Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, México
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Abstract |
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Keywords: catalysis/directed evolution/enzyme activation/selection method/triosephosphate isomerase
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Introduction |
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Triosephosphate isomerase is an enzyme that acts as a homodimer in all known species studied so far. The degree of sequence conservation from archea to eukaryontes is remarkable. Each of the subunits conforming the dimer has an independent active site, but the reaction is not simultaneous in both subunits (Schnackerz and Gracy, 1991). The dimer interface is a very localized region composed mainly of loop 3, which interdigitates into the pocket of the active site of the other subunit, interacting mainly with residues at loops 1 and 4. The insertion of loop 3 from subunit 1 into the cavity of subunit 2 pulls loop 1 from subunit 2 into the active site cavity, forming a salt bridge between residues Lys13 and Glu97 (Wierenga et al., 1991a
,b
). The formation of this salt bridge pulls loop 4 in such a way that the catalytic histidine, His95, is positioned correctly for catalysis.Wierenga's group designed a monomeric variant of triosephosphate isomerase from Trypanosoma brucei (Tb) by shortening loop 3, the main component of dimerization interface (Borchert et al., 1994
). This loop was mostly hydrophobic and seven out of its 15 residues were replaced by more hydrophilic residues. The shortened version of triosephosphate isomerase resulted in a monomeric version of the enzyme called MonoTIM, which was soluble and stable but with only one thousandth of the original activity in the dimeric protein (Schliebs et al., 1996
). Other attempts to obtain a monomeric TIM were directed to destabilize the interaction of the loop 3 with the other subunit by electrostatic repulsion, through the introduction of positively charged residues at the tip of loop 3 (Schliebs et al., 1997
). The protein obtained was a monomer but its activity was as low as the original MonoTIM. In human TIM, Mainfroid et al. (1996) tried to destabilize the dimeric interface by perturbing a hydrophobic interaction in which Met14 participates and a salt bridge between Arg98 and Glu77 from the other subunit by mutating Met14 to Gln and Arg98 to Gln. Only the double mutant was shown to be a monomeric protein, but the activity and stability were considerably reduced.
In the present work we attempted a combination of rational design with the advances in protein directed evolution (Crameri and Stemmer, 1995; Crameri et al., 1996
; Arnold and Volkov, 1999
) to obtain a version of monoTIM, which remained monomeric but with an increased activity. Based on the hypothesis that the loss of activity is due to an increased entropy in the catalytic loops promoted by the lack of interaction with the other subunit (Schliebs et al., 1996
; Thanki et al., 1997
), it would be feasible to attain an active monomeric protein by replacing intersubunit interactions by intrasubunit interactions that would reduce the entropy of the catalytic loops and/or fill in the space occupied by the other subunit, thus decreasing the activation energy for the reaction. This looks feasible also in the light of the mentioned evidence that the active site of each subunits works independently of the other (Schnackerz and Gracy, 1991
). Therefore, dimerization should not be a condition for activity. The energetic cost of positioning His95 for catalysis upon removal of the other subunit is translated into an increase in the activation energy for the reaction with a resulting decrease in kcat. An approach to overcome this effect would be to fill in the hole left by the other subunit. This could be accomplished by increasing the size of side chains and the size of the loops near loop 1. We therefore decided to mutagenize loop 2 directly by randomized oligonucleotides, allowing the size of the loop to be increased by up to two amino acids. In parallel, we decided to follow a completely random approach by performing error-prone polymerase chain reaction (PCR) of the entire gene. In both cases we did a genetic selection to obtain the more active mutants.
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Materials and methods |
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Restriction enzymes, high-fidelity Expand, T4 DNA ligase and alkaline phosphatase were purchased from Boehringer Mannheim.
Reagents
Buffers and reagents such as triethanolamine, Tris HCl, EDTA, NaCl, dithiothreitol, ß-nicotinamide adenine dinucleotide reduced and oxidized forms, -glycerophosphate dehydrogenase and glyceraldehyde 3-P dehydrogenase were purchased from Sigma.
Trypanosoma brucei TIM gene
The gene for the engineered triosephosphate isomerase from Trypanosoma brucei (MonoTIM) was generously provided by Wierenga's group at EMBL. This gene was originally cloned through NdeI sites in the pET3a expression vector from Novagen (Madison, WI).
Genetic selection
In order to evaluate the large libraries to be generated by either mutagenesis strategy, it was necessary to have a strain capable of differentiating active from inactive tpi genes. We obtained the strain AA200 from the ATCC (Anderson and Cooper, 1970) and tested it for selection. This strain was obtained by the action of chemical mutagens and showed a high rate of reversion. Therefore, we decided to construct our own Escherichia coli mutant strain, VR101, by interrupting the tpi gene in the E.coli JM101 strain. The procedure was as follows: the tpi gene encoding for TPI was obtained by PCR from E.coli chromosomal DNA with suitable primers to clone it in pUC18. In this construction the gene was interrupted at the unique MluI site by inserting a cassette containing the neomycin phosphotransferase gene (nptII) responsible for kanamycin resistance obtained from pBSL98 plasmid (Alexeyev et al., 1995
). The BamHI/HindIII fragment was subcloned in the temperature-sensitive plasmid pMAK705, which carries the chloramphenicol acetyltransferase (CAT) gene (Hamilton et al., 1989
). The method proceeds by homologous recombination between the gene on the chromosome and homologous sequences carried on the plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into the host, it is possible to select for integration of the plasmid into the chromosome at 44°C in the presence of kanamycin. Subsequent growth of these cointegrates at 30°C leads to a second recombination event, resulting in their resolution by the lost of Cmr and the preservation of Kmr phenotype (Hamilton et al., 1989
). Once that the auxotroph strain had been obtained, it was validated for its lack of growth in minimal medium supplemented with glucose as the only carbon source as well as the complementation of the function by transforming it with a plasmid carrying the WT tpi gene as well as the MonoTIM gene. Conditions for selection between these two extreme activities were established.
Random mutagenesis
The gene of MonoTIM with its ShineDelgarno was amplified from the pET3aMonoTIM plasmid by PCR using high-fidelity Expand and the following oligonucleotides in order to introduce restriction sites EcoRI and HindIII suitable for cloning in pUC18: 5'-AGCACCAAAGCTTATTGACACATGAAGGAAAATCA-3' and 5'-TAATTTTGTTGAATTCTAAGAAGGAGATATACATATGTCC-3'. The PCR product was cloned into the expression vector pUC18 in order to have it under the control of the lac promoter. A single round of error-prone PCR was carried out by the method described by Cadwell and Joyce (1992, 1994) in a Stratagene Robocycler 40. Approximately 8 µg of the mutagenized gene and 4 µg of pUC18 plasmid were digested with EcoRI and HindIII (the vector was furthermore digested with PstI to ensure complete destruction of the polylinker and to reduce the number of clones without insert). The 2.8 kb and 700 bp fragments obtained were purified by 1% agarose gel electrophoresis and extracted from the agarose using a QIAquick gel extraction kit (Qiagen, Germany). A 1 pmol amount of purified vector was ligated to the mutagenized gene in a 1:8 ratio at 16°C for 18 h in a final volume of 200 µl. The ligation was precipitated with butanol and resuspended in 20 µl of distilled water. Half of this library was used to electroporate competent E.coli XL1Blue cells. The size of the library was quantified by plating 1 µl of transformation on Luria plates supplemented with ampicillin and tetracycline. The rest of the transformation was used to inoculate 30 ml of LB medium supplemented with antibiotics, from which the plasmid DNA library was purified using a High Pure Plasmid Isolation kit from Boehringer Mannheim. A 1 µg amount of the library DNA was electrotransformed in competent tpi E.coli JM101 cells and plated on different media: (1) LB supplemented with kanamycin and ampicillin to estimate the size of the library, (2) a selective medium consisting of minimal medium supplemented with 0.2% glucose as the only carbon source, thiamine, kanamycin and ampicillin and (3) the same minimal medium as before but supplemented further with 0.006% of casamino acids. Controls of cells transformed with the original pUC18-MonoTIM, pUC18 and pUC18-WT Tb TIM, were treated under the same conditions to compare the phenotypes present in the library. There was a clear difference in the colony size between 12 h and 40 h for the tpi E.coli cells transformed with pUC18-MonoTIM and pUC18-WT Tb TIM. The larger colonies from the library obtained in the different selection media were picked out, their plasmid DNA purified and the EcoRIHindIII fragment subcloned in pUC18. The transformation and selection were repeated and the plasmid DNA of the newly selected clones were sequenced using a Perkin-Elmer ABI PRISM 377 DNA sequencer using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit from Perkin-Elmer Applied Biosystems (Foster City, CA), following the procedure described by the provider. Two independent colonies were further characterized by following the growth curves of the colonies in triplicate in minimal medium supplemented with the antibiotics.
Mutagenesis directed to loop 2
Randomization of loop 2 was carried out by PCR using a the megaprimer method (Sarker and Sommer, 1990). Three spiked oligonucleotides (Ner et al., 1988
; Hermes et al., 1989
), that randomized 6, 7 and 8 codons respectively, were synthesized. In the first round of PCR the oligo 5'-TAATTTTGTTGAATTCTAAGAAGGAGATATACATATGTCC-3' was used with an equimolecular mixture of the oligos 5'-CCTTCGTCATGGC-(SNN)68-GGCCACTACGCATTG-3' in order to introduce variability in the six residues at and flanking loop 2, as well as with the possibility of obtaining loops one or two residues longer. The 200 bp library megaprimer thus obtained was purified through a 1.5% agarose gel and the DNA was extracted using the QIAquick gel extraction kit (Qiagen). A second round of PCR was carried out by using the MonoTIM gene cloned into pET3a vector from Novagen as template and the Megaprimer generated in the first round of PCR and oligo 5'-AGCACCAAAGCTTATTGACACATGAA-GGAAAATCA-3' as primers. A Stratagene Robocycler 40 was used for both PCR rounds, for 25 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 2 min, with a final extension cycle at 72°C for 10 min. The PCR product was extracted with phenolchloroform, precipitated with butanol and resuspended in a suitable buffer for digestion with EcoRI and HindIII. Digestion proceeded overnight (O/N) at 37°C. The ~800 bp digested PCR product was purified by electrophoresis on a 1.2% low melting point agarose gel, from which the DNA band was extracted using a QIAquick gel extraction kit (Qiagen). A 1 pmol amount of pUC18 vector digested as described before was ligated to 8 pmol of digested purified PCR product at 16°C O/N in a volume of 200 µl. The ligation was treated as above and 16 colonies were randomly picked out from the first transformation in XL1Blue cells (without selection) in order to corroborate the variability introduced in the library by sequencing the DNA. From the genetic selection (transformation in tpi JM101 E.coli cells and growth in minimal medium), 20 colonies were selected by colony size. The DNAs of these colonies were purified with a High Pure Plasmid Isolation kit from Boehringer Mannheim. A mixture of the DNAs obtained was digested with EcoRI and HindIII and the DNA fragments containing the tpi structural gene were subcloned in pUC18 to repeat the genetic selection. Ten colonies were picked out from this selection and further characterized by DNA sequencing and growth curves.
Purification of mutant proteins
One fast-growing variant from each strategy was subcloned in the overexpression vector pET3a from Novagen using the HindIII and NdeI sites and transformed in BL21DE3 E.coli cells. The cells were grown on minimal medium at 30°C and induced with 0.5 mM IPTG at OD 0.4. The growth continued O/N. The cells were spun down at 10 400 g for 20 min and the pellet of cells was resuspended in a 100 mM HEPES, 20 mM NaCl, 1 mM EDTA, 0.5 mM DTT buffer at pH 8.0 (buffer A) and sonicated for seven cycles of 30 s on a Branson Sonifier 450. The cell debris were removed by centrifugation at 12 100 g for 30 min and the supernatant was precipitated with 3070% ammonium sulfate. The 70% pellet was resuspended and dialyzed against buffer A. The dialyzate was centrifuged at 12 100 g for 30 min to remove any aggregated protein and loaded on a Bio-Rex 70 column, 100200 mesh, from Bio-Rad Laboratories (Richmond, CA), previously equilibrated with the same buffer. The column was washed with 23 volumes of buffer A, before starting elution with a two volumes gradient of NaCl from 20 to 120 mM. The elution profile was followed by measuring the UV absorption at 280 nm and the fractions were checked for purity by 15% denaturing polyacrylamide gel electrophoresis, using Coomassie Brilliant Blue to visualize the protein bands. The pure fractions were consolidated and precipitated with ammonium sulfate for storage at 4°C. Under these conditions, the proteins were stable for at least 8 months.
Determination of protein concentration
The ammonium sulfate precipitate of the mutant proteins was dialyzed against two changes of 20 mM triethanolamine (TEA), 1 mM EDTA, 50 mM NaCl, 0.5 mM DTT buffer at pH 7.6 and filtered after dialysis through a 0.22 µm sterile Acrodisc from Gelman Science (Ann Arbor, MI) to remove any aggregated protein. The protein concentration was measured by the Bradford method (Bradford, 1976) using a Bio-Rad Protein Assay Kit against a standard curve of BSA. The UV spectra of the same protein solutions were measured to determine
1% (280 nm) for each mutant protein.
FPLC
The purified enzymes were analyzed by size exclusion chromatography to determine the nature of oligomerization against a gel filtration standard mixture from Bio-Rad Laboratories in an Äkta FPLC system with a size-exclusion Superose 12 column from Amersham Pharmacia Biotech (Uppsala, Sweden).
Activity assay
The enzymatic activity of the mutant proteins was followed at 25°C as the change in absorbance at 340 nm due to the oxidation of NADH in a coupled-enzyme assay as described by Plaut and Knowles (1972). Briefly, the reaction conditions for the forward reaction forming dihydroxyacetone phosphate were 100 mM TEA, 5 mM EDTA buffer at pH 7.60. The substrate, glyceraldehyde-3-phosphate, was prepared from DL-glyceraldehyde-3-phosphate diethyl acetal monobarium salt as described by the supplier (Sigma Chemical). The concentration of the D-glyceraldehyde-3-phosphate was determined enzymatically (Plaut and Knowles, 1972) and varied from 0.08 to 5 mM to determine the kinetic parameters kcat and Km. The coupling enzyme
-glycerophosphate dehydrogenase and the cofactor NADH were also obtained from Sigma Chemical and used at 0.023 µg/ml and 0.22 mM concentrations, respectively. All monomeric mutants have a much lower activity than the dimeric wild-type (WT) Trypanosoma brucei TIM; therefore, 50300 ng/ml of enzyme were used, instead of the 1 ng/ml normally used for the WT enzyme (Borchert et al., 1993b
; Gao et al., 1998
). One activity unit (U) is defined as the conversion of 1 µmol of substrate per minute at 25°C. A molar extinction coefficient of 6.22x103 M1 cm1 is used for NADH at 340 nm.
Effect of protein concentration on Vmax
In order to evaluate if there was any protein concentration effect on Vmax, the activity assay was repeated varying the amount of the mutant proteins from 12 to 100 ng/ml keeping the substrate concentration constant at 3.8 mM to ensure saturating conditions. To establish if a loss of activity observed upon dilution of the enzyme stock solution was related to dissociation of some dimeric form of the enzyme, we compared the remaining activity against time of a diluted stock solution (8 µg/ml of RMM0-1 mutant TIM) in the presence and absence of 100 µg/ml of BSA.
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Results |
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A single round of random mutagenesis of the entire Tb MonoTIM gene yielded a library of 5x106 colonies. The analysis of 10 clones picked out without selection for activity showed that the mutation rate obtained was 0.41%, lower than the 0.6% value reported for the method employed (Cadwell and Joyce, 1992), but consistent with the rate that we usually obtain in our laboratory. The analysis of these clones demonstrates that there was no bias in the mutagenesis methods, finding 34 mutations at the nucleotide level distributed randomly across the gene.
Under selective conditions, there were tens of colonies that stood out over a background of tiny colonies. Three clones were picked out from each of two selective media (i.e. minimal medium with or without casamino acids). The clones obtained were checked by PCR for the absence of loop 3, to rule out contamination with the WT Tb TIM gene. One of the faster growing clones from the selection without casamino acids and all three clones selected with casamino acids came from the MonoTIM gene. The DNA fragments comprising the tpi structural gene of these four variants were subcloned in pUC18 and transformed in the selective strain to select finally one clone from each scheme, which were streaked on LB plates supplemented with ampicillin and kanamycin. Independent colonies of each clone were taken to follow their growth rate in liquid minimal medium in triplicate. As can be observed in Figure 1, clone RMM0-1 transformed with the gene coming from the selection without casamino acids grows significantly faster than clone RMMC-1 selected in minimal medium (MM) with casamino acids.
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The DNA sequence analysis of the two clones selected showed that both of them had four mutations at the nucleotide level giving rise to three mutations at the amino acid level. The most active clone, RMM0-1, shows the amino acid substitutions S43P, T44A, A179T, whereas the less active mutant, RMMC-1, shows S43P, T44S, N208S. A silent C/A mutation was also observed at position 91 for the less active mutant.
It is significant that both selected mutants had replacements at positions 43 and 44 and even more significant that both converged in replacing residue 43 to a Pro, especially considering that a different codon is used for Pro in each mutant (i.e. they are independent mutations). This suggests that these mutations are crucial for the increased activity of the monomeric enzyme in this context. It is important to mention that mutations at these positions were not found in any of the 10 clones sequenced from the non-selective medium, discarding the possibility of PCR hot spots.
Randomization of loop 2
The randomization of six residues at and around loop 2 with NNG/C triplets has a complexity of 1x109 when the loop is held of equal size. However, for a loop 2 + one amino acid, the complexity of the library is more than an order of magnitude larger and if we consider loop 2 + two amino acids, this number increases to 1x1012. The size of the library generated by randomizing loop 2 of monoTIM was 1x107 clones. Therefore, only 102 for loop 2 and 105 for loop 2 + two of the randomized sequence space were explored. Sixteen clones were randomly picked out to determine the variability of the library. Sequence analysis shows the expected distribution of mutations (Figure 2), including insertions. Under selective conditions with casamino acids there were hundreds of larger colonies that showed up against a background of tiny ones. Twenty of these larger colonies were picked out and their plasmid DNA was purified, mixed, digested and cloned in pUC18 and transformed into the selection strain. Ten of the larger colonies were picked out from this retransformation and further characterized. Their DNA sequences showed that there were only six out of the 10 colonies which were independent; they are shown in Figure 3
. It is noteworthy that all the selected mutants have a Pro residue at position 43 and an Arg residue at position 44, replacements that were not found in the clones without selection. There is furthermore a consensus in some of the other mutated positions, such as the presence of a polar residue at position 46, a methionine at position 47 and a hydrophobic residue at position 48. Note also that none of the selected mutants had a larger loop 2, in spite of the fact that these variants were well represented in the library (Figure 2
). Although a very limited sequence space was explored from all the possibilities of larger loops, the analysis of faster growing clones shows that some of the mutated positions do not have consensus and do not seem to be crucial for activity. The same could be expected for a more active variant coming from those with a longer loop 2; therefore, we speculate that we would have found an active mutant with a longer loop 2 if indeed this was favorable for activity.
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Growth curves of tpi JM101 E.coli cells complemented with the different mutants
Culture growth in minimal medium supplemented with casamino acids at 30°C was followed for each independent clone and the growth curves were compared. As can be observed in Figure 1, there was a clear difference in the growth rates of the two mutants obtained from the random mutagenesis of the entire gene. The six independent colonies from the randomization of loop 2 scheme grow in between these two, with the exception of clone LMMC-30, which grows more slowly than RMMC-1. Clone LMMC-23, which has the sequence S43P T44R V46T H47M L48I at loop 2, seems to grow slightly better than the others, so it was selected for further characterization.
Purification and characterization of mutant MonoTIM
The mutant proteins RMM0-1 and LMMC-23 were purified for kinetic characterization. The purity of each mutant was demonstrated by the presence of a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) (data not shown). It is noteworthy that the RMM0-1 mutant was considerably more soluble than the LMMC-23 mutant. The existence of a consensus sequence in loop 2 for the more active clones suggests that it has been nearly optimized in the context of monoTIM and therefore the differences in solubility between the RMM0-1 and LMMC-23 mutants may rely on the mutation at position 179, present in the RMM0-1 mutant. This position is in the middle of helix 6 and is exposed to the solvent, and therefore a replacement by a more polar residue may be favorable for solubility.
The initial rate of formation of dihydroxyacetone from glyceraldehyde-3-P was measured at 25°C, using 100 ng of each mutant enzyme per assay, whereas for MonoTIM 1 µg per assay was used owing to its low activity. The results for kcat and Km are shown in Figure 4 and Table I
. Both mutations have a considerably higher kcat than the parental enzyme (~11-fold) and 4-fold smaller Km, which results in a net gain in catalytic efficiency of 44-fold for both mutants.
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Discussion |
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The combination of directed evolution with a genetic selection system was demonstrated once again to be useful for selecting those mutants that have gained activity with respect to the parental monoTIM gene (Cadwell and Joyce, 1992; Arnold and Volkov, 1999
). However, interpretation of results has to be done carefully. We observed a subtle difference in the growth rate of our clones and this difference is probably due to the difference in solubility of the expressed proteins rather than a difference in activity.
Several attempts based on protein engineering have been made to destabilize the dimeric interface in TIM, always resulting in an accompanying loss of activity (Borchert et al., 1994, 1995b
; Mainfroid et al., 1996
; Schliebs et al., 1997
; Garza et al., 1998
; Perez et al., 1999
). The structural analysis of some of the mutants generated in the presence and absence of inhibitors suggests that the loss of activity is associated with an entropy cost of keeping the catalytic residues in place in the context of monomeric proteins with more flexible loops (Borchert et al., 1995a
; Schliebs et al., 1996
, 1997
). We hypothesize that the creation of new intrasubunit interactions that reduce the entropy of the loops, pulling loops 1 and 4 into the catalytic cavity should be the basis to regenerate a wild-type activity in a monomeric context. Our results show that a limited design (directed only to loop 2) combined with random mutagenesis and a selection method found an easy solution, i.e. to reduce the entropy of loop 2 by introducing a Pro residue at the beginning of the loop. In this sense, one would expect that the design of a monoTIM following the same strategy employed by Wierenga et al. (shortening of loop 3) (Borchert et al., 1994
) would have been more successful if initially done on one of the genes that contain Pro at the equivalent position of residue 43.
The results of both strategies employed to generate variability in monoTIM converged in mutating Ala43 to Pro and also in mutating the next position, 44. The resulting mutants have the same specific activity independently of the other changes that occurred. Preliminary recombination experiments by gene shuffling (Stemmer, 1994a,b
) and STEP (Zhao et al., 1998
) of a mixture of the selected clones from the two strategies have not been successful in further increasing the specific activity of the enzyme, suggesting that point mutations may not be able to increase the activity further in the context of the monomeric enzyme. The ongoing determination of the X-ray crystal structure of the RMM0-1 mutant will yield an insight into the structural changes occurring upon mutation and how they correlate with the increased activity. Our efforts in the near future will be directed to re-engineering all or some of the catalytic loops of monoTIM, based on the knowledge gained with the crystallographic structure. The strategies to be used will probably comprise a combination of directed evolution with protein engineering in order to jump to a different hill in the activity landscape.
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Notes |
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Acknowledgments |
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References |
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Received May 18, 2000; revised November 1, 2000; accepted December 1, 2000.