Proc. Nati. Acad. Sci. USAVol. 83, pp. 1084-1088, February 1986Medical Sciences

Cloning and expression of a cDNA coding for the anticoagulanthirudin from the bloodsucking leech, Hirudo medicinalis

(thrombosis/antithrombin/recombinant DNA/coagulation)

R. P. HARVEY*t, E. DEGRYSE*, L. STEFANI*, F. SCHAMBER*, J.-P. CAZENAVEt, M. COURTNEY*§,P. TOLSTOSHEV*¶, AND J.-P. LECOCQ**Transgtne, S.A., 11, rue de Molsheim, 67000 Strasbourg, France; and tCentre R6gional de Transfusion Sanguine, Strasbourg, France

Communicated by J. Bracket, October 24, 1985

ABSTRACT Cloned cDNAs have been isolated that encodea variant of hirudin, a potent thrombin inhibitor that issecreted by the salivary glands of the medicinal leech, Hirudomedicinals. This variant probably corresponds to a form thathas been purified from leech heads but differs in amino acidsequence from the hirudin purified from whole leeches. Thereare at least three hirudin transcripts detectable in leech RNAsthat are different in size, site of synthesis, inducibility bystarvation, and relationship to hirudin activity. The newhirudin variant predicted by the cDNA and the heterodispersetranscription products suggest a hirudin protein family. Thehirudin cDNA was expressed in Esckerichia coli under thecontrol of the bacteriophage X PL promoter. The recombinantproduct is biologically active, inhibiting the cleavage by throm-bin of fibrinogen and a synthetic tripeptide substrate.

Leech hirudin is the most potent natural inhibitor of coagu-lation known (1-4). A very stable noncovalent 1:1 complexis rapidly and specifically formed with a-thrombin, therebyabolishing its ability to cleave fibrinogen (4). To date there isno evidence that it can interact with other components of thehuman coagulation cascade (5, 6).

Hirudin is a polypeptide of 65 amino acids that is stable toextremes ofpH and heat (4). It contains six cysteine residuesgrouped in the NH2-terminal half of the protein, an acidicCOOH-terminal half, and one sulfated tyrosine (7). A hirudinform with isoleucine at the NH2 terminus was first purifiedfrom leech heads (H. medicinalis) (4, 8) in which activity wasfound to be concentrated in the salivary glands. Subsequent-ly, Bagdy et al. (9) adopted new purification schemes usingwhole leeches instead of heads, yielding a form with Val-Valas the first two NH2-terminal amino acids. The amino acidsequence of the "whole body form" has been determined byindependent groups (8, 10), and valine residues at positions1 and 2 have been confirmed. Both forms had a specificactivity of around 8000-10,000 antithrombin units/mg. How-ever, more recently, Baskova et al. (11) described twodistinct hirudins: a highly active form in heads with an Ile-1NH2 terminus and an inactive form in bodies (pseudohirudin)with a Val-Val NH2 terminus. The potency and specificity ofhirudin have generated interest in its possible use as a clinicalreagent in treatment of thrombotic diseases, but a detailedpharmacological assessment is prevented by the cost andsupply of purified material. In animal studies (12-14), hirudinwas shown to be pharmacodynamically inert apart from itsanticoagulant activity. It has extremely low toxicity (LD50 >500,000 antithrombin units/kg in rats; ref. 12), appears to benonantigenic, and is eliminated almost completely via thekidneys in a biologically active form (12). It is effective inpreventing venous thrombosis, vascular shunt occlusion, and

thrombin-induced disseminated intravascular coagulation inrats (12). Endotoxin-induced disseminated intravascular co-agulation is prevented in newly weaned pigs (15).

Purification of large quantities of hirudin from leeches forfurther clinical testing or eventual clinical use is highlyimpractical, but this problem can potentially be solved byrecombinant DNA technology. In addition, cloning of thegene(s) for hirudin should help to resolve questions aboutdifferent hirudin forms and possible precursor proteins.Here we report the cloning of a cDNA encoding one variant

of H. medicinalis hirudin and its expression in Escherichiacoli to yield a biologically active product.

MATERIALS AND METHODSLeeches. Live Hirudo medicinalis were purchased from

Ricarimpex (Audenge, France) and kept in aerated watercontaining 0.63 mM NaCl, 0.07 mM CaCl2, 0.05 mM MgSO4,and 0.05 mM KCl at ambient temperature. They were fed oncitrate-treated rabbit blood from an inflated porcine bladder.Fed leeches were kept in a separate aquarium.

Protein Extracts. Frozen leech segments were homoge-nized in phosphate-buffered saline (0.1 M Na3PO4, pH7.0/0.15 M NaCl) with a Polytron homogenizer (Brinkmann),and particulate material was sedimented. For bacterial ex-tracts, cells were disrupted by sonication in TGE buffer (25mM Tris-HCl, pH 8/50 mM glucose/10 mM EDTA) andcleared by centrifugation.RNA Extraction. Powdered, frozen leech sections were

added to lx NETS buffer (0.1 M NaCl/1 mM EDTA/10 mMTris HCl, pH 7.5/0.5% sodium dodecyl sulfate) containing50% phenol at 95°C and were homogenized immediately.After a cooling period, phases were separated by centrifu-gation. The phenol phase was reextracted with 2x NETS,and the aqueous phase was reextracted with phenol. Nucleicacid was precipitated from the pooled aqueous phases with 2vol of ethanol. After centrifugation, the pellet was dissolvedin H20, and the solution was adjusted to 2.5 M LiCl and keptat 4°C overnight. To pellet precipitated RNA, the solutionwas underlaid with 0.25 vol of 3 M LiCl and centrifuged at15,000 x g for 10 min at 4°C. The pellet was dissolved in H20and reprecipitated with ethanol.

Hirudin Activity. Antithrombin activity in leech or bacte-rial extracts was measured in a clotting assay (4) usingcitrated human platelet-poor plasma as a fibrinogen source orin a colorimetric assay using the thrombin chromogenicsubstrate Tos-Gly-Pro-Arg-p-nitroanilide (Chromozym TH,Boehringer Mannheim; Tos = tosyl) (7). Standard curves

Abbreviation: kb, kilobases.tPresent address: Department of Biochemistry and Molecular Biol-ogy, Harvard University, 7 Divinity Avenue, Cambridge MA 02138.§To whom all reprint requests should be addressed.Present address: Biotechnology Australia, Pty Ltd., 28 BarcooStreet, East Roseville, NSW 2069, Australia.

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Proc. Natl. Acad. Sci. USA 83 (1986) 1085

were established with standardized bovine thrombin (Roche,Neuilly-sur-Seine, France) in the case of the clotting assayand with standardized hirudin (a gift from F. Markwardt) inthe case of the chromogenic assay.cDNA Cloning. cDNA banks were constructed in pBR322

by standard procedures (16). Screening of banks with oligo-nucleotides was as described (16), with the stringency ofwashes for the 48-mer probe being 0.3 M NaCl/0.03 Msodium citrate/0.1% NaDodSO4 at 500C. Oligonucleotideswere synthesized by the phosphotriester method on aninorganic support (17). DNA sequence analysis of clones wasperformed by the dideoxy chain-termination method (18)after subcloning into an M13 vector.Thrombin-Sepharose. Thrombin (61 National Institutes of

Health units/mg) was bound to CNBr-activated Sepharosebeads (Pharmacia) by using the manufacturer's recommend-ed protocol. For affinity selection ofbacterial hirudin, throm-bin-Sepharose was added, in batch, to bacterial extracts sothat all activity was bound. The Sepharose was sedimentedby gravity, washed twice with excess 0.5 M NaCl, and theneluted with 4 vol of 0.1 M 4-aminobenzamidine/25 mM HCl.All incubations and elutions were at ambient temperature for10 min. Carrier bovine serum albumin (30 pg/ml) was addedto the eluted proteins, and 4-aminobenzamidine was removedby dialysis against 25 mM HCl and then H20. [35S]Cysteine-labeled proteins were analyzed on 15% NaDodSO4/polyac-rylamide gels (19) after reduction/denaturation in 2.3%NaDodSO4/6.25% 2-mercaptoethanol at 100°C.

RESULTS

Cloning ofHirudin cDNAs. ComplementaryDNA banks wereconstructed from mRNA purified from the crudely dissectedhead region of starved leeches. Hirudin clones were identifiedby screening with a long synthetic oligonucleotide (16), thedesign of which was based upon the published amino acidsequence ofhirudin (8, 9) and on codon usage data from insects,the closest evolutionary relatives of segmented worms forwhich extensive sequence data were available. A 48-mer (5'CTGAGGCTTAGGAGTACCCTGGCCGGTGACGCACTG-GTTCTTCTCGCC 3') was synthesized, corresponding to ami-no acids 34-49 of the hirudin sequence (4).Two cDNA banks of 43,000 and 21,000 independent

transformants were screened with the radiolabeled oligonu-cleotide and subsequently with confirmed hirudin cDNAinserts. In total, nine positive clones were isolated. Thecomplete DNA sequence of the longest clone, pTG717, isshown in Fig. 1. Translation in one reading frame predicts aprotein sequence of86% homology with the published hirudinsequence (8, 9). The 5' terminus of this cDNA is 21 nucleo-tides upstream from the beginning of the hirudin codingsequence, and no initiator methionine codon is found beforethe first amino acid of the mature protein. The seven aminoacid NH2-terminal extension predicted by the cDNA buttruncated by the cloning process is hydrophobic and ends inalanine, an amino acid commonly occurring at the NH2-terminal side of the signal peptide cleavage site (20). There-fore, we assume that it represents part of a signal peptidesequence that is removed from a precursor form of hirudin.Hirudin Variants. The isolation ofpTG717 (Fig. 1) confirs

the existence and defines the sequence of a new hirudin withan isoleucine NH2 terminus, as predicted by NH2-terminalanalysis of highly purified hirudin from leech heads (4). Wepropose a simple nomenclature to distinguish the two (nowconfirmed) variants of hirudin: HV-1 (hirudin variant 1) forthe sequenced protein with the Val-Val terminus and HV-2 forthe form with the isoleucine NH2 terminus and encoded byour cDNA clone. There are nine amino acid differencesbetween HV-1 and HV-2 (Fig. 1). The positions of the sixcysteine residues are constant. It is evident that HV-1 and

1 21a GCA ATC TGC GTG TCT CAA GCA

b Ala Ile Cys Val Ser Gln Ala

22 66a ATT ACT TAC ACT GAT TGT ACA GM TCG GGT CM MT TTG TGC CTC

1 15b Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu

1 2C Val Val ---------------------------------------------------

67 1lla TGC GAG GGA AGC MT GTT TGC GGT AM GGC MT MG TGC ATA TTG

16 30b Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ile Leu

24C ------------Gln------------------------

112 156a GGT TCT MT GGA MG GGC MC CM TGT GTC ACT GGC GAA GGT ACA

31 45b Gly Ser Asn Gly Lys Gly Asn Gln Cys Val Thr Gly Glu Gly Thr

33 35 36C ------- Asp --- Glu Lys -----------------------------------

157 201a CCG MC CCT GAA AGC CAT MT MC GGC GAT TTC GAA GM ATT CCA

46 60b Pro Asn Pro Glu Ser His Asn Asn Gly Asp Phe Glu Glu Ile Pro

47 49 53C --- Lys --- Gln ------------Asp ---------------------------

202 256a GAA GAA TAT TTA CM TGAAMATGAAAGAATATCMTCATAGAGAATTTTGATTT

61 65b Glu Glu Tyr Leu Gln

257 316a MAAACATTTCCATAGCTMGCTATTTACCMTAAATAMTTMTTTTTCCATTGMTCT

317 376a CMTCATATTTACTCTCMTCATATTCAGCTATTTACCAATMATAMTTMTTTTTCCA

377a TGA

FIG. 1. DNA sequence of the insert ofpTG717 (row a), the aminoacid sequence predicted by pTG717 in the reading frame that bearshomology to the published hirudin sequence (6, 9) (row b), and theresidues in the published hirudin sequence that differ from thesequence in row b (row c) are shown.

HV-2 are not related as precursor and product, as suggestedfor the Ile-1 (head) and Val-Val (body) forms observed byBaskova et al. (11).To screen for cDNAs encoding HV-1, an 18-mer oligonu-

cleotide probe (5' TCTAATGGAAAGGGCAAC 3') wassynthesized spanning amino acids 32-37 of HV-2 and match-ing exactly the pTG717 DNA sequence. In this region thereare three amino acid changes out of six between HV-1 andHV-2 (Fig. 1), and under stringent conditions, such a probeshould not hybridize to an HV-1 cDNA. Indeed, all of thehirudin cDNA isolates (selected at low stringency) hybrid-ized to this probe, indicating absence of HV-1 cDNAs in theleech-head cDNA bank. Hence, pTG717 probably representsthe major transcript in leech heads (salivary glands).To investigate hirudin gene transcription or activity in

other tissues that could possibly be attributed to HV-1, wemade specific dissections of fed and starved leeches toseparate the salivary glands from the rest of the body. Threeleech segments were studied: a head section and two bodysections, head proximal (Bi) and head distal (B2). Solubleprotein from crude homogenates was assayed for antithrom-bin activity by using a synthetic chromogenic peptide sub-strate (7), and total RNA was extracted for blotting analysis.In these dissections, most hirudin activity was found in thehead section (Fig. 2 Upper). In RNA blotting analysis ofleechRNAs using pTG717 insert as probe, a complex situation wasobserved (Fig. 2 Lower). In the head segments of starvedleeches (containing salivary glands), a predominant RNA of630 bases was detected. This species, while still synthesizedin newly fed leeches, was induced approximately 10-fold byprolonged starvation and not detected in the body segments.

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HIRUDIN ACTIVITY* IN LEECH SECTIONS

FED STARVEDH HEAD 470 U (85%) 536 U (97%)Bi BODY (UPPER) 24 U ( 4%) 4 U (.7%)B2 BODY (LOWER) 62 U (11%) 12 U ( 2%)

*NIH-ANTITHROMBIN UNITS/LEECH

1 2 3 4 5 6.. .. ....... ... ,^. ...... ~~... .... .... .~~~~~~~~~~~~~~... ....

1 2 3 4kb

.. ..

.:

.:

:::.... ..:

.. .. ..* .: ... .. .. . . . ..14.2 -:

* 4 = i

3,8 =.

kb

' - 4.2

- 1.8

- 0.63

FIG. 2. RNA analysis and antithrombin activity in leech sections.(Upper) Antithrombin activity of extracts prepared from dissectedsegments for fed and starved leeches. Activity was measured byusing a chromogenic oligopeptide thrombin substrate (7). Units areNational Institutes of Health antithrombin units (4) and are averagedfor two leeches. (Lower) RNA-blotting analysis (21) of total RNAsfrom leech sections indicated in Upper. For each sample, 1 ,ug and10 ,ug ofRNA were electrophoresed on a 15% formaldehyde/agarosegel. Lanes: 1, 2, and 3, RNAs from the H, B1, and B2 segments,respectively, of starved leeches; 4, 5, and 6, RNAs from the H, B1,and B2 segments, respectively, of fed leeches. Hybridization was toradiolabeled pTG717 insert.

Despite the fact that minimal hirudin activity was found inthe leech body, relatively abundant transcripts of 1.8 kilo-bases (kb) and 4.2 kb were detected in both body sections andalso the head segment (Fig. 2 Upper). These were not inducedby starvation. Hybridization of the probe to all RNA specieswas stable at high stringency (0.075 M NaCl/0.0075 sodiumcitrate/0.1% NaDodSO4 at 65°C).

It is clear from this analysis that the biology of expressionof hirudin or hirudin-related peptides is more complex than ispredicted by the available protein data (see Discussion).

Southern Transfer Analysis of Leech DNA. The complexityof the hirudin genetic loci was studied by Southern transferanalysis on leech DNA. Blots of restriction enzymes digestsof genomic DNA from whole leeches were analyzed byhybridization with the pTG717 insert (Fig. 3). The resultantbanding patterns were complex and stable at high stringency.These observations are consistent with the analysis ofhirudintranscription products (above), the most likely interpretationbeing that several hirudin genes exist that also may be mosaicin structure. Not excluded are the possibilities that hirudinpseudogenes were being detected with the pTG717 probe(23), or that a single complex gene was transcribed butdifferentially spliced in different tissues to generate distinctRNA products (for example, see ref. 24).

Expression of HV-2 in E. coli. As an initial step in theproduction ofrecombinant hirudin for evaluation as a clinicalreagent and to study the biological properties of the differenthirudin variants, we expressed HV-2 cDNA in E. coli. Ofconsideration in selecting an expression host suitable for aparticular protein are post-translational modifications thatmay be crucial for biological activity. Hirudin is not glycosyl-ated but has one sulfated tyrosine at position 63 (25, 26).Native hirudin is 88% sulfated (7) and upon desulfation retainsat least 45% ofits original activity. The exact biological functionof the tyrosine sulfate in hirudin is unknown, but the fact that

FIG. 3. Southern transfer analysis (22) of leech genomic DNA.Samples ofDNA (10,ug) were digested with restriction enzymes andelectrophoresed on a standard 0.8% agarose gel. Hybridization wasto the radiolabeled pTG717 insert. Washing was at 650C in 0.075 MNaCl/0.0075 M sodium citrate/0.1% NaDodSO4. DNA was digestedwith endonucleases EcoRI (lane 1), HindIII (lane 2), BamHI (lane 3),and Bgi II (lane 4), respectively. Sizes offragments were determinedby reference to phage X EcoRI/HindIII DNA markers stained withethidium bromide.

it is not an absolute requirement for activity indicated thatexpression of biologically active hirudin could be engineered inE. coli systems.The hirudin HV-2 coding sequence was inserted into the E.

coli expression vector ptg927, a derivative of ptg920 (27).This sequence consisted of a fragment of ptg717 (Hinfl-AhaIII) and a synthetic oligonucleotide encoding the first sevenNH2-terminal amino acids. This hirudin expression construct(pTG720) was designed to express the native molecule (plusNH2-terminal methionine) under inducible control from thephage X PL promoter by the host-encoded temperature-sensitive phage X repressor, cI857. Translation was initiatedat the phage X cII ribosome binding site. The E. coli hoststrain used was TGE900 (27).Antithrombin activity was specifically induced in ptg720-

transformed cells after a temperature shift to 370C (Fig. 4).This activity was detected in lysates by using two differentclotting assays (4) and also in thrombin assays by using achromogenic substrate. Like native hirudin, the bacterialactivity was resistant to heating at 700C at pH 2.8 for 15 min.The activity could be eliminated completely from bacteriallysates by incubation with thrombin-coupled Sepharosebeads. Newly synthesized proteins were visualized onNaDodSO4/polyacrylamide gels after pulse-labeling bacteri-al cultures with [35S]cysteine (Fig. 4 Lower). Synthesis of agroup of low molecular mass products (7000-12,000 daltons)was induced on a temperature shift to 370C. These proteinswere absent in control cultures not containing ptg720 (notshown). Most ofthe induced group ofbands remained solubleafter treatment of the bacterial lysates at 700C and pH 2.8(Fig. 4 Lower, lane 2), which denatured and precipitated 90%6of bacterial proteins, but did not affect hirudin.To determine which of the induced bands were bacterial

hirudin, we used an affinity technique (28) to purify theradiolabeled hirudin from bacterial lysates. [35S]Cysteine-pulsed and acid/heat-treated bacterial lysates were incubatedwith thrombin-coupled Sepharose under conditions where allhirudin activity was bound. The Sepharose was washed inhigh-salt solution (0.5 M NaCl) to remove nonspecifically

Proc. Natl. Acad. Sci. USA 83 (1986)

Britain:

Proc. Natl. Acad. Sci. USA 83 (1986) 1087

2.0 ....

.00~~~~~~~~

1.5~~~~~~~~~~~~~~~~~~~~~~~.

o o'

~X1.02

0.5

2 4 6 8

Time after induction, hr

1 2 3 4

kd

-43

-25.7

~ ~ ~ 8.

12.3

FIG. 4. Antithrombin activity and gel analysis of E. coli extracts

containing ptg720. (Upper) Growth characteristics and accumulation

of antithrombin activity (7) after induction of cultures at 370C.

Cultures are grown at 30'C to an OD6w of 0.3 before induction.(Lower) 15% NaDodSO4/polyacrylamide gel analysis and affinityselection on thrombin-Sepharose. Lanes: 1, total soluble proteinfrom pTG720-containing cultures pulse-labeled with [35 ]cysteine, 4hr after induction at 370C; 2, acid/heat-treated, [35S]cysteine-pulsedbacterial proteins [total proteins (as in lane 1) were heated to 700C atpH 2.8 for 15 min, and the insoluble protein sedimented]; 3 and 4,different loadings of acid/heat-treated [35S]cysteine-labeled proteins(as in lane 2) that had been bound to thrombin-Sepharose and elutedwith 0.1 M 4-aminobenzamidine (see text).

bound material, and then strongly bound protein was elutedby competition with 0.1 M 4-aminobenzamidine, a lowmolecular mass synthetic thrombin inhibitor (28). Afterextensive dialysis to remove 4-aminobenzamidine, the elutedmaterial still retained antithrombin activity in a chromogenicassay. Lanes 3 and 4 in Fig. 4 Lower show the sample elutedfrom the thrombin-Sepharose. It is clear that the completeseries of bands (at least four), ranging in apparent mass from7000-12,000 daltons, was selectively bound and eluted fromthrombin-Sepharose. Therefore, we believe that this wholeseries represents the bacterial hirudin.The behavior of the recombinant protein on denaturing/

reducing polyacrylamide gels (e.g., in 6 M urea) was similarto that of purified native hirudin (not shown). The heteroge-neity observed in the bacterial hirudin could be due tospecific protease degradation, post-translational modifica-tion (29), or isomeric conformations of hirudin that areresistant to reduction/denaturation.

DISCUSSIONConditions of abnormal hemostasis (thrombosis) account forextremely high morbidity and mortality in advanced coun-tries (30, 31). Current therapy or prophylaxis of thromboticconditions concentrates first on drugs that inhibit thrombinformation and platelet activation/aggregation, and second ondrugs that accelerate thrombolysis (for a review, see ref. 32).In particular, heparin is widely used in conditions wherethrombin production is responsible for the development orextension of a thrombus (for example, venous thromboem-bolism) (33). However, heparin is a highly heterogeneoussubstance with many biological effects (32). Low molecularweight fractions of heparin show a reduced tendency to causehemorrhage and thrombopoenia (34), but there is clearly aneed for more specific, versatile, and less toxic anticoagulantdrugs.To this end we have cloned cDNAs that encode one variant

ofH. medicinalis hirudin and have expressed the cDNA in E.coli to yield a bacterial product with anticoagulant properties.The amino acid sequence predicted by our cDNA differsconsiderably from the sequence of hirudin purified fromwhole leeches (HV-1) and, thereby, specifies a new hirudinvariant (HV-2).RNA-blotting analysis indicates hirudin transcripts distinct

in size, sites of synthesis, and mode of regulation. The630-nucleotide species induced by starvation and expressedin the leech head is produced by the salivary glands andtranslated into the hirudin activity classically observed to beconcentrated in these glands (4). It is also likely that this is themRNA that we have isolated as cDNA (pTG717 from a leechhead cDNA bank), being the predominant hirudin mRNA inpreparations of RNA from starved leech heads.The presence of other transcripts (1.8 and 4.2 kb) synthe-

sized throughout the whole leech from tissues other than thesalivary glands is puzzling. These transcripts are not inducedby starvation. It is not yet evident that they are translated intoactive hirudin proteins because we find only low antithrom-bin activity in leech bodies. However, Baskova et al. (11) findthat the hirudin form located in leech bodies (pseudohirudin)is inactive. It is possible that the body transcripts aretranslated into precursor hirudins, requiring for activation astimulus not yet known. Alternatively, the activity of thebody hirudins may not (or only weakly) be detected by theantithrombin assays used.

Circumstantial data suggest that the HV-1 variant is foundin the leech body (4, 8). In support of this, we have found nocDNAs specifying HV-1 in our head cDNA bank. HV-1 hasbeen claimed to be of high specific activity (8), but very lowhirudin activity is found in leech bodies (ref. 12 and thisstudy). The cloning of the body transcripts should resolvemany of these ambiguities and provide a basis for analysis ofthe specificities and mode of activation of hirudin variants.

In limited animal trials, native hirudin is effective inpreventing experimental venous thrombosis and disseminat-ed intravascular coagulation. Production of large amounts ofbacterial hirudin will provide a means for further assessmentof this molecule for human use.

We thank Prof. F. Markwardt, Dr. T. S. Edgington, and Dr. D.Meyer for valuable discussions and Prof. P. Chambon and Prof. P.Kourilsky for their continued interest in this work. We are gratefulto Prof. Chapeville for suggestions in the preliminary stage of the

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1088 Medical Sciences: Harvey et al.

project. We are also thankful to F. Daul for the artwork and I. Batraand N. Monfrini for expert secretarial assistance.

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Proc. Natl. Acad. Sci. USA 83 (1986)