Expression in Escherichia coli of a Chemically Synthesized Gene for the Hormone Somatostatin Keiichi Itakura; Tadaaki Hirose; Roberto Crea; Arthur D. Riggs; Herbert L. Heyneker; Francisco Bolivar; Herbert W. Boyer Science, New Series, Vol. 198, No. 4321 (Dee. 9, 1977), 1056-1063. Stable URL: hhup//links,jstor-org/sici?sici=0036-8075% 281977 1209% 203 %3A 198%3A.4321 %3C 1056% 3AEIECOA%3E2.0,CO%3B2-D ‘Your use of the ISTOR archive indicates your acceptance of JSTOR’s Terms and Conditions of Use, available at hhup:/www.jstororg/about/terms.html. JSTOR’s Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. 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Be 1 PE re, ge, ee Epil 6 te More 9 Soe tapes at ob 1 te ere aa isi Sat a Bae er minaret Soe ie cchratne fe caganon es upmar ear Peet pecs 1 ie Sco ceo OR ee ear tee ere a cape a aan Soars tel, ene ci me Net ge fo eae otc Eloy he Cece ESL eeameniners Soins oreee 19 uly 197; evn 20 September 1977 Expression in Escherichia coli of a Chemically Synthesized Gene for the Hormone Somatostatin Abstract. A gene for somatostatin, a mammalian peptide (14 amino acid residues) hormone, was synthesized by chemical methods. This gene was fused to the Esche- richia coli B-galactosidase gene on the plasmid pBR322. Transformation of E. coli with the chimeric plasmid DNA led to the syrthesis of a polypeptide including the sequence of amino acids corresponding to somatostatin, In vitro, active somarosta~ tin was specifically cleaved from the large chimeric protein by treatment with ey- ‘anagen bromide. This represents the frst synthesis ofa functional polypeptide prod- uct from a gene of chemically synthesized origin. The chemical synthesis of DNA and recombinant DNA methods provide the technology for the design and synthesis of genes that ean be fused to plasmid ele- ments for expression in Escherichia coli ‘oF other bacteria. As a model system we have designed and synthesized a gene for the small polypeptide hormone, soma- tostatin (Figs. 1 and 2). The major con siderations in the choice of this hormone were its small size and known amino acid sequence (/), sensitive radioimmune and biological assay's 2), and its intrinsic bio- logical interest (2), Somatostatin is a tet- radecapeptide: it was originally discov- cred in ovine hypothalamic extracts but subsequently was also found in signifi {cant quantities in other species and other tissues @). Somatostatin inhibits the se- cretion of a number of hormones, inelud- ing growth hormone, insulin, and gluca- ‘gon. The effect of somatostatin on the se- cretion of these hormones has attracted attention to its potential therapeutic val- ue in acromegaly. acute pancreatitis, and insulin-dependent diabetes, ‘The overall construction of the soma- tostatin gene and plasmid was designed to result in the in vivo synthesis of a pre: cursor form of somatostatin (see Fig. 1) The precursor protein would not be ex- pected to have biological activity, but ‘could be converted to a functional form by cyanogen bromide cleavage () after cellular extraction. The synthetic soma- tostatin gene was fused to the lac operon because the controlling sites ofthis oper- ‘on are well characterized, Given the amino acid sequence of somatostatin, one can design from the genetic code a short DNA fragment con: taining the information for its 14 amino acids (Fig. 2), The degeneracy of the code allows for a large number of pos- sible sequences that could code for the same 14 amino acids. Therefore, the choice of codons was somewhat arbi trary except for the following restric~ tions. Fist, amino acid codons known to be favored in E, col for expression of the ‘MS? genome were used where appropri ate (5). Second, since the complete se- quence would be constructed from a ‘number of overlapping fragments, the fragments were designed to eliminate un- desirable inter- and intramolecular pait- ing. And third, GC-rich (uanine-cyto- sine) followed by AT-rich (adenine thymine) sequences were avoided since they might terminate transcription (6), Eight oligonucleotides, varying in length from 11 to 16 nucleotides, labeled SCIENCE, VOL. 198in Fig. 2 as A through H, were synthe- sized by the triester method (7). In addi tion to the 14 codons for the structural information of somatostatin, several oth- cr features were built into the nucleotide sequence. First, to facilitate insertion in- to plasmid DNA, the 5” ends have single- stranded cohesive termini for the Eco RI and Bam HI restriction endonucleases. Second, a methionine codon precedes the normal NH,-terminal amino acid of somatostatin, and the COOH-terminal codon is followed by two nonsense co dons. In the cloning and expression of the synthetic somatostatin gene we used (0 plasmids. Each plasmid has an Eco RI substrate site at a different region of the Beealactosidase structural gene (see Figs. 3 and 4). The insertion of the syn- thetic somatostatin DNA fragment into the Eco RI sites of these plasmids brings the expression of the genetic information in that fragment under control of the Ine ‘operon controlling elements. After the insertion of the somatostatin fragment into these plasmids, translation should result in a somatostatin polypeptide pre- ceded either by ten amino acids (SOM1) or by vietally the whole f-ea- Sor! Alo Gly Cys us E.coli Loe Operon ONA | boc PO. $-¢at pBRS22 Plasmid ONA 5x01 Mp DAR tes Got Frogments GENETIC CODE Chemicot DNA Synthesis Somatostotin Gene In vivo ’ som | Ho Che -Ser- Tne Bre In vitro Cyanogen Bromide } Clavoge Active Somatostatin Fig. 1, Sehematie outine ofthe experimental plan, The gene for somatostatin, made by chem ical DNA synthesis, was fused to the E, coli B-galactosidase gene on the plasmid pBR3Z2. After transformation into Ecol, the chimeric plasmid directs the synthesis of chimere protein that, canbe specifically cleaved in vitro at methionine resis by evanogen bromide to Yield active ‘mammalian peptide hormone Asn Phe Phe Trp tys the Phe Thr Ser Cys Stop. Stop fom Ht AA sAATICAT (2) GCIGGTIGTAAGAACTICIITIGGAAGACTITCACTICGIGTIGATAG (C} (0) GTACCGACCAACAT TCT TGAAGAAAA CC TTC TGAAAGT GAAGCACAAC TATCCTAGg Fig. 2. Chemical synthesis ofthe somatostatin gene. (a) Eight oligodeoxyribonucleotides, Deled A through H, were synthesized by the ‘Modified tester method (7, 23) The codons ate indicated, and their corresponding amino seids ate given, The eight fragments were de- Signed to have at least five nucleotide com- plementary overlaps to ensure ecient joining by T4 DNA ligase. (6) Recent improvements in the synthesis of fully protected timers, Which constitute codon Blocks and are the basic units for building longer oligodeoxyr bonucleotdes, With an excess of 12 mmole), the coupling rection with 2 (2 mmole) went almost to completion in 60 minutes with the aid of a powerful coupling reagent, 2.4.6 Uisopropylbenzenesulfonyl. tetrazolide (TPSte," 4 mmole) (2). The S-protecting [Eup was removed with 2 percent benzene Sulfonic acid, and the Shydroxyl dimer § ould be separated trom an excess of 3 Dhosphodiester monomer by simple solvent extraction with aqueous NaHCO, solution in CCL, The fully protected trimer block was brepared successively from the Shydroxyl Gimer §, 12 mmole), and TPSTe (4 mmole) 8nd isolated by chromatography on silica ge (24). These improvements simplify the pori- Cation step and lead to an increase in the overall yields of trimer blocks ind toa decrease inthe working ime by at least a factor of 2). The eight oligodeoxyribonucleotides then ° «woshe 2 . 8 8 ° reste. pwn Pol ow a N Ne $ a —tEsTt_» Trimer = Protected Bose DMT = 4,4'-dimethoxytrity! 9 (©)-OME or she @)-c1 OcH,cH,cN were synthesized from the trimers by published procedures (7). The final products, after removal ofall protecting groups, were purified by high- Pressure ligui chromatography on Permaphase AAX (25). The purity of each oligomer was checked by homochromatography on thin-layer DEAE-cellulose and also by gl electrophoresis in 20 percent acrylamide (slab) afte labeling ofthe oligomers with [y>"PJATP inthe presence of polynucleotide kinase, One major labeled product was obtained from each DNA fragment 9 DECEMBER 1977 1087+991 9¥1¥991911190191919¥WV1911¥1929219¥9¥VL Y¥9912¥91L¥999¥LL¥9. Oe BY aE STE Potxgs ewosd 06} rk vousebip ty 3 wouo8n NO FL omneip somone 15 ip 1 oo ope 7 ge +, ge =? sxop at y 6 oat yo @F owed jog) esrouned Yay pow xece \ bi Pot x ore ound 2) Twrnarn fy etna % reyes SCIENCE, VOL. 158Jactosidase subunit structure (pSOMII3). ‘The plasmid construction scheme (Fig, 3) begins with plasmid pBR322, a well-characterized cloning vehicle (8). ‘The lac elements were introduced to this, plasmid by insertion of an Hae II restric- tion endonuclease fragment (208 nucle- tides) carrying the lac promoter, ca- tabolite-gene-activator-protein binding site, operator, ribosome binding site, and the first seven amino codons of the fg lactosidase structural gene (®) (Figs. 3 and 4). The Hae III fragment was de- rived from AplacS DNA. The Eco Rl cleaved pBR322 plasmid, which had its termini repaired with T4 DNA polymer- ase and deoxyribonucleotide triphos- phates, was blunt-end ligated to the Hae II fragment to create Eco RI termi nial the insertion points. Joining of these Hae Ill and repaired Eco RI termini gen- crate the Eco RI restriction site (Figs. 3 and 4) at each terminus. Transformants of E. coli RRI (8) with this DNA were selected for resistance to tetracycline Fig 3 (facing page, lft). Construction of recombinant plasmids. Plas (Te) and ampicillin (Ap) on S-bromo-4- chloro-indolylgalactoside (X-gal) medi- uum (10). On this indicator medium, colo- nies constitutive for the synthesis of galactosidase by virtue of the increased ‘number of lac operators titrating repre: sor, are identified by their blue color. Two orientations of the Hae III fragment are possible, but these were distin- guished by the asymmetric location of an ha testeiction site inthe fragment. Plas- ‘mid pBH10 was further modified to elim. inate the Eco RI endonuclease site distal to the lac operator (pBH20) The eight chemically synthesized oli- godeoxyribonucleotides (Fig. 2) were la beled at the 5" termini with [yP]ATP (adenosine triphophatase) by T4 polynu- leotide kinase and joined with T4 DNA ligase. Through hydrogen bonding be- tween the overlapping fragments, the somatostatin gene selfassembles and eventually polymerizes into larger mole cules because of the cohesive restriction site termini. The ligated products were Aesired somatostatin DNA fragment inserted (da ‘weated with Eco RI and Bam HI restric- tion endonucleases to generate the soma- tostatin gene (Fig. 2). The synthetic somatostatin gene frag- ment with Eco RI and Bam HI termini ‘was ligated to the pBH20 plasmid, pre- Viously treated with the Eco RI and Bam HI restriction endonucleases and alkaline phosphatase. The treatment With alkaline phosphatase provides a molecular selection for plasmids car- rying the inserted fragment (11). Ampi- Gillinresistant transformants. obtained With this igated DNA were screened for tetracycline sensitivity, and several were examined for the insertion of an Eco RI- Bam HI fragment of the appropriate Both strands of the Beo RI-Bam HI fragments of plasmids from two clones. were analyzed by a nucleotide sequence analysis (/2) starting from the Bam Hl tnd Eco RI sites. The sequence analysis, was extended into the lac-controlling le ‘ments; the lac fragment sequence was in- not showin), Be mid pBR322 was used asthe parental plasmid), Plasmid DNA GS yg) was digested with the restriction endonuclease Eco RI. The reaction ‘was terminated by extraction with a mixture of phenol and ehoro orm; the DNA was precipitated with ethanol and resuspended in $0 lof T4 DNA polymerase btfer 26). The reaction was started by the {nition of 2 units of T4 DNA polymerase. The reaction (held for 30, ‘minutes at37°C) was terminated by extraction with phenol and chiro form and precipitation with ethanol. The Aplacs DNA (34) as di ested withthe endonuclease Hae Ii1 (8) The digested pBR322 DNA. ‘was bluntend ligated withthe Hae Ul-dgested AplacS DNA in final Yolume of 40 jl with TDNA ligase (hydroxslapatite fraction) (27) in 20 mM trie HCI (pH 7.6, 10 mM MgCl, 10 mi dithiothreitol, and (0.8 mM ATP for 12 hours at 12°C. The ligated DNA mixture was di Iyzed against 10 mM ts: HICI (pH 7.6) and used to transform E. col strain RR (8). Transformants were selected for tetracycline resist: ance (Te?) and ampicillin resistance (AP) on antibiotic (20 gm) ‘minimal X-gal (40 pin) medium 00). Colonies constitutive Jor the Synthesis of fgalacosidase were identified by thee blue color. After Sindependently isolated blue colonies were screened, three of them were found to contain plasmids with two Eco RI sites separated bY approximately 200 base pairs 28). Plasmid pBHIO was shown to carry the fragment inthe desired orientation, thats, lac transcription going into the Te" gene ofthe plasmid, Pasmid pBH was further modified {o eliminate the Eco RI site distal to the lac operator and plasmid ‘pBH20 was obtained (29). The nucleatide sequence from the Eo RI Site into the lac-control region of pBH20 (data not shown), was con- firmed. This plasmid was used for cloning the syathetic somatostatin. gene. Plasmid pBH20 (10 yg) was digested with endonucleases Eco RI and Bam HI and ireated with bacterial alkaline phosphatase (0.1 unit of BAPF, Worthington), and incubation was continue for 10 ‘minutes at 68°C, The reaction mixtures were extracted with mixture fof phenol and chloroform, and the DNA was precipitated with ethanol (20). Somatostatin DNA (30 ul ofa solution containing + wg!) was, ligated” "with the Bam HI-Eco Rl, alkaline phosphatave-treated BH20 DNA ina total volume of $0 ul with the use of 4 ufits of TH DNA ligase for 2 hours at 22°C (1). Ina. contcol experiment, Bam HI-Eco RI alkaline phosphatase-treated pBH20 DNA ws liga edn the absence of somatostatin DNA tinder similar conditions. Both Preparations were used to transform E.coli RI. Transformants were Selected on minimal X-gal antibiotic plates, Ten Te” transformants Were isolated. Inthe control experiment no transformants were ob {ained. Four out of the ten transformants contained plasmids with both an Eco RI and a Bam HI site. The size of the small Eco RI- Bam HI fragment of these recombinant plasmids ws in ll four i stances similar to the size ofthe in vitro prepared somatostatin DNA. Base sequence analysis (12) revealed thatthe plasmid pSOMI had the 9 DECEMBER 1977 {ause of the fale to detect somatostatin activity Irom eultres car fying plasmid pSOMI, a plasmid was canatrated in which the som fostatin gene could be located at the COOH- terminus of the f-zalc tosidase'gene, keeping the translation in phase. For the construction ‘of such s plasmid, SOM! (30 ag) was digested with restriction en- zymes Eco Rl and PstI. A preparative $ percent polyacrylamide gel Was used to separate the large Pst I-Eeo RI fragment that caries the Somatostatin gene from the small fragment carrying the lac contro elements (72). In a sie way plasmid pBRS22 DNA (50 ja) was Aigested with Ps Land Eco RI restriction endonucleases, and the to resulting DNA fragments were purified by preparative electrophoresis fona $ percent polyacrylamide gel. The small Pst I-Fco RI fragment From pBRS22 (I yg) was ligated with the large Pst I-Eco RIDNA, ment (54g) ffom pSOMI. The ligated mixture was used to form £. col RRI. and transformants were selected for Ap’ on Xl medium. Almost all the Apr transformants (95 percent) gave white Colonies (no lac operator) on X-gal indicator plates, The resulting Plasmid, PSOMIT, was used in the construction of plasmid pSOMIL- S.A mixture ofS pg of pSOMIT DNA and 5 ug of Apacs DNA vas, ested with Feo RI. The DNA was extracted sith a mixture of phe- nol and chloroform; the extract wis precipitated by ethanol, and the precipitate was resuspended in T4 DNA ligase butfer (30 yl) in the Presence of T DNA ligase (I unit). The ligated mixture was used {0 transform. col stain RRI. Transformants were selected for Ap’ om X-gal plates containing ampicilin and screened for constitutive fa lactostdase production. Approximately 2 percent of the colonies were blue (such as pSOMII-1 and 11-2). Restriction enzyme analysis of plasmid DNA obtained from these clones revesled that al the plas- ‘mids carried a new eo RI fragment of approximately 4.4 mega fons, which carries the lae operon contol sites and most of the B Balactosidase gene (13, 14). Two orientations of the Eco Rl fragment fare possible. and the asymmetric location of Hind II restriction in this fragment can indicate which plasmids had transcription po" ceeding into the somatostatin gene. The clones carrying plismids PSOMII-3, pSOMIL-S, pSOMIL-, and pSOMII-7 contained the Eco RI fragment in this orientation. Fig 4 (facing page righ. Nucleotide sequences ofthe lac-somatosta tin plasmids, The nucleotide sequence of the la contro elements, B- salactosidase structural gene, and the synthetically derived soma tostatin DNA. are depicted (9.14.27) along with the restriction endo- nuclease substrate sites. The nucleotide sequence of pSOMI. as de- Picted, was confirmed (iegends to Figs. 3 and 5). The nucleotide se {quence of pSOMIL-S was inferred from published data, 13, 14,27), ‘The amino acid sequence of somatostatin is italicized The amino ack sequence numbers of Bigalactosidase tein bracketstact, and in one case, pSOMI, the nucle tide sequence of both strands were in- dependently determined, each giving the sequence shown in Fig. 3. In the other cease, the sequence was identical except for a base pair deletion (A-T) at a posi- tion equivalent (0 the junetion of the B-C oligonucleotides in the original DNA fragment. The basis for the deletion is unclear. The standard radioimmune assays (RIA) for somatostatin 2) were modified by decreasing the assay volume and by using phosphate buffer (Fig, 6). This ‘modification proved suitable for the de- tection of somatostatin in E, coli ex- tracts. Bacterial cell pellets, extracts, or ‘cultures were treated overnight in 70 per- cent formic acid containing cyanogen bromide (5 mg/m). Formic acid and ¢ anogen bromide were removed und vacuum over KOH before the assay. Ini- tial experiments with extracts of E.coli strain RRI (the recipient strain) /0) in- dicated that less than 10 pg of somatosta- tin could easily be detected in the pres- ence of 16 yg or more of cyanogen bro- mide-treated bacterial protein. More 5. Ligation and acrylamide gel analysis of somatostatin DNA. The S-OH termini of the chemically synthesized fragments A through H (Fig. 2a) were labeled and phosphorylated sepa- rately, Just prior tothe kinase reetion, 28 ag of [y=PIATP (~ 1500 cimmole) (72) was evapo fated t9 dryness in 0.-ml Eppendort tubes, The fragment (Sg) was incubated with? units af ‘TDNA kinase thydroxslapatte faction, 2900 unit} 2), in 70 mM ts-HCl, pH 7.6, 10 mM MgCh,,and’S mM dithiothreitol in total volume of 150 al for 20 minutes at 37°C. To ensure maximum phosphorylation ofthe Iragments fr ligation purposes, 10g of a mixture consisting (OF 70 mM trissHCT, pH 7.6, 10 mM MgCl, § mM dithiothreitol, 0.8 mM ATP, and 2 units oF INA kinsse were added, and incubation continued for an additional 0 minutes at 37°C. The fragments (250 ngs) were stored at ~20°C without further treatment. Kinase-treated fragments AB. Eand F125 ugeach) were ligated ins foal volume of $0 lin 20 mM tris-HCl (pit 7-6), Wi mM MgCl, 10 mf ihiothritol, 0-5 mM ATP. and 2 units of T4 DNA ligase (hydroxyl. apatite fraction, 400 nit (26), f0F U6 hours at 4. Fragments C, D, G, and It were ligated tinder similar conditions, Samples (2 al) were removed for analysis by eletraphoresis ona 10 pereent polyacrylamide gel and subsequent autoradiography (16) (lanes land 2, respectively) "The fast migrating materal represents nreacted DNA fagnents. Material migrating with the bromophenol blue dye (BPS) is the monomeric form of the ligated fragments. The slowest iigrting material represents dimers, which form by virtue ofthe cohesive ends, ofthe ligated fragments A, B, E, and F (lane I) and C, D, G, and H (lane 2). The dimers can be cleaved by restriction endonuclease Eco RI or Ham HI, respectively (data not shown). The two half mole wiles (ligated A'» B+ E + Fand ligated C + D + G ~ H) were joined by an addtional i tion step cared out ina final volume of 10 gl at C for 16 hours. A sample Il} was removed for analysis lane 3), The reaction mise was heated for 15 minutes at °C to inactivate the TS DNA ligase. The heat treatment docs not affect the migration patter of the DNA misture (ne ‘). Enough restriction endonuclease Bam HI was added lo the feaction mixture to eleave the imultmerte fom ofthe somatostatin DNA in 30 minites a 37°C (ane $) Afler the addition of NaCI to 3 concentration of 100 mM, the DNA was digested with Eco RI endonuclease (ane 6) “The restriction endonuclease digestions were terminated by phenol-chloroform extraction of the DNA. The somatostatin DNA fragment wis purifed from unreacted and partially ligated DNA fragments by preparative electrophoresis on 10 percent polyacrylamide gel. The band indicated with an arrow (ane 7) was excised Irom the gel, andthe DNA was luted by sing the {el into small poses and extracting the DNA with elution buffer (0.5M ammonium acetate, TO mM MgCl, 0.1 mMf EDTA, and 0.1 percent sodium dodecd1 sulfate) overnight at 65°C (12), ‘The DNA wat precipitated with two volumes of ethanol, centrifuged, redissolved in 200 ul of 10 mf wis HCI Ht 7-6), and dialyzed against the same buffer, resulting in a somatostatin DNA, concentration of ym than 2 jg of protein from formic acid treated bacterial extracts interfered ‘Somewhat by increasing the background, bbut cyanogen bromide cleavage greatly reduced this interference. Reconstruc- tion experiments showed that somatosta- tin is stable in cyanogen bromide-treated extracts. ‘The DNA sequence analysis of SOM! indicated that the clone carrying this plasmid should produce a peptide containing somatostatin. However, to date all attempts to detect somatostatin radioimmune activity from extracts of cell pellets or culture supernatants have been unsuccessful. Negative results were also obtained when the growing culture was added directly to 70 percent formic acid and cyanogen bromide. We calculate that £, coli RRI (pSOM1) con- tains less than six molecules of soma tostatin per cell. In a reconstruction ex- periment we have observed that exoge- nous. somatostatin is degraded very rapidly by £, coli RRI extracts. The fail> ure to find somatostatin activity might be accounted for by intracellular degrada tion by endogenous proteolytic er zymes, If the failure to detect somatostatin ac- tivity from pSOMI was due to pro- teolytic degradation of the small protein (Fig. 4), attachment to a large protein might stabilize it. The P-galactosidase structural gene has an Eco RI site near the COOH-terminus (13). The available {data on the amino acid sequence of this protein (/3, 14) suggested that it would be possible to insert the Eco RI-Bam HI somatostatin gene into the site and main- tain the proper reading frame for the cor- rect translation of the somatostatin gene Fig. 4) The construction of this plasmid is outlined in Fig, 3. The Eco RI-Pst frag- ment of the pSOMI plasmid, with the Tae-controlling element, was’ removed and replaced with the Eco RI-Pst frag- ‘ment of pBR32 to produce the plas- mid pSOMII. The Eco RI fragment of AplacS, carrying the lac operon control region and most of the f-galactosidase structural gene, was inserted into the Eco RI site of pSOMII. Two orienta tions of the Eco RI lac fragment of AplacS were expected. One of these ori- fentations would maintain the proper reading frame into the somatostatin ‘gene, the other would not. ‘A number of independently isolated clones (with plasmid designations pSOMII-2 and pSOMII-3) were ana lyzed for somatostatin activity, as de- scribed above. In constrast tothe results, of experiments with pSOM1, four clones (PSOMIL3, 11-5, 11-6, and 11-7) were SCIENCE, VOL, 156found to have easily detectable soma tostatin radioimmune activity (Fig. 6, a and b). Restriction fragment analysis re- vealed that pSOMII-3, pSOMIL-s, pSOMII-6, and pSOMII-7 had the de- sired orientation of the Inc operon, whereas pSOMII-2 and 11-4 had the op- posite orientation. Thus, there is a per- fect correlation between the correct i= 2/8 Mcroiters entation of the Ine operon and the pro= duction of somatostatin radioimmune activity, The design of the somatostatin plas- ‘mid predicts that the synthesis of soma tostatin would be under the control of the lac operon, The lac repressor gene is not included in the plasmid, and the re- cipient strain (E. coli RR}) contains the wild-type chromosomal lac repressor gene, which produces only 10 t0 20 re- pressor molecules per eell (5). The plas- ‘mid copy number (and therefore the ‘number of lac operators) is approximate ly 20 to 30 per cell and complete repres- sion is impossible. The specific activity of somatostatin in E. coli RR] (pSOMLI- 3) was increased by IPTG, an inducer of the lac operon (Table 1). As expected, the level of induction was low, varying from 2.4. t© Told. In experiment 7 (Table 1), the @ activity (4), a measure of the first 92 amino acids of f-galactosi- dase, also was induced by a factor of 2 In several experiments (Table 1 and oth- lr experiments not shown), no soma tostatin radioimmune activity was de- tected prior to cyanogen bromide cleav- age of the total cellular protein. Since the antiserum used in the radioimmune as- say, $39, requires a free NH,-terminal alanine, no activity was expected prior to cyanogen bromide cleavage. After cleavage by cyanogen bromide, cell ex- tracts were chromatographed on Seph- adex G-50 in 50 percent acetic acid (Fig. 6c). In this system, somatostatin is well separated from excluded large peptides and fully included small molecules. Only extracts of clones positive for somatosta~ tin exhibited radioimmune activity in the column fractions, and this activity elutes, in the same position as chemically syn- thesized somatostatin The strains carrying the Beo RI lac op- cron fragment (such as pSOMII-2 and pSOMII-3) segregate with respect to the plasmid phenotype. For example, after 100. 2° + ry € + 50 , 80 30 Ze Ss. i a oS 5 ee ‘Sia & ) mn 3S eee = [ek R20} + i a * ach fe 2 “tral “Wit : Pee seTu eB wo . Clone Number (p Som 1I-) Fig. 6, Radioimmune assay for somatostatin activity. The assay used is ‘modification of existing methods 2). [Tye"Somatostatin (agit from W. Vale) was iodinated by a chloramine T procedure 2). To assay for somatostatin, the sample, usualy in 70 percent formic acid containing ‘cyanogen bromide S mg/m, was died ina conical polypropslene tube {0.7 ml, Sarstedt over moist KOH under vacuum. Then 20 pot PASA buffer (75 mMf NaCl 75 mf sodium phosphate (pH 7.2; bovine serum albumin (I gin): and sodium azide (0.2 maim was added, followed bby 40a of af I}somatostain mixture ad 20 of a LO-Fod dilation in PBSA of rabbit antiserum to somatostatin 39 2) obtained from W. Vale. The [*I}somatostatin mixture contained (per milter of PRSA, buffer) 250 ug of normal abbit gamma pobulin Antibodies, ne.) 1500 nits of Trasylol (Calbiochem), and about 100,000 counts of rye" Stjsomatostatin, After atleast i hours at room temperate, 0333 ml of goat antibody to rabbit gamma globulin (Antibodies, Ine., = 03) in PBSA butfer was added to the sample tubes, The misture was inc Dated for2hoursat 87°C, cooled to °C. then centrifuged st 10,0004 for ‘minutes. The supernatant was removed, and the radioactivity in the pel Jet was counted ina gamma counter. With the amount of antiserum wed 20 percent ofthe counts wis precipitated with no unlabeled competing Somatostatin. The Background with infinite somatostatin 200 ng) was Usually 3 percent. One-half maximum competition was obtained with 0p of somatosiatin. (a) Competition by tacterial extracts. Strains E.coli RRI (pSOMI1-5) and E.coli RRI (pSOMIL-#) were grown a 3PCto $ x 10Fcelml in L broth. Then IPTG was added toa concen tration of | mM and growth continued for2 hours. Portions (Imi were 9 DECEMBER 197 centrifuged fora few seconds in an Eppendorf centrifuge, and the pel- lets were suspended in S00 yi of 70 percent formic acid containing eyanogen bromide (5 maim). After approximately 24 hours at room temperature, the samples were diluted tenfold in water, and the indie ted volumes were assaved in triplicate for somatostatin, By isthe ratio of (llsomatostatin bound in the presence of sample to tht hound in the absence of competing somatostatin, Eaeh point isthe average of triplicate tubes. The protein content of the undiluted san ples were determined to be 2.2 myml for E. coll RRI (pSOMII-S) And 1-5 mgiml for E.coli RRI (pSOM-).(b) The intial sereening of clones for somatostatin. Cyanogen bromide-treated extracts of II clones (such as pSOMI-2 and pSOMII-3) were made as described above for (a). A sample GO sof each extract was taken in tilicate for radioimmune assay. The range of assay points is indicated. The values for picograms of somatostatin were fea from a standard curve ‘obtained as part ofthe same experiment. fe) Gel tration of evanogen bromide-treated extracts. Formic eid and cyanogen treated extracts ofthe positive clones (11-3, 1F5, 11, and 11-7) were pooled (otal volume, 250), dried. and resuspended in 0.1 ml of 80 percent acetic acid. PHjLeveine was added, and the sample was applied to column (0.7 by 47 em) of Sephadex 6-50 in $0 percent acetic acid, Portions (60 gl) ofthe column Factions were assayed for somatostatin, Pooled ‘negative clone extracts (11-2, 11-4, and I-11) were treated identically. ‘nthe same column known somatostatin Beckman Instruments, Ine.) lites as indicated (3),Table 1. Somatostatin radioimmune specifi activity. Abbreviations: LB, ria broth, IPTG, isopropylthiogalactoside: CNBr, cyanogen bromide: SS, somatostatin, Protein was measured by the method of Bradford (22). Medium Strain Experiment Le a iB cB i co. c cB cB i VB + alycerol* LB + glycerol i cB iB LB “Vogel-Honnes minimal medium pls alyeor about 15 generations, about one-half of the E. coli RRI (pSOMII-3) culture was constitutive for B-galactosidase, that is, carried the lac operator, and about hall fof the nonconstitutive colonies were ampicillinssensitive, Strains positive (SOM11-3) and negative (pSOMII-2) for somatostatin are unstable, and, therefore, the growth disadvantage pre- sumably comes from the overproduction of the large but incomplete and inactive galactosidase. The yield of somatostatin has varied from 0,001 to 0.03 percent of the total cellular protein (Table 1) prob- ably as the result ofthe selection for cells in culture having plasmids with a deleted luc region. The highest yields of soma- tostatin have been from preparations where growth was started from a single Ap-resistant, constitutive colony. Even in these cases, 30 percent of the cells at harvest had deletions ofthe lac region. Several moderate scale (up to 10 iters) attempts have been made to purify so- matostatin from E. coli strain RRI (PSOM1I-3). The initial” purification scheme was based on known purification properties of P-galactosidase followed by purification of the cyanogen bromide cleavage products of the chimeric pro- tein, However, essentially all of the somatostatin activity found in the erude extract is insoluble and is found in the pellet from the first low speed cent tion. The activity can be solubilized in 70 percent formic acid, 6M guanidinium hy- Grochloride, 8M urea, or 2 percent So- dium docecyl sulfate, Somatostatin ac tivity has been enriched approximately 100-fold from the cellular debris by cy= fanogen bromide cleavage, and subse ‘quent alcohol extraction and chromatog raphy on Sephadex G-0 in $0 percent acetic acid 1062 IPTG CNBr mM Simei t =01 + 28 io Recent improvements in the chemical synthesis of DNA provide the opportu nity to synthesize quickly DNA with bio- logical interest for genetic manipulation and experimentation. As illustrated ear- lier (6, 12), in vitro recombinant DNA techniques and molecular cloning en- hhance the experimental value of chem- ically synthesized DNA. There are two well-established methods for the syn- thesis of DNA. The phosphodiester method of Klorana and co-workers (/8) and the more recently developed modi- fied phosphotriester method (7). Both ‘methods are capable of producing fune- tional DNA (16, 17, 19, 20); however, the triester method is probably faster. More- lover, a method for rapidly synthesizing trimer blocks (codons) as building units for longer oligodeoxyribonucleotides 21) (Fig. 2b) has increased the speed of the triester method. From the trimer block library, a hexadecadeoxyribonu- cleotide now ean be obtained ina week. ‘We have established here that the DNA ‘made with this improvement is function- al ‘The data establishing the synthesis of ‘a polypeptide containing the somatosta- tin amino acid sequence are summarized as follows. (i) Somatostatin radioimmune activity is present in E. coli cells having the plasmid pSOM1I-3, whieh contains a Somatostatin gene of proven correct Se quence anil has the correct orientation of the lae Eco RI DNA fragment. Cells with the related plasmid pSOMII-2, which has the same somatostatin gene ‘but an opposite orientation of the Ine Feo RI fragment, produce no detectable somatostatin activity. (i) As predicted by the design scheme, no detectable somatostatin radioimmune activity is ob- served until after cyanogen bromide treatment of the cell extract. somatostatin activity is under egatrol of the lac operon as evidenced by induction by IPTG, an inducer of the lac operon. iv) The somatostatin activity eochro- matographs with known somatostatin on Sephadex G-50. (v) The DNA sequence of the cloned somatostatin gene is eor- rect, If translation is out of phase, pep: tide willbe made whichis diferent from somatostatin at every positon. Radio- immune activity is detected indicating that a peptide closely related to soma- tostatin is made, and translation must be in phase, Since translation occurs in phase, the genetic code dictates that a peptide with the exact sequence of soms- tostain is made. (vi) Partially purified samples have been independently as sayed by W. Vale (Salk Institute). He has confirmed our radioimmune activity ‘with both antiserum $39, whichis direct- ced by the NHterminal, and with an Serum S201 which interacts mainly with Somatostatin positions 6 through 14. (vi) Finally, the above samples of B. col RRI (pSOMII-3) extract inhibit the re- lease of growth hormone from rat pitui- tary cells, whereas samples of E. col RRI (pSOMII-2) prepared in parallel and with identical protein concentration hhave no effet on growth hormone re- lease (22) ‘Our results represent the fist success in achieving expression (that is, tran- scription into RNA and translation of that RNA into a protein of a designed amino acid sequence) of a gene of chem- ically symthesized origin. The large num- ber of plasmid molecules per cell results in substantial amount (atleast 3 per- cent) of the cellular protein as the frga- lactosidase-somatostatin hybrid. This molecule appears to be relatively resist ant to endogenous proteolytic activity. ‘There is evidence that abnormally short galactosidase peptides are degraded in . coli (4) suggesting that the hybrid protein molecule expected from the fist Somatostatn-lac plasmid (pSOM1) is al- so rapidly degraded. The symhesis of many gratuitous proteins in. col whether large enzymes or smaller poly” Peptides, may be undetectable for this reason. In cases where the amino acid composition of the protein is apprope- fle, the precursor technique described here can be employed. This approach could possibly be extended by taking ad- vantage of proteolytic enzymes with amino acid sequence specifety “The amount of somatostatin. synthe- sized was variable and about a factor of 10 less than the maximum predicted Yield. This variability could be inter- preted in several ways. Protein degradae SCIENCE, VOL. 198tion by endogenous proteases, the inabi- ity to Gly solubilize the chimeric pro- tein, and the selection of altered plasmids could allbe contributing factors to the variability in yield. Although re- combinant’ DNA. experiments - with chemically synthesized DNA are inher- ently Tess hazardous than those_ with DNA from natural sources, consid- eration should be given to the possible toxicity ofthe peptide product. A major factor inthe choice of somatostatin was its proven low toxicity G). In addition, the experiment was deliberately de- signed to have the cells produce not free somatostatin but rather a precursor, which would be expected to be relatively inaetive. The cloning and growth of cell cultures were performed in a P-3 con- tainment facility Kent Iraxura, ‘TaDAAKI HIROSE RoweRro CRE Anriu D. Ricos Division of Biology. City of Hope National Medical Center, Duarte, California 91010 Heracier L. HEYNEKER* FRaNcisco BOLIVAR? Herneer W. Boyer Department of Biochemistry and Biophysics, University of California, San Francisco 9143 teresa Note 1 tao ty Be ions to ba iets 1 PUR riae Re finite bear Pas ee Std Ete a « Geis ee dances Se : ES a eet al, Netwre (London) 260, 00 7 6 evea1 Ror 6 Lae Tat sae Since K iol N, Kaas CP. Bah, RH ST T8EF Tigti) Kk Thakura, N. Katagir S. Narang. Bah, KWo. Blot Chen 254 Pea 4. Evtotea 8 1 Rodrigues PJ. Greene, MC Belloc HL Hegreder i W.cbover 9H. Grou 8. Falkow, Gene 3, 98 1977) 9. We Gert Gala. ajo. Maxam, in Protein Ligand ntfacions” W. Sund Sa of Eis" (De Gruyter, Bertin, 1975. pp. 1 Pir cera tama (Cold Spring Harbor Laboratory, Cold Spine iene aay es SRE Rs eas reshs fests Sat ei 8 Ss Po et oS a ag Rome Ser A 1 ag Solna ta Fl Betti iietrl eli Bt 9 DECEMBER 197 1S. We Gler aad Mule, Pro. Nat fear Si STi 16, HSE ear at Nae London) 263,768 tote 17 RAs Sui. Homan 18. HG Rhorande Pure Appl. Chem. 17, 49 Oe 19. B29 Gendt a, rc. Na. Acad. Set Gs eS 29. 1, Koran, personal communication 2B 1 Hine Cts Kein peo, 3 WAS tical aston BER Br {ord Anal Bachem 72,248 (196) 23. YSiaipat Hoi 8 A Nore C List Mle aa es. 9 SSH 24. Eat Wig. chem: nd. 8, 25, HER. Hay... sbi, RC. Wilas, Gorman Sih, 88 3). 2s, Reet Saat de Std FC Loews, iota en Cease ipa tei 7, Ree Bighon J Atos WM Bares, WS, Rent, Slne 3458 28, Thepontona aye ote Ha Inen 9) slows for fhe detrmiation of he of Famed ie igen ess 2», TAR scope by referent Keo Rd {nl tection wih RNA polymerase of te Sth The dibs Wenge he Tew Trane ater Bd NR ern NA Pe ates wn Ee Kt naa ‘lume of ial for minutes a3HC, The rene= ‘greg sped by henna Co te ‘es wn St acne ng satan of 3 mf Sal ae ip) Maha NAG, Pilates 9 ines ne econ tre wat toned tye aaajon of EDTA Tam) Se us HCl ba ware tupthe BRA was xed it phen fam ected wih canal a Ts rtrd Cees ee Benassi Si Pexilenats cLishacee Sh aie cana, iiooseae nian enecamerane Shariah Sp ackccrcunte Preven seiflenton of the Eso RlcBam Ht Sadat ee ae So cee amtorped ih NeW low eiecy by near eee ee Rie armen earned ae eee ee Sho nomeeiesetce tea ee Stat as a Fee ney ma 5 EN et eae itaiie, We tank L, Sve. Lu, 1 Shh, ediaectre eter ‘design and execution of the project: We also src EES ta ug oe ce eu de : ee eh mene nome ioe ice nee tet SSRIS eee Bach SMES Sap aa 2 November 1977 Cytidine 3',5'-Monophosphate (Cyclic CMP) Formation in Mammalian Tissues Abstract. Mammalian tissues possess the capacity to synthesize eytidine 3',5'- ‘monophosphate (cyclic CMP) via the enzymatic conversion of eytidine 5'-triphos- hate to cyclic CMP by cytidylate cyclase. Cyclic CMP formation occurs best in the presence of manganese or iron, at neutral pH, at 37°C, in the absence of detergents, land with whole tissue homogenate fractions. Thus, mammalian tissues are capable of synthesizing not only eyclic AMP and eyelie GMP, but also eyclic CMP. Adenosine 3',$'-monophosphate (cy- clic AMP) and guanosine 3',5'-mono- phosphate (cyclic GMP) are purine cy- clic nucleotides that are generally ‘thought to influence or regulate numer- ‘ous cell functions and biological events. In many instances, however, alterations in cell function cannot be accounted for by corresponding or concomitant altera- tions in the tissue concentrations of ei- ther of the two purine cyclic nucleotides. Therefore, the existence of other endog: enous regulatory molecules is constant ly being sought. Cytidine 35'-mono- phosphate (cyclic CMP) was first identi- fied in cells (leukemia L.-1210) by Bloch, who demonstrated also that the addi tion of exogenous eyclic CMP to L- 1210 cells in culture abolishes the char- acteristic temperature-dependent lag phase and stimulates the resumption of growth or proliferation of these leukemic cells (1, 2). These experimental findings suggest that cyclic CMP, a pyrimidine cyclic nucleotide, may play a biologic role in the control of proliferation of leu- emie cells ‘Shortly after the discovery of the natu- ral occurrence of cyclic CMP in certain leukemic cells, an enzyme system ca- pable of forming cyclic CMP from its naturally occurring substrate was found in murine myeloid leukemic tumors and in normal mouse liver and spleen (3). Thus, our experimental findings on the capacity of mammalian tissues to synthe size eyelic CMP support those of Bloch on the identification of cyclic CMP in ‘malignant cells. ‘The properties and biologic impor- tance of cytidylate cyclase in normal and malignant mammalian tissues were recently reported briefly (4). At the same time Cailla and Delage reported on the 1088