From the Laboratoire de Biologie Moléculaire du Cycle Cellulaire, INSERM U309, Institut Albert Bonniot, Faculté de Médecine, Domaine de la Merci, 38706 La Tronche Cedex, France
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ABSTRACT |
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Individual anti-H10 monoclonal antibodies were screened in an immunolocalization assay to isolate clones able to recognize H10 in a differentiation-dependent manner using a murine erythroleukemia cell line. Two clones were selected, one recognizing H10 only in differentiating cells (clone 27 antibody), and the other recognizing the protein constitutively (clone 34 antibody). Both antibodies recognized a restricted region of the protein located at the N-terminal part of the globular domain. Amino acids 24-30, essential for the recognition of the protein by the clone 27 antibody, are extremely conserved in all known H10-like proteins from sea urchin to human. Within these residues, proline 26, responsible for a bend in this region, plays a particularly important role in the epitope recognition. The region involved in the protein recognition by clone 34 antibody is larger and encompasses amino acids 20-30. However, proline 26 does not play an essential role in the structure of this epitope. Detailed analysis of the differential recognition of H10 in chromatin during cell differentiation and proliferation suggests that the modification of chromatin structure as well as that of H10 conformation can account for this effect. Indeed, in vitro study of H10-four-way junction DNA interaction showed that the N-terminal tail domain of the protein can influence the recognition of H10 by these antibodies when the protein interacts with DNA. The two monoclonal antibodies described here therefore seem to be valuable tools for investigating fine modulations in chromatin structure and the concomitant changes occurring in the conformation of the protein.
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INTRODUCTION |
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Linker histone is an abundant basic protein present in almost all eukaryotes. The protein is involved in the formation of higher order structure in the chromatin (1) and the maintenance of the overall chromatin compaction (2). In general, linker histone has a tripartite structure: a central globular domain flanked by N- and C-terminal tail domains (3). The globular domain binds the linker DNA and interacts with the nucleosome where DNA enters and exits the nucleosome (4, 5). Unlike core histones, linker histones diverge significantly in sequence and structure (6, 7).
Numerous developmentally regulated variants of linker histone have been defined. These variants can be subdivided in three major groups in vertebrates as a function of their expression during development and cell differentiation (8). First, an embryonic form of linker histone is present during the oogenesis and the early development in amphibians. Replication-dependent types are present in all tissues during the life of the organism, and finally the differentiation-specific group accumulates in differentiating cells. Some members of this later group are tissue- and species-specific, like histones H5 and H1t. Others, like histone H10, are widely expressed in many tissues and in almost all vertebrates (9).
Previously, we have shown that there is a tight correlation between the type of linker histone expressed and the proliferative capacities of cells during early Xenopus development. Histone H10 appears relatively late and concomitant with a dramatic decrease in the cell proliferation during the tail bud-tadpole transition period (10). Therefore, crucial periods in development can be characterized by a transition in the linker-histone variants within chromatin. Nothing is known concerning the role of these variants in specific organization of the chromatin structure. Immunolocalization of these proteins using specific polyclonal and monoclonal antibodies provided interesting information concerning the distribution of a given linker histone variant in the nucleus (11-13). However, chromatin organization is extremely dynamic and is subject to permanent remodeling. One of the most striking examples of this phenomenon is early embryonic development. Indeed, transition periods have been defined during development that are characterized by the modification of both chromatin constituents and the proliferative capacities of cells (8). Moreover, later during development and in adult tissues, chromatin remodeling continues as adult type linker histones accumulate in cells (9). It is therefore of great importance to understand the nature of these remodeling processes and to evaluate their role in the expression of specific genetic programs.
The aim of this work was the identification of monoclonal antibodies raised against histone H10, showing specific abilities in recognizing this protein in chromatin. Individual anti-H10 monoclonal antibodies were screened in an immunolocalization assay to isolate clones able to recognize H10 in a differentiation-dependent manner. Antibodies characterized in this work appeared to be probes that are useful for monitoring chromatin structure modifications occurring concomitantly with regulatory events such as the onset of a differentiation program and the arrest of cell proliferation.
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MATERIALS AND METHODS |
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Cell Culture-- Murine erythroleukemia (MEL)1 cells from clone G9, a subclone of F4NW0, were maintained in culture in minimum essential medium (Life Technologies, Inc.) containing 10% fetal calf serum (14). To induce differentiation, MEL cells in exponential growth were treated with hexamethylene-bis-acetamide (Sigma) as described previously (14). Clone 6 cells, a rat embryonic fibroblast cell line transformed by ras, were maintained in RPMI 1640 (Boehringer) supplemented with 5% fetal calf serum and glutamin 4 mM and grown in a humidified atmosphere of 95% air, 5% CO2 normally at 37 °C or shifted to 32 °C to induce the cell growth arrest (15).
Purification of Nuclei and Oligonucleosomes-- Nuclei were extracted from untreated or hexamethylene-bis-acetamide-treated MEL cells. Cells were collected and washed with phosphate-buffered saline. After centrifugation at 200 × g for 5 min, cells were lyzed in a lysis buffer containing 15 mM Tris-HCl, pH 7.4, 60 mM KCl, 15 mM NaCl, 0.65 mM spermidine, 2 mM EDTA, 0.5 mM EGTA, 0.34 M sucrose, 0.05% (v/v) Triton X-100, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride. After centrifugation at 1500 × g for 5 min, nuclei were rinsed with the same buffer lacking EDTA, EGTA, and Triton (buffer D).
To obtain chromatin, nuclei in buffer D were digested by micrococcal nuclease (Boehringer, 5 units/107 nuclei) for 7 min at 37 °C. Oligonucleosomes were fractionated on linear sucrose gradients in 1 mM phosphate buffer, pH 7, 80 mM NaCl, 0.2 mM EDTA.Preparation of Digested and Recombinant Histone
H10--
Mouse full-length histone H10
cDNA (16) or cDNAs corresponding to mutated H10
were cloned in pET expression vector (Novagen). All mutations and
deletions were performed by polymerase chain reaction. Briefly, for
N-terminal deletion mutants, oligonucleotides (33 bases) containing the
desired sequence (removing an increasing number of amino acids from the
N-terminal part of the protein, see Fig. 1) and an additional NdeI restriction site were used to amplify cDNAs by
polymerase chain reaction. The 3 primer had the sequence of the stop
codon region and an additional XhoI site. The proline-valine
mutant as well as the 24-30 deletion mutant were produced according to the method described in Refs. 17 and 18. Polymerase chain reaction
products were digested with NdeI and XhoI and
cloned, and their sequences were verified.
Antibodies-- Anti-H10 antibodies were monoclonal antibodies produced in our laboratory as described previously (19). Antibodies used in this work were from clones 27E 8E10 (clone 27 antibody) and 34B10H4 (clone 34 antibody). For immunostaining, hybridoma supernatant was used, and for gel shift assays, Igs were purified as follows. Ascites were induced in BALB/c mice after the intraperitoneal injection of hybridoma cells (5 × 106 cells/mouse). Ascitic fluid was collected and centrifuged at 3000 × g for 10 min, and the supernatant was collected. Igs were then purified as described (20). Briefly, albumin and other non-Ig proteins were first precipitated with caprilyc acid. Then Igs were precipitated by ammonium sulfate.
Anti-H1 antibodies were elicited in rabbits. Primary injection and boost were performed using H1-yeast tRNA complex (3:1 w/w). Rabbits were subcutaneously injected at multiple sites with 100 µg of ox liver H1-1 and H1-2 (1 ml phosphate-buffered saline/Freund's adjuvant, v/v). Specific IgG was successively purified by protein A-Sepharose 4B chromatography and by affinity chromatography using H1-coupled cyanogen bromide-activated Sepharose 4B.Protein Electrophoresis and Immunodetection-- H1 histones were analyzed by SDS-15% polyacrylamide gel electrophoresis (21). Proteins were transferred to a Hybond C extra membrane (Amersham Corp.) at 24 V (0.2 A) for 1 h with a semi-dry electrotransfer apparatus. The membrane was blocked in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl (Tris-buffered saline) containing 3% bovine serum albumin. The membrane was incubated with specific antibodies and then with an horseradish peroxidase-linked sheep anti-IgG. Detection was performed by enhanced chemiluminescence kit (ECL, Amersham Corp.). Oligonucleosomes fractionated on sucrose gradient were dotted on Hybond C extra membrane (10 µg of chromatin for each fraction) and subjected to immunodetection as above.
Gel Shift Assays--
The four-way junction DNA was obtained by
annealing four oligonucleotides synthesized according to the sequence
published by Teo et al. (22). The four oligonucleotides were
annealed in 10 mM Tris-HCl, 1 mM EDTA, and 50 mM NaCl, gel purified, and labeled by polynucleotide kinase
and [-32P]ATP.
Total RNA Extraction and Northern Blot Analysis-- RNAs were extracted from cells and analyzed by Northern blot by methods described by Khochbin et al. (14).
Immunostaining--
Cells were collected and fixed with 3%
paraformaldehyde in phosphate-buffered saline for 20 min at room
temperature, permeabilized with 0.25% (v/v) Triton X-100 (Sigma), then
incubated with monoclonal antibody against H10. The second
antibody was a goat fluorescein isothiocyanate-conjugated F(ab)2 fragment against mouse IgG Fc fragment (Jackson
Immunoresearch Lab). After immunolabeling, DNA was stained with
DNA-specific fluorochrome Hoechst 33258 (Sigma). When immunostaining
was carried out on nuclei, purified nuclei were obtained as described
above, resuspended in 20 mM Tris, pH 7.4, 6 mM
MgCl2 containing either 100 mM, 200 mM or 300 mM NaCl, at room temperature for 30 min then fixed with 3% paraformaldehyde in the same buffer for 1 h at +4 °C. Nuclei were then doubly stained according to the
procedure described above.
Analysis by Flow Cytofluorimetry-- The doubly stained cells were analyzed in a FACStar Plus (Becton Dickinson) using a dual laser configuration: the Hoechst fluorescence was excited at 340-360 nm by the first argon laser, and the fluorescein isothiocyanate fluorescence at 488 nm by the second argon laser. The fluorescence intensities were collected in a list mode. To determine the mean specific H10 fluorescence per cell we used ProCyt®, a computer program developed in our laboratory (available on request) (24).
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RESULTS |
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Screening of Monoclonal Antibodies for H10 Recognition in Chromatin-- Previously, we have reported the preparation of monoclonal antibodies raised against histone H10 (19). In this work we undertook a screening of these antibodies for their ability to recognize the protein in chromatin. The first screening was performed using immunodetection of the protein in undifferentiated MEL cells. This work allowed us to define two clones: one (34B10H4) able to recognize H10 in chromatin and the second one (27E8E10) not able to bind the protein in this environment (both antibodies recognizing H10 in cellular or nuclear extracts with high specificity; data not shown). For simplicity, in the text we will refer to 34B10H4 and 27E8E10, as clone 34 and clone 27 antibodies, respectively. Preliminary mapping based on the recognition of peptides obtained by partial cleavage of H10 by cyanogen bromide (cleaving the protein at methionine 31) showed that the N-terminal part of the protein is essential for recognition; neither antibody recognized the protein fragment containing the amino acids 31-193 (Fig. 1A). We therefore focused our attention on the N-terminal part of histone H10. A series of experiments were planned to map precisely the epitopes recognized by these two antibodies and to elucidate the basis for the differential recognition of the protein in chromatin.
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Precise Epitope Mapping--
Mouse H10 cDNAs
encoding the wild type protein or proteins bearing mutations affecting
the N-terminal tail and the globular domain were cloned into
prokaryotic expression vectors to obtain purified recombinant proteins
(Fig. 1B). The wild type recombinant protein was efficiently
recognized by both antibodies, and moreover, the complete deletion of
the N-terminal tail domain did not affect protein recognition (Fig.
1C, 20). However, the removal of 18 AA from
the N-terminal part of the globular domain completely abolished the
binding of these antibodies (Fig. 1C,
38). A
portion of the protein covering the AA 20-38 region therefore plays an essential role in the recognition of the protein by both
antibodies.
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Interaction of H10 with Four-way Junction DNA and the Recognition of the Target Epitope by Clone 34 and 27 Antibodies-- The high affinity binding of H1 to four-way junction DNA (23, 28) allows the study of the specific aspects of H1-DNA interactions (29). We took advantage of this model to determine how the defined target epitopes are recognized by our antibodies when H10 interacts with DNA. Conditions for a complete shift of the labeled four-way junction DNA upon the addition of recombinant H10 (wild type or mutated) were determined, excesses of the clone 27 and 34 antibodies were added to the mixture, and the shift was examined. Upon the addition of the clone 34 antibody, three different cases were observed: 1) a fraction of DNA-H10 complex interacts with the antibody and is super-shifted (Fig. 3A, WT panels, lanes +H10+Ab); 2) a fraction of DNA-H10 complex is not recognized by the antibody; and 3) a fraction is dissociated as indicated by the release of free DNA.
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Differential Recognition of H10 within Chromatin during the Induced Differentiation of MEL Cells-- MEL cells are virus-transformed erythroid precursors able to undergo a differentiation program under the action of a large variety of chemical inducers (30). We used this differentiation model to monitor H10 recognition by our antibodies during cell differentiation. Uninduced MEL cells or cells treated with the inducer (4 mM hexamethylene-bis-acetamide) for 6, 8, 16, 24, 32, and 48 h were fixed, and the immunofluorescence was monitored by flow cytofluorimetry after immunostaining with clone 27 and 34 antibodies. In uninduced MEL cells, whereas H10 is efficiently recognized by the clone 34 antibody, the protein is not recognized by clone 27 antibody (Fig. 4A, 0 h). In these cells, clone 27 antibody-related immunofluorescence corresponds to the background fluorescence, which is observed when anti-H10 antibody is omitted (not shown).
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Modification of the H10 Recognition during the P53-mediated Arrest of Cell Proliferation-- Clone 6 cells are rat embryonic fibroblasts transformed by ras and a thermosensitive P53 mutant. At 37 °C, P53 is in a mutated conformation that is responsible for the appearance of a transformed phenotype. At 32 °C, P53 exhibits the property of the wild type protein and triggers an arrest of cell proliferation (15). We used this system to monitor H10 accessibility during this process. A flow cytofluorimetric analysis of H10 immunolabeling using clone 34 antibody was performed (Fig. 6A). H10 was detected by indirect immunofluorescence (y axis) and DNA by Hoechst fluorescence (x axis). DNA fluorescence reflects the position of cells in the cell cycle (G1 cells are around channel 60, G2 cells are found around channel 120, and S phase cells are in between). A general increase of H10 immunofluorescence is observed during the cell cycle indicating the normal doubling of cell constituents when cells accomplish DNA replication and enter the G2/M phases of the cell cycle (Fig. 6A, panel 37 °C). However, 8 h after the transfer of cells at 32 °C, a clear increase in the H10 immunofluorescence intensity was observed, specifically visible in the G2/M cell populations (32 °C panel, dot plot representation, compare 0 and 8 h). It is precisely in this phase of the cell cycle that the first accumulation of cells is observed (Fig. 6A, note the accumulation of cells in the G2/M phase of the cell cycle, 32 °C panel, 8 h histogram). After 16 h at 32 °C, these cells enter the G1 phase and stop proliferating. An increase in the H10 immunofluorescence is visible in these arrested cells (panel 32 °C, lane 16 h). After 24 h at 32 °C, almost all cells are in the G0/G1 phase of the cell cycle, and a clear increase of the H10 related immunofluorescence intensity is observed in these cells compared with the control cells kept at 37 °C (panel 32 °C, lane 24 h). Clone 27 antibody did not recognize H10 either in proliferating cells or in arrested cells (data not shown).
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Differential Pattern of H10 Immunolocalization by Clone 27 and Clone 34 Antibodies-- To know if the differential recognition of H10 by these antibodies correlates also with a specific pattern of immunodetection, we performed a microscopic analysis of immunolabeled nuclei. Xenopus embryos were first used in this experiment to examine the situation in an in vivo context. H10 accumulates relatively late during the Xenopus development, and the first detectable accumulation of the protein is tissue-specific, observed in the nervous tissue, somites, and the cement gland. Later during development, at tadpole stage, the accumulation of the protein was observed in many different tissues (10). The analysis of the immunolabeled cells observed on a section of the cement gland (for example, Fig. 7A, arrowhead) shows that nuclei are immunolabeled with both antibodies and that, moreover, foci could be observed in clone 27 antibody-immunolabeled nuclei (Fig. 7B, bottom left panel) in contrast to a relatively homogenous labeling for nuclei labeled by the clone 34 antibody. Nuclei of cells forming the neighboring tissue are negative for H10 detection (Fig. 7B, compare the anti-H10 column with the Hoechst column). The same pattern of immunofluorescence was observed when we examined the pattern of clone 27 and 34 antibody immunolabeling in nuclei of differentiated MEL (data not shown). These observations suggest that the recognition of H10 by clone 27 antibody occurs only on restricted regions that could be sites of specific chromatin remodeling.
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DISCUSSION |
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In this work we have precisely mapped a region of histone H10 located at the entry of the globular domain and involved in the recognition of the protein by two monoclonal antibodies. One of them, clone 34 antibody, recognizes the protein dependent on the AA 20-30 region. Recognition by the second antibody (clone 27 antibody) has been shown to be dependent on only 7 AA (AA 24-30) within this region. Moreover, an essential role of the proline 26 has been illustrated by site-directed mutagenesis. Indeed, the replacement of this proline by a valine completely abolished the recognition of the protein. These data suggest that the proline-mediated structure is important for the recognition of the protein by this antibody. Interestingly, this antibody does not recognize H10 within the chromatin of undifferentiated MEL cells. However, after the commitment in the differentiation program, H10 becomes recognizable. The AA 20-30-dependent recognition of the protein by clone 34 antibody is efficient in uninduced as well as in differentiated MEL cells.
Two explanations can be proposed for the differential recognition of H10 by the clone 27 antibody during cell differentiation. First, modification of the chromatin structure in differentiated cells can render H10 accessible to this antibody. However, clone 34 antibody, for which the recognition of H10 is also highly dependent on the AA 24-30 region, binds the protein in uninduced cells as well as in differentiated cells. Therefore, a simple modification of the accessibility of the 24-30 region cannot satisfactorily account for the differentiation-dependent reactivity of H10 toward the clone 27 antibody. The proline-valine replacement experiment suggests that recognition by clone 27 antibody could be dependent on a structure in the AA 24-30 region. Therefore, a modification of the structure of the N-terminal domain of H10 occurring during the induced differentiation of MEL cells could be a reason for the observed differential recognition by the clone 27 antibody.
The importance of the N-terminal tail of H10 in the
recognition of the protein by our antibodies is also suggested by
analyzing their ability to recognize H10-four-way junction
DNA complex in vitro. At least two different kinds of
H10-DNA complexes have been found. One is able to interact
with the antibody and is supershifted in a gel retardation assay. In
the rest of the population of H10-DNA complexes, the
addition of the antibody creates a competition between the antibody and
DNA for interaction with H10. This competition is
accompanied with a release of DNA from the complex. The observed
displacement of the DNA is enhanced when the N-terminal tail is
shortened. The removal of 10 AA or the whole N-terminal tail domain
(20) facilitated greatly the displacement of DNA by the clone 34 antibody. This facilitated displacement could be due to a decrease in
the strength of DNA-protein interaction or to a better recognition of
the protein by the antibody. The first possibility is unlikely because
the removal of 10 AA, although it dramatically increases the
dissociation of the H10-DNA complex, does not eliminate any
of the 58 lysine/arginine residues present in the protein or any of the
conserved residues showed to be involved in the interaction of H1 with
DNA (29). Moreover, the shortening of the N-terminal tail domain does
not affect the clone 27 antibody-dependent dissociation of
the complex. However, it influences the formation of the ternary
complex.
These data suggest that the N-terminal tail domain of histone H10, which is nonessential for the recognition of the free protein by our antibodies, can influence the protein recognition when it interacts with DNA and strengthens the possibility of a differential recognition of the protein in chromatin due to a modification of the N-terminal tail conformation during critical stages of cell life.
A different pattern of the immunolabeling is also observed when we compared clone 34 and clone 27 antibody immunostained nuclei. Clone 27 antibody is able to reveal foci of immunoreactivity within the nuclei. Under the same conditions clone 34 antibody shows a more homogenous nuclear labeling. The appearance of foci after the immunolabeling by clone 27 antibody is observed in different Xenopus tissues, as well as in cells in culture (not shown). Because in MEL cells, labeling by clone 27 antibody is differentiation-dependent, one can assume that these foci of H10 immunolabeling correspond to sites of specific chromatin remodeling, rendering the N-terminal part of H10 recognizable by the clone 27 antibody.
The salt treatment experiment (Fig. 4C) showed that the recognition of H10 by clone 34 antibody is more sensitive to modification of chromatin structure than that of clone 27 antibody. Clone 34 antibody shows an enhanced H10 recognition after the P53-mediated arrest of cell proliferation, whereas in these cells (cycling or arrested), H10 is not recognized by clone 27 antibody. These observations suggest that chromatin remodeling events of a different nature are associated with cell arrest and differentiation.
Several reports described the use of monoclonal and polyclonal
antibodies raised against different parts of histone H1, H5, and
H10, as well as the use of immobilized proteases to
investigate the accessibility of the linker histones in chromatin. The
majority of these works showed a lower accessibility of the globular
domain compared with the N- and C-terminal tail domains of linker
histone in the chromatin (32, 33). Therefore, recognition of H1s by antibodies raised against the globular domain of the protein are expected to be much more sensitive to different chromatin remodeling events than that of antibodies raised against tail domains. The work
presented here shows that modifications of the chromatin structure
occur at precise periods during different important cellular events,
such as the commitment of cells in a particular differentiation program
or the arrest of cell proliferation. The clone 27 antibody can
immunolabel H10 in MEL cells precisely at the onset of
-globin gene expression during the induced differentiation. This
observation shows that a modification of the chromatin structure occurs
concomitant with the reinitiation of erythropoiesis. The clone 34 antibody allowed us to show a modification of the chromatin structure
occurring during a restricted period of the cell cycle before the
arrest of cell proliferation. The described antibodies therefore seem to be valuable tools for monitoring precise timing of chromatin remodeling associated with different regulatory events controlling the
cell fate and enable investigation of these modifications in more
detail.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Jean Jacques Lawrence, the head of INSERM U309, for supporting this work and to Drs. Stefan Dimitrov and Kym Duncliffe for critical reading of the manuscript. We are also grateful to Dr. C. Gache for providing us with sea urchins.
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FOOTNOTES |
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* 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. Tel.:
33-4-76-54-95-83; Fax: 33-4-76-54-95-95; E-mail:
khochbin{at}ujf-grenoble.fr.
1 The abbreviations used are: MEL, murine erythroleukemia; AA, amino acid(s).
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REFERENCES |
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