Proc. Nati. Acad. Sci. USAVol. 91, pp. 3593-3597, April 1994Biochemistry

A recombinant bisphosphoglycerate mutase variant with acidphosphatase homology degrades 2,3-diphosphoglycerateMARIE-CLAUDE GAREL, NICOLE AROUS, MARIE-CLAUDE CALVIN, CONSTANTIN TIGELIU CRAESCU,JEAN ROSA, AND RAYMONDE ROSAInstitut National de la Sante et de la Recherche M6dicale, U.91, H6pital Henri Mondor, 94010 Cr6teil, France

Communicated by M. F. Perutz, January 3, 1994 (receivedfor review September 6, 1993)

ABSTRACT To date no definite and undisputed treatmenthas been found for sickle cell anemia, which is characterized bypolymerization of a deoxygenated hemoglobin mutant (HbS)giving rise to deformed erythrocytes and vasoocclusive com-plications. Since the erythrocyte glycerate 2,3-bisphosphate(2,3-DPG) has been shown to facilitate this polymerization, onetherapeutic approach would be to decrease the intraerythro-cytic level of2,3-DPG by increasing the phosphatase activity ofthe bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose,we have investigated the role of Gly-13, which is located in theactive site sequence Arg'-His"'-Gly"1-Glu12-Gly13 in humanBPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg*sequence of the distantly related acid phosphatases, whichcatalyze asBPGM similar phosphoryl transfers but to a greaterextent. We hypothesized that the conserved Arg* residue inacid phosphatase sequences facilitates the phosphoryl transfer.Consequently, in human BPGM, we replaced by site-directedmutagenesis the corresponding amino acid residue Gly'3 withan Arg or a Lys. In another experiment, we replaced Gly'3 withSer, the amino acid present at the corresponding position of thehomologous yeast phosphoglycerate mutase (n-phosphoglycer-ate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly'3 to Serdid not modify the synthase activity, whereas the mutase andthe phosphatase were 2-fold increased or decreased, respec-tively. However, replacing Gly'3 with Arg enhanced phospha-tase activity 28.6-fold, whereas synthase and mutase activitieswere 10-fold decreased. The presence of a Lys in position 13gave rise to a smaller increase in phosphatase activity (6.5-fold)but an identical decrease in synthase and mutase activities.Taken together these results support the hypothesis that apositively charged amino acid residue in position 13, especiallyArg, greatly activates the phosphoryl transfer to water. Theseresults also provide elements for locating the conserved Arg*residue in the active site of acid phosphatases and facilitatingthe phosphoryl transfer. The implications for genetic therapyof sickle cell disease are discussed.

Some enzymes displaying phosphatase activities such ashuman prostatic acid phosphatase (1, 2), lysosomal (3) andyeast (4) acid phosphatases, erythrocyte bisphosphoglycer-ate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomu-tase, EC 5.4.2.4) (5-7), glycolytic phosphoglycerate mutase(PGM; D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1)(7, 8), and hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (fructose-2,6-bisphosphate 2-phosphatase;D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC3.1.3.46) (1) possess a His residue in their active site, whichis transiently phosphorylated during the course of the phos-phoryl transfer. It was suggested that most such enzymesinvolved in the binding of phosphate esters should have atleast one cationic group suitably disposed near the active site

His in order to facilitate the orientation and binding of thephosphate group and consequently activate the phosphoryltransfer to water (2).

Little is known about the three-dimensional structure andparticularly localization of the active site residues in acidphosphatases. Conversely, the structure of the yeast PGMhas been determined by x-ray diffraction analysis by usingcrystals soaked in 3-phosphoglycerate (3-PG). This latterenzyme shares 5Ow sequence identity with human BPGMand catalyzes the same three reactions (synthase, mutase,and phosphatase), although at substantially different rates.These activities are catalyzed at the unique active site ofBPGM. To date and in spite of recent crystallization ofhuman BPGM (9), no crystallographic data have been ob-tained for this enzyme. Consequently, the amino acids in-volved in the active site ofhuman BPGM have been deducedby comparison with the structure of the yeast PGM (8).Amino acid residues of the active site were highly conservedbetween BPGM and PGM. The different catalytic ratesobserved for these two homologous enzymes could be ex-plained by the nonconserved residues in their active site.Among them, Gly13 in BPGM and the homologous Ser11 in theyeastPGM have been postulated to play an important role (7,8).

Synthase and phosphatase activities of BPGM catalyze,respectively, the synthesis and degradation of glycerate2,3-bisphosphate (2,3-DPG), the main allosteric effector ofhemoglobin. In spite of the presence of acid phosphatases inerythrocytes, the degradation of2,3-DPG is very low becausethese acid phosphatases cannot degrade 2,3-DPG and thephosphatase activity ofBPGM is very slow (1000-fold lowerthan the synthase activity and lower than that of acid phos-phatases). Consequently, 2,3-DPG is present in high concen-trations in erythrocytes.Our purpose being to decrease 2,3-DPG level in erythro-

cytes by increasing the BPGM phosphatase activity, we havecompared amino acid sequences of several acid phosphatasespossessing a His residue in their active site and especially thesequences of a fragment common to these enzymes andhuman BPGM (Fig. 1) (4). This fragment, corresponding tothe amino acid sequence Arg9-His10-Gly11-Glu12-Gly13 in hu-man BPGM, is homologous to the amino acid sequenceArg7-His8-Gly9-Gln10-Ser'1 in yeast PGM, which is localizedin the active site and contains the phosphorylatable His (7, 8,10). From this analysis, we have postulated that the con-served Arg residue, denoted Arg* in the acid phosphataseconsensus sequence and which corresponds to the Gly13 inBPGM, could be a good candidate as a cationic group forenhancing the phosphoryl transfer. We have therefore sub-stituted Arg for Gly13 in human BPGM by site-directedmutagenesis. We have also substituted Lys for Gly13 toevaluate the role of another positively charged amino acid at

Abbreviations: 2,3-DPG, glycerate 2,3-bisphosphate; 3-PG, glycer-ate 3-phosphate; BPGM, bisphosphoglycerate mutase; PGM, phos-phoglycerate mutase.

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Hu-BPGM Arg9 His10 GIyll G1u12 G1y13 tion was performed on an HPLC column of Fractogel TSKAF blue (Merck) at room temperature as reported (17).

HU-PGM-M Arg His Gly G1u Thr Enzyme and 2,3-DPG Assays. Synthase and mutase activ-

Ye-PGM Arg7 His8 GIy9 Gin10 Ser11 ities were assayed according to methods already reported(18). When synthase activity was assayed directly on the E.

F-2,6-P2ase Arg His Gly Glu Ser coli crude extracts, this assay being directly related toNADHproduction by glyceraldehyde phosphate dehydrogenase, thereaction could be partially masked by the intrinsic NADH

Hu-P-ACP Arg His Gly Asp Arg* oxidase activity ofE. coli. It was, therefore, necessary to add

Hu-L-ACP Arg His GIl ASP Arg* 2 mg of antimycin A and 1 mmol ofKCN to 1 ml ofthe assaysystem as specific inhibitors of E. coli NADH oxidase. The

Ye-ACP1 Arg His Gly Ser Arg* phosphatase activity was measured by coupling the reactionwith phosphoglycerate kinase, glyceraldehyde phosphate

Ye-ACP3 Arg His Gly Glu Arg* dehydrogenase, triosephosphate isomerase, and glycerolYe-ACP5 Arg His Gly GIu Arg* phosphate dehydrogenase. In this sequence of reactions, the

degradation of 1 mol of 3-PG is coupled with the oxidation ofFIG. 1. Conserved peptide sequence in human BPGM (Hu- 2 mol of NADH. Each sample was checked against a control

BPGM), human muscle PGM (Hu-PGM-M), yeast PGM (Ye-PGM), deprived of 2,3-DPG. In the case of 2-phosphoglycolatehepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F- stimulation of the phosphatase reaction, 1 mM 2-phospho-2,6-P2ase), human prostatic acid phosphatase (Hu-P-ACP), human glycolate was added to the standard assay system and be-lysosomal acid phosphatase (Hu-L-ACP), yeast acid phosphatase P1 cause of its potent effect (19) incubation was performed for(Ye-ACP1), yeast acid phosphatase P3 (Ye-ACP3), and yeast acid 15 min instead of 60 min and 0.4 mg of the enzyme was usedphosphatase PS (Ye-ACP5). instead of 4 mg. The amount of 2,3-DPG was measured

this position in the active site. Another substitution was enzymatically on the deproteinized extracts by techniquesperformed by replacing Gly" with Ser to analyze the modi- previously reported (18), and its amount was related to thefications of the catalytic properties ofBPGM when this Gly13 ratio of protein in the lysate measured according to Lowry etis replaced by the amino acid residue present in the homol- al. (20).ogous yeast PGM. Electrophoresis. PAGE was performed in the presence ofSDS according to Laemmli (21). The gels were stained with

Coomassie blue R250 and the amount of expressed wild-typeMATERIALS AND METHODS or mutant BPGM was determined by densitometric scanning

Materials. Except when specified otherwise the reagents of the gels.used for the buffers were obtained from Merck. All substrates Thermostability Studies. The purified variants were incu-and commercial enzymes were purchased from Boehringer bated at 550C for 30 min in 10 mM Tris HCl buffer (pH 7.5)Mannheim except for NADH, which was a product of Sigma containing 1 mM EDTA, 1 mM 2-mercaptoethanol, and 1 mgas were Trizma base (Tris), bovine serum albumin, and of bovine serum albumin per ml. The incubation was stoppeddithiothreitol. Acrylamide and bisacrylamide were supplied by placing the tubes in ice water. After centrifugation,by Fluka. Isopropyl alcohol was obtained from Prolabo antibody consumption was determined as previously re-(Paris). Rabbit antiserum directed against human erythrocyte ported (22).BPGM was obtained according to methods previously re- Modeling of the Enzymes. Molecular modeling of the en-ported (11). Oligonucleotides were synthesized by the phos- zymes was performed using the UNIX version of the BRUGELphoramidite method on an Applied Biosystems DNA syn- software package as described (10). Modeling of the three-thesizer (model 381A) followed by purification by electro- dimensional conformation of the human BPGM enzyme wasphoresis on a 20%6 polyacrylamide gel containing 7 M urea. made by using the known crystallographic structure of the

Site-Directed Mutagenesis. The procedure used for oligo- homologous yeast PGM as a starting structure (8).nucleotide-directed site-specific mutagenesis was based onthe method described by Taylor et al. (12) using a kit RESULTSdeveloped by Amersham and 20-mer oligonucleotides encod-ing single amino acid mutations at residue 13. At the end of Expression of the Mutated BPGM Enzymes. The site-theprocedure,single-stranded ofputativemut directed mutagenesis experiments were performed as de-

the procedure, sngle-stranded unAs of putative mutant scribed. For all the BPGM mutants, the entire coding regionphages were prepared and sequenced by the dideoxynucle- of the cDNA was sequenced by using specific internalotide cham-termination method (13) to confirm the desired oligonucleotides in order to confirm that no additional mu-mutation. The complete sequence of the mutated BPGM tation was introduced during mutagenesis.insert was then checked entirely between the restriction sites Large amounts of the wild type and the three mutants ofused for subcloning in the expression vector pKK223-3 human BPGM (Gly13 to Arg, Gly13 to Lys, and Gly13 to Ser)(Pharmacia) as described (14). For all these procedures, the were expressed in E. coli by using the expression vectorbasic cloning methods described by Maniatis et al. were used pKK223-3 as described for the wild-type human BPGM (14).(15). Each BPGM variant and the wild-type enzyme were pro-

Preparation of Overexpressed Mutants and Wild-Type Hu- duced in E. coli in the same amount (-5.5% of the bacterialman BPGM. Overnight cultures of Escherichia coli contain- protein). As demonstrated for the wild-type enzyme, theing an expression plasmid of either mutant or wild-type growth rate of bacteria was decreased every time 2,3-DPGenzyme were grown in L broth medium containing ampicillin was synthesized by the recombinant enzyme (14). Such an(100 ug/ml) and then diluted 1:100 for the expression cul- inhibition of the growth rate of bacteria was not observedtures. Induction of protein expression was performed with during expression of the Gly13 to Arg and Gly13 to Lysisopropyl /3-D-thiogalactoside as described (14). variants, which produced very low levels of 2,3-DPG as

Purification of the mutant and wild-type enzymes was described below. In contrast, this inhibition was similar toperformed as described (16) except that the lysate was not that of the wild-type enzyme when the Gly13 to Ser variant,heated before chromatography and the first step of purifica- which could synthesize 2,3-DPG, is expressed.

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is

a b c d e

FIG. 2. Coomassie blue-stained SDS/15% polyacrylamide gel ofpurified BPGM Gly13 to Arg variant used in this study. Lanes: a,

molecular mass markers (from top) of 94, 67, 43, 30, and 14.4 kDa;b, purified wild-type human BPGM expressed in E. coli; c, total cellextract of E. coli expressing the Gly13 to Arg variant after induction;d, partially purified Gly13 to Arg variant after the first blue Fractogelcolumn chromatography; e, purified Gly13 to Arg variant after thesecond step of purification on a Fractogel TSK column. The mutatedoligonucleotide for production of the Gly13 to Arg variant by site-directed mutagenesis according to the method described by Taylor etal (12) has the sequence 5'-CATGGAGAGCGTGCTTGAATT-3'.Construction of the expression vector, expression of the wild-typeand mutant BPGM, and their purification have been described (14,16). Induction ofthe tac promoter ofthe expression vector was madewith 0.5 mM isopropyl f-D-thiogalactoside during 3 h at 37°C.

Neither synthase activity nor detectable amounts of 2,3-DPG could be found in crude extracts of nontransformed E.coli XL1-B bacteria. Consequently, we measured these val-ues in the lysate of the bacteria expressing the differentrecombinant enzymes. When bacteria expressed the Gly13 toArg and Gly13 to Lys variants, we detected a low synthaseactivity (0.038 and 0.11 unit per mg of protein, respectively)as well as a low 2,3-DPG production (0.0016 and 0.0015 umolof 2,3-DPG per mg of protein, respectively). In contrast, a

synthase activity (0.41 unit per mg of protein) similar to thatobtained with the wild-type enzyme (0.45 unit per mg ofprotein) was present in the crude extracts of E. coli trans-formed with the plasmid bearing the Gly13 to Ser mutation,giving rise to production of 0.027 ,umol of 2,3-DPG per mg ofprotein (control, 0.054 umol of 2,3-DPG per mg of protein).

All the variants were purified to homogeneity (14, 16). Asingle polypeptide band with a molecular mass of 30 kDa wasobtained by SDS/PAGE for each purified enzyme afterCoomassie blue staining as shown in Fig. 2 for the Gly13 toArg variant. All the variants were stable when incubated at55°C for 30 min.

Catalytic Properties of Gly'3 Variants. The catalytic prop-

erties of the three variants are summarized in Table 1 andcompared to those of normal human BPGM expressed in E.coli. When Gly'3 was replaced by Ser, a slight decrease(2-fold) of the phosphatase activity was observed and therewas a lower capacity of its stimulation by 2-phosphoglyco-late. Simultaneously the synthase activity was normal and themutase activity was 2-fold increased. By contrast, when Argor Lys replaced Glyl3 a very strong increase in the phospha-tase activity (28.6- and 6.5-fold, respectively) was observed,whereas phosphatase stimulation by 2-phosphoglycolate wasdecreased. These two variants (Glyl3 to Arg and Glyl3 to Lys)showed decreased synthase (10- and 8.6-fold, respectively)and mutase (10- and 6.4-fold, respectively) activities. Itshould be noted that the control values of the phosphataseactivities and Km values were different from those previouslyreported (16) because of the differences in the two assaytechniques.The Michaelis constants for the substrates in the three

reactions catalyzed by wild-type and mutant human BPGMwere measured (16) and are summarized in Table 2. The Kmfor 3-PG in the synthase activity and for 2-PG in the mutaseactivity were slightly modified (2-fold).The Michaelis constants for the binding of 2,3-DPG were

2- to 6.7-fold decreased in the basic phosphatase activity andthe mutase activity of the three variants except for a slightincrease in the mutase activity for the Gly'3 to Arg variant.In contrast, a moderate increase was observed for the Gly13to Arg and Gly13 to Lys variants in the 2-phosphoglycolate-stimulated phosphatase activity (3.5- and 2.5-fold, respec-tively). The Km value for the 1,3-DPG in the synthase activitycould not be determined because of the great instability ofthis substrate.

DISCUSSION

In this report, we show that replacement by site-directedmutagenesis of Glyl3 by Ser in human BPGM did not greatlymodify the catalytic properties of the mutant enzyme ascompared to the wild-type BPGM. The slightly decreasedphosphatase activities as well as increased mutase activitymimic the PGM enzyme, and these results are in accord withthose obtained for the corresponding yeast PGM Ser" to Glyvariant recently produced by White and Fothergill-Gilmore(23) (as shown in Fig. 1, the BPGM Glyl3 residue correspondsto the yeast PGM Ser" residue). X-ray diffraction analysis ofyeast PGM crystals soaked in 3-PG has shown that the Ser"residue could directly interact with substrate (8). Our resultsof Km for the Gly13 to Ser BPGM variant show that themonophosphoglycerate affinities are only slightly modifiedwhile the affinity for 2,3-DPG increases. Such results arecomparable to those obtained for the Ser" to Gly PGMvariant, which showed a greatly decreased (10-fold) affinityfor 2,3-DPG. These results afford evidence for interactionsbetween 2,3-DPG and the serine residue located at a homol-ogous position in the active sites of the human BPGM and

Table 1. Specific activities for purified wild-type, Gly13 to Arg, Gly13 to Lys, and Gly13 to Ser variants of humanBPGM expressed in E. coli

Specific activity, units per mg of protein

Enzyme Reaction Wild type Gly13 to Arg Gly13 to Lys Gly13 to Ser

Synthase 1,3-DPG - 2,3-DPG 16.3 1.5 1.9 15.8Mutase 2-PG 3-PG 8.5 0.8 1.3 17.3Stimulatedphosphatase 2,3-DPG 3-PG+Pi 7.6 1.3 0.3 4.7

Unstimulatedphosphatase 2,3-DPG - 3-PG+Pi 2.45 x 10-2 70.2 x 10-2 15.9 x 10-2 1.34 x 10-2

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Table 2. Km values for substrates of the three reactions catalyzed by purified wild-type, Gly13 toArg, Gly13 to Lys, and Gly13 to Ser variants of human BPGM expressed in E. coli

Ki, mM

Enzyme Wild type Gly13 to Arg Gly13 to Lys Gly13 to Ser

Synthase3-PG 80 35 52 71

Mutase2-PG 15 8.6 29 18.52,3-DPG 125 416 65 25

Unstimulated phosphatase2,3-DPG 3570 680 530 1040

Stimulated phosphatase2,3-DPG 41 142 104 25

Km values were determined according to the methods previously reported (16) except for thephosphatase activities, whose determination is described in Materials and Methods. Calculations werefrom Lineweaver-Burke plots. Measurements are averages of at least three determinations.

yeast PGM enzymes and suggest that this residue is notinvolved in the binding of monophosphoglycerates.

In contrast, as postulated, replacement of Gly'3 in humanBPGM by a positively charged amino acid residue greatlymodifies the relative ratios ofthe three reactions. For the twoGlyl3 to Arg and Gly13 to Lys BPGM variants, the synthaseand mutase reactions are greatly decreased. On the contrary,the basic phosphatase activity is markedly increased for theGlyl3 to Arg variant and to a lesser extent by the Gly'3 to Lysvariant. Such results show that in human BPGM, an Argresidue present at position 13 strongly increases the phos-phoryl transfer to a water molecule. Nevertheless, the pres-ence in the active site of a monophosphoglycerate (3-PG)during the mutase reaction or 2-phosphoglycolate in thestimulated phosphatase reaction, inhibited this phosphoryltransfer. In this paper, the Km values for monophosphoglyc-erates and 2,3-DPG show that their affinities are slightly(2-fold) modified. Nevertheless, for the Gly13 to Arg andGly13 to Lys variants, it is remarkable that the 2,3-DPGaffinities are greatly increased in the phosphatase activity,indicating that the presence of Arg3 or Lys'3 residues led tobetter interactions with 2,3-DPG. However, the presence of2-phosphoglycolate in the active site in the stimulated phos-

H 187

FIG. 3. View of the active site of human BPGM modeled on thebasis of crystallographic coordinates of PGM (8, 10). Only theresidues considered to play a major role in ligand binding are shown.

phatase reaction inhibited the binding of2,3-DPG as revealedby the increased Km values.The Gly'3 to Arg mutation in human BPGM greatly in-

creasing the phosphatase activity suggests that the guanidiumgroup ofArgl3 stabilizes the transition state ofthe transferredphosphate, promoting its transfer from His10 to water. So, theessential feature of the proposed mechanism of action of acidphosphatases using a phosphohistidine intermediate is ex-perimentally demonstrated in this paper with human BPGM.The structure of human BPGM was modeled (10) usingcrystallographic data of the well-known homologous yeastPGM and amino acid residues involved in the active site havebeen proposed. Some ofthem are represented in Fig. 3. It canbe seen that the Glyl3 residue is located in the neighborhoodof the phosphorylatable His10 residue. Replacement of thisGly'3 by Arg followed by energy minimization (data notshown) indicates that the new guanidium group could pointtoward the active site and occupies a position located in theneighborhood of His'0 and Cys'2 residues. In such a position,it can be postulated that the new guanidium group couldinteract with the substrates.

In conclusion, if the peptide that has a sequence similar tothat of BPGM performs a similar function in all the acidphosphatases, it is reasonable to assume that, as in BPGM,it contains the active site (phospho)His residue and that theconserved Arg* residue is located in their active site. Inaddition, it is likely that activation of the phosphoryl transferto a water molecule obtained in this report for human BPGMwith the Gly'3 to Arg mutation is not unique to this enzymebut represents a common mechanism for other phosphatasespossessing His residues in their active site. It would beinteresting to make the corresponding mutation in the yeastPGM (7) and fructose-2,6-bisphosphatase (1) to show thatsuch a mutation also activates the phosphatase activity.Only in vitro modulation of 2,3-DPG level has been per-

formed to study its effect on polymerization of deoxyHbS(24). The recent production by several laboratories (25-27)and ourselves (28) of a transgenic mouse model of sickle celldisease (29-33) offers the opportunity to evaluate the poten-tial role of a decreased 2,3-DPG level in vivo on erythrocytesickling by expressing the Gly'3 to Arg BPGM variant in theirsickle erythrocytes.

We thank Dr. N. Blumenfeld for assistance in revision of theEnglish. We are grateful to A. M. Dulac, R. Quintel, and J. M. Massefor preparation of the manuscript and the figures. This work wassupported by grants from the Institut National de la Sante et de laRecherche Medicale and the Centre National de la RechercheScientifique.

1. Bazan, J. F., Fletterick, R. J. & Pilkis, S. J. (1989) Proc. Natl.Acad. Sci. USA 86, 9642-9646.

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2. Van Etten, R. L. (1982) Ann. N. Y. Acad. Sci. 390, 27-51.3. Pohlmann, R., Krentler, C., Schmidt, B., Schr6der, W.,

Lorkowski, G., Culley, J., Mersmann, G., Geier, C., Waheed,A., Gottschalk, S., Grzeschik, K.-H., Hasilik, A. & Figura,K. V. (1988) EMBO J. 7, 2343-2350.

4. Van Etten, R. L., Davidson, R., Stevis, P. E., MacArthur, H.& Moore, D. L. (1991) J. Biol. Chem. 266, 2313-2319.

5. Rapoport, S. & Luebering, J. J. (1950) J. Biol. Chem. 183,507-516.

6. Rosa, R., Audit, I. & Rosa, J. (1975) Biochimie 57, 1059-1063.7. Fothergill-Gilmore, L. A. & Watson, H. C. (1989) Adv. En-

zymol. 62, 227-313.8. Winn, S. I., Watson, H. C., Harkins, R. W. & Fothergill,

L. A. (1981) Philos. Trans. R. Soc. London Ser. B 293,121-130.9. Cherfils, J., Rosa, R., Garel, M. C., Calvin, M. C., Rosa, J. &

Janin, J. (1991) J. Mol. Biol. 218, 269-270.10. Craescu, C. T., Schaad, O., Garel, M. C., Rosa, R. & Edel-

stein, S. (1992) Biochimie 74, 519-526.11. Dubart, A., Romeo, P. H., Tsapis, A., Goossens, M., Rosa, R.

& Rosa, J. (1984) Biochem. Biophys. Res. Commun. 120,441-447.

12. Taylor, J. W., Ott, J. & Eckstein, F. (1985) Nucleic Acids Res.13, 8765-8785.

13. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl.Acad. Sci. USA 74, 5463-5467.

14. Garel, M. C., Joulin, V., Le Boulch, P., Calvin, M. C., Prehu,M. O., Arous, N., Longin, R., Rosa, R., Rosa, J. & Cohen-Solal, M. (1989) J. Biol. Chem.' 264, 18966-18972.

15. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) MolecularCloning: A Laboratory Manual (Cold Spring Harbor Lab.Press, Plainview, NY).

16. Calvin, M. C., Blouquit, Y., Garel, M. C., Prehu, M. O.,Cohen-Solal, M., Rosa, J. & Rosa, R. (1990) Biochimie 72,337-343.

17. Garel, M.-C., Lemarchandel, V., Calvin, M.-C., Arous, N.,

Craescu, C. T., Prehu, M.-O., Rosa, J. & Rosa, R. (1993) Eur.J. Biochem. 213, 493-500.

18. Rosa, R., Prdhu, M. O., Beuzard, Y. & Rosa, J. (1978) J. Clin.Invest. 62, 907-915.

19. Rose, Z. B. & Liebowitz, J. (1970) J. Biol. Chem. 245, 3232-3241.

20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.(1951) J. Biol. Chem. 193, 265-268.

21. Laemmli, U. K. (1970) Nature (London) 227, 680-685.22. Rosa, R., Prehu, M.-O., Albrecht-Ellmer, K. & Calvin, M.-C.

(1983) Biochim. Biophys. Acta 742, 243-249.23. White, M. F. & Fothergill-Gilmore, L. A. (1992) Eur. J. Bio-

chem. 207, 709-714.24. Poillon, W. N. & Kim, B. C. (1990) Blood 76, 1028-1036.25. Greaves, D. R., Fraser, P., Vidal, M. A., Hedges, M. T.,

Ropers, D., Luzzatto, L. & Grosveld, F. (1990) Nature (Lon-don) 343, 183-185.

26. Ryan, T. M., Townes, T. M., Reilly, M. P., Asakura, T.,Palmiter, R. D., Brinster, R. L. & Behringer, R. R. (1990)Science 247, 566-568.

27. Rubin, E. M., Witkowska, H. E., Spandler, E., Gurtin, P.,Lubin, B. H., Mohandas, N. & Clift, S. M. (1991) J. Clin.Invest. 87, 639-647.

28. Trudel, M., Saadane, N., Garel, M. C., Bardakdjian-Michau,J., Blouquit, Y., Guerquin-Kern, J. L., Rouyer-Fessard, P.,Vidaud, D., Pachnis, A., Romeo, P. H., Beuzard, Y. & Cos-tantini, F. (1991) EMBO J. 10, 3157-3165.

29. Ingram, V. M. (1956) Nature (London) 178, 792-794.30. Wishner, B. C., Ward, K. B., Lattman, E. E. & Love, W. E.

(1975) J. Mol. Biol. 98, 179-194.31. Dykes, W., Crepeau, R. H. & Edelstein, S. J. (1979) J. Mol.

Biol. 130, 451-472.32. Eaton, W. A. & Hofrichter, J. (1987) Blood 70, 1245-1266.33. Luzzatto, L. & Goodfellow, P. (1989) Nature (London) 337,

17-18.

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