THE USE OF PSG5 AS A TEMPERATURE-SENSITIVE PLASMID

r ilk'6~21 ~3.6 111111c125__II~;11111.2 L.4-MICROCOPY ,RESIOLUTION TEST CHARTBUREAU OF STANDARS -1963-ANATIONAL IS T(CONVENTION. A~y one .r miore p~rjons- M' d/or i -Company.), rarn -qCOMMONWEALTH OF AUSTRtALIAPatents Act 1952-1969CONVENTION APPLICATION FOR A PATENTinert(n fu 11) Name or Names ofWA~pp, Icant Orfollowed by;V0. Hre insert Titlehereby apply for the grant of a Patent for an invention entitled: THE USE OF.pSG5 AS,A T.EMPERAT.URE=,SENS.TL3-E..P.LASMID..............H3 inset1er r R1 uberfs) oI basicwhich is described in the accompanying complete specification. This application is aConvention application and is based on the application P.3.8. numberedO Name of basfc Country orCnuntrits, A for a paten or similar poeto aei (4e~rl..epbi f..~x~naddress for service Is Messs. Edwd. Waters OurQueen Street, Melbourne, Victoria, Austraiai.Sons, Patent Attorneys,DATEI fh y of. _Ar~ 1 ture Signa-(5 ofHOE CHER.,AKTIiBNGESEbLS HAFT Company andSignatures of its Officers asnprescri bed byYTo,. Registered *Patent. Attorney~FormrCOMMONWEALTH OF ,AUSTRALIAPatents Act 1952DECLARATION IN SUPPORT OF A CONVENTION APPLICATIONUNEPATXXFOR A PATENT.In support of the Convention application made-under Part XVI,,of the Patents Act 1952 by HOECHST AKTIENGESELLSCHAFT of BrUningstrasse, D-6230 Frankfurt/Main 80, Federal Republic ofGermany for a patent for an invention entitled:THE USE OF PSG5 AS A TEMPERATURE-SENSITIVE PLASMIDUlrich Tergau, Am Dornbusch 3, D-6239 Eppstein/Taunus,'eFranz Lapice, Sandweg 2, D-6233 Kelkheim (Taunus),Federal Republic of Germanydo solemnly and sincerely declare as follows:We are authorized by HOECHST AKTIENGESELLSCHAFT the applicantfor the patent to make this declaration on its behalf.2. The basic application as defined by Section 141 of the Act was0. e made in the Federal Republic of Germanyunder No. P 38 09 692.7 .on March 23, 1988by HOECHST AKTIENGESELLSCHAFT33 Wolfgang W~ohlleben, Menzelstra~e 1, D-4800 Bielefeldb) GUnter Muth, Talbr~ckenstra3e 91, D-4800 Bielefeldc) Alfred Ptihler, Am Waldschl~bchen 2, D-4800 Bielefeldd) Bernhard NuBbaumer, Hansastra~e 15, D-4900 Bielefelda) d) Federal Republic of Germanyis/are the actual inventor(s) of the invention and the facts uponHOECHST AKTIENGESELLSCHAFT~which 4ris entitled to make the application are as follows:HOECHST AKTIENGESELLSCHAFTThe said is th~e assignee of the saidWolfgang Wohlleben, Gflnter Muth, Alfred PUhler, Bernhard NuI~baumerThe basic application referred to in paragraph 2 of thisDeclarAtIon was the first application made in A Conventioncountry in re-spect of the invention the subject of the application.,at Frankfurt/Main, Federal Republic of GermanyDECLARZD, 4. this 23rd day of February 1989PatenitsHOECHST AXTI8NGESELLSQHAFTTo, the Qc0,amiss!.porxer PAT ~iO .Prokurist Auhrized Sipney_ppa: Tergai iV.LapiceS1. IN i (12) PATENT ABSTRACT (11) Document No. AU-A-31582/89(19) AUSTRALIAN PATENT OFFICE(54) TitleTHE USE OF PSG5 AS A TEMPERATURE-SENSITIVE PLASMID(51)4 International Patent Classification(s)C12N 015/00 C12P 019/34 C12N 001/20(21) Application No. 31582/89 (22) Application Date: 22.03.89Priority Data(31) Number (32) Date (33) Country3809692 23.03.88 DE FEDERAL REPUBLIC OF GERMANY(43) Publication Date: 28.09.89(71) Applicant(s)HOECHST AKTIENGESELLSCHAFT(72) Inventor(s)WOLFGANG WOHLLEBEN; GUNTER MUTH; ALFRED PUHLER; BERNHARDNUSSBAUMER(74) Attorney or AgentWATERMARK MELBOURNE(57) ClaimWhen a plasmid which has the replicon of pSG5, containinga marker,, is constructed and used to transform a com-patible host strain, and then the temperature is raisedabove the threshold, the only transformants obtainedafter selection for the marker are those which have theplasmid integrated, in whole or in part, into the genome.Since such integrations essentially take place only inhomologous regions of the genome, this method is suitablefor finding such homologous regions in the genome of thehost strain.The use of pSG5 as a temperature-sensitive plasmid.3. The use of the pSG5 replicon for finding IS elementsand transposons.4. A method for the preparation of Streptomycetesmutants, for the isolation of the mutated genes andfor the isolation of the corresponding wild-typegene, which comprises isolating from the startingStreptomycetes strain the complete DNA, convertingit into short fragments, integrating these into ahybrid plasmid which has the pSG5 replicon, contain-~I(11)31582/89 -2-ing a marker, and transforming the resulting hybridplasmid population into the starting strain, select-ing the transformants by selection for the marker,eliminating the hybrid plasmids by increasing thetemperature above the threshold, and selecting themutants by renewed selection for the marker.~d4COMMONWEALTH, Of AUSTRALIAPAT'ENTS ACT 1952-69Form COMPLETE SPECIFICATION(ORIGINAL)ClassI nt. ClassApplication Number:Lodged,.Com~plete Specification Lodged:Accepted:Published:Priority:'Related Art:,Name o-f Applicant:HOECHST AKTIENGESELLSCHAFTAddress oft Applicant:501 Bruningstrasse, D-6230 Frankfurt/Main Federal Republic of GermanyActual Inventor:WOLFGANG WOHLLE.BENj GUJNTER MUTH, BERNHARD ALFRED PUHLER NIJSSBAUMERandAddress for Service,:FtDWD. 50- QUEEN WATERS, STREET, SONS,MELBOURNE, AUSTRALIA, 3000.Complete Specification for Ihe inventiont entitled:THE USE OF pSG5 AS A TEMPERATUkt-SENSITIVE P-1ASMIDThe following statement is a full description of this invefftionr, including the besd methiodof perforrip091t known 'to.6-I:HWCiHST AKTIENGESLiUSCHAFT HOE 88/F 069 Dr.KL/rhDescriptionThe use of pSG5 as a temperature-sensitive plasmidEuropean Patent (EP-B) No. 0,158,872 discloses theStreptomycetes plasmid pSG5 which can be isolated from aculture of Streptomyces ghanaensis DSM 2932. This plasmidis suitable for the preparation of hybrid vectors, forexample for what are called shuttle vectors, which can,S 0 because of an incorporated E. coli replicon, be multi-S' plied in E. coli strains. Vectors of this type aredisclosed, for example, in the European Patent Applica-Stion with the publication No. (EP-A) 158,201.It has now been found that pSG5 is temperature-sensitive.The property of temperature-sensitivity for pSG5 is15 surprising because as yet no natural Streptomycetesplasmids having this property have been disclosed. SincepSG5 and the hybrid plasmids prepared with its repliconI exhibit a wide host range, the new use of this plasmidaccording to the invention provides those skilled in thei t t20 art with a large number of possibilities:When a plasmid which has the replicon of pSGS, containinga marker, is constructed and used to transform a com-patible host strain, and then the temperature is raisedabove the threshold, the only transformants obtainedafter selection for the marker are those which have thepP plasmid integrated, in whole or in part, into the genome.Since such integrations essentially take place only inhomologous regions of the genome, this method is suitablefor finding such homologous regions in the genome of thehost strain.EP-A 0,243,856 discloses a method for the preparation ofmutants, which comprises isolating from the startingtMW12 *i strain the complete DNA, converting it into short frag-ments, integrating these into a plasmid which contains amarker, is temperature-sensitive and replicates in thestarting strain, and transforming the resulting hybridpopulation into the starting strain, selecting thetransformants by selection for the marker, eliminatingthe hybrid plasmids by increasing the temperature abovethe threshold of the temperature-sensitive plasmid, andselecting the mutants by renewed selection for themarker.ttThe term \"short\" fragments denotes DNA sections which areobtained with restriction enzymes which cut many times,such as Sau3A or TaXI, but also with mechanical methodssetfte(ultrasound, shearing) and which contain neither thepromoter region nor the translation stop signals.Because the only cells surviving after elimination of theS°'plasmids in a selection for the marker are those whichhave taken up the plasmid DNA into their chromosome(which preferably takes place via the homologous DNA'20 integrated in the plasmid), the mutants are obtaineddirectly.S~ The plasmid pSG5 is mentioned in EP-A 0,243,856 as asuitable starting plasmid. However, it is now possibleaccording to the invention to dispense with mutation togive temperature-sensitive replication mutants when thisplasmid is used. 'his means that not only are the muta-tion and the particularly elaborate selection dispensed-77-with, but also the risk of generating undesired multiplemutations is avoided. The plasmid pSG5 is thus particu-larly well suited for this method.The temperature-sensitivity of pSG5 is manifested in sucha way that this plasmid becomes unstable and is no longerreplicated at temperatures at or above 360C. The upperlimit of the utilizable temperature range depends on thehost cell: it is, for example, 38°C in S. venezuelae, 390C-3-in S. lividans and 45PC with S. ghanaensis. When cultureswhich contain pSG5 or a derivative of this plasmid are0C or above, the \"plasmidincubated at a temperature of 36becomes diluted out\" and is no longer detectable after afew generations.The use, according to the invention, of the pSG5 repliconcan thus be utilized to find genes which are homologousto an existent gene. Thus a utilizable alternative tosetting up a gene bank and screening with labeled DNAprobes is available. This alternative is especiallyvaluable when the result obtained from hybridization hasbeen conclusive. Another advantage of this method isa 0010 0 *0 o 00oS\"not that it is possible to avoid working with radioactivesubstances.It is also possible correspondingly to find insertionelements (IS elements) and transposons which have beentaken up into the genome of the host strain investigated:If, specifically, the plasmid which has been providedonly with the marker and contains no other inserted DNAis used, then homologous regions are available only if ano 0o oZ, o4° IS element or transposon has been transferred from thechromosome to the plasmid before raising the temperature.i Hence selection for the marker makes it possible to findsuch DNA elements.The said investigations can be carried out in accordancewith the method for isolating mutants described inEP-A 0,1243,856.*l Thus the invention is associated with a number ofadvantages:1. There already exists a family of vectors which isbased on the pSG5 plasmid and, because of the wide hostrange, has a large variety of possible uses and which hasvarious selection markers such as resistance to neomycin,thiostrepton, kanamycin (EP-A 0,158,201) and gentamicin(EP-A 0,248,207) as well as color markers such as melaninI 4and other colioring substances or pigments (EP-A 0,257,416and 0,257,417).2. It is possible, by use of a plasmid which belongs tothe said family of vectors and contains a marker whichcan be selected in E. coli, to extend the method dis-closed in EP-A 0,243,856 to the use of cosmid banks andthus to the isolation of large DNA sections of about kb: if the marker which can be selected in E. coli isintegrated into the host chromosome, the genome of themutants produced in this way can be transferred into aCacosmid gene bank. Selection for the marker (expedientlyat the same time as the marker intrinsic to the cosmid)then immediately leads to the cosmid clone of interest.ae£4 IIn this way the extremely elaborate screening by hybridi-a- t 4zation which was hitherto necessary is dispensed with. Init C,contrast to the method disclosed in EP-A 0,243,856, it isIIthus possible to detect in one step large gene regions inthe neighborhood of the mutated gene. Gene clusters canI-s I4' 4be isolated in this way, that is to say, for example, theat 44 a 4'igenes for an entire biosynthetic pathway. Nor is there4 44any longer a need for cleavage sites suitable for res-4. t 4triction enzymes 4' Ito be present in the vicinity of themutated gene.It4 4The invention is explained in detail in the examples4 CCwhich follow. Unless indicated otherwise, percentage '4 4( 44dataC 14herein relate to weight.ZXampbl 1: Complete DN3A isolationj 0.1 g of mycelium from a 3-day old homogenized Strepto-mycetes culture of the strain S. ghanaensis (ATCC 14672;US Patent 3,674,866), which produces no melanin (mel-),is pelleted in a 1.5 ml Eppendorf reaction tube in anEppendorf centrifuge for 1 mih and then washed once withml of TE (10 mM tris-HCl, 1 mM EDTA (pH 8) containingsucrose). The pellet is then resuspended in 0.5 ml oflysozyme solution (0.3 M sucrose, 25 mM tris-HCl (pH 8),mM EDTA, 10 mg/ml lysozyme) and incubated at 370C forI5 min. 0.2 ml of 5% strength SDS solution is added andthen the solution is thoroughly mixed and incubated atfor 10 min and subsequently cooled again to roomtemperature. Then 100 pl of phenol/chloroform (5 g ofphenol, 5 ml of chloroform, 5 mg of 8-hydroxyquinoline,1 ml of 0.1 M tris (pH are added, and the suspensionis mixed cautiously on a shaker ((R)Vortex) until it ishomogeneous. The mixture is then centrifuged in anEppendorf centrifuge for 5 min, and the upper aqueousphase is transferred into a new reaction tube. 70 pl of3 M unbuffered sodium acetate and 700 pl of isopropanolare added to the DNA-containing solution. After mixingand incubation at room temperature for 15 minutes, theDNA is pelleted by centrifugation (5 min in an Eppendorfcentrifuge), and the supernatant is removed quantitative-ly. The DNA is resuspended in 300 pl of TE and thenincubated with 10 pl of RNase solution (50 pg of RNase/mlof H20) at 370C for 45 min. The RNase is inactivated by100 pl of phenol/chloroform, and the denatured proteinsare pelleted (5 min in an Eppendorf centrifuge). The DNA-containing solution is again treated with isopropanolS, a(addition of 30 pl of 3 M sodium acetate and 400 pl ofisopropanol, incubation at room temperature for 15 min).The DNA pellet obtained after centrifugation is washed5 twice with 70% strength ethanol and again pelleted. Afterthe DNA has been dried it is taken up in 300 pl of TE andused for the further steps.tEample 2i Cleavage of the complete DNA with Sau3A1 pg of DNA is incubated in cleavage buffer (50 mM tris-HC1 (pH 10 mM MgC1250 mM NaCl) in the presence of0. 1 unit of Sau3A (manufactured by BRL-Gibco, Karlsruhe) at370C for 1 h. The reaction is stopped by phenol treatment,and the DNA is purified by ethanol precipitation.0oo 0~ Sso i Stemperature aa 6Eampl:e 3: Cloning of fragments of complete DNA into theplasmid pGM4pGM4 (EP-A 0,257,416 and 0,257,417) is completely linear-ized with BamHI in analogy to Example 2. The two DNAsamples are mixed in cleavage buffer, heated to 70oC andadjusted to the ligase reaction conditions by addition ofmercaptoethanol (final conc. 10 mM) and ATP (0.1 mM). Themixture is incubated in the presence of 1 unit of T4 DNAligase (Boehringer Mannheim) at 140C for 12 h. The mixtureis then transformed into protoplasts of the startingstrain and plated out on regeneration plates. After about20 h, a top layer of soft agar which contains sufficientthiostrepton for the final concentration in the plate tobe 50 yg/ml is placed on the latter.Example 4: Generation and selection of mutantsThe transformants aro incubated in S medium (Hopwood et\"Genetic Manipulation of Streptomyces, a LaboratoryManual\The John Innes Foundation, Norwich 1985) in ashake culture at 280C for about 36 h. The temperature isthen raised to 390C and incubation is continued at thisfor about 36 h. The culture is harvestedsterile, washed in TE 10% sucrose and incubated incomplete medium containing thiostrepton (20 mg/1) at 390Cfor a further 48 h. The mutants generated in this way arethen plated out and characterized {incubation always at39oC).Example 5: Isolation of the mutated DNAThe DNA is isolated as in Example 1 from the mutants, iscut with a restriction enzyme which has no cleavage sitesin the integrated plasmid, and is religated with T4 DNAligase. This results in the integrated plasmid, which nowcontains the mutated gene, being formed as the onlycyclic DNA capable of replication. Retransform~tion intoa suitable strain such as S. lividans is followed by4 e0ection Tor thios-trepton resistance and isolation ofthe plasm4d which harbors the mutated gene from thetxransformfants.The following publishel Australian patent at'plications,which are hereby incorporated by reference, czrrespond tothe above-mentioned European specifications:BP-B 0,158,872 AU-A 40,599/8SEP-A 0,158,201 AU-A 40,600/85I' 0W-A 0,257,416 AU-A 76,803/87EP-A 0,257,417 r09 AU-A 76,802/870i 0o ~00000 8s0 000.40\"4)LU~1: II IoTHE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:1. The use of pSG5 as a temperature-sensitive plasmid.2. The use of the pSG5 replicon for the preparation oftemperature-sensitive hybrid plasmids.3. The use of the pSG5 replicon for finding IS elementsand transposons.4. A method for the preparation of Streptomycetesmutants, for the isolation of the mutated genes andfor the isolation of the corresponding wild-typegene, which comprises isolating from the startingStreptomycetes strain the complete DNA, convertingit into short fragments, integrating these into ahybrid plasmid which has the pSG5 replicon, contain-ing a marker, and transforming the resulting hybridplasmid population into the starting strain, select-ing the transformants by selection for the marker,eliminating the hybrid plasmids by increasing thetemperature above the threshold, and selecting themutants by renewed selection for the marker.The method as claimed in claim 4, wherein themutated genes are isolated from the selectedmutants.6. The method as claimed in claim 5, wherein thecorresponding wild-type genes are isolated using themutated genes.7. The method as claimed in claim 5, wherein the hybridplasmid having the pSG5 replicon contains a markerwhich can be selected in E. coli, and wherein acosmid bank is used in the isolation of the mutatedgenes.DATED HOECHST AKTIENGESELLSCHAFTthis 21st day of March 1989.EDWD. WATERS SONSPATENT ATTORNEYSMELBOURNE. VIC. 3000.t