24). All buffers contained
1 mM phenylmethylsulfonyl fluoride. Briefly, nuclei (50 A260 units/ml) were digested with micrococcal nuclease (15 A260 units/ml for 25 min at
37 °C), collected by centrifugation, and then resuspended in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA. Following
centrifugation the soluble chromatin fraction (fraction SE) and low
salt insoluble chromatin fraction (nuclear skeleton, PE fraction) were
isolated. The chromatin fragments of fraction SE were further
fractionated by the addition of NaCl to 150 mM. Following
centrifugation, chromatin fractions P150 (pellet) and S150
(salt-soluble chromatin) were isolated.
Organomercury Column Affinity Chromatography--
Chicken
erythrocyte chromatin fraction S150 was dialyzed against buffer A (10 mM Tris-HCl, pH 7.5, 25 mM KCl, 25 mM NaCl, 5 mM sodium butyrate, 0.1 mM phenylmethylsulfonyl fluoride, and 5 mM
EDTA, pH 7.5) and then applied to an organomercurial column (Affi-Gel
501; Bio-Rad) that was pre-equilibrated with buffer A. The column
(1.5 × 8 cm) was then washed with buffer A (flow rate, 60 ml/h)
to remove unbound chromatin fragments until the absorbance at 260 nm
returned to base line. This was followed by washing the column with 0.5 M NaCl in buffer A (buffer B) until the absorbance at 260 nm returned to base line. The bound nucleosomes were eluted by 10 mM DTT in buffer A. The released material was monitored by
measuring absorbance at 260 nm (17). The fractions containing the
unbound material and 0.5 M NaCl eluted material were
pooled; the fractions eluted with DTT were pooled. For analysis of
histones in the unbound and bound fractions, the fractions were
acid-extracted by the addition of 4 N sulfuric acid to a final concentration of 0.4 N. Before lyophilization, the
supernatants were dialyzed overnight against 0.1 M acetic
acid and then against two changes of double-distilled
H2O.
Reaction of Nucleosomes with [3H]Iodoacetic
Acid--
Ten µl/ml [3H]iodoacetic acid (NEN Life
Science Products, 204.9 mCi/mmol) containing 2.50 µCi was added to
the chromatin fraction SE, S150, P150, and PE (2 A260/ml) in buffer E (10 mM
Tris-HCl, pH 8.2, 1 mM EDTA) and allowed to incubate at
room temperature for 1 h in the dark. The chromatin fraction was
applied directly to a hydroxylapatite column. The histones were
isolated by acid extraction as described above. Histones were
electrophoretically resolved on SDS-polyacrylamide gel electrophoresis.
Following staining with Coomassie Blue, the gel pieces containing a
histone band were disrupted in hydrogen peroxide and then counted in 5 ml of scintillation fluid.
Hydroxylapatite Chromatography--
The chromatin fraction was
mixed with hydroxylapatite HTP gel powder (Bio-Rad) at a ratio of 1 mg
of DNA to 0.25 g of hydroxylapatite as described previously (27).
The column was washed with 0.63 M NaCl in 0.1 M
potassium phosphate buffer, pH 6.7, to remove histone H1, H5, and
non-histone chromosomal proteins before applying a linear gradient of
NaCl (0.63 to 2 M NaCl in 0.1 M potassium phosphate buffer, pH 6.7) at a flow rate of 35 ml/h as described previously (27).
DNA Preparation and Southern Blot Hybridization--
DNA was
prepared from the different chromatin fractions as described previously
(24). For electrophoresis, equal amounts of DNA were dissolved in DNA
sample loading buffer, and the samples were loaded onto 1% agarose
minigels containing 0.5 µg of ethidium bromide/ml. The DNA was
transferred to Hybond-N+ nylon transfer membrane and hybridized to
radiolabeled probes as described previously (28). The cloned probes
used were pCBG 14.4, a unique intronic sequence of chicken Polyacrylamide Gel Electrophoresis and Western Blotting--
AUT
(acetic acid, 6.7 M urea, 0.375% (w/v) Triton X-100) and
SDS-15% polyacrylamide gel electrophoresis were performed as described
elsewhere (24). Antiacetylated H3 and antiacetylated H4 antibodies
generously supplied by Dr. D. Allis were used to detect acetylated
species of H3 and H4 (29-31) in Western blot experiments using a
protocol described previously (32).
State of Acetylation of Dynamically Acetylated Histones in Chicken
Immature and Mature Erythrocytes--
Histone acetylation is rapidly
reversible in immature erythrocytes (3, 4, 7). To find the steady state
of acetylated H3 and H4, histones of immature and mature erythrocyte
chromatin fractions S150 and PE were electrophoretically resolved on
AUT-polyacrylamide gels, transferred to nitrocellulose, and
immunochemically stained with either antiacetylated H4 antibodies or
antiacetylated H3 antibodies. Both antibodies detect the
multiacetylated forms of H4 and H3, with the antibody to acetylated H3
showing a strong preference for the highest acetylated isoforms of H3
(30). We have shown previously that highly acetylated histones are
found in chromatin fractions S150 and PE, but not in fraction P150 (7, 24). Thus, this latter fraction was not analyzed. Fig.
1 shows that the steady state levels of
highly acetylated H3 and H4 isoforms were low in fractions S150 and PE
from immature erythrocytes. However, the steady state levels of the
highly acetylated H3 and H4 isoforms in fractions S150 and PE were
markedly increased when immature erythrocytes were incubated in the
presence of sodium butyrate, a histone deacetylase inhibitor, for 60 min (Fig. 1). These results suggest that the rate of deacetylation is
so rapid in immature erythrocytes that, once dynamically acetylated H3 and H4 reach a highly acetylated state, they are rapidly deacetylated (4); the net result is a low steady state level of highly acetylated histone isoforms in untreated immature cells.
Histone Acetylation Is Required to Maintain the Unfolded
Nucleosome Structure Associated with Transcribing DNA*
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-D-ribofuranosylbenzimidazole (DRB)2 (75 µM)
(24, 25), or camptothecin (20 µM) (26) followed by a
90-min incubation with or without 30 mM sodium
butyrate.
globin
gene; pChV2.5B/H, which contains the gene coding for chicken histone H5
and flanking sequences; and pVTG412, that recognizes the 5' region of
the chicken vitellogenin gene (24).
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (52K):
[in a new window]
Fig. 1.
Level of acetylated H3 and H4 histones in
immature and mature erythrocyte chromatin fractions S150 and PE.
A, histones were acid-extracted from chromatin fractions S150
(S) or PE (P) of immature (IE) or
mature (ME) cells that were either untreated ( 
) or
incubated in the presence of sodium butyrate for 1 h (+). The
histones (9 µg) were electrophoretically resolved on an AUT-15%
polyacrylamide gel. The gel was stained with Coomassie Blue.
B and C, the proteins were electrophoretically
transferred to nitrocellulose and immunochemically stained with
antiacetylated H3 (B) or antiacetylated H4 (C)
antibodies. The mono-, di-, tri-, and tetra-acetylated isoforms of H4
are marked as 1, 2, 3, and
4, respectively. Note that the content of H1 and H5 in
fraction S150 is typically lower than that of the other chromatin
fractions (65).
Mercury-Agarose Column Fractionation of Salt-soluble Immature Erythrocyte Chromatin-- The chromatin fraction S150 was isolated from chicken immature erythrocytes that were untreated or incubated with sodium butyrate for 90 min. The S150 chromatin fractions were applied to mercury-agarose columns (Fig. 2). Analysis of the proteins released from the mercury column with DTT showed that histones were present in the S150 chromatin fraction from butyrate incubated cells (Fig. 2C), but absent in the S150 fraction from untreated cells (Fig. 2B). The major proteins in the latter fraction were the cysteine-containing high mobility group proteins 1 and 2 (Fig. 2B). These results suggested that unfolded nucleosomes were absent or at very low levels in the S150 chromatin fraction from untreated immature erythrocytes. However, the unfolded nucleosome appeared to be present in immature erythroid cells that were incubated with butyrate. This result suggested that when deacetylation of dynamically acetylated histones was halted, the transcriptionally active nucleosomes was prevented from reverting to a thiol-nonreactive state.
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-globin (not shown) DNA, but
not repressed vitellogenin DNA (Fig. 4).
These results show that mercury-bound nucleosomes were associated with
transcriptionally active DNA.
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Hydroxylapatite Dissociation Chromatography Analysis of Immature Erythrocyte-soluble and Nuclear Skeleton-associated Chromatin Labeled with [3H]Iodoacetic Acid-- The characterization of the thiol-reactive, unfolded nucleosome of transcribing chromatin described by Allfrey and colleagues has been done solely with soluble chromatin fragments. However, most transcribing chromatin is associated with the residual nuclear material (fraction PE), the nuclear skeleton. In immature erythrocytes approximately 75% of the transcribed DNA sequences are associated with the nuclear skeleton (7). To test the reactivity of the thiol group (Cys-110) of H3 in chromatin from butyrate-incubated immature erythrocytes, fraction SE and PE chromatin fragments were incubated with [3H]iodoacetic acid. Fig. 5, B and C, shows that H3 was labeled in SE and PE chromatin. In contrast to the results obtained with fraction SE and PE chromatin, the H3 of chromatin fraction P150, which contained repressed DNA, was not labeled (data not shown).
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Hydroxylapatite Dissociation Chromatography Analysis of Mature Erythrocyte Chromatin Labeled with [3H]Iodoacetic Acid-- Transcriptional elongation is arrested in mature erythrocytes. To find if hyperacetylating histones associated with transcriptionally competent chromatin (3, 34) was sufficient to observe a thiol-reactive H3, chromatin fractions S150 and PE from mature cells untreated or incubated with sodium butyrate were labeled with [3H]iodoacetate. Figs. 8 and 9 show that labeled H3 was not observed in the mature erythrocyte S150 and PE chromatin fractions. These results and those with immature erythrocyte chromatin suggest that histone acetylation is required but not sufficient for formation and/or stabilization of nucleosomes with thiol-reactive H3.
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Effect of Inhibitors of Transcription on the Thiol Reactivity of H3 in Nucleosomes-- The absence of thiol-reactive nucleosomes in butyrate-treated mature erythrocytes indicates that both transcription elongation and highly acetylated histones are required to form and maintain the unfolded nucleosome conformation. To test whether inhibition of transcription elongation has an effect on the H3 thiol reactivity of immature erythrocyte nucleosomes, immature erythrocytes were incubated with inhibitors of transcription before the addition of sodium butyrate. Camptothecin is an inhibitor of topoisomerase I and has been reported to stimulate initiation but inhibit elongation by RNA polymerase II (26, 35, 36). A thiol-reactive H3 was not detected in the SE and PE chromatin of immature cells treated with DRB or camptothecin followed by butyrate (Figs. 8 and 10) (25). These results show that both highly acetylated histones and elongation are needed to detect the thiol-reactive H3 in immature erythrocyte chromatin.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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We show that highly acetylated histones are required to maintain the unfolded, thiol-reactive structure of transcribing nucleosomes. The thiol-reactive nucleosome is not detected in the chromatin of transcriptionally active immature erythrocytes where the steady state level of highly acetylated histones is low. But when deacetylation of the highly acetylated histones is prevented by incubating immature erythrocytes with histone deacetylase inhibitors, the thiol-reactive nucleosome is detected. The rate of deacetylation in immature erythrocytes is such that the highly acetylated H3 and H4 isoforms are short lived (4). In contrast, the thiol-reactive nucleosome is detected in the chromatin of mammalian cells without the use of histone deacetylase inhibitors (37) (unpublished observations). These observations suggest that the net activities of the histone acetyltransferases and deacetylases decide the longevity of the unfolded nucleosome. In transcribing regions where the rate of histone deacetylation exceeds the rate of acetylation, the unfolded nucleosome structure will be short lived and will rapidly revert to a thiol nonreactive state following passage of the RNA polymerase. In yeast the converse is the case. The unfolded nucleosome structure associated with a specific gene persists well after the transcription of that gene has been arrested (38). The rates of histone acetylation and deacetylation are very slow in yeast, but the high steady state of highly acetylated histones argues that the rate of histone acetylation exceeds the rate of deacetylation (39, 40).
Histone acetylation, however, is not sufficient to generate the unfolded nucleosome structure; transcription elongation is required. Thiol-reactive nucleosomes could not be found in the chromatin of chicken mature erythrocytes. Transcription may be initiated in these mature cells, but RNA polymerases are paused at the 5' end of the transcribed genes (41, 42). The treatment of immature erythrocytes with camptothecin may mimic the mature erythrocyte situation, that is, transcription initiation occurs but elongation does not (26). Thus, although dynamic histone acetylation and initiation are occurring in these cells at transcriptionally competent/active loci, without elongation the thiol-reactive nucleosome is not formed.
Our results suggest that unfolded transcribing nucleosomes are associated with highly acetylated H3-H4 tetramers. In agreement with the studies of Allfrey and colleagues (19), we found that immature erythrocyte chromatin fragments bound to mercury agarose are enriched in highly acetylated H3 and H4. Furthermore, Sterner et al. (20) showed that the thiol-reactive H3 of unfolded mammalian nucleosomes is hyperacetylated. Acetylation of H3 and H4 may maintain the unfolded nucleosome conformation by breaking interactions between the histone N-terminal tail and nucleosomal DNA. The N-terminal tail of H4 is not mobile in nucleosomes, and there is evidence that the H4 N-terminal tail makes intranucleosomal contacts (43). Indeed, His-18 in the N-terminal region of H4 cross-links to nucleotides 57, 66, and 93 from the 5' end of nucleosomal DNA (44, 45). This position in the nucleosomal DNA corresponds to where the nucleosomal DNA is sharply bent or kinked. In active gene chromatin and in chromosomal domains containing hyperacetylated histones, the cross-linking between His-18 of H4 and nucleosomal DNA in situ is greatly diminished (44, 46, 47). Moreover, site 60 from the end of nucleosomal DNA of hyperacetylated nucleosomes has an increased susceptibility to DNase I (48). These observations strongly suggest that acetylation at lysines located in N-terminal tail of H4 may have important functions in altering histone-DNA contacts and nucleosome structure. Further, hyperacetylation of the H3-H4 tetramer reduces the linking number change per nucleosome, that is, negative DNA supercoils constrained in unmodified nucleosomes are partially released in nucleosomes with hyperacetylated histones (49, 50).
The destabilizing effect that histone acetylation has on H3-H4 tetramer-DNA interactions in transcribing nucleosomes is seen in hydroxylapatite dissociation chromatography. The H3 of thiol-reactive nucleosomes dissociated from hydroxylapatite bound S150 or SE chromatin after the dissociation of the H2A-H2B dimers but before the bulk of the H3-H4 tetramers. In a previous study we monitored the dissociation of labeled ([3H]acetate) dynamically acetylated histones from hydroxylapatite-bound chromatin of immature erythrocytes. The dissociation of the labeled ([3H]acetate) highly acetylated H3-H4 tetramers coincided exactly with that of labeled ([3H]iodoacetate) H3 (27). These observations with SE chromatin from chicken immature erythrocytes suggest that the interaction between highly acetylated H3-H4 tetramer and DNA of transcribing nucleosomes is weaker than that of typical nucleosomes. Analysis of mercury-agarose bound nucleosomes by electron spectroscopic imaging also indicated that the H3-H4 tetramer of unfolded nucleosomes is disrupted (18). The disruption of the tetramer in transcribing nucleosomes may facilitate subsequent rounds of elongation.
Transcribing chromatin is associated with the insoluble residual nuclear material (fraction PE) which contains the nuclear matrix. The PE chromatin from butyrate treated immature erythrocytes had 76% of the active DNA and 74% of the acetate-labeled tetraacetylated H4 (7). Further, the PE fraction retained 75-85% of the nuclear histone acetyltransferase and histone deacetylase activity (7, 13). The thiol-reactive nucleosome was detected in PE chromatin of butyrate-treated immature erythrocytes, but not in the PE chromatin of untreated immature erythrocytes or mature erythrocytes. Further, inhibition of transcription with camptothecin or DRB prevented the detection of the unfolded nucleosome. Thus, the results obtained with PE chromatin were similar to those observed with S150 or SE chromatin; both hyperacetylated histones and elongation are required to detect the unfolded nucleosome in fraction PE.
There is increasing evidence that the transcription machinery is associated with the nuclear matrix and that for chromatin to be transcribed it is spooled through the anchored large RNA polymerase complex (51-54). We have proposed that histone acetyltransferase and deacetylase are localized in these transcription foci (13, 14). Recent studies show that coactivators (CBP/p300, ACTR, and GCN5) and proteins associated with TATA-binding protein (TAFII250) have histone acetyltransferase activity (55-60). Histone deacetylases (HDAC-1 and HDAC-2) are associated with corepressors (mSin3A and N-CoR) and the nuclear matrix bound transcription factor YY1 (61-64). These studies suggest that the basal transcription machinery and transcription factors recruit histone acetyltransferases and histone deacetylases to sites of transcription at the nuclear matrix. Nucleosome structure will be perturbed when the chromatin fiber is passed through the fixed RNA polymerase (transcriptosome) (53). While in a highly acetylated state, the unfolded nucleosome structure will persist, helping subsequent rounds of transcription. Our results are consistent with the idea that the nucleosome is a dynamic structure conforming its structure to facilitate movement of chromatin through the RNA polymerase II elongation complex, with dynamic histone acetylation having a major role in modulating the unfolded structure of transcribing nucleosomes.
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FOOTNOTES |
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* This work was supported in part by the Medical Research Council of Canada (MRC) Grant MT-9186 (to J. R. D.), a United States Army Breast Cancer Postdoctoral Research Fellowship DAM17-96-1-6269 (to L. T. H.), and a MRC Senior Scientist Award (to J. R. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Manitoba R3E 0W3, Canada. Tel.: 204-789-3215;
Fax: 204-789-3900; E-mail: Davie{at}cc.umanitoba.ca.
1 J. A. Ridsdale, P. Fredette, and J. R. Davie, unpublished observations.
2
The abbreviations used are: DRB,
5,6-dichloro-1--D-ribofuranosylbenzimidazole; DTT,
dithiothreitol.
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REFERENCES |
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References
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