Lysine residues of Epstein–Barr virus-encoded nuclear antigen 2 do not confer secondary modifications via ubiquitin or SUMO-like proteins but modulate transcriptional activation

Annette Hille1, Akua Badu-Antwi1, Daniela Holzer1 and Friedrich A. Grässer1

Institut für Mikrobiologie und Hygiene, Abteilung Virologie, Haus 47, Universitätskliniken, 66421 Homburg/Saar, Germany1

Author for correspondence: Friedrich Grässer. Fax +49 6841 162 3980. e-mail graesser{at}uniklinik-saarland.de


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Epstein–Barr virus nuclear antigen 2 (EBNA2) is essential for transformation through activation of viral and cellular genes. Within 487 residues, EBNA2 contains six lysine (K) residues (positions 335, 357, 359, 363, 366 and 480), which were mutated to arginine (R) residues, either individually or in combination, and tested for subcellular localization, mobility by SDS–PAGE and transactivation of three promoters. All mutants featuring the K480R mutation within the nuclear localization signal were partially cytoplasmic with a reduced level of transactivation of the latent membrane protein 1 (LMP1) promoter (-327 to +40). The K366R mutation also showed a decrease in transactivation of a promoter consisting only of 12 recombination signal-binding protein-J{kappa}-binding sites, while all mutants with the K335R exchange showed a markedly elevated transactivation with the -327 to +40 construct and all mutants showed slightly reduced transactivation with a -634 to +40 LMP1 promoter. None of the mutants exhibited altered migration in SDS–PAGE, excluding secondary modification, i.e. through SUMO-like proteins.


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Epstein–Barr virus (EBV) is associated with various tumours and transforms primary B cells in vitro (reviewed by Rickinson & Kieff, 1996 ). EBV nuclear antigen 2 (EBNA2) is essential for the induction of the transformed phenotype (Kempkes et al., 1995 ) by transactivation of viral [latent membrane protein 1 (LMP1) and LMP2] as well as cellular (CD21, CD23 and c-fgr) genes (reviewed by Bornkamm & Hammerschmidt, 2001 ). It targets responsive promoters by binding to recombination signal-binding protein-J{kappa} (RBP-J{kappa}), the ets family proteins Spi-1/Spi-B and the hnRNP-D/AUF1 protein (Laux et al., 1994a ; Ling et al., 1994 ; Zimber-Strobl et al., 1994 ; Fuentes-Panana et al., 2000 ). Activation of the LMP1 promoter also involves an ATF/CRE element (Sjøblom et al., 1998 ). The two subtypes of the virus differ in their ability to transform cells (Rickinson et al., 1987 ): type 1 EBV (encoding EBNA2A) induces LCLs with higher efficiency than type 2 EBV (encoding EBNA2B) due to differences in EBNA2 (Hammerschmidt & Sugden, 1989 ). EBNA2A consists of 487 amino acids (aa) and important functional domains are conserved within EBNA2A, EBNA2B and the herpesvirus papio (HVP) EBNA2 (Cohen et al., 1991 ; Ling et al., 1993 ; Ling & Hayward, 1995 ). Regarding transformation and transactivation, eight prolines, the tryptophan–tryptophan–proline324 motif (Cohen et al., 1991 ; Ling & Hayward, 1995 ), which confers binding to RBP-J{kappa} and parts of the conserved region between residues 361 and 425 as well as the acidic domain around aa 460, are essential, while the glycine–arginine repeat is critical but not essential (Tong et al., 1994 , 1995 ). The conserved C terminus can be deleted, although it contains a nuclear localization signal (NLS), most likely because a second NLS encompassing the glycine–arginine repeat within aa 341–355 can substitute for the C-terminal NLS (Ling et al., 1993 ).

Within 487 residues, EBNA2A contains six lysine (K) residues (positions 335, 357, 359, 363, 366 and 480), of which residues 366 and 480 are conserved within EBNA2A, EBNA2B and the HVP homologue, while K359 is conserved between EBNA2A and EBNA2B (Ling et al., 1993 ). EBNA2A has a calculated molecular mass of about 52 kDa but migrates with an apparent molecular mass of 85 kDa in SDS–PAGE. This discordant behaviour cannot be attributed to phosphorylation (Grässer et al., 1992 ) but could be due to the presence of about 30% proline residues. Another possibility might be a covalent modification via lysine residues of EBNA2A with molecules like ubiquitin (Bradbury, 1992 ), SUMO (Mahajan et al., 1997 ; Saitoh & Hinchey, 2000 ) or a SUMO-related protein(s) (Hochstrasser, 2000 ). In particular, modification of certain proteins by SUMO-1 is necessary for subcellular localization (Mahajan et al., 1997 ); for instance, modification by a SUMO-related protein might be used to direct EBNA2 to certain target proteins or even to the nucleus. To test for SUMO-1 modification, EBNA2 was immunoprecipitated from EBV-positive Raji cells using EBV-negative BJAB cells as a control (Kremmer et al., 1995 ); the resulting blot, however, only yielded a signal with EBNA2-specific monoclonal antibodies (mAbs) but not with a commercially available SUMO-1 specific antiserum (Santa Cruz). Likewise, a SUMO-1-specific goat serum (a kind gift of Frauke Melchior, Max-Planck Institute, Munich) did not detect a signal of SUMO-modified EBNA2 when EBNA2 immunopurified from Raji cell extracts was tested (data not shown). Since at least three additional SUMO-1-related proteins (Melchior, 2000 ) that might also modify EBNA2 exist, we chose to mutate the lysine residues of EBNA2 to study this question.

To demonstrate that the mutation of lysine residues does not change the apparent molecular mass of EBNA2A, the lysine residues of EBNA2 were mutated to arginine, either individually or in combination, as shown in Fig. 1(a), and tested for expression in 293gp cells and in EBV-negative BJAB and BL41 cells. In addition, a serine to alanine exchange within the C-terminal NLS (S479,485->A) was generated. Mutation was achieved by site-directed mutagenesis, which introduced a new restriction site in addition to the desired mutation, using Pfu Turbo DNA Polymerase (Stratagene). Mutations were verified by DNA sequencing. EBNA2 was expressed from vectors pSG5 (Stratagene) or pEGFP-C1 (Clontech), either untagged or with an enhanced green fluorescent protein (EGFP) tag, respectively. Briefly, 106 293gp cells were transfected with 5 µg of expression vector by the calcium phosphate method, 107 B lymphocytes were electroporated with 10 µg EBNA2 plasmid, 4 µg reporter plasmid and 1 µg pEGFP vector (when appropriate) at 950 µF and 250 V with a Bio-Rad Gene Pulser, exactly as described previously (Voss et al., 2001 ). Western blot analysis of transfected 293gp cells using a set of non-fusion EBNA2 constructs (Fig. 1b) showed that all constructs expressed EBNA2 to comparable levels, while the EBNA2–EGFP fusion proteins varied somewhat in their expression level. As shown in Fig. 1(c), none of these constructs, including the EBNA2 K335–480R construct (all lysine residues mutated to arginine), exhibited a change in mobility following SDS–PAGE, indicating that secondary modifications, when present, were not conferred to EBNA2 through the lysine residues, i.e. linking SUMO-like peptides or ubiquitin through isopeptide bonds. In the EGFP–EBNA2 K335–480R construct, all lysine residues were mutated and the N-terminal NH2 group of EBNA2 was also masked by fusion to EGFP, thus excluding a theoretical modification through the N-terminal NH2 group.



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Fig. 1. (a) Schematic representation of the EBNA2 protein. The mutations of the different lysine (K) to arginine (R) residues are indicated below the diagram, other regions important for function are shown above the diagram. (b) Expression of EBNA2 mutants as non-fusion proteins using vector pSG5 in 293gp cells. Cell extracts were analysed by Western blotting using the EBNA2-specific mAb R3 (Kremmer et al., 1995 ). Mutations are indicated below the blot. The position of the molecular mass marker proteins (in kDa) is indicated on the left. (c) Expression of EGFP-tagged EBNA2 mutants in BJAB cells. Detection of EBNA2 by Western blot was as described in (b).

 
To investigate the subcellular localization of the lysine mutants of EBNA2, two NLS, one corresponding to a ‘canonical’ NLS at the extreme C terminus and one between aa 341 and 355, were described (Cohen et al., 1991 ; Ling et al., 1993 ). EGFP–EBNA2 mutants (3 µg) were transfected into 106 HeLa or 293gp cells using Superfect (Qiagen) with efficiencies ranging from 10 to 20%. When K480 within the C-terminal NLS was mutated individually, the resulting EGFP–EBNA2 K480R construct exhibited both diffuse cytoplasmic as well as diffuse nuclear staining, with about 5% of cells showing only a nuclear signal. Likewise, all additional mutants that contained the K480R exchange showed nuclear plus cytoplasmic staining, again with about 5% of the cells yielding only nuclear signals. In no case staining was found to be exclusively cytoplasmic. Conversely, all mutants in which K480 was left unchanged were exclusively nuclear. Mutation of the serine residues at positions 479 and 485 to alanine in the C-terminal NLS had an even more pronounced effect on localization than mutation of K480 (Fig. 2d), in that we observed a diffuse nuclear as well as cytoplasmic stain in virtually all transfected cells. Also, as observed for wild-type EBNA2, the nucleoli were not stained with any mutant (Fig. 2b) (data not shown). The EBNA2 constructs without the EGFP region showed a similar subcellular distribution in BHK or 293gp cells when stained using EBNA2-specific mAbs (data not shown). If sumoylation was the critical step for nuclear translocation, than the K480R mutation should yield a stronger cytoplasmic signal than any other substitution within the NLS.



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Fig. 2. Subcellular localization of EBNA2 lysine mutants. HeLa cells were transfected with the EGFP–EBNA2 constructs; nuclei were visualized using DAPI (2,4-diamidino-2-phenylindole). Immunofluorescence results are shown on the left panel; the EBNA2 construct is indicated for each stain. The right panel shows the corresponding nuclear stain of the same cells using DAPI.

 
In an attempt to identify the nuclear transporter for EBNA2, a two hybrid screen was carried out in Saccharomyces cerevisiae, strain Y190, employing the Matchmaker Two-Hybrid System 2 (Clontech) according to the manufacturer’s manual. For this purpose, EBNA2 without the acidic domain (EBNA2{Delta}aa437–477) in vector pAS2-1 was used as bait in a two hybrid screen of a human lymphocyte-derived cDNA library (Clontech). Interacting clones were segregated with cycloheximide and mated with yeast strain Y187 containing pAS2-1, the bait plasmid pAS-EBNA2{Delta}aa437–477 or the bait plasmid without an NLS (pAS-EBNA2{Delta}aa437–487), respectively. Of the interacting proteins, none was specific only for the construct featuring the NLS. Thus, we were not able to identify a nuclear transporter by this approach. One of the clones isolated contained part of the DP103 protein (DDX20/Gemin 3), previously identified by our laboratory as an EBNA2-interacting factor (Grundhoff et al., 1999 ), showing that the bait plasmid was in a biologically active conformation. We also tested the nuclear import adapters Rch1{alpha}2, NPI-1{alpha}1, importin {alpha}3 and karyopherin {alpha}3 for an interaction with the EBNA2 bait pAS-EBNA2{Delta}aa437–477 in the yeast two hybrid system but were not able to detect binding to EBNA2.

EBNA2 transactivation was tested either with the EBNA2-responsive, viral LMP1 promoter from position -634 to +40, pgLRS(-634)CAT (Sjøblom et al., 1998 ), the LMP1 promoter LLO from position -327 to +40 (Laux et al., 1994b ) or a multimerized RBP-J{kappa}-binding site in front of a minimal promoter driving the firefly luciferase gene pGA981-16 (Strobl et al., 1997 ) in conjunction with the appropriate control reporter plasmids. We assayed both the non-fusion EBNA2 mutants as well as the EGFP–EBNA2 mutants for transactivation in BJAB cells; in the former case, a plasmid expressing EGFP was co-transfected to normalize FACS analysis for transfection efficiency. The amount of EBNA2 used was chosen to be below the saturation level, as a titration curve had shown that maximal activity of EBNA2 was reached at 10–12 µg DNA/107 cells (data not shown). Thus, 107 BJAB cells were transfected by electroporation with 2 µg promoter plasmid, 4 µg EGFP–EBNA2 vector or 4 µg EBNA2 construct in conjunction with 1 µg EGFP expression plasmid. After 48 h, 106 cells were harvested and luciferase or CAT activity was determined. With the remaining cells, FACS or Western blot analysis was performed to estimate transfection efficiency. Both kinds of constructs yielded comparable results. Because normalization can be carried out without co-transfection of the EGFP vector, a larger series of transfections employing the EBNA2–EGFP constructs was performed to allow for better statistical analysis.

Activation of the LMP1 promoter by EBNA2 is mediated through both RBP-J{kappa} and Spi-1/Spi-B. We chose to test the influence of the lysine residues first using a promoter construct that features 12 RBP-J{kappa}-binding sites in front of a minimal promoter together with the luciferase gene (Strobl et al., 1997 ). The activation by wild-type EBNA2 was set to a relative level of 100% and compared to the activity obtained with the mutants as shown in Fig. 3(a). Exchange of only the conserved lysine residues, K480 or K366, slightly decreased activation. Since these two mutants were expressed at a lower level than wild-type EBNA2, this reduction of specific transactivation potential was relatively small, in line with previous results that deletion of the C-terminal NLS had been shown to increase the expression of the LMP1 protein (Cohen et al., 1991 ). In contrast, all combinations that included the K335R mutation stimulated the expression of the LMP1 promoter as compared to wild-type EBNA2. When all but the C-terminal lysine residue were mutated, we observed a 1·5-fold stimulation in the expression of the LMP1 promoter and the mutation with all lysine residues (EBNA2K335–480R) resulted in an approximately 2·5-fold stimulation.



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Fig. 3. Transactivation of different promoter constructs driving the firefly luciferase or CAT gene by various EGFP–EBNA2 lysine mutants. Experiments were carried out in duplicate or triplicate for each mutant assayed. Values shown represent the mean value of at least seven independent assays. The value obtained using wild-type (WT) EBNA2 was set to 100%; in the control reaction, empty vector pEGFP-C1 was employed. The different EBNA2 mutants used are given below the columns. (a) Transactivation of a promoter consisting of 12 RBP-J{kappa}-binding sites arranged in tandem in front of a minimal herpes simplex virus type 1 thymidine kinase promoter (Strobl et al., 1997 ). (b) Transactivation of the viral LMP1 promoter extending from -327 to +40 (Laux et al., 1994b ). (c) Transactivation of the LMP1 promoter extending from -634 to +40 (Sjøblom et al., 1998 ).

 
When we used the LMP1 promoter extending from -327 to +40 (Laux et al., 1994b ), we still observed a slight increase with the K335R mutant, wild-type levels of activation with the K335–366R mutant and a slight decrease with the K335–480R mutant (all lysine residues mutated), as shown in Fig. 3(b). While a slight stimulatory effect of the K335R mutation on the -327 to +40 promoter construct was seen, we observed a decrease with a construct including sequences located at -634 to +40 (Fig. 3c) (Sjøblom et al., 1998 ). The mutation K480R, which affects subcellular localization, decreased transactivation to 30%. This was surprising in that the deletion of the complete C terminus had been reported to stimulate LMP1 protein expression by EBNA2 (Cohen et al., 1991 ). Likewise, all mutants that included the K335R mutation and that showed an elevated transactivation with the 12 RBP-J{kappa} promoters exhibited the same, or a somewhat decreased, transactivation with the larger promoter construct when compared to wild-type EBNA2. In addition to RBP-J{kappa}, the effects of EBNA2 on the LMP1 promoter are also mediated through Spi-1/Spi-B (Laux et al., 1994a ; Zhao & Sample, 2000 ), ATF/CRE (Sjøblom et al., 1998 ) and, possibly, by other factors. While our data indicate that stimulation through RBP-J{kappa} might be down-modulated through the lysine residues, interaction with additional factors such as Spi-1/Spi-B or ATF/CRE might be up-regulated by these residues.

In summary, only the lysine to arginine mutation at position 480 and the serine to arginine mutations at positions 479 and 485 resulted in a discernible effect on subcellular localization. Of the other conserved lysine residues at positions 359 and 366, mutation of either residue had no effect on the functions of the protein, while mutation at position 335 increased activity when we used a promoter containing 12 RBP-J{kappa}-sites; this might indicate that this lysine residue (position 335) somehow affects binding to RBP-J{kappa}.


   Acknowledgments
 
We thank Anna Sjøblom, Gerhard Laux and Lothar Strobl for the generous gift of reporter plasmids and Peter Hahn and Marc D. Voss for help with the art work. Supported by the Deutsche Forschungsgemeinschaft (DFG) through the Sonderforschungsbereich 399.


   References
Top
Abstract
Main text
References
 
Bornkamm, G. W. & Hammerschmidt, W. (2001). Molecular virology of Epstein–Barr virus. Philosophical Transactions of the Royal Society of London B Biological Sciences 356, 437-459.

Bradbury, E. M. (1992). Reversible histone modifications and the chromosome cell cycle. Bioessays 14, 9-16.[Medline]

Cohen, J. I., Wang, F. & Kieff, E. (1991). Epstein–Barr virus nuclear protein 2 mutations define essential domains for transformation and transactivation. Journal of Virology 65, 2545-2554.[Medline]

Fuentes-Panana, E. M., Peng, R., Brewer, G., Tan, J. & Ling, P. D. (2000). Regulation of the Epstein–Barr virus C promoter by AUF1 and the cyclic AMP/protein kinase A signaling pathway. Journal of Virology 74, 8166-8175.[Abstract/Free Full Text]

Grässer, F. A., Göttel, S., Haiss, P., Boldyreff, B., Issinger, O. G. & Mueller-Lantzsch, N. (1992). Phosphorylation of the Epstein–Barr virus nuclear antigen 2. Biochemical and Biophysical Research Communications 186, 1694-1701.[Medline]

Grundhoff, A. T., Kremmer, E., Tureci, O., Glieden, A., Gindorf, C., Atz, J., Mueller-Lantzsch, N., Schubach, W. H. & Grässer, F. A. (1999). Characterization of DP103, a novel DEAD box protein that binds to the Epstein–Barr virus nuclear proteins EBNA2 and EBNA3C. Journal of Biological Chemistry 274, 19136-19144.[Abstract/Free Full Text]

Hammerschmidt, W. & Sugden, B. (1989). Genetic analysis of immortalizing functions of Epstein–Barr virus in human B lymphocytes. Nature 340, 393-397.[Medline]

Hochstrasser, M. (2000). Biochemistry. All in the ubiquitin family. Science 289, 563-564.[Free Full Text]

Kempkes, B., Spitkovsky, D., Jansen-Dürr, P., Ellwart, J. W., Kremmer, E., Delecluse, H. J., Rottenberger, C., Bornkamm, G. W. & Hammerschmidt, W. (1995). B-cell proliferation and induction of early G1-regulating proteins by Epstein–Barr virus mutants conditional for EBNA2. EMBO Journal 14, 88-96.[Abstract]

Kremmer, E., Kranz, B., Hille, A., Klein, K., Eulitz, M., Hoffmann-Fezer, G., Feiden, W., Herrmann, K., Delecluse, H.-J., Delsol, G., Bornkamm, G. W., Mueller-Lantzsch, N. & Grässer, F. A. (1995). Rat monoclonal antibodies differentiating between the Epstein–Barr virus nuclear antigens 2A (EBNA2A) and 2B (EBNA2B). Virology 208, 336-342.[Medline]

Laux, G., Adam, B., Strobl, L. J. & Moreau-Gachelin, F. (1994a). The Spi-1/PU.1 and Spi-B ets family transcription factors and the recombination signal binding protein RBP-J{kappa} interact with an Epstein–Barr virus nuclear antigen 2 responsive cis-element. EMBO Journal 13, 5624-5632.[Abstract]

Laux, G., Dugrillon, F., Eckert, C., Adam, B., Zimber-Strobl, U. & Bornkamm, G. W. (1994b). Identification and characterization of an Epstein–Barr virus nuclear antigen 2-responsive cis element in the bidirectional promoter region of latent membrane protein and terminal protein 2 genes. Journal of Virology 68, 6947-6958.[Abstract]

Ling, P. D. & Hayward, S. D. (1995). Contribution of conserved amino acids in mediating the interaction between EBNA2 and CBF1/RBPJ{kappa}. Journal of Virology 69, 1944-1950.[Abstract]

Ling, P. D., Ryon, J. J. & Hayward, S. D. (1993). EBNA-2 of herpesvirus papio diverges significantly from the type A and type B EBNA-2 proteins of Epstein–Barr virus but retains an efficient transactivation domain with a conserved hydrophobic motif. Journal of Virology 67, 2990-3003.[Abstract]

Ling, P. D., Hsieh, J. J., Ruf, I. K., Rawlins, D. R. & Hayward, S. D. (1994). EBNA-2 upregulation of Epstein–Barr virus latency promoters and the cellular CD23 promoter utilizes a common targeting intermediate, CBF1. Journal of Virology 68, 5375-5383.[Abstract]

Mahajan, R., Delphin, C., Guan, T., Gerace, L. & Melchior, F. (1997). A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88, 97-107.[Medline]

Melchior, F. (2000). SUMO – nonclassical ubiquitin. Annual Review of Cell and Developmental Biology 16, 591-626.[Medline]

Rickinson, A. B. & Kieff, E. (1996). Epstein–Barr Virus. In Fields Virology , pp. 2397-2446. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia:Lippincott–Raven.

Rickinson, A. B., Young, L. S. & Rowe, M. (1987). Influence of the Epstein–Barr virus nuclear antigen EBNA 2 on the growth phenotype of virus-transformed B cells. Journal of Virology 61, 1310-1317.[Medline]

Saitoh, H. & Hinchey, J. (2000). Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. Journal of Biological Chemistry 275, 6252-6258.[Abstract/Free Full Text]

Sjøblom, A., Yang, W., Palmqvist, L., Jansson, A. & Rymo, L. (1998). An ATF/CRE element mediates both EBNA2-dependent and EBNA2-independent activation of the Epstein–Barr virus LMP1 gene promoter. Journal of Virology 72, 1365-1376.[Abstract/Free Full Text]

Strobl, L. J., Hofelmayr, H., Stein, C., Marschall, G., Brielmeier, M., Laux, G., Bornkamm, G. W. & Zimber-Strobl, U. (1997). Both Epstein–Barr viral nuclear antigen 2 (EBNA2) and activated Notch1 transactivate genes by interacting with the cellular protein RBP-J{kappa}. Immunobiology 198, 299-306.[Medline]

Tong, X., Yalamanchili, R., Harada, S. & Kieff, E. (1994). The EBNA-2 arginine–glycine domain is critical but not essential for B-lymphocyte growth transformation; the rest of region 3 lacks essential interactive domains. Journal of Virology 68, 6188-6197.[Abstract]

Tong, X., Wang, F., Thut, C. J. & Kieff, E. (1995). The Epstein–Barr virus nuclear protein 2 acidic domain can interact with TFIIB, TAF40, and RPA70 but not with TATA-binding protein. Journal of Virology 69, 585-588.[Abstract]

Voss, M. D., Hille, A., Barth, S., Spurk, A., Hennrich, F., Holzer, D., Mueller-Lantzsch, N., Kremmer, E. & Grässer, F. A. (2001). Functional cooperation of the Epstein–Barr virus nuclear antigen 2 and the survival motor neuron protein in transactivation of the viral LMP1 promoter. Journal of Virology 75, 11781-11790.[Abstract/Free Full Text]

Zhao, B. & Sample, C. E. (2000). Epstein–Barr virus nuclear antigen 3C activates the latent membrane protein 1 promoter in the presence of Epstein–Barr virus nuclear antigen 2 through sequences encompassing an spi-1/spi-B binding site. Journal of Virology 74, 5151-5160.[Abstract/Free Full Text]

Zimber-Strobl, U., Strobl, L. J., Meitinger, C., Hinrichs, R., Sakai, T., Furukawa, T., Honjo, T. & Bornkamm, G. W. (1994). Epstein–Barr virus nuclear antigen 2 exerts its transactivating function through interaction with recombination signal binding protein RBP-J{kappa}, the homologue of Drosophila suppressor of hairless. EMBO Journal 13, 4973-4982.[Abstract]

Received 3 October 2001; accepted 18 January 2002.



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