1 Bioprocessing Technology Centre, and
2 Departments of Medicine and
3 Biochemistry, National University of Singapore, Singapore 119260, Republic of Singapore
Correspondence to: Correspondence to: Z.-J. Yao, National Center for Human Genome Research, BDA, #3-707 North Yongchang Road, Beijing 100176, China
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Abstract |
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Keywords: autoantibody, anti-double-stranded DNA antibody, mimotope, phage peptide library
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Introduction |
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Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disease characterized by the deposition of autoantibodies and immune complex, which eventually leads to tissue damage. Since 1957, the antinuclear antibody (ANA) test had been one of the most important tests used to evaluate patients for the presence of connective tissue disorders (1). The ANA screening test detects primarily a group of antibodies against the nuclear antigens that are present in abundance, e.g. double-stranded (ds) DNA, Sm, nuclear ribonucleoprotein, SSA and SSB. Among them, the anti-dsDNA test is the most widely used serological hallmark for SLE, and its titer correlates positively with disease activity in both humans and mice. Moreover, antibodies to dsDNA are not only a diagnostic marker but also a pathogenic factor for SLE. A number of studies have shown that anti-DNA antibodies cross-react with a variety of antigens, including intracellular and extracellular components (24). Thus, direct binding of anti-DNA antibodies to glomerular basement membrane was suggested as the major step in the development of glomerulonephritis (5). On the other hand, although reports have demonstrated that anti-DNA antibodies have the characteristics of antibodies arising from an antigen-selected immune response (68), the actual role of DNA in eliciting the production of anti-DNA antibodies is not yet clear. It remains uncertain which antigen triggers the production of these antibodies.
The technology of phage-displayed random peptide libraries (RPL) (911) has recently become a powerful technique for elucidating proteinprotein/peptide interactions. Many publications have already shown the potency of this procedure by establishing the binding characteristics between antigenantibody, signaling moleculereceptor and substrateenzyme. In particular, this technology offers a number of advantages in the research area of autoimmune diseases. Firstly, the RPL screening method can be used to map any epitope (mimotope) without prior knowledge of the protein antigen. Secondly, since several reports have shown that polyclonal antibodies could be used as screening ligates, this means that autoimmune patient sera could be used as a source for isolating screening ligates, thus providing a unique advantage of linking the RPL approach to a disease-related antigenic structure directly.
We present here the application of RPL in searching a peptide mimic of dsDNA antigenic structure by using anti-dsDNA antibodies obtained from SLE patient sera. We have successfully identified a peptide motif which could mimic the antigenicity of native human placenta dsDNA. Investigations in specificity revealed that this structure was shared by denatured human placenta single-stranded (ss) DNA, natural phage genomic ssDNA and native calf liver RNA. Moreover, when the peptide was used to immunize rabbits, the harvested anti-peptide rabbit serum could not only recognize the peptide itself, but also ss and dsDNAs.
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Methods |
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The presence and absence of antibodies to dsDNA in the SLE sera and control sera were assayed by the QUANTA Lite dsDNA ELISA test kit (INOVA Diagnostics, San Diego, CA). The assay procedures provided by the manufacturer were followed. Briefly, each serum was diluted 1:100 with sample diluent and introduced into dsDNA-coated ELISA microtiter wells (100 µl/well) in duplicate. After incubating for 30 min at room temperature, the wells were emptied and washed with wash buffer. Horseradish peroxidase-conjugated anti-human IgG (raised in goat) was then added and incubated for another 30 min at room temperature. The wells were then emptied, washed and colored by adding 3,3',5,5'-tetramethylbenzidine (TMB) chromogen. After stopping the color reaction by adding 100 µl of ELISA stopping solution to each well, absorbance was read at 450 nm with a reference wavelength 620 nm.
Preparation of dsDNA-coupled CNBr-activated Sepharose column
CNBr-activated Sepharose 4B gel was purchased from Pharmacia (Uppsala, Sweden). The protocol provided by the manufacturer for treatment of the gel and for coupling of protein to the gel was used. Briefly, 1.5 g of the gel was soaked in 50 ml of 1 mM HCl for at least 30 min at room temperature and allowed to swell. The swollen gel was then washed 3 times with about 300 ml 1 mM HCl by suspending the gel in a beaker and filtering the gel suspension in a flask. Human placenta dsDNA (Sigma, St Louis, MO) was dissolved in 1xSSC (150 mM NaCl/15 mM sodium citrate, pH 7.0) at a concentration of 3 mg/ml and further diluted in coupling buffer (0.1 M Na2CO3/0.5 M NaCl, pH 8.3) to 0.5 mg/ml. The dried gel was resuspended in 6 ml of this dsDNA solution and incubated at 4°C overnight with constant rotation to keep the gel in suspension (~2 mg of dsDNA was retained with the gel by measuring the difference of OD260 before and after the incubation). After washing away the unbound dsDNA with coupling buffer, the gel was resuspended in 0.1 M TrisHCl, pH 8.0 and incubated for 2 h at room temperature to block the uncoupled area. After the blocking incubation, the gel was successively washed with acetate buffer (0.1 M sodium acetate, 0.5 M NaCl), pH 4.0 followed by 0.1 M TrisHCl, pH 8.0 (3 times), with 10 mM Tris, pH 7.5 (at least 8 times), with 0.1 N HClglycine, pH 2.5 (at least 8 times), with 0.1 M triethylamine, pH 11.5 (at least 8 times) and finally with 10 mM sodium phosphate buffer, pH 7.0 until the pH of filtrate was 7.0. The gel was then packed into a chromatography column (Pharmacia) and kept at 4°C for use.
Preparation of the screening ligate
The preparation of screening ligate has been described elsewhere (12), but with some modifications in this study. Briefly, 1 ml of pooled positive sera was loaded on a GammaBind Sepharose (Pharmacia) column. After incubating for 30 min at room temperature, the column was washed to remove unbound fractions with 0.1 M sodium phosphate buffer, pH 7.0 until the baseline reached zero. The bound IgG fraction was then eluted with 0.5 M acetic acid, pH 2.7 and neutralized immediately with 2 M Tris. The IgG-containing fractions were then pooled together and dialyzed against 10 mM phosphate buffer, pH 7.0. Subsequently, the `mono-specific' anti-dsDNA fraction was purified using the human placenta dsDNA-coupled CNBr-activated Sepharose 4B gel column prepared as described above. The column was washed extensively with washing buffer (10 mM sodium phosphate, pH 7.0) overnight before use. The dialyzed IgG fraction was then loaded onto the affinity column and incubated for 30 min at room temperature. After incubation, the unbound IgG fraction was washed away with the washing buffer, while the bound IgG was eluted with 0.1 M sodium phosphate buffer, pH 7.0. The affinity-purified anti-dsDNA IgG was dialyzed for 4 h against 0.1 M sodium borate buffer, pH 8.8 and biotinylated subsequently by the succinimide method (13).
Biopanning of phage-displayed RPL by anti-dsDNA antibodies
A 15mer peptide library displayed on gene VIII product of fd phage (14) was used in this study. This library was generously provided by Professor G. P. Smith (University of Missouri, Columbia, MO) The basic methods of screening were adapted from the procedures as described before (12,15). Three rounds of screening were performed with the biotinylated anti-dsDNA antibodies. For the first round of selection, 10 µg of biotinylated anti-dsDNA antibodies in 400 µl of 0.5xTBST (25 mM Tris, 75 mM NaCl and 0.05% Tween 20, pH 7.5) containing 1 mg/ml dialyzed BSA were immobilized onto a streptavidin-coated Petri dish by incubation for 2 h at room temperature. In subsequent rounds of selection, 1 µg of biotinylated anti-dsDNA antibodies was used. To saturate the biotin binding sites on the dish, 4 µl of 10 mM free biotin was added and incubated for 1 h at room temperature. The unbound antibodies and biotin were removed by washing 6 times with 0.5xTBST. Phage suspensions (5 µl of library for the first round and 100 µl of output phages for the second and third rounds and 4 µl of 10 mM biotin in 400 µl of 0.5xTBST) were added and incubated for 4 h at room temperature. After the removal of unbound phages by washing 10 times with 0.5xTBST, the bound phages were recovered using elution buffer (0.1 N HClglycine, pH 2.2, 1 mg/ml BSA, 0.1 mg/ml phenol red). The eluates were immediately neutralized using 2 M Tris.
Eluted phages were amplified in Escherichia coli K91Kan and used as input in the subsequent rounds of selection. After three rounds of selection, phage clones harvested from the third round output were randomly selected and propagated in 1.5 ml of terrific broth [1.2% (w/v) bacto-tryptone, 2.4% (w/v) yeast extract, 0.4% (v/v) glycerol, 17 mM KH2PO4 and 72 mM K2HPO4] supplemented with 20 µg/ml tetracycline at 37°C for 20 h. The cultures were clarified at 2500 g for 10 min, and the supernatants were recovered for the selection of anti-dsDNA antibody binding clones and subsequent DNA sequencing.
Test of biopanning enrichment and selection of phage clones reactive with anti-dsDNA antibodies by ELISA assay
Preparation of ELISA plates.
ELISA microtiter wells were coated overnight at 4°C with 100 µl/well affinity-purified anti-dsDNA antibodies at a concentration of 5 µg/ml in coating buffer (50 mM NaHCO3, pH 9.6). After incubation, the wells were washed 3 times with 0.5xTBST and blocked with Blotto (5% skimmed milk in 0.5xTBST) for 60 min at 37°C. After washing again, the well strips were stored at 4°C for use.
Test of biopanning enrichment.
Phages from the library and the output phages of three rounds were diluted with Blotto to 1x1011 transducing units (TU)/ml. The phage suspensions were introduced into the anti-dsDNA antibody-coated microtiter wells and incubated for 2 h at 37°C. The unbound phages were then removed and the wells were washed 3 times with 0.5xTBST. This was followed by incubation with biotinylated anti-fd phage antibodies (Sigma) diluted with Blotto at a ratio of 1/1000 for 2 h at 37°C. After another round of extensive washing, horseradish peroxidase-conjugated streptavidin (Sigma), 1/1000 diluted with Blotto, was then added and incubated for 1 h at 37°C. Finally, following another washing step, the plates were treated with o-phenylenediamine dihydrochloride (OPD) for color development. Absorbance was measured at 492 nm using 620 nm as reference wavelength in an ELISA reader (Dynex, Middlesex, UK).
Selection of anti-dsDNA antibody-reactive phage clones.
Phage supernatants prepared as described above were added to the wells (100 µl/well) of the ELISA selection plates and allowed to bind for 2 h at 37°C. The subsequent treatments for ELISA assay were exactly the same as described above. A phage clone bearing a 15mer phagotope DLHRYSWKTQGDDRE selected previously from the 15mer phage library by mAb MAb47 which is specific for dengue virus NS1 protein (unpublished data) was processed in parallel as a negative control and its reading was referenced as background binding. Those phage clones showing more than twice the reading of background binding at 492 nm were selected as positive clones.
DNA sequencing
Clarified culture supernatants of positive clones selected as mentioned above were transferred to new Eppendorf tubes and the phages were recovered by 20% (w/w) PEG/NaCl solution (16.7%/3.3 M). After incubation for 4 h at 4°C, phages were pelleted at 10,000 g for 10 min. The phage pellets were then re-suspended in 100 µl of TBS. Viral DNA was extracted from 30 µl of phage suspension with phenol/chloroform/water solution. DNA in the aqueous layer was then precipitated by ethanol and used as a template for cycling sequencing with the primer: 5'-CTGAAGAGAGTCAAAAGC-3'. The sequencing was carried out in an automated DNA sequencer (model 373) from Perkin-Elmer Applied Biosystems (Foster City, CA). The amino acid sequences of the inserts were deduced from the DNA sequences.
Peptide synthesis
Peptides were synthesized by Fmoc chemistry using an eight-branched multiple antigen peptides (MAP) resin. Fmoc amino acids and MAP resins were purchased from Bachem (San Jose, CA) and AnaSpec (Torrance, CA). Peptide synthesis was carried out in our laboratory in an automatic peptide synthesizer, model 433A (Perkin-Elmer Applied Biosystems). The motif peptide MAP-RLTSSLRYNP and a control peptide MAP-TLPNRSYLSR that is a randomly scrambled sequence of the motif peptide sequence were synthesized. The purity of the peptides was 90% checked by reverse-phase HPLC and the compositions were confirmed by amino acid analysis.
Serum binding to synthetic peptides
Motif peptide MAP-RLTSSLRYNP was first dissolved in DMSO at a concentration of 100 mg/ml (w/v) and then diluted to 10 mg/ml with water (stock solution). The stock solution was further diluted 100 times with coating buffer to 0.1 mg/ml for coating ELISA microtiter wells. Control peptide MAP-TLPNRSYLSR was dissolved in coating buffer at a concentration of 0.1 mg/ml. The peptide solutions were added to microtiter wells (100 µl/well) and incubated at 4°C overnight. The wells were washed and blocked with Blotto. The sera diluted 1/100 with Blotto were added to the peptide-coated wells and incubated for 2 h at room temperature. After washing, horseradish peroxidase-conjugated anti-human IgG was added and incubated for 1 h at room temperature. The plates were then washed and colored by OPD chromogen as described above.
Competitive binding inhibition assay
Human placenta DNA, ss fd phage genomic DNA and calf liver RNA were 2-fold serially diluted starting from 400 µg/ml. fd phage DNA was extracted using ssPHAGE DNA Spin kit (BIO101, La Jolla, CA), and the other two DNA and RNA were purchased from Sigma. A set of dilutions of human placenta DNA and a set of dilutions of calf liver RNA were heated at 95°C for 3 min and rapidly cooled on ice to obtain denatured ssDNA and RNA. To be comparable, all dilutions of DNAs and RNAs were cooled on ice before mixing equally in volume with a cold dilution of a panel of SLE sera. The final concentrations of DNAs and RNAs were from 200 to 0.39 µg/ml and the dilution of SLE sera was 1/100. After mixing together, they were left at 4°C for 2 h and then transferred to a motif peptide-coated ELISA plate in duplicate (100 µl/well) for 30 min at 4°C. After subsequent incubation with horseradish peroxidase-conjugated anti-human IgG for 30 min at room temperature, the plates were processed as described above. The percentage inhibition of sera binding to peptide by the inhibitors was calculated according to the following equation: 100x[1 (OD492 with inhibitor background OD492)/(OD492 without inhibitor background OD492)].
Immunization
The immunization was performed by mixing equal volumes of Freund's adjuvant with peptide MAP-RLTSSLRYNP solution at a dose of 0.5 mg of peptide per injection. The emulsion was injected s.c. at multiple points in the back region of the rabbits. Complete Freund's adjuvant was used in the first injection, whereas incomplete adjuvant was used in booster injections. Booster injections were carried out on days 14, 28, 42 and 56 after the first injection. Blood samples were taken before immunization and on the days when the booster injections were performed. The last sample of serum was obtained on day 70.
ELISA binding reactivity and competitive inhibition assay of anti-peptide rabbit serum
Anti-peptide rabbit serum was tested for its ELISA binding reactivities with both motif and control peptide-coated plates and the QUANTA Lite ds and ssDNA ELISA plates. The rabbit sera taken before and after the peptide immunization were 2-fold serially diluted with Blotto from 1/100 to 1/6400, and added to the peptide- and DNA-coated wells separately. Each dilution was assayed in duplicate. The sera were allowed to bind for 1 h at room temperature. After washing away the sera with 0.5xTBST, horseradish peroxidase-conjugated anti-rabbit IgG, 1/1000 diluted with Blotto, was added and incubated for 30 min at room temperature. The wells were washed, and colored by OPD chromogen and absorbance was measured as described above.
To demonstrate the specificity of anti-peptide serum, competitive binding inhibition ELISA assay was also performed with anti-peptide rabbit serum and the inhibitors DNAs and RNAs as with SLE sera described above. The experiment and the concentrations of inhibitor DNAs and RNAs remained the same, but the final dilution of anti-peptide serum was 1/300 in Blotto and the secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG, 1/1000 diluted in Blotto.
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Results |
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Immunogenic motif peptide MAP-RLTSSLRYNP and anti-peptide serum
Since motif peptide MAP-RLTSSLRYNP showed highly sensitive and specific binding reactivity with SLE sera in ELISA, it is very interesting to demonstrate its antigenic and immunogenic properties on animals. If the motif peptide is indeed a mimic of dsDNA antigenic determinant, the antibody against this peptide should recognize both the peptide and dsDNA. Therefore, we used rabbits to demonstrate the immunogenicity of the motif peptide. The anti-peptide rabbit serum showed high ELISA binding reactivities with the motif peptide and also with dsDNA (Fig. 6A and B), suggesting that the peptide does mimic an antigenic determinant of dsDNA and this antigenic determinant is shared by ssDNA as mentioned above since the serum could also recognize ssDNA (Fig. 6B
). On the other hand, the anti-peptide serum did not bind to the control peptide (Fig. 6A
), suggesting that the serum is specific for the motif peptide sequence.
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Discussion |
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In this study, we have identified a peptide mimic for dsDNA using RPL and SLE patient sera. This peptide mimic, with the sequence RLTSSLRYNP, exhibited a close correlation (correlation coefficient, r = 0.809, P < 0.0001) with dsDNA in the binding reactivities with human serum antibody to dsDNA (Fig. 4). This result suggests the possibility of using the peptide mimic in the diagnosis of SLE. By using the peptide mimic in an ELISA format, it was established that autoantibodies to dsDNA were detected in 37 of the 42 SLE+ sera (Fig. 3
). More importantly and interestingly, the peptide mimic was able to bind competitively SLE anti-DNA antibodies in vitro not only with dsDNA but also with ssDNA (Fig. 5
). These findings, on the one hand, suggest that the peptide mimic is an antigenic structure shared by both ss and dsDNAs, and, on the other hand, reflect that the motif peptide binding SLE anti-dsDNA antibodies are cross-reactive. This type of antigenic structures is believed to encompass exposed bases as well as the phosphodiester backbone (16,17). With the same spices of DNA, human placenta DNA, more binding structures were exhibited to the antibody on denatured ssDNA than on native dsDNA. This feature most probably results from the conformational changes of ds to ssDNA that allow the antibody to bind exposed regions of single strandedness (24) that are normally masked by the dsDNA helix structure. Alternatively, the small molecule of phage ssDNA exhibited more binding sites than the large molecule of human placenta ss and dsDNAs when they were applied at the same amount, suggesting that the binding site occurs variably on both species of DNAs and the number of the binding site is not directly proportional to the length of DNA molecule. Another interesting finding is the inhibition on motif peptide and SLE sera binding by native calf liver RNA. It has been reported that only anti-DNA antibodies eluted from the kidney of MRL-lpr/lpr mice bound with RNA, the antibodies in MRL-lpr/lpr mouse serum did not; moreover, the most characteristic feature of nephritogenic autoantibody was polyreactivitythe ability of individual antibodies to bind to multiple antigens (18). We did not address the inhibition by other autoantigens other than polynucleotide antigens in this study and the pathogenic potential of the idiotypic anti-dsDNA antibodies that bind the motif peptide needs to be further investigated. The potential of the motif peptide to be a drug candidate for SLE could be true only when the peptide binding anti-dsDNA antibodies are tested as a pathogenic idiotype. It is certain, however, that this peptide mimic provides us with a unique opportunity to investigate the antigenic structure of DNA and the DNAanti-DNA antibody interaction.
The origins of antibodies to DNA in SLE are not yet clear. Native mammalian DNA is poorly immunogenic and has never been found to be able to induce anti-DNA antibodies (25,26). Although some bacterial DNAs have been demonstrated to be immunogenic in normal mouse, they do not elicit production of antibodies that cross-react with mammalian DNA (27,28). Complexes of ssDNA and Escherichia coli dsDNA non-covalently bound to a cationic protein such as methylated serum albumin have been reported to be able to induce antibodies with specificity only for ssDNA and Escherichia coli dsDNA respectively, but not for native mammalian DNA, and the complexes when prepared with mammalian dsDNA have not been immunogenic (29,30). Similarly, although anti-DNA antibodies produced after polyoma BK virus infection and immunization were specific for native DNA, these induced anti-DNA antibodies were still different from autoimmune anti-DNA antibodies (31,32). Until recently, anti-dsDNA antibodies have been successfully induced by a complex of native calf thymus DNA and a highly immunogenic DNA binding peptide, Fus1 (33). The structural and functional characteristics of the induced anti-DNA antibodies were similar in all aspects to autoimmune anti-DNA antibodies (33,34). The common feature in these studies is that DNA seemed more immunogenic in non-autoimmune animals only when complexed with immunogenic protein and peptide.
On the other hand, nevertheless, pneumococcal polysaccharide antigens have been described to be able to elicit an immune response that produced some idiotypic antibodies cross-reactive with DNA (35). More recently, a peptide surrogate for dsDNA, which was identified from a phage peptide library using a murine monoclonal anti-dsDNA antibody (19), has induced the production of anti-dsDNA antibodies in non-autoimmune BALB/c mice. Moreover, the immunized mice also developed antibodies against some other lupus antigens and Ig deposition was present in renal glomeruli (36). We could also demonstrate in this study that a peptide surrogate identified from a phage peptide library using anti-dsDNA antibodies in SLE sera has the same antigenic structure with DNA and native RNA, and induced anti-DNA antibody production in rabbits. These studies suggest that DNA antigen is not indispensable for the production of anti-DNA antibodies. Therefore, which antigen induces spontaneous production of anti-DNA antibodies in the autoimmunity of SLE remains unclear.
As such, our second goal of the project is to attempt to identify the `genuine autoantigen(s)' that may elicit the production of anti-DNA antibodies in SLE. Since the RPL approach can map and identify epitopes of antigens without prior knowledge of their structures, the peptide motifs/sequence(s) selected by biopanning could provide clues for a known antigen by using an online homology search program (e.g. Blitz). The competition with DNA for binding anti-DNA antibodies in SLE sera and the ability to induce immune response of producing anti-DNA antibodies (Figs 6 and 7) suggest that the motif peptide mimics an antigenic structure of SLE autoantigen, that may be or may not be DNA. To our knowledge, this is the first report of the identification of a peptide mimic for DNA by polyclonal antibodies separated directly from SLE patient sera. Motif peptide RLTSSLRYNP exhibits homology with a chromosome-associated polypeptide, but it does not show any similarity with antigens of suspected infectious agents, such as pneumococcal (35) or EpsteinBarr viral antigens (37,38). An online similarity search is useful only for linear epitopes; furthermore, the amino acid residues of a linear epitope must be well defined. Otherwise, an online search is usually fruitless. In the case of motif peptide RLTSSLRYNP, it may represent a mimotope rather than a linear epitope of `genuine autoantigen(s)'. If this is the case, then it is difficult to identify the causative antigen(s) for SLE using this approach. On the other hand, we are further characterizing this motif for defining more critical residues and for shortening the sequence if possible. Thus, we could not rule out the possibility that the pathogenesis is still microbial in origin. With more sero-epidemiological studies of SLE and more peptide epitopes/mimotopes of dsDNA, it may enhance the chance of identifying potential putative antigens for anti-DNA antibodies.
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Acknowledgments |
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Abbreviations |
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ANA antinuclear antibody |
ds double-stranded |
RPL random peptide library |
SLE systemic lupus erythematosus |
ss single-stranded |
TU transducing unit |
MAP multiple antigen peptides |
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Notes |
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5 Present address: National Center for Human Genome Research, BDA, #3-707 North Yongchang Road, Beijing 100176, China
Received 14 June 2000, accepted 7 November 2000.
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References |
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