Proc. Nati. Acad. Sci. USA Vol. 83, pp. 1084-1088, February 1986 Medical Sciences Cloning and expression of a cDNA coding for the anticoagulant hirudin 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 encode thrombin-induced disseminated intravascular coagulation in a variant of hirudin, a potent thrombin inhibitor that is rats (12). Endotoxin-induced disseminated intravascular co- secreted by the salivary glands of the medicinal leech, Hirudo agulation is prevented in newly weaned pigs (15). medicinals. This variant probably corresponds to a form that Purification of large quantities of hirudin from leeches for has been purified from leech heads but differs in amino acid further clinical testing or eventual clinical use is highly sequence from the hirudin purified from whole leeches. There impractical, but this problem can potentially be solved by are at least three hirudin transcripts detectable in leech RNAs recombinant DNA technology. In addition, cloning of the that are different in size, site of synthesis, inducibility by gene(s) for hirudin should help to resolve questions about starvation, and relationship to hirudin activity. The new different hirudin forms and possible precursor proteins. hirudin variant predicted by the cDNA and the heterodisperse Here we report the cloning ofa cDNA encoding one variant transcription products suggest a hirudin protein family. The of H. medicinalis hirudin and its expression in Escherichia hirudin cDNA was expressed in Esckerichia coli under the coli to yield a biologically active product. control of the bacteriophage X PL promoter. The recombinant product is biologically active, inhibiting the cleavage by throm- MATERIALS AND METHODS bin of fibrinogen and a synthetic tripeptide substrate. Leeches. Live Hirudo medicinalis were purchased from Leech hirudin is the most potent natural inhibitor of coagu- Ricarimpex (Audenge, France) and kept in aerated water lation known (1-4). A very stable noncovalent 1:1 complex containing 0.63 mM NaCl, 0.07 mM CaCl2, 0.05 mM MgSO4, is rapidly and specifically formed with a-thrombin, thereby and 0.05 mM KCl at ambient temperature. They were fed on abolishing its ability to cleave fibrinogen (4). To date there is citrate-treated rabbit blood from an inflated porcine bladder. no evidence that it can interact with other components ofthe Fed leeches were kept in a separate aquarium. human coagulation cascade (5, 6). Protein Extracts. Frozen leech segments were homoge- Hirudin is a polypeptide of65 amino acids that is stable to nized in phosphate-buffered saline (0.1 M Na3PO4, pH extremes ofpH and heat (4). It contains six cysteine residues 7.0/0.15 M NaCl) with a Polytron homogenizer (Brinkmann), grouped in the NH2-terminal half of the protein, an acidic and particulate material was sedimented. For bacterial ex- COOH-terminal half, and one sulfated tyrosine (7). A hirudin tracts, cells were disrupted by sonication in TGE buffer (25 form with isoleucine at the NH2 terminus was first purified mM Tris-HCl, pH 8/50 mM glucose/10 mM EDTA) and from leech heads (H. medicinalis) (4, 8) in which activity was cleared by centrifugation. found to be concentrated in the salivary glands. Subsequent- RNA Extraction. Powdered, frozen leech sections were ly, Bagdy et al. (9) adopted new purification schemes using added to lx NETS buffer (0.1 M NaCl/1 mM EDTA/10 mM whole leeches instead ofheads, yielding a form with Val-Val Tris HCl, pH 7.5/0.5% sodium dodecyl sulfate) containing as the first two NH2-terminal amino acids. The amino acid 50% phenol at 95°C and were homogenized immediately. sequence ofthe "whole body form" has been determined by After a cooling period, phases were separated by centrifu- independent groups (8, 10), and valine residues at positions gation. The phenol phase was reextracted with 2x NETS, 1 and 2 have been confirmed. Both forms had a specific and the aqueous phase was reextracted with phenol. Nucleic activity of around 8000-10,000 antithrombin units/mg. How- acid was precipitated from the pooled aqueous phases with 2 ever, more recently, Baskova et al. (11) described two vol of ethanol. After centrifugation, the pellet was dissolved distinct hirudins: a highly active form in heads with an Ile-1 in H20, and the solution was adjusted to 2.5 M LiCl and kept NH2 terminus and an inactive form in bodies (pseudohirudin) at 4°C overnight. To pellet precipitated RNA, the solution with a Val-Val NH2 terminus. The potency and specificity of was underlaid with 0.25 vol of 3 M LiCl and centrifuged at hirudin have generated interest in its possible use as a clinical 15,000 x g for 10 min at 4°C. The pellet was dissolved in H20 reagent in treatment of thrombotic diseases, but a detailed and reprecipitated with ethanol. pharmacological assessment is prevented by the cost and Hirudin Activity. Antithrombin activity in leech or bacte- supply ofpurified material. In animal studies (12-14), hirudin rial extracts was measured in a clotting assay (4) using was shown to be pharmacodynamically inert apart from its citrated human platelet-poor plasma as a fibrinogen source or anticoagulant activity. It has extremely low toxicity (LD50 > in a colorimetric assay using the thrombin chromogenic 500,000 antithrombin units/kg in rats; ref. 12), appears to be substrate Tos-Gly-Pro-Arg-p-nitroanilide (Chromozym TH, nonantigenic, and is eliminated almost completely via the Boehringer Mannheim; Tos = tosyl) (7). Standard curves kidneys in a biologically active form (12). It is effective in preventing venous thrombosis, vascular shunt occlusion, and Abbreviation: kb, kilobases. tPresent address: Department of Biochemistry and Molecular Biol- ogy, Harvard University, 7 Divinity Avenue, Cambridge MA 02138. The publication costs of this article were defrayed in part by page charge §To whom all reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" Present address: Biotechnology Australia, Pty Ltd., 28 Barcoo in accordance with 18 U.S.C. §1734 solely to indicate this fact. Street, East Roseville, NSW 2069, Australia.

1084 Downloaded by guest on September 28, 2021 Medical Sciences: Harvey et al. Proc. Natl. Acad. Sci. USA 83 (1986) 1085 were established with standardized bovine thrombin (Roche, 1 21 Neuilly-sur-Seine, France) in the case of the clotting assay a GCA ATC TGC GTG TCT CAA GCA and with standardized hirudin (a gift from F. Markwardt) in b Ala Ile Cys Val Ser Gln Ala the case of the chromogenic assay. 22 66 cDNA Cloning. cDNA banks were constructed in pBR322 a ATT ACT TAC ACT GAT TGT ACA GM TCG GGT CM MT TTG TGC CTC 1 15 by standard procedures (16). Screening of banks with oligo- b Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu nucleotides was as described (16), with the stringency of 1 2 washes for the 48-mer probe being 0.3 M NaCl/0.03 M C Val Val ------sodium citrate/0.1% NaDodSO4 at 500C. Oligonucleotides 67 1ll were synthesized by the phosphotriester method on an a TGC GAG GGA AGC MT GTT TGC GGT AM GGC MT MG TGC ATA TTG 16 30 inorganic support (17). DNA sequence analysis ofclones was b Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ile Leu performed by the dideoxy chain-termination method (18) 24 after subcloning into an M13 vector. C ------Gln------Thrombin-Sepharose. Thrombin (61 National Institutes of 112 156 a GGT TCT MT GGA MG GGC MC CM TGT GTC ACT GGC GAA GGT ACA Health units/mg) was bound to CNBr-activated Sepharose 31 45 beads (Pharmacia) by using the manufacturer's recommend- b Gly Ser Asn Gly Lys Gly Asn Gln Cys Val Thr Gly Glu Gly Thr ed protocol. For affinity selection ofbacterial hirudin, throm- 33 35 36 bin-Sepharose was added, in batch, to bacterial extracts so C ------Asp --- Glu Lys ------that all activity was bound. The Sepharose was sedimented 157 201 a CCG MC CCT GAA AGC CAT MT MC GGC GAT TTC GAA GM ATT CCA by gravity, washed twice with excess 0.5 M NaCl, and then 46 60 eluted with 4 vol of 0.1 M 4-aminobenzamidine/25 mM HCl. b Pro Asn Pro Glu Ser His Asn Asn Gly Asp Phe Glu Glu Ile Pro All incubations and elutions were at ambient temperature for 47 49 53 10 min. Carrier bovine serum albumin (30 pg/ml) was added C --- Lys --- Gln ------Asp ------eluted and 4-aminobenzamidine was removed 202 256 to the proteins, a GAA GAA TAT TTA CM TGAAMATGAAAGAATATCMTCATAGAGAATTTTGATTT by dialysis against 25 mM HCl and then H20. [35S]Cysteine- 61 65 labeled proteins were analyzed on 15% NaDodSO4/polyac- b Glu Glu Tyr Leu Gln rylamide gels (19) after reduction/denaturation in 2.3% 257 316 NaDodSO4/6.25% 2-mercaptoethanol at 100°C. a MAAACATTTCCATAGCTMGCTATTTACCMTAAATAMTTMTTTTTCCATTGMTCT 317 376 RESULTS a CMTCATATTTACTCTCMTCATATTCAGCTATTTACCAATMATAMTTMTTTTTCCA 377 Cloning ofHirudin cDNAs. Complementary DNAbanks were a TGA constructed from mRNA purified from the crudely dissected head region of starved leeches. Hirudin clones were identified FIG. 1. DNA sequence ofthe insert ofpTG717 (row a), the amino by screening with a long synthetic oligonucleotide (16), the acid sequence predicted by pTG717 in the reading frame that bears design of which was based upon the published amino acid homology to the published hirudin sequence (6, 9) (row b), and the sequence ofhirudin (8, 9) and on codon usage datafrom insects, residues in the published hirudin sequence that differ from the the closest evolutionary relatives of segmented worms for sequence in row b (row c) are shown. which extensive sequence data were available. A 48-mer (5' CTGAGGCTTAGGAGTACCCTGGCCGGTGACGCACTG- HV-2 are not related as precursor and product, as suggested GTTCTTCTCGCC 3') was synthesized, corresponding to ami- for the Ile-1 (head) and Val-Val (body) forms observed by no acids 34-49 of the hirudin sequence (4). Baskova et al. (11). Two cDNA banks of 43,000 and 21,000 independent To screen for cDNAs encoding HV-1, an 18-mer oligonu- transformants were screened with the radiolabeled oligonu- cleotide probe (5' TCTAATGGAAAGGGCAAC 3') was cleotide and subsequently with confirmed hirudin cDNA synthesized spanning amino acids 32-37 of HV-2 and match- inserts. In total, nine positive clones were isolated. The ing exactly the pTG717 DNA sequence. In this region there complete DNA sequence of the longest clone, pTG717, is are three amino acid changes out of six between HV-1 and shown in Fig. 1. Translation in one reading frame predicts a HV-2 (Fig. 1), and under stringent conditions, such a probe protein sequence of86% homology with the published hirudin should not hybridize to an HV-1 cDNA. Indeed, all of the sequence (8, 9). The 5' terminus of this cDNA is 21 nucleo- hirudin cDNA isolates (selected at low stringency) hybrid- tides upstream from the beginning of the hirudin coding ized to this probe, indicating absence of HV-1 cDNAs in the sequence, and no initiator methionine codon is found before leech-head cDNA bank. Hence, pTG717 probably represents the first amino acid of the mature protein. The seven amino the major transcript in leech heads (salivary glands). acid NH2-terminal extension predicted by the cDNA but To investigate hirudin gene transcription or activity in truncated by the cloning process is hydrophobic and ends in other tissues that could possibly be attributed to HV-1, we alanine, an amino acid commonly occurring at the NH2- made specific dissections of fed and starved leeches to terminal side of the signal peptide cleavage site (20). There- separate the salivary glands from the rest of the body. Three fore, we assume that it represents part of a signal peptide leech segments were studied: a head section and two body sequence that is removed from a precursor form of hirudin. sections, head proximal (Bi) and head distal (B2). Soluble Hirudin Variants. The isolation ofpTG717 (Fig. 1) confirs protein from crude homogenates was assayed for antithrom- the existence and defines the sequence of a new hirudin with bin activity by using a synthetic chromogenic peptide sub- an isoleucine NH2 terminus, as predicted by NH2-terminal strate (7), and total RNA was extracted for blotting analysis. analysis of highly purified hirudin from leech heads (4). We In these dissections, most hirudin activity was found in the propose a simple nomenclature to distinguish the two (now head section (Fig. 2 Upper). In RNA blotting analysis ofleech confirmed) variants of hirudin: HV-1 (hirudin variant 1) for RNAs using pTG717 insert as probe, a complex situation was the sequenced protein with the Val-Val terminus and HV-2 for observed (Fig. 2 Lower). In the head segments of starved the form with the isoleucine NH2 terminus and encoded by leeches (containing salivary glands), a predominant RNA of our cDNA clone. There are nine amino acid differences 630 bases was detected. This species, while still synthesized between HV-1 and HV-2 (Fig. 1). The positions of the six in newly fed leeches, was induced approximately 10-fold by cysteine residues are constant. It is evident that HV-1 and prolonged starvation and not detected in the body segments. Downloaded by guest on September 28, 2021 1086 Medical Sciences: Harvey et al. Proc. Natl. Acad. Sci. USA 83 (1986) HIRUDIN ACTIVITY* IN LEECH SECTIONS 1 2 3 4 FED STARVED kb H 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 14.2 -:* .: ...... 1 2 3 4 5 6 kb * 4 = i ...... 3,8 =...... ,^...... ' - 4.2 ...~~~~~~~~~~~~~~.....~~... - 1.8 - 0.63

Britain:

FIG. 3. Southern transfer analysis (22) of leech genomic DNA. FIG. 2. RNA analysis and antithrombin activity in leech sections. Samples ofDNA (10,ug) were digested with restriction enzymes and (Upper) Antithrombin activity of extracts prepared from dissected electrophoresed on a standard 0.8% agarose gel. Hybridization was segments for fed and starved leeches. Activity was measured by to the radiolabeled pTG717 insert. Washing was at 650C in 0.075 M using a chromogenic oligopeptide thrombin substrate (7). Units are NaCl/0.0075 M sodium citrate/0.1% NaDodSO4. DNA was digested National Institutes ofHealth antithrombin units (4) and are averaged with endonucleases EcoRI (lane 1), HindIII (lane 2), BamHI (lane 3), for two leeches. (Lower) RNA-blotting analysis (21) of total RNAs and Bgi II (lane 4), respectively. Sizes offragments were determined from leech sections indicated in Upper. For each sample, 1 ,ug and by reference to phage X EcoRI/HindIII DNA markers stained with 10 ,ug ofRNA were electrophoresed on a 15% formaldehyde/agarose ethidium bromide. gel. 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, it is not an absolute requirement for activity indicated that and B2 segments, respectively, of fed leeches. Hybridization was to expression ofbiologically active hirudin could be engineered in radiolabeled pTG717 insert. E. coli systems. The hirudin HV-2 coding sequence was inserted into the E. Despite the fact that minimal hirudin activity was found in coli expression vector ptg927, a derivative of ptg920 (27). the leech body, relatively abundant transcripts of 1.8 kilo- This sequence consisted of a fragment of ptg717 (Hinfl-Aha bases (kb) and 4.2 kb were detected in both body sections and III) and a synthetic oligonucleotide encoding the first seven also the head segment (Fig. 2 Upper). These were not induced NH2-terminal amino acids. This hirudin expression construct by starvation. Hybridization of the probe to all RNA species (pTG720) was designed to express the native molecule (plus was stable at high stringency (0.075 M NaCl/0.0075 sodium NH2-terminal methionine) under inducible control from the citrate/0.1% NaDodSO4 at 65°C). phage X PL promoter by the host-encoded temperature- It is clear from this analysis that the biology of expression sensitive phage X repressor, cI857. Translation was initiated of hirudin or hirudin-related peptides is more complex than is at the phage X cII ribosome binding site. The E. coli host predicted by the available protein data (see Discussion). strain used was TGE900 (27). Southern Transfer Analysis of Leech DNA. The complexity Antithrombin activity was specifically induced in ptg720- of the hirudin genetic loci was studied by Southern transfer transformed cells after a temperature shift to 370C (Fig. 4). analysis on leech DNA. Blots of restriction enzymes digests This activity was detected in lysates by using two different of genomic DNA from whole leeches were analyzed by clotting assays (4) and also in thrombin assays by using a hybridization with the pTG717 insert (Fig. 3). The resultant chromogenic substrate. Like native hirudin, the bacterial banding patterns were complex and stable at high stringency. activity was resistant to heating at 700C at pH 2.8 for 15 min. These observations are consistent with the analysis ofhirudin The activity could be eliminated completely from bacterial transcription products (above), the most likely interpretation lysates by incubation with thrombin-coupled Sepharose being that several hirudin genes exist that also may be mosaic beads. Newly synthesized proteins were visualized on in structure. Not excluded are the possibilities that hirudin NaDodSO4/polyacrylamide gels after pulse-labeling bacteri- pseudogenes were being detected with the pTG717 probe al cultures with [35S]cysteine (Fig. 4 Lower). Synthesis of a (23), or that a single complex gene was transcribed but group of low molecular mass products (7000-12,000 daltons) differentially spliced in different tissues to generate distinct was induced on a temperature shift to 370C. These proteins RNA products (for example, see ref. 24). were absent in control cultures not containing ptg720 (not Expression of HV-2 in E. coli. As an initial step in the shown). Most ofthe induced group ofbands remained soluble production ofrecombinant hirudin for evaluation as a clinical after treatment of the bacterial lysates at 700C and pH 2.8 reagent and to study the biological properties of the different (Fig. 4 Lower, lane 2), which denatured and precipitated 90%6 hirudin variants, we expressed HV-2 cDNA in E. coli. Of of bacterial proteins, but did not affect hirudin. consideration in selecting an expression host suitable for a To determine which of the induced bands were bacterial particular protein are post-translational modifications that hirudin, we used an affinity technique (28) to purify the may be crucial for biological activity. Hirudin is not glycosyl- radiolabeled hirudin from bacterial lysates. [35S]Cysteine- ated but has one sulfated tyrosine at position 63 (25, 26). pulsed and acid/heat-treated bacterial lysates were incubated Native hirudin is 88% sulfated (7) and upon desulfation retains with thrombin-coupled Sepharose under conditions where all at least 45% ofits original activity. The exact biological function hirudin activity was bound. The Sepharose was washed in of the tyrosine sulfate in hirudin is unknown, but the fact that high-salt solution (0.5 M NaCl) to remove nonspecifically Downloaded by guest on September 28, 2021 Medical Sciences: Harvey et al. Proc. Natl. Acad. Sci. USA 83 (1986) 1087 reducing polyacrylamide gels (e.g., in 6 M urea) was similar to that of purified native hirudin (not shown). The heteroge- neity observed in the bacterial hirudin could be due to - - specific protease degradation, post-translational modifica- tion (29), or isomeric conformations of hirudin that are resistant to reduction/denaturation. .0 0~~~~~~~~ DISCUSSION ~X 1.02 Conditions of abnormal hemostasis (thrombosis) account for extremely high morbidity and mortality in advanced coun- 0.5 tries (30, 31). Current therapy or prophylaxis of thrombotic conditions concentrates first on drugs that inhibit thrombin 2.0 .... formation and platelet activation/aggregation, and second on 2 4 6 8 drugs that accelerate thrombolysis (for a review, see ref. 32). Time after induction, hr In particular, heparin is widely used in conditions where 1.5~~~~~~~~~~~~~~~~~~~~~~~. thrombin production is responsible for the development or extension of a thrombus (for example, venous thromboem- bolism) (33). However, heparin is a highly heterogeneous 1 2 3 4 substance with many biological effects (32). Low molecular weight fractions ofheparin show a reduced tendency to cause kd hemorrhage and thrombopoenia (34), but there is clearly a need for more specific, versatile, and less toxic anticoagulant drugs. o o' To this end we have cloned cDNAs that encode one variant -43 ofH. medicinalis hirudin and have expressed the cDNA in E. colito yield a bacterial product with anticoagulant properties. The amino acid sequence predicted by our cDNA differs considerably from the sequence of hirudin purified from whole leeches (HV-1) and, thereby, specifies a new hirudin -25.7 variant (HV-2). RNA-blotting analysis indicates hirudin transcripts distinct in size, sites of synthesis, and mode of regulation. The ~ ~ ~ 8. 630-nucleotide species induced by starvation and expressed 12.3 in the leech head is produced by the salivary glands and translated into the hirudin activity classically observed to be concentrated in these glands (4). It is also likely that this is the mRNA that we have isolated as cDNA (pTG717 from a leech head cDNA bank), being the predominant hirudin mRNA in preparations 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 the salivary glands is puzzling. These transcripts are not induced by starvation. It is not yet evident that they are translated into active hirudin proteins because we find only low antithrom- FIG. 4. Antithrombin activity and gel analysis of E. coli extracts bin activity in leech bodies. However, Baskova et al. (11) find containing ptg720. (Upper) Growth characteristics and accumulation that the hirudin form located in leech bodies (pseudohirudin) of antithrombin activity (7) after induction of cultures at 370C. It is possible that the body transcripts are Cultures are grown at 30'C to an OD6w of 0.3 before induction. is inactive. (Lower) 15% NaDodSO4/polyacrylamide gel analysis and affinity translated into precursor hirudins, requiring for activation a selection on thrombin-Sepharose. Lanes: 1, total soluble protein stimulus not yet known. Alternatively, the activity of the from pTG720-containing cultures pulse-labeled with [35 ]cysteine, 4 body hirudins may not (or only weakly) be detected by the hr after induction at 370C; 2, acid/heat-treated, [35S]cysteine-pulsed antithrombin assays used. bacterial proteins [total proteins (as in lane 1) were heated to 700C at Circumstantial data suggest that the HV-1 variant is found pH 2.8 for 15 min, and the insoluble protein sedimented]; 3 and 4, in the leech body (4, 8). In support of this, we have found no different loadings of acid/heat-treated [35S]cysteine-labeled proteins cDNAs specifying HV-1 in our head cDNA bank. HV-1 has (as in lane 2) that had been bound to thrombin-Sepharose and eluted been claimed to be of high specific activity (8), but very low with 0.1 M 4-aminobenzamidine (see text). hirudin activity is found in leech bodies (ref. 12 and this study). The cloning of the body transcripts should resolve bound material, and then strongly bound protein was eluted many of these ambiguities and provide a basis for analysis of by competition with 0.1 M 4-aminobenzamidine, a low the specificities and mode of activation of hirudin variants. molecular mass synthetic thrombin inhibitor (28). After In limited animal trials, native hirudin is effective in extensive dialysis to remove 4-aminobenzamidine, the eluted preventing experimental venous thrombosis and disseminat- material still retained antithrombin activity in a chromogenic ed intravascular coagulation. Production of large amounts of assay. Lanes 3 and 4 in Fig. 4 Lower show the sample eluted bacterial hirudin will provide a means for further assessment from the thrombin-Sepharose. It is clear that the complete of this molecule for human use. series of bands (at least four), ranging in apparent mass from 7000-12,000 daltons, was selectively bound and eluted from We thank Prof. F. Markwardt, Dr. T. S. Edgington, and Dr. D. thrombin-Sepharose. Therefore, we believe that this whole Meyer for valuable discussions and Prof. P. Chambon and Prof. P. series represents the bacterial hirudin. Kourilsky for their continued interest in this work. We are grateful The behavior of the recombinant protein on denaturing/ to Prof. Chapeville for suggestions in the preliminary stage of the Downloaded by guest on September 28, 2021 1088 Medical Sciences: Harvey et al. Proc. Natl. Acad. Sci. USA 83 (1986) project. We are also thankful to F. Daul for the artwork and I. Batra 18. Messing, J. (1983) Methods Enzymol. 101, 20-78. and N. 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(1982) Nucleic Acids Res. 10, 7439-7448. 485-490. Downloaded by guest on September 28, 2021