BIOCHEMICAL
Vol. 181. No. 2. 1991 December
16, 1991
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 707-713
MOLECULARCLONINGANDNU~TIDESEQ~CEOF SmmCES GRISEUS TRYPSIN
GENE
Jee Cheon Kim, Seung Hee Cha*, Seong Tae Jeong, San Kon Oh* and Si Myung Byun’ Department of Life Science, Korea Advanced Institute of Science and Technology 373-l Kuseong-Dong, Yuseong-Gu, Taejon, Korea *Agency for Defense Development, P.O. Box 35 Yuseong, Taejon, Korea Received
November
5,
1991
SUMMARY Streptomymgriseus trypsin (E.G. 3.4.21.4) isoneof themajorextra&l~protei.nase, which is secreki by S. @cur. The gene encoding S. grirem trypsin wasisolated from a S. grisew genomic library by using a synthetic oligonuckotide probe. Fragments containing the gene for S. griseus ttypsin were characterized by hybridization and demonstration of proteolytic activity in S. lividan. Deduced amino acid sequencefrom the nucleotide sequencesuggeststhat S. griseus hypsin is produced as a precursor, consisting of three portions; an aminoterminal pre sequence (32 amino acid residues), a pro sequence(4 residues), and the matute trypsin. The S. griseus trypsin consists of 223 amino acids wrth a computed molecular weight of 23,112. The existence of proline at the pro and mature junction Press, Inc. suggeststhat the processing of S. grirezkr trypsin is non-autocatalytic. 0 1991 Academic
Stmplmnyces griseus, an organism used for the commercial production of pronase, sect&s many extracellular proteins (14). Srregzovnycesgrireus trypsin (SGT) is a bacterial serine proteinase that, curiously, is more similar to mammalian proteinase than to S. griseus protease A and B, two serine proteinases from the samebacterium (25). The general similarity of the substratebinding regions of SGT andbovinetrypsin(BT)isconsistentwi~Ihe~~oftheitinteractionswithsubstratesandinfiibitors. SGT cleaves the oxidized B chain of insulin in the same manner as BT, aswell as the synthetic substrate N-&enzoyl-kuginmeethylester(20). It~bepurifisdonthesameaffulitycolumnasBT(28). Also, it is inhiiited by the same pmkinase inhibitors (19). Although the structure of SGT hasbeen extensively studied, the gene encoding SGT has not been characterized. This report describes the structure of a S. griseus gene which is responsible for the exptession of SGT. The nucleotide sequence suggeststhat proteinase is initially secmted as a pmcursor which is p~toremoveaveryshort~~peptide@ropeptide)fromthematureproteinase. We propose the genetic designation SprT for the unmapped gene for SGT. ‘To whom unrespondence should be addressed. . . Abbrevta&ns used are: SOT, Sfnzptmyces g&em trypti, BT, bovine trypsin; kbp, kilobase pair(s); SDS, sodium dodecyl sulfate, PAGE, polyacrylamide electrophoresis. OCO6-291x/91
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Strains andPlasm& S. grkeur A’ICC 10137, S. lividans TK24, andpIJ702 (12) wereobtained from theKoreanCollech’onfortheTypeCultures,GeneticEngineesingCenter,~~Insti~~ofScienceand Tcchnolog , Taejon, Korea. EFcherichiacoli JM107orIM109 wereusedforalltransformation. pUC18 and pUC1 B werepurchased from Bethesda Besearch Labomtories, Inc. Media, Growth, and Transformation: Growth of Smpmnyces mycelium for the isolation of DNA or the preparation of protoplast has been described previously (12). Transformants were screened for proteol ‘c activity on YD plates (5) containing 30 pg of thiostrepton per ml, 0.5 %(w/v) glycine, and 2 %(wPv) skim nnlk. Competent cells of E, coli strains for tmnsformation were prepared asdescribed by Hanahan (7). E. coli transfommnts were grown on LB plates (26) containing 100 pg ampicillin. Materiakz Oligonucleotides were synthesized by the solid phase phosphoramidate methods with a Beckman system I plus DNA syntheskr. Enzymes for digesting and modifying DNA werepurchased from KGSCO Biotech (Seongnam, Korea) and used in W with the recommendations of the supplier. The radioisotopes [&%i]dATP and [T-~*P]ATP were from Amersham International. Consauction of Genomic Library: Chromosomal DNA of S. g&em ATCC 10137, prepared as descrii previously (12), was digested to completion with ECORI-Hind III and tiactionated by electrophoresis on a 0.8 96 agarose gel. DNA fragments in sire 6.8 kilobase pairs (kbp) were isolated from the agarose gel. The plasmid vector pUC18 and pUC19 were digested with &oRI-Hind III and tmated with calf intestinal alkaline phosphatase @o&ringer Mannheim Biochemicals). The S. grisem DNA fragments (0.5 pg) and vectors (0.2 pg) were seqmtially ligated in a tinal volume of 50 d as described previously (26). Approximately 7,500 transformants of E. coli IMlCt7 were obtained from each ligation reaction. Subcloniug of Proteinase Gene Fragments: A hybrid Smptomyms - E. coli vector wasconstructed by ligating pII702, which had been linearized with Sac I, into the Sue I site of pUCl8. The resultant vector, pUI718-2, wascapable of replicating in S. liridan and E. coli. The multiple cloning sites of this vector, originated from pUC18, were used for subchming DNA fragments of the proteinase gene. Gther fragments were adapted parually with ECORI linker to tkcilitate ligahon into the ECORI site. Hybridization: A 23mer oligonucleotide (5’AAC GC(GC) GAC GAG TGG ATC CAG GT- 3’) was designed from an amino sequence (NADEWIQV) of SGT. For use as a hybridization probe, the oligonucleotide was end-labeled by T4 polynuclcotide kinase and [-$*P]ATP. Digested genomic or plasmid DNA was elecuophorescd and directly hybridized (24). The S. griseur genomic library was screenedby colony hybridization as described previously (26). DNA Sequencing: DNA sequencingwasperformed by the dideoxy-chain termination method (27) using Sequenase(U.S.BiochemicaIs). SubclonesforsequencingwerepreparedinthepUC19orpUC18,and the dideoxy sequencing reaction were run by 40 forward or reverseprimer (New England Biolabs). To reduce compressions, sequencing reactions were carried out 42 “C with dlTP substituted for dGTP or C residues were modified with methoxylamine and sodium bisulfite after the sequencing reaction (1). Also, in some experiments, 10 % formamide (10) wasadded to 6 96 polyacrylamide7M urea gel.
!Screening for Trypsii Gene by Hybridization: An oligonucleotide probe was designed from one of the SGT amino acid sequencesby using the known codon bias for Streptmycces species(9). ‘Ihe utility of the nucleotide probe was demonstrated by hybridization to genomic DNA of S. griseus. As anticipated, the probe hybridized to a fragment generated by EkoRI-Hind IlI (6.8 kbp), but no such hybridization wasobserved with DNA from S. Zitidans. By using the oligonucleotide probe, plasmids containing spfl were isolated from agenomic DNA librsry prepamd from S. gliseur DNA. Gf4,500 E. coli transformants that werescreenedby colony blot hybridization, 9 were detectedby oligonucleotide probe and isolated for tkther characterization. These colonies contained single classof plasmids, based on restriction analysis. As expected, basedon the hybridizationofgenomic DNA, the plasmids contained a 6.8 kbp EcoRI-Hind III fmgment. CharacterizationofTkypsinGene: TheDNAf@mentsisolatedbyhybridirationscreeningweretested forexpressionofproteolyticactivity. The 6.8kbp EcoRI-Hind IIIliagmentswereligatedintothe ECORIHindID site of thevector pUI718-2, to allow transformation of S. hi&m, with selection forthiostrepton
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I
J
Fig. 1. Restrictionendonuclease mapsandsequencing strategyfor the1.8kbpBglI fragment contamlngspfl. (A) The thick line indicatesthe minimumrestriction fragment capableof hybridizingto the oligonucleotide probe. (B) Thearrowsindicatethedirectionand lengthof the sequence determmed by thedideoxychainterminationmethods.The thick lineindicatesthespfl ding region. The organizationof thestructuralgene,with pre-propeptide(36aminoacid,-) andmatureproteinase (223aminoacids,q ), is shownbelow map.Abbreviations: Bm, BomKI; BgI, BglI; BgII, Bgm, EC,EcoRI; Hd, HWIII; Kp, &@I; Pv, ⅈ Sa,SolI; SC,SacI; Sm,SmuI.
red.ance. Transformants containing this construction were then tested on a milk plate containing 0.5 5%glycine for secretion of protekse. A clear zone, although of very small size, representing the degmdation of milk proteins, surrounded each transformant that contained &oRI-Hind III fragment. The clear zoneswere not found around S. fividans coIonies which contained either pIJ7U2 only or no plasmid. The intact proteinase gene could be delimited to a 1.8 kbp Bgl I fragment. This functionally active subclone contained the region which hybridized to the nuckotide probe (Fig. 1.). To identify the protein correqonding to SGT in 5. lividan, the activity staining of proteinase was pedormed (Fig. 2.). The proteinase activity wasfound only in culture supematant of S. Zivih TIC24 harboringpSGTwhichispUJ718-2derivativescontaininga1.8kbpBgZIfkagment. Thesodiumdodecyl sulMepolyaqlamidegelelectrophoresis (SDS-PAGE) of extmceklarproteins produced by 5. lividans TIC24 harboring pSGT showedthat the protein responsible for the activity had the same molecular weight asthe reference SGT purified from ActinaseE. This result wasconsistent with the result of SDS-PAGE of immunopmcipitants between rabbit anti-SGT immunoglobulin G and extracellular proteins (Data not shown). DNA !kquence of Trypsin Gene: The sequencing strategy for the spfl is shown (Fig. 1.). Analysis of the sequencerevealed an open reading frame (nucleotide 525 - 1304), encoding 259 amino acids, that is preceded by a ribosomal binding site (GAAAGAAGG), which is complementary to a squence close to th~3’ end of the 16s rRNA of S. grireur (17), assumed to be the spfl coding sequence (Fig. 3.). The predicted sequenceof SGT differed from the published amino acid sequence (21). It should be corrected by the insertion of two serine residues near position 76 (Ser) according to the numbering ofachymohypsin (25). Four residues, Gln74-Ser75-Ser76-Ser77 in the current sequence, were proposed initially as Gln75-Ser76Gly77Ala79 (25). The N-termimd 32 amino acids resemble a typical 709
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A
B 1
M123M
3
2
Fig. 2. Sodiumdodecyl sulfate- polyacrylamidegel electrophoresis (SDS-PAGE) of extracellular proteinsproducedby 5. ZivrdansTX24 harboringeither pJJ702or pSGT. Extracellularproteinswereobtainedasfollows:Approximately, 0.1 ml sporesolutionsper 100 ml YEME medium,containing10pglmlof thiostrepton,wereinoculatedandculturedfor 4-5days at 30 “C. After filtering the supematants, the supematants were precipitatedwith ammonium sulfateto 60 96saturation.Theprecipitantsweredissolvedanddiiyxed throughagainstthe 1mM HCl and then concentratedwith Centriprep-10(Amicon). Approximately 0.5 ml of 100X concentratedsamples were further lyophilixed with SpeedVat Concentrator(SavantIndustry Inc.,Farmingdale,NY), dissolvedin 20 pl of 1X SDSloadingbuffer, and boiledfor 10 minutes at 85 “C. Thesupematants weresubjected to gelelectrophoresis. Coomassie brilliantbluestaining (A) andactivity staining(B) in a SDS-polyacrylamide gelcontaining0.2 A casein(10)areshown. The molecularmassof the SGT in a SDS-polyacrylamide gel wasestimatedto be about45 killodaltons.A andB: laneM, molecularmassmarkersareindicatedin killodaltons;from top to bottom,bovineserumalbumin,ovalbumm,glyceraldehyde-3-phos@ate dehydrogenase,. carbonic anhydrase,phenylmethanesulfonyl fluonde treatedbovme trypsm, soybeantrypsm mmbitor, bovmec&ctalbumin;lane1, partialpurifiedSGTfrom Actinase-E(KalcenChemicalCo., Tokyo, Japan),preparedasdescribed previously(15) andtreatedwith phenylmethanesulfonyl fluoride; lane2and3, extracellularprotemsproduced by S.lividu~ TK24 harboringeitherplJ702orpSGT.
prolcqotic
signal peptkk
4 positively charged amino acids, followed by a 16 amino acids long
hydrophobicstretchanda smallsidechainaminoacidsat theputative signal@&se X-Al&Q)
cleavagesite(Ala-
(22). The remainingshortsequence betweenthe signalprwzssingsiteand mature amino
tertninusappearstorepresentapqeptide.Thegenomicdesignationofspfl, basedontheinterpretation of the DNA sequencedata,is shownschematically(Fig. 1.). The translationstopuxion at the endof the codingregion is followed by inverted repeatsequence which could form stable hairpin loop. This structure, which is similar to those characterized in other Streptomyces genes (1 l), is believed to be involved with terminationtranscription. An inverted repeat wasfound2oObasepairspriortoinitiationcodonofSprT. Pre&ingt.hisstructtnewasapotentialcoding region, with the characteristics of Sfrepmnycescodonusage,which wasfollowed in frameby a TGA stopcoclon. If the invertedrepeatsequence precedingthe SpJTcodingregionrepresents atranscriptionaI terminator,thentheentiretranscriptionunit of spr’I wouldbe defined. This corroboratesthe functional activity of the Bgl I subclone. DISCUSSION
The base composition of the qfl coding sequenceis 72.4 96 G+C, in good agreement with the avemgeG+Ccontentof SfrepfonycesDNA@). ThishighG+Ccontentresultsinanextremelybiased usage of synonymous codons with 96.5 46 of spfl codons possessingG or C in the third position, as 710
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1 CCCCOOCATCOCCCCCCCCACCCOCAACATCTACCCCCCCT 91 COCCCCCCCOOCCCGCCGCGAACTCCGCCCCCCCCCCACC 181 CTGCAGCTCCTACCGGOCCTCCCG~C~CCCTCCGGCA 271 -20.0 GccccccTCCccC~TAC~ACA~A~TCC~GCCGTA~T~TTCCG~C~~CGTCCC~GTCCG~TGACC~ 361 CGTCCTGGACGGGCCGTOGTOOCCCOTACCACCTTGGCTT~CCG~TGTGCGCCTTGT~CAG~C~CC~TTCCCGA~T~~G~TC
451
TGCATGACCATOCCGTCGCGCCCCGTCGGGOTTCCCACAGCGAC~CCC~C~~GM~CMTC~TGMG~~TC~C RBS MKHFLR -10 541 GTGCGCTOAAGAGATGCTCCCTCGCCGT~C~CCGTffiC~T~~GT~T~CCTC~GCC~T~C~CCTC~C~CCCC~CC ALKRCSVAVATVAIAVVGLQPVTASA+APNPt 631 CCGTCGTCCCCOCMCCCCCCCCGCCCACGGCGAGTTCCCCTT~TGGTC~GCTCTC~T~CTG~CGG~CCCTCTA~CC~~ CGGALYAQD VVGGTRAAQGEFPPHVRLSHG 721 ACATCGTCCTCACCGCGGCCCACTGCGTGAGCGGATCGGC IVLTAAHCVSGSGNNTSITATGGVVDLQSS 811 CCAGCGCCGTCAAGGTCCGCTCCACCAAGGTCCTCCAGGCCCC SAVKVRSTKVLQAPGYNGTGKDWALIKLAQ 901 AGCCCATCMCCAGCCCACGCTGMGATCGC~C~C~CCGC~A~C~~~~TTCACCGT~C~~~~C~C~~ PINQPTLKIATTTAYNQGTFTVAGWGANRE 991 AGGGCGGCAGCCAOCAGCGCTACCTGCTCAAGGCCMCGTCCCGTTCGTCTCCGA~CCGC~GC~CTC~CGTAC~CMCGAG~~ GGSQQRYLLKANVPPVSDAACRSAYGNELV 1081 TGGCCMCGAGGAGATCTGCGC~GACACCGG ANEEICAGYPDTGGVDTCQGD SGGPMFRKD 1171 AC~CGCCGACGAGTGGATCC~TCCTCACCTCGC NADEWIQVGIVSWGYGCARPGYPGVYTEVS 1261 CGACCTTCCCTTCCGCCATCGCCTCGCCCGCCGCCCGCA~GCTC~A~CA~TAC~CACCC~CCGCTCC~GGTGAT~CCCCTGATC TFASAIASAARTL* 135j -32.0 ACCGCCGCCOCCCGTCCCTCTTCTCCGGCCCCCCCCCGCCTGCT 1441 GCGCOTGGACCGGGTGGTAGTCACCGAACTCGACGOATG 1531 AGCCGGGGCCGACCCCGCCCGCCATGGCCCCCTCGTCGCCG~TCGT~C~AC~ACT~CTG~CGTGAGCGT~TGACGCCCTCGTGCT~A~AG~ 1621 TGAGGGTCATGTGCCAGGCGMCTCCCCGGGCC~GGCACC~TGA~TGA~ACGAGACGACGAGGGGCCCGGCA~CCTCGA~TTG~ 1711 GCTCGGCCTGGTGCTCGTCGCCCTCGTAGAGCATCCGGAAC 1801 TTCCGGGCCOCCATCGAGCACGTCTGOCCGGCGGTCACCG 1892
Fig. Nucleotidesequence of sprT. (GenBanlcaccession No. M64471) Thededuced amino acidsequence, whichis numberedfrom the terminiof the matureproteinase,is shownunderthe nucleotidesequence. Stopcodonisindicatedby asterisk.Theprocessing sitesfor thesignalpeptide (downwardarrow)andthematureproteinase (upwardarrow)areshown.Bases involved with the putative+osome bindingsite(RF%)areindicatedby underline.The invertedrepeatsequences areoverhned. The free.energychanges (kilocaloriesper mole)of the hypotheticalhair@ loops werecaktdatedasdesertbed previously(31). DNA sequence whichhybridizedto theohgonucleotideprobeis doubleunderlined.The putative-35and-10regions,which showsimilarityto the consensus sequence of Streptontycesspecies (13), areindicatedby underline.
hasbeencbervd in other Sfnzpturnyca genes(10). Also noticeableis a genemlandpronounced preference(129:28) for a third positionC over G wheresynonymousaxtons allow sucha choice. In comparison,thebasecompositionoutddeoftheproposedopenreadingfrsunesisuniformlydistributed with eachtmckotide triplet. ‘IIre maintermnce of a biasedcodonusagethroughouteachcodingregion 711
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and -02 of the triplet perk&city outside of the reading frames strengthens the assignment of initiation and termination codons (3). Recent studies with secreted proteinazs from both gram-positive and gmm-negative bacteria, in&ding Bacilhs species(16,32), Lysobactor enzymogens (29), Neisseria gonorrhoeae (23), Sermtia (18),Strepcomycesspe~ies(4,9),Vibriocholera(8), and ZJre~wagu#i~(3O),haveshownthatallof these bacterial proteinase are synthesized as precursors, although the pro region varies in its amino or carboxyl terminal location. All of these precursors contain extra signal sequencesand pro sequences, except Semua protease (18) which lacks such signal sequence. The processing of each precursor proteinase is probably at&catalytic, since the amino acid sequencenear the scissile bond should provide agoodsubstrate. TheputativeprosequenceofspflisAla-Pro-Asn-Pro,whichisveryshortascompared to other bacterial prosequences. The p mcessing of SGT precursor is probably non-autocatalytic, since the amino acid sequencenear the pro and mature junction is not a good substrateof SGT itself. Another proteinase, one of the component of pronasc, is probably involved in proessing of SGT. In practice, pronase contains the component that hydrolyzes an artificial substrate, L-plhe-~nitroanilide (0.6 increase of optical density at 420 nm per mg of pronase within 30 minutes). The function of the propeptide is still unclear. It may play an essential role in guiding the folding of proproteinase molecule into the proper conformation necessaryfor activity suchas has been observed with subtilisin (32) and +tic protease (29). In caseof spfl, the pro sequencemay not play a role in guiding the folding of pro enzyme because of its small size, but probably play a function to keep the precursor inactive, as is the casewith bovine trypsinogen whosepro sequenceis Val-Asp-AspAsp-Asp Lys. The non-autocatalytic junction of the precursor and Longisporus trypsii inhibitor that is secreted by S. Zivihns 66 (2) maybe responsible for the low proteinase activity in culture supernatant of S. lividanr TK24 harboring pSGT. Further investigation will be directed toward unraveling the activation mechanism of the proteinase precursor and elucidating the exact function of the pro sequence. REFERENCES 1. Ambartsumyan, N.S. and Mazo, A.M. (1980) FEBS L&t. 114,265-268. 2. Be&a, T.R., Fomwald, J.A., Gorniak, J.G., Rosenberg, M., Stickler, J.E., and Taylor, D.P. (1988) PCl- Int. Appl. WO 88 01,278. 3. Bibb, M.J., Fmdlay, P.R., and Johnson, M.W. (1984) Gene 30, 157-166. 4. Chang, P.C., Kuo, T.-C., Tsugita, A., and Lee, Y .-H.W. (1990) Gene 88,87-95 5. Foor, F., Roberts, G.R., Morin, N., Snyder, L., Hwang., M., Gibbons, P.M., Pamdiso, M.J., Stotish, R.L., Ruby, C.L., Wolanski, B., and Suer&r, S.L. (1985) Gene 39, 11-16. 6. Gladek, A. and Zakrzewska, J. (1984) FEMS Micmbiol. Lett. 24,73-76. 7. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580. 8. H&e, C.C. andFinkelstein, R.A. (1991) J. Bacterial. 173,3311-3317. 9. Henderson. G.. Krvgsman, P., Liu, C.J., Darvey, C.C., and Malek, L.T. (1987) J. Bacterial. 169,3778-3784.
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Hensen, C. and Dowdle, E.B. (1980) Anal. B&hem. 102,196202. Hopwood, D.A., Bibb, M.J., Charter, K.F,, Janseen, G.R., Ma&u%%, F., and Smith, C.P. (1986) In Regulation of Gene Expression - 25 Years Gn (Booth, L.R. and Higgins, C.F. Eds.), pp. 251-276. Cambridge University Press, Cambridge. 12. H wood, D.A.,Bibb, M.J., Charter, K.F.,Kieser,T.,Bruton, C.J., Kieser, H.M., L & . , D.J., Smith, C.P., Ward, J.M., and Schrempf, H. (1985) Genetic Manipulation of Strepwmym: A Laboratory Manual. The John Innes Foundation, Norwrch. 13. Horii, H.. Ishizaki, T., Pa&, S.-Y., Manome, T., and Murooka, Y. (1990) J. Bact&i01: 172,36443653. 14. Jur&k. L,, Jhonson, P., Olafson, R.W., and Smillie, L.B. (1971) Can. J. Biochem. 49,548-56%
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15. Ju&ek, L., Jhonson,P., Olafson, R.W., and Smillie, L.B. (1971) Can. J. B&hem. 49, 1195-1201. 16. Kaneko, R., Koyama, N., Tsai, Y.C., Juang,R.-Y., Yoda, K., and Yamasaki,M. (1989) J. Bacterial. 171,5232-5236. 17. Kim,E.,Kim,H.,Kang,K.H,,Kho, Y.H.,andPark,Y.-H. (1991) NucleicAcidsRes. D, 1149. 18. Nakahama,K., Yoshimum,K., Marumoto, R., Kikuchi, M., Lee, L.S., Has, T., and Mat&ma, H. (1986) Nucleic Acids Res. 14,5843-5855. 19. Nagata,K. and Yoshida,N. (1983) J. Biochem. (Tokyo) 93,909-919. 20. Olafson, R.W., Jurdsek,L., Carpenter,M.R., and Smillie, L.B. (1975) Biochemistry 14, 1168- 1177. 21. Olafson, R.W. and Smillie, L.B. (1975) Biochemistry 14,1161-1167. 22. PerlmaqD. andHalvorson, H.O. (1983) J. Mol. Biol. 200, 523-551. 23. Pokier, J., Halter, R., Beyreuther, K., andMeyer, T.F. (1987) Nature 325,458-462. 24. Purrcllo, M. andBalaq L. (1983) Anal. Biochem. l28,393-397. 25. Read,R.J. andJames,M.N.G. (1988) J. Mol. Biol. 200, 523-551. 26. Sambrook,J., Fritsch, E.F., and Ma&is, T. (1989) Molecular Cloning: A Laboratory Mammal (2ndEdition). Cold Spring Harbor Labomtory, Cold Spring Harbor, NY. 27. Sanger,F., Nicklen, S., and Co&on, A.R. (1977) Proc. Natl. Acad. Sci. USA 74,5463-5467. 28. Shimma,K. and Kasai, K.-I. (1984) J. Chromatogr. 315, 161-166. 29. S&m, J.L. and Agard, D.A. (1989) Nature 341,462-464. 30. Terada, I., Kwon, S.-T., Miyata, Y., Matsuzawa, H., and Ohta, T. (1990) J. Biol. Chem. 265,6576-6581. 31. Turner, D.H., Sugimoto,N., Saeger,J.A., Longfellow, C.E., Freier, SM., and Kierzek, R. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, 123-133. 32. Zhu, X., Ohta, Y., Jordan,F., and Inouye, M. (1989) Nature 339,483-484.
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Vol. 181. No. 2. 1991 December
16, 1991
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 707-713
MOLECULARCLONINGANDNU~TIDESEQ~CEOF SmmCES GRISEUS TRYPSIN
GENE
Jee Cheon Kim, Seung Hee Cha*, Seong Tae Jeong, San Kon Oh* and Si Myung Byun’ Department of Life Science, Korea Advanced Institute of Science and Technology 373-l Kuseong-Dong, Yuseong-Gu, Taejon, Korea *Agency for Defense Development, P.O. Box 35 Yuseong, Taejon, Korea Received
November
5,
1991
SUMMARY Streptomymgriseus trypsin (E.G. 3.4.21.4) isoneof themajorextra&l~protei.nase, which is secreki by S. @cur. The gene encoding S. grirem trypsin wasisolated from a S. grisew genomic library by using a synthetic oligonuckotide probe. Fragments containing the gene for S. griseus ttypsin were characterized by hybridization and demonstration of proteolytic activity in S. lividan. Deduced amino acid sequencefrom the nucleotide sequencesuggeststhat S. griseus hypsin is produced as a precursor, consisting of three portions; an aminoterminal pre sequence (32 amino acid residues), a pro sequence(4 residues), and the matute trypsin. The S. griseus trypsin consists of 223 amino acids wrth a computed molecular weight of 23,112. The existence of proline at the pro and mature junction Press, Inc. suggeststhat the processing of S. grirezkr trypsin is non-autocatalytic. 0 1991 Academic
Stmplmnyces griseus, an organism used for the commercial production of pronase, sect&s many extracellular proteins (14). Srregzovnycesgrireus trypsin (SGT) is a bacterial serine proteinase that, curiously, is more similar to mammalian proteinase than to S. griseus protease A and B, two serine proteinases from the samebacterium (25). The general similarity of the substratebinding regions of SGT andbovinetrypsin(BT)isconsistentwi~Ihe~~oftheitinteractionswithsubstratesandinfiibitors. SGT cleaves the oxidized B chain of insulin in the same manner as BT, aswell as the synthetic substrate N-&enzoyl-kuginmeethylester(20). It~bepurifisdonthesameaffulitycolumnasBT(28). Also, it is inhiiited by the same pmkinase inhibitors (19). Although the structure of SGT hasbeen extensively studied, the gene encoding SGT has not been characterized. This report describes the structure of a S. griseus gene which is responsible for the exptession of SGT. The nucleotide sequence suggeststhat proteinase is initially secmted as a pmcursor which is p~toremoveaveryshort~~peptide@ropeptide)fromthematureproteinase. We propose the genetic designation SprT for the unmapped gene for SGT. ‘To whom unrespondence should be addressed. . . Abbrevta&ns used are: SOT, Sfnzptmyces g&em trypti, BT, bovine trypsin; kbp, kilobase pair(s); SDS, sodium dodecyl sulfate, PAGE, polyacrylamide electrophoresis. OCO6-291x/91
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Strains andPlasm& S. grkeur A’ICC 10137, S. lividans TK24, andpIJ702 (12) wereobtained from theKoreanCollech’onfortheTypeCultures,GeneticEngineesingCenter,~~Insti~~ofScienceand Tcchnolog , Taejon, Korea. EFcherichiacoli JM107orIM109 wereusedforalltransformation. pUC18 and pUC1 B werepurchased from Bethesda Besearch Labomtories, Inc. Media, Growth, and Transformation: Growth of Smpmnyces mycelium for the isolation of DNA or the preparation of protoplast has been described previously (12). Transformants were screened for proteol ‘c activity on YD plates (5) containing 30 pg of thiostrepton per ml, 0.5 %(w/v) glycine, and 2 %(wPv) skim nnlk. Competent cells of E, coli strains for tmnsformation were prepared asdescribed by Hanahan (7). E. coli transfommnts were grown on LB plates (26) containing 100 pg ampicillin. Materiakz Oligonucleotides were synthesized by the solid phase phosphoramidate methods with a Beckman system I plus DNA syntheskr. Enzymes for digesting and modifying DNA werepurchased from KGSCO Biotech (Seongnam, Korea) and used in W with the recommendations of the supplier. The radioisotopes [&%i]dATP and [T-~*P]ATP were from Amersham International. Consauction of Genomic Library: Chromosomal DNA of S. g&em ATCC 10137, prepared as descrii previously (12), was digested to completion with ECORI-Hind III and tiactionated by electrophoresis on a 0.8 96 agarose gel. DNA fragments in sire 6.8 kilobase pairs (kbp) were isolated from the agarose gel. The plasmid vector pUC18 and pUC19 were digested with &oRI-Hind III and tmated with calf intestinal alkaline phosphatase @o&ringer Mannheim Biochemicals). The S. grisem DNA fragments (0.5 pg) and vectors (0.2 pg) were seqmtially ligated in a tinal volume of 50 d as described previously (26). Approximately 7,500 transformants of E. coli IMlCt7 were obtained from each ligation reaction. Subcloniug of Proteinase Gene Fragments: A hybrid Smptomyms - E. coli vector wasconstructed by ligating pII702, which had been linearized with Sac I, into the Sue I site of pUCl8. The resultant vector, pUI718-2, wascapable of replicating in S. liridan and E. coli. The multiple cloning sites of this vector, originated from pUC18, were used for subchming DNA fragments of the proteinase gene. Gther fragments were adapted parually with ECORI linker to tkcilitate ligahon into the ECORI site. Hybridization: A 23mer oligonucleotide (5’AAC GC(GC) GAC GAG TGG ATC CAG GT- 3’) was designed from an amino sequence (NADEWIQV) of SGT. For use as a hybridization probe, the oligonucleotide was end-labeled by T4 polynuclcotide kinase and [-$*P]ATP. Digested genomic or plasmid DNA was elecuophorescd and directly hybridized (24). The S. griseur genomic library was screenedby colony hybridization as described previously (26). DNA Sequencing: DNA sequencingwasperformed by the dideoxy-chain termination method (27) using Sequenase(U.S.BiochemicaIs). SubclonesforsequencingwerepreparedinthepUC19orpUC18,and the dideoxy sequencing reaction were run by 40 forward or reverseprimer (New England Biolabs). To reduce compressions, sequencing reactions were carried out 42 “C with dlTP substituted for dGTP or C residues were modified with methoxylamine and sodium bisulfite after the sequencing reaction (1). Also, in some experiments, 10 % formamide (10) wasadded to 6 96 polyacrylamide7M urea gel.
!Screening for Trypsii Gene by Hybridization: An oligonucleotide probe was designed from one of the SGT amino acid sequencesby using the known codon bias for Streptmycces species(9). ‘Ihe utility of the nucleotide probe was demonstrated by hybridization to genomic DNA of S. griseus. As anticipated, the probe hybridized to a fragment generated by EkoRI-Hind IlI (6.8 kbp), but no such hybridization wasobserved with DNA from S. Zitidans. By using the oligonucleotide probe, plasmids containing spfl were isolated from agenomic DNA librsry prepamd from S. gliseur DNA. Gf4,500 E. coli transformants that werescreenedby colony blot hybridization, 9 were detectedby oligonucleotide probe and isolated for tkther characterization. These colonies contained single classof plasmids, based on restriction analysis. As expected, basedon the hybridizationofgenomic DNA, the plasmids contained a 6.8 kbp EcoRI-Hind III fmgment. CharacterizationofTkypsinGene: TheDNAf@mentsisolatedbyhybridirationscreeningweretested forexpressionofproteolyticactivity. The 6.8kbp EcoRI-Hind IIIliagmentswereligatedintothe ECORIHindID site of thevector pUI718-2, to allow transformation of S. hi&m, with selection forthiostrepton
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Fig. 1. Restrictionendonuclease mapsandsequencing strategyfor the1.8kbpBglI fragment contamlngspfl. (A) The thick line indicatesthe minimumrestriction fragment capableof hybridizingto the oligonucleotide probe. (B) Thearrowsindicatethedirectionand lengthof the sequence determmed by thedideoxychainterminationmethods.The thick lineindicatesthespfl ding region. The organizationof thestructuralgene,with pre-propeptide(36aminoacid,-) andmatureproteinase (223aminoacids,q ), is shownbelow map.Abbreviations: Bm, BomKI; BgI, BglI; BgII, Bgm, EC,EcoRI; Hd, HWIII; Kp, &@I; Pv, ⅈ Sa,SolI; SC,SacI; Sm,SmuI.
red.ance. Transformants containing this construction were then tested on a milk plate containing 0.5 5%glycine for secretion of protekse. A clear zone, although of very small size, representing the degmdation of milk proteins, surrounded each transformant that contained &oRI-Hind III fragment. The clear zoneswere not found around S. fividans coIonies which contained either pIJ7U2 only or no plasmid. The intact proteinase gene could be delimited to a 1.8 kbp Bgl I fragment. This functionally active subclone contained the region which hybridized to the nuckotide probe (Fig. 1.). To identify the protein correqonding to SGT in 5. lividan, the activity staining of proteinase was pedormed (Fig. 2.). The proteinase activity wasfound only in culture supematant of S. Zivih TIC24 harboringpSGTwhichispUJ718-2derivativescontaininga1.8kbpBgZIfkagment. Thesodiumdodecyl sulMepolyaqlamidegelelectrophoresis (SDS-PAGE) of extmceklarproteins produced by 5. lividans TIC24 harboring pSGT showedthat the protein responsible for the activity had the same molecular weight asthe reference SGT purified from ActinaseE. This result wasconsistent with the result of SDS-PAGE of immunopmcipitants between rabbit anti-SGT immunoglobulin G and extracellular proteins (Data not shown). DNA !kquence of Trypsin Gene: The sequencing strategy for the spfl is shown (Fig. 1.). Analysis of the sequencerevealed an open reading frame (nucleotide 525 - 1304), encoding 259 amino acids, that is preceded by a ribosomal binding site (GAAAGAAGG), which is complementary to a squence close to th~3’ end of the 16s rRNA of S. grireur (17), assumed to be the spfl coding sequence (Fig. 3.). The predicted sequenceof SGT differed from the published amino acid sequence (21). It should be corrected by the insertion of two serine residues near position 76 (Ser) according to the numbering ofachymohypsin (25). Four residues, Gln74-Ser75-Ser76-Ser77 in the current sequence, were proposed initially as Gln75-Ser76Gly77Ala79 (25). The N-termimd 32 amino acids resemble a typical 709
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A
B 1
M123M
3
2
Fig. 2. Sodiumdodecyl sulfate- polyacrylamidegel electrophoresis (SDS-PAGE) of extracellular proteinsproducedby 5. ZivrdansTX24 harboringeither pJJ702or pSGT. Extracellularproteinswereobtainedasfollows:Approximately, 0.1 ml sporesolutionsper 100 ml YEME medium,containing10pglmlof thiostrepton,wereinoculatedandculturedfor 4-5days at 30 “C. After filtering the supematants, the supematants were precipitatedwith ammonium sulfateto 60 96saturation.Theprecipitantsweredissolvedanddiiyxed throughagainstthe 1mM HCl and then concentratedwith Centriprep-10(Amicon). Approximately 0.5 ml of 100X concentratedsamples were further lyophilixed with SpeedVat Concentrator(SavantIndustry Inc.,Farmingdale,NY), dissolvedin 20 pl of 1X SDSloadingbuffer, and boiledfor 10 minutes at 85 “C. Thesupematants weresubjected to gelelectrophoresis. Coomassie brilliantbluestaining (A) andactivity staining(B) in a SDS-polyacrylamide gelcontaining0.2 A casein(10)areshown. The molecularmassof the SGT in a SDS-polyacrylamide gel wasestimatedto be about45 killodaltons.A andB: laneM, molecularmassmarkersareindicatedin killodaltons;from top to bottom,bovineserumalbumin,ovalbumm,glyceraldehyde-3-phos@ate dehydrogenase,. carbonic anhydrase,phenylmethanesulfonyl fluonde treatedbovme trypsm, soybeantrypsm mmbitor, bovmec&ctalbumin;lane1, partialpurifiedSGTfrom Actinase-E(KalcenChemicalCo., Tokyo, Japan),preparedasdescribed previously(15) andtreatedwith phenylmethanesulfonyl fluoride; lane2and3, extracellularprotemsproduced by S.lividu~ TK24 harboringeitherplJ702orpSGT.
prolcqotic
signal peptkk
4 positively charged amino acids, followed by a 16 amino acids long
hydrophobicstretchanda smallsidechainaminoacidsat theputative signal@&se X-Al&Q)
cleavagesite(Ala-
(22). The remainingshortsequence betweenthe signalprwzssingsiteand mature amino
tertninusappearstorepresentapqeptide.Thegenomicdesignationofspfl, basedontheinterpretation of the DNA sequencedata,is shownschematically(Fig. 1.). The translationstopuxion at the endof the codingregion is followed by inverted repeatsequence which could form stable hairpin loop. This structure, which is similar to those characterized in other Streptomyces genes (1 l), is believed to be involved with terminationtranscription. An inverted repeat wasfound2oObasepairspriortoinitiationcodonofSprT. Pre&ingt.hisstructtnewasapotentialcoding region, with the characteristics of Sfrepmnycescodonusage,which wasfollowed in frameby a TGA stopcoclon. If the invertedrepeatsequence precedingthe SpJTcodingregionrepresents atranscriptionaI terminator,thentheentiretranscriptionunit of spr’I wouldbe defined. This corroboratesthe functional activity of the Bgl I subclone. DISCUSSION
The base composition of the qfl coding sequenceis 72.4 96 G+C, in good agreement with the avemgeG+Ccontentof SfrepfonycesDNA@). ThishighG+Ccontentresultsinanextremelybiased usage of synonymous codons with 96.5 46 of spfl codons possessingG or C in the third position, as 710
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1 CCCCOOCATCOCCCCCCCCACCCOCAACATCTACCCCCCCT 91 COCCCCCCCOOCCCGCCGCGAACTCCGCCCCCCCCCCACC 181 CTGCAGCTCCTACCGGOCCTCCCG~C~CCCTCCGGCA 271 -20.0 GccccccTCCccC~TAC~ACA~A~TCC~GCCGTA~T~TTCCG~C~~CGTCCC~GTCCG~TGACC~ 361 CGTCCTGGACGGGCCGTOGTOOCCCOTACCACCTTGGCTT~CCG~TGTGCGCCTTGT~CAG~C~CC~TTCCCGA~T~~G~TC
451
TGCATGACCATOCCGTCGCGCCCCGTCGGGOTTCCCACAGCGAC~CCC~C~~GM~CMTC~TGMG~~TC~C RBS MKHFLR -10 541 GTGCGCTOAAGAGATGCTCCCTCGCCGT~C~CCGTffiC~T~~GT~T~CCTC~GCC~T~C~CCTC~C~CCCC~CC ALKRCSVAVATVAIAVVGLQPVTASA+APNPt 631 CCGTCGTCCCCOCMCCCCCCCCGCCCACGGCGAGTTCCCCTT~TGGTC~GCTCTC~T~CTG~CGG~CCCTCTA~CC~~ CGGALYAQD VVGGTRAAQGEFPPHVRLSHG 721 ACATCGTCCTCACCGCGGCCCACTGCGTGAGCGGATCGGC IVLTAAHCVSGSGNNTSITATGGVVDLQSS 811 CCAGCGCCGTCAAGGTCCGCTCCACCAAGGTCCTCCAGGCCCC SAVKVRSTKVLQAPGYNGTGKDWALIKLAQ 901 AGCCCATCMCCAGCCCACGCTGMGATCGC~C~C~CCGC~A~C~~~~TTCACCGT~C~~~~C~C~~ PINQPTLKIATTTAYNQGTFTVAGWGANRE 991 AGGGCGGCAGCCAOCAGCGCTACCTGCTCAAGGCCMCGTCCCGTTCGTCTCCGA~CCGC~GC~CTC~CGTAC~CMCGAG~~ GGSQQRYLLKANVPPVSDAACRSAYGNELV 1081 TGGCCMCGAGGAGATCTGCGC~GACACCGG ANEEICAGYPDTGGVDTCQGD SGGPMFRKD 1171 AC~CGCCGACGAGTGGATCC~TCCTCACCTCGC NADEWIQVGIVSWGYGCARPGYPGVYTEVS 1261 CGACCTTCCCTTCCGCCATCGCCTCGCCCGCCGCCCGCA~GCTC~A~CA~TAC~CACCC~CCGCTCC~GGTGAT~CCCCTGATC TFASAIASAARTL* 135j -32.0 ACCGCCGCCOCCCGTCCCTCTTCTCCGGCCCCCCCCCGCCTGCT 1441 GCGCOTGGACCGGGTGGTAGTCACCGAACTCGACGOATG 1531 AGCCGGGGCCGACCCCGCCCGCCATGGCCCCCTCGTCGCCG~TCGT~C~AC~ACT~CTG~CGTGAGCGT~TGACGCCCTCGTGCT~A~AG~ 1621 TGAGGGTCATGTGCCAGGCGMCTCCCCGGGCC~GGCACC~TGA~TGA~ACGAGACGACGAGGGGCCCGGCA~CCTCGA~TTG~ 1711 GCTCGGCCTGGTGCTCGTCGCCCTCGTAGAGCATCCGGAAC 1801 TTCCGGGCCOCCATCGAGCACGTCTGOCCGGCGGTCACCG 1892
Fig. Nucleotidesequence of sprT. (GenBanlcaccession No. M64471) Thededuced amino acidsequence, whichis numberedfrom the terminiof the matureproteinase,is shownunderthe nucleotidesequence. Stopcodonisindicatedby asterisk.Theprocessing sitesfor thesignalpeptide (downwardarrow)andthematureproteinase (upwardarrow)areshown.Bases involved with the putative+osome bindingsite(RF%)areindicatedby underline.The invertedrepeatsequences areoverhned. The free.energychanges (kilocaloriesper mole)of the hypotheticalhair@ loops werecaktdatedasdesertbed previously(31). DNA sequence whichhybridizedto theohgonucleotideprobeis doubleunderlined.The putative-35and-10regions,which showsimilarityto the consensus sequence of Streptontycesspecies (13), areindicatedby underline.
hasbeencbervd in other Sfnzpturnyca genes(10). Also noticeableis a genemlandpronounced preference(129:28) for a third positionC over G wheresynonymousaxtons allow sucha choice. In comparison,thebasecompositionoutddeoftheproposedopenreadingfrsunesisuniformlydistributed with eachtmckotide triplet. ‘IIre maintermnce of a biasedcodonusagethroughouteachcodingregion 711
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and -02 of the triplet perk&city outside of the reading frames strengthens the assignment of initiation and termination codons (3). Recent studies with secreted proteinazs from both gram-positive and gmm-negative bacteria, in&ding Bacilhs species(16,32), Lysobactor enzymogens (29), Neisseria gonorrhoeae (23), Sermtia (18),Strepcomycesspe~ies(4,9),Vibriocholera(8), and ZJre~wagu#i~(3O),haveshownthatallof these bacterial proteinase are synthesized as precursors, although the pro region varies in its amino or carboxyl terminal location. All of these precursors contain extra signal sequencesand pro sequences, except Semua protease (18) which lacks such signal sequence. The processing of each precursor proteinase is probably at&catalytic, since the amino acid sequencenear the scissile bond should provide agoodsubstrate. TheputativeprosequenceofspflisAla-Pro-Asn-Pro,whichisveryshortascompared to other bacterial prosequences. The p mcessing of SGT precursor is probably non-autocatalytic, since the amino acid sequencenear the pro and mature junction is not a good substrateof SGT itself. Another proteinase, one of the component of pronasc, is probably involved in proessing of SGT. In practice, pronase contains the component that hydrolyzes an artificial substrate, L-plhe-~nitroanilide (0.6 increase of optical density at 420 nm per mg of pronase within 30 minutes). The function of the propeptide is still unclear. It may play an essential role in guiding the folding of proproteinase molecule into the proper conformation necessaryfor activity suchas has been observed with subtilisin (32) and +tic protease (29). In caseof spfl, the pro sequencemay not play a role in guiding the folding of pro enzyme because of its small size, but probably play a function to keep the precursor inactive, as is the casewith bovine trypsinogen whosepro sequenceis Val-Asp-AspAsp-Asp Lys. The non-autocatalytic junction of the precursor and Longisporus trypsii inhibitor that is secreted by S. Zivihns 66 (2) maybe responsible for the low proteinase activity in culture supernatant of S. lividanr TK24 harboring pSGT. Further investigation will be directed toward unraveling the activation mechanism of the proteinase precursor and elucidating the exact function of the pro sequence. REFERENCES 1. Ambartsumyan, N.S. and Mazo, A.M. (1980) FEBS L&t. 114,265-268. 2. Be&a, T.R., Fomwald, J.A., Gorniak, J.G., Rosenberg, M., Stickler, J.E., and Taylor, D.P. (1988) PCl- Int. Appl. WO 88 01,278. 3. Bibb, M.J., Fmdlay, P.R., and Johnson, M.W. (1984) Gene 30, 157-166. 4. Chang, P.C., Kuo, T.-C., Tsugita, A., and Lee, Y .-H.W. (1990) Gene 88,87-95 5. Foor, F., Roberts, G.R., Morin, N., Snyder, L., Hwang., M., Gibbons, P.M., Pamdiso, M.J., Stotish, R.L., Ruby, C.L., Wolanski, B., and Suer&r, S.L. (1985) Gene 39, 11-16. 6. Gladek, A. and Zakrzewska, J. (1984) FEMS Micmbiol. Lett. 24,73-76. 7. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580. 8. H&e, C.C. andFinkelstein, R.A. (1991) J. Bacterial. 173,3311-3317. 9. Henderson. G.. Krvgsman, P., Liu, C.J., Darvey, C.C., and Malek, L.T. (1987) J. Bacterial. 169,3778-3784.
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Hensen, C. and Dowdle, E.B. (1980) Anal. B&hem. 102,196202. Hopwood, D.A., Bibb, M.J., Charter, K.F,, Janseen, G.R., Ma&u%%, F., and Smith, C.P. (1986) In Regulation of Gene Expression - 25 Years Gn (Booth, L.R. and Higgins, C.F. Eds.), pp. 251-276. Cambridge University Press, Cambridge. 12. H wood, D.A.,Bibb, M.J., Charter, K.F.,Kieser,T.,Bruton, C.J., Kieser, H.M., L & . , D.J., Smith, C.P., Ward, J.M., and Schrempf, H. (1985) Genetic Manipulation of Strepwmym: A Laboratory Manual. The John Innes Foundation, Norwrch. 13. Horii, H.. Ishizaki, T., Pa&, S.-Y., Manome, T., and Murooka, Y. (1990) J. Bact&i01: 172,36443653. 14. Jur&k. L,, Jhonson, P., Olafson, R.W., and Smillie, L.B. (1971) Can. J. Biochem. 49,548-56%
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15. Ju&ek, L., Jhonson,P., Olafson, R.W., and Smillie, L.B. (1971) Can. J. B&hem. 49, 1195-1201. 16. Kaneko, R., Koyama, N., Tsai, Y.C., Juang,R.-Y., Yoda, K., and Yamasaki,M. (1989) J. Bacterial. 171,5232-5236. 17. Kim,E.,Kim,H.,Kang,K.H,,Kho, Y.H.,andPark,Y.-H. (1991) NucleicAcidsRes. D, 1149. 18. Nakahama,K., Yoshimum,K., Marumoto, R., Kikuchi, M., Lee, L.S., Has, T., and Mat&ma, H. (1986) Nucleic Acids Res. 14,5843-5855. 19. Nagata,K. and Yoshida,N. (1983) J. Biochem. (Tokyo) 93,909-919. 20. Olafson, R.W., Jurdsek,L., Carpenter,M.R., and Smillie, L.B. (1975) Biochemistry 14, 1168- 1177. 21. Olafson, R.W. and Smillie, L.B. (1975) Biochemistry 14,1161-1167. 22. PerlmaqD. andHalvorson, H.O. (1983) J. Mol. Biol. 200, 523-551. 23. Pokier, J., Halter, R., Beyreuther, K., andMeyer, T.F. (1987) Nature 325,458-462. 24. Purrcllo, M. andBalaq L. (1983) Anal. Biochem. l28,393-397. 25. Read,R.J. andJames,M.N.G. (1988) J. Mol. Biol. 200, 523-551. 26. Sambrook,J., Fritsch, E.F., and Ma&is, T. (1989) Molecular Cloning: A Laboratory Mammal (2ndEdition). Cold Spring Harbor Labomtory, Cold Spring Harbor, NY. 27. Sanger,F., Nicklen, S., and Co&on, A.R. (1977) Proc. Natl. Acad. Sci. USA 74,5463-5467. 28. Shimma,K. and Kasai, K.-I. (1984) J. Chromatogr. 315, 161-166. 29. S&m, J.L. and Agard, D.A. (1989) Nature 341,462-464. 30. Terada, I., Kwon, S.-T., Miyata, Y., Matsuzawa, H., and Ohta, T. (1990) J. Biol. Chem. 265,6576-6581. 31. Turner, D.H., Sugimoto,N., Saeger,J.A., Longfellow, C.E., Freier, SM., and Kierzek, R. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, 123-133. 32. Zhu, X., Ohta, Y., Jordan,F., and Inouye, M. (1989) Nature 339,483-484.
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