NZ203864A

NZ203864A - Recombinant dna cloning vector for use in streptomyces and e.coli

Info

Publication number: NZ203864A
Application number: NZ203864A
Authority: NZ
Inventors: C L Hershberger; J L Larson
Original assignee: Lilly Co Eli
Priority date: 1982-04-16
Filing date: 1983-04-12
Publication date: 1986-08-08
Also published as: PT76536A; GB2118947A; ES521468A0; ATE54327T1; JPS58189198A; ES8500995A1; DK162083D0; AU1347683A; PL241511A1; IL68349A0; AU570631B2; GB8310069D0; IE830848L; GR78559B; ZA832526B; DE3381699D1; KR840004451A; FI831245A0; DK162083A; GB2118947B

Abstract

There is described a recombinant DNA cloning vector useful in Streptomyces and E. coli, comprising a) a functional origin of replication- containing restriction fragment of plasmid SCP2 or SCP2*, b) a restriction fragment comprising an E. coli origin of replication, c) one or more DNA segments that confer resistance to at least one antibiotic when transformed into a cell of E. coli, said cell being sensitive to the antibiotic for which resistance is conferred, and d) one or more DNA segments that independently confer either or both of the Streptomyces tra function or resistance to at least one antibiotic when transformed into a cell of Streptomyces, said cell being sensitive to the antibiotic for which resistance is conferred. Transformants and a method for detecting transformants of the vector are also disclosed.

Description

<div class="application article clearfix" id="description"> 203864 * Priority Date(s>. tC-.M.tt. Complete Specification Filed: Class: MOW wywwv'"'*' 7 y C # IS/GO... .f.?2 I Publication Date: P.O. Journal, No: vc 8 AUGJ986 /<? * (g) CVUo\£|"7k NEW ZEALAND HATHNTS ACT, I"5. No.: Daie: COMPLETE SPECIFICATION CHIMERIC CLONING VECTORS FOR USE IN STREPTOMYCES AND E. COLI AND RESTRICTION FRAGMENTS COMPRISED THEREIN + /We, ELI LILLY AND COMPANY, a corporation of the State of Indiana, U.S.A., having a principal place of business at 307 East McCarty Street, City of Indianapolis, State of Indiana, United States of America hereby declare the invention fcr which I we pray that a patent may be granted to nw/us, and the method by which it is to be performed, to be particularly described in and by the following statement:-- - 1 (followed by page la) v% -la' Chimei-ic Cloning Vectors For- Use In The present invention relates to selectable novel recombinant DNA cloning vectors comprising a 5 functional origin of replication-containing restriction fragment of plasmid SCP2 or SCP2* and a functional origin of replication-containing and antibiotic resistance-conferring restriction fragment of a plasmid that is functional in E. coli. The invention also 10 relates to transformants and a method for detecting transformants of the aforementioned vectors. The vectors to which this invention relates are particularly useful because they are small, versatile, and can be transformed and selected in Streptomyces or 15 E. coli. Since over half of the clinically important antibiotics are produced by Streptomyces strains, it is desirable to develop cloning systems and vectors that are applicable to that industrially important group. Such vectors allow for the cloning of genes into Strepto-2o myces both for increasing the yields of known antibiotics as well as for the production of new antibiotics and antibiotic derivatives. The method of the present invention provides for the convenient selection of transformants. Since 25 transformation is a very low frequency event, such a functional test is a practical necessity for determining which cell(s), of among the millions of cells, has acquired the plasmid DNA. This is important because DNA sequences that are non-selectable can be 30 inserted into the vectors and, upon transformation, "J *3. 3 6 ' - V../ v *-5TOA -2- cells containing the vector and the particular DNA sequence of interest can be isolated by appropriate phenotypic selection. For purposes of the present invention as 5 disclosed and claimed herein, the following terms are as defined below.. Plasmid pLRl or pLR4 3.4kb BamHI Restriction Fragment - the same 3.4kb BamHI neomycin resistance-conferring fragment contained in plasmid pIJ2. AmpR - the ampicillin resistant phenotype. O Amp - the ampicillin sensitive phenotype. Tet - the tetracycline resistant phenotype. C Tet - the tetracycline sensitive phenotype. R CM - the chloramphenicol resistant phenotype, 15 CMS - the chloramphenicol sensitive phenotype. Neo - the neomycin resistant phenotype. c Neo - the neomycin sensitive phenotype. £ Thio - the thiostrepton resistant phenotype. C Thio - the thiostrepton sensitive phenotype. _ _ R S R S 20 Neo , Neo , Thio , and Thio refer only to results of tests in Streptomyces as used in this dis- R S R S R S closure. Amp , Amp , Tet , Tet , Cm and Cm refer only to results of tests in E. coli as used in this disclosure. 25 The present invention specifically relates to recombinant DNA cloning vectors comprising: a) a functional origin of replication-containing restriction fragment of plasmid SCP2 or SCP2*, 30 ■r-:?, "ii f< X 5 7 7 3A -3- b) a restriction fragment comprising an E. coli origin of replication, c) one or more DNA segments that confer resistance to at least one antibiotic when trans- 5 formed into a cell of E. coli, said cell being sensitive to the antibiotic for which resistance is conferred, and d) one or more DNA segments that independently confer either or both of the Streptomyces 3_0 tra function or resistance to at least one antibiotic when transformed into a cell of Streptomyces, said cell being sensitive to the antibiotic for which resistance is conferred. ]_5 The recombinant; DNA cloning vectors are made by a process which comprises ligating a functional origin of replication-containing restriction fragment of plasmid SCP2 or SCP2* and one or more DNA sequences comprising: 20 a) a restriction fragment comprising an E. coli origin of replication, b) one or more DNA segments that confer resistance to at least one antibiotic when transformed into a cell of E. coli, said cell 25 being sensitive to the antibiotic for which resistance is conferred, and c) one or more DNA segments that independently confer either or both of the Streptomyces tra function or resistance to at least one 3° antibiotic when transformed into a cell of 2 038^4 X 5773A -4- Streptomyces, said cell being sensitive to the antibiotic for which resistance is conferred . The invention further comprises transformants 5 and a method for detecting transformants of the aforementioned vectors comprising: 1) mixing Streptomyces cells, under transforming conditions, with a recombinant DNA cloning vector, said vector comprising 10 a) an origin of replication and P gene- containing restriction fragment of plasmid SCP2 or SCP2*, and b) a non-lethal DNA sequence cloned into the EcoRI restriction site of said P 15 gene, and 2) growing said Streptomyces cells on a lawn of an indicator Streptomyces strain and selecting colonies that show the M pock phenotype. The vectors of the present invention are 20 best constructed by ligating an origin of replication-containing and Streptomyces tra function-conferring restriction fragment of plasmid SCP2 or SCP2* into an E. coli origin of replication-containing and antibiotic resistance-conferring restriction fragment of an E. 25 coli plasmid. Plasmids SCP2 and SCP2*, from which origins of replication are constructed, are each ^31kb and show similar restriction patterns. Plasmid SCP2* arose as a spontaneous mutant of plasmid SCP2 and codes for a selectable colony pock morphology. Although the 30 pock is distinguishable from that of plasmid SCP2, in other ways plasmids SCP2 and SCP2* are virtually identical. % 03 2>£> X 5773ft -5- Since the present disclosure teaches that the Streptomyces tra function and the origin of replication of plasmids SCP2 and SCP2* are within their respective ^5.4kb EcoRI-Sall restriction fragments, a 5 variety of different origin of replication-containing and Streptomyces tra function-conferring fragments can be generated. This is accomplished by digestion with restriction enzymes that cut outside the %5.4kb EcoRI-Sall region. A detailed restriction site map of plasmid XO SCP2* (and thus also plasmid SCP2) is presented in Figure 1 of the accompanying drawings. Plasmids SCP2 and SCP2* can be conventionally isolated respectively from the Streptomyces coelicolor A3 (2) and Streptomyces coelicolor MHO strains deposited X5 and made part of the permanent stock culture collection of the Northern Regional Research Laboratory, Peoria, Illinois. Streptomyces coelicolor A3(2) is available to the public as a preferred source and stock reservoir of plasmid SCP2 under the accession number 15042. 20 Streptomyces coelicolor M110 is available to the public as a preferred source and stock reservoir of plasmid SCP2* under the accession number 15041. Many tra function-conferring and origin of replication-containing restriction fragments of plasmids 25 SCP2 and SCP2* can be constructed. Those specifically exemplified, for illustrative purposes, include the ^5.4kb EcoRI-Sall, the ^6.Okb Sail, the ^19kb EcoRI-Hindlll, and the ^31kb EcoRI restriction fragments of plasmid SCP2* and the ^31kb BglH restriction fragment 30 of plasmid SCP2. The aforementioned plasmid SCP2* and SCP2 fragments were respectively ligated to an origin X 577-^A -6- of replication-containing and antibiotic resistance-conferring fragment of E. coli plasmids pBR32 5 and pBR322. Those skilled in the art will recognize that although not required, it is convenient for both the 5 DNA segment that confers antibiotic resistance in E. coli and the E. coli origin of replication to comprise a restriction fragment of the same E. coli plasmid. Thus, for convenience and ease of construction, the ^31kb EcoRI fragment of plasmid SCP2* and the "Lo ^6kb EcoRI fragment of plasmid pBR325 were ligated to form illustrative plasmids pJL120 and pJL121. Recombinant plasmids of two orientations result because the fragments can be ligated in either direction. Similarly, ligation of the SCP2* ^6.0kb Sail fragment -^5 and the ^6k&-SalI fragment of pBR3 25 results in the illustrative plasmids pJL180 and pJLl81; ligation of the SCP2* ^5.4kb EcoRI-Sall fragment and the M.8kb EcoRI-Sall fragment of pBR325 results in the illustrative plasmid pJLl25; and ligation of the SCP2 BamHI 2 0 digest and the 'x-4.4kb BamHI fragment of plasmid pBR322 results in the illustrative plasmid pJLll4. All of the aforementioned vectors are readily selectable in each of E. coli and Streptomyces. For example, in E. coli, plasmids pJL120 and pJL121 confer 22 ampicillin and tetracycline resistance; plasmids pJL18 0 and pJLl81 confer ampicillin and chloramphenicol resistance; and plasmids pJL125 and pJL114 confer only ampiciliin resistance. Therefore, the vectors are conventionally selectable in the E. coli host system by adding the appropriate antibiotic to the culture medium. 30 The aforementioned vectors also produce the 'pock' phenotype and therefore are conventionally selectable in Streptomyces. The 'pock' phenotype is an assayable trait and known phenomenon (Bibb and Hopwood, 1981, J. Gen. Microbiol. 126:427) associated with lethal zygosis and the tra function (tra = genes coding for sexual transmissability) of Streptomyces sex factors Three distinct 'pock' morphologies are associated with transformants, when plated on an appropriate indicator strain, of plasmids SCP2, SCP2*, and SCP2 and SCP2* derivatives. The colony morphology identified with the wild-type SCP2 and the mutant SCP2* are respectively designated herein as P and P*. A third and heretofore unknown pock morphology results from cloning into the EcoRI restriction .site of SCP2 or SCP2*. Such an insertion inactivates the P gene and unexpectedly results in a morphologically distinguishable minipock phenotype, designated herein as M, when transformants are appropriately plated. "Minipock" is a pock of significantly smaller size than pocks caused by either SCP2 or SCP2*. The present invention thus provides a novel method for detecting transformants comprising: 1) mixing Streptomyces cells, under transforming conditions, with a recombinant DNA cloning vector, said vector comprising a) an origin of replication and P gene-containing restriction fragment of plasmid SCP2 or SCP2*, and b) a non-lethal DNA sequence cloned into the EcoRI restriction site of said P gene and X 5773A -8- ^ :pt 2) growing said Streptomyces cells on a lawn of an indicator Streptomyces strain and selecting colonies that show the M pock phenotype. Only transformed Streptomyces cells will show 5 the M pock phenotype and therefore transformants can be readily identified and selected. Those skilled in the art will quickly recognize, from the above description of the present pJL vectors, that plasmids pJL120, pJL121, and pJL125 code for M phenotype, that plasmids 10 pJL18 0 and pJL181 code for P* phenotype, and that plasmid pJL114 codes for P phenotype. Appropriate indicator strains for expression of the pock phenotype are known and include the various SCP2 and SCP2* strains as illustrated in the Examples below. The ]_5 present vectors are thus selectable and extremely useful in Streptomyces. The aforementioned plasmids can also be provided with a DNA segment that confers antibiotic resistance in Streptomyces. Such derivatives, specif-2o ically exemplified for illustrative purposes by plasmids pJL190 and pJL195, express an additional selectable phenotype. Plasmid pJL190 was constructed by ligating the neomycin resistance-conferring ^7.7kb EcoRI-Hindlll fragment of plasmid pLR4 to the ^19kb EcoRI-Hindlll 25 fragment of plasmid pJL121. Plasmid pJL195 was constructed by ligating the pLR4 ^7.5kb EcoRI-partial Sail fragment to the ^5.4kb EcoRI-Sail fragment of plasmid pJL125. The latter pJL125 plasmid comprises the largest (5.4kb) EcoRI-Sall fragment of plasmid pSCP2* 30 and was constructed by Sail deletion of plasmid pJL121. Illustrative plasmids pJL190 and pJL195, in addition to neomycin resistance, also express the M phenotype as di scus sed above. -X-5773A- -9- 203864 10 Plasmid pLR4, the source of the neomycin resistance conferring fragments, is ^7.7kb and is constructed by ligating BamHI-treated plasmids pBR322 and pLRl. Plasmid pLRl is ^14.8kb and is constructed by ligating HindIII-treated plasmid pIJ2, disclosed in Thompson et al., 1980, Nature 286:525, to Hindlll-treated plasmid pBR322. As is readily apparent to those skilled in the art, both plasmids pLR4 and pLRl contain the same neomycin resistance gene and thus either plasmid can be used for constructing the aforementioned pJL neomycin resistant vectors. These are the class of plasmid vectors which are designated as pJL and which have a gene for neomycin resistance.. An additional neomycin resistance-conferring 15 plasmid, designated as pJLl92, was isolated as a spontaneous mutant of plasmid pJL190 resident in Streptomyces griseofuscus. Plasmid pJLl92 specifies resistance to elevated levels of neomycin and therefore comprises a novel neomycin resistance gene which is 20 distinguishable from the resistance gene comprised in plasmids pJL190, pJL195, pIJ2, pLR4, and pLRl. In a similar manner, an additional neomycin resistance-conferring plasmid, designated as pJL199, was isolated as a spontaneous mutant of plasmid pJLl95. Those 25 skilled in the art will recognize that the novel neomycin resistance gene of plasmid pJLl92 or pJL199 can be readily excised and ligated to other vectors. The gene allows for improved and more efficient selecr tion of transformants. As in the case of plasmids pJL190 and pJL195, transformants of plasmids pJL192 and pJL199 express the M phenotype when plated on an appropriate indicator strain. n r"- 30 jr1 \ o jUi.V?85>J 2 03 8 6 10 HWA -10- Plasmid pJL192 can be conventionally isolated from E. coli K12 C600Rjc~M^-/pJL192, a strain deposited and made part of the permanent stock culture collection of the Northern Regional Research Laboratory, Peoria, Illinois. It is available to the public as a stock reservoir and preferred source of plasmid pJL192 under the accession number B-15040. A DNA segment that confers resistance to antibiotic thiostrepton, exemplified by the ^1.35kb BamHI restriction fragment of plasmid pLR2, can also be used with or substituted for the neomycin resistance-conferring segment. Plasmid pLR2, the source of the thiostrepton resistance conferring fragment, is 'vlS.Tkb and is constructed by ligating Hindlll treated plasmid pIJ6, disclosed in Thompson et al. , 1980, Nature 286:525, to Hindlll treated plasmid pBR322. Plasmid pLR2 is functional in E. coli and therefore can be amplified and isolated conveniently for subsequent manipulation. 2Q For convenience and ease of construction, the thiostrepton resistance conferring 'v-l.SSkb BamHI fragment of plasmid pLR2 was ligated into the BamHI restriction site of plasmid pBR328 to form plasmid pJLl93. The 'V'lkb Bell restriction fragment of pJL193 25 contains the thiostrepton resistance-conferring DNA segment. Therefore, ligation, as described in Examples 52-56, results in vectors that are within the scope of the present invention. Various plasmid SCP2 and SCP2* restriction fragments can be used for purposes of constructing the present invention provided that the origin of repli- 30 -X '5773ft -11- cation contained in their respective ^5.4kb EcoRI-Sall restriction fragments is present. Such additional plasmid SCP2 and SCP2* restriction fragments include, but are not limited to, the ^kb Sail, ^15kb PstI, ,v<23kb 5 BglH, v,15kb BamHI, ^14kb EcoRI-PstI, ^13kb EcoRI- BamHI, and ^15kb Pstl-BamHI fragments. These fragments contain the Streptomyces tra function and can be ligated to a functional E. coli origin of replication-containing and antibiotic resistance-conferring re-o striction fragment of an E. coli plasmid. Such E. coli plasmids include, for example, plasmids pBR322, pBR324, pBR325, pBR327, pBR328 and the like. Therefore, the present invention is not limited to the us.e of either plasmid pBR322 or pBR325 as exemplified in ^5 several pJL constructions. Although the neomycin and thiostrepton antibiotic resistance-conferring DNA segments exemplified herein are respectively the '^7.7kb EcoRI-Hindlll and the ^7.5kb EcoRI-partial Sail fragments of plasmid 20 pLR4 and the pLR2 ^1.35kb BamHI and the pJLl93 ^lkb Bell fragments, those skilled in the art can construct and use other DNA segments that also confer resistance to neomycin or thiostrepton. Other neomycin resistance-conferring DNA segments of plasmid pLRl include, for 25 example, the ^3.4kb BamHI restriction fragment, the -v3.5kb PstI restriction fragment, and the larger of the Sstl-Kpnl subfragments of the ^3.4kb BamHI restriction fragment. Other thiostrepton resistance-conferring segments include, for example, the -vlSkb PstI fragment 2Q of plasmid pLR2. Still other DNA segments conferring resistance to the same or to different antibiotics such OJOU985mj X-SX73A -12- as, for example, hygromycin, viomycin, tylosin, erythromycin and the like, can also be constructed and used by those skilled in the art. In addition, various functional derivatives of the above described antibiotic 5 resistance-conferring DNA segments can be constructed by adding, eliminating, or substituting nucleotides in accordance with the genetic code. Ligation of the aforementioned derivatives, or any of the other antibiotic resistance-conferring 10 DNA segments, to a vector comprising an E coli antibiotic resistance-conferring DNA segment, an E. coli origin of replication-containing restriction fragment, and also an origin of replication-containing restriction fragment of plasmids SCP2 or SCP2*, results in ^5 plasmids that are within the scope of the present invention. Therefore, an antibiotic resistance-con-ferring DNA segment can be used as a selectable marker in place of the Streptomyces tra function and associated pock phenotype. Thus, the present vectors are not 20 limited to the use of tra alone or in combination with •an antibiotic resistance-conferring DNA segment. In addition, a particular antibiotic resistance-conferring DNA segment is not limited to a single position on the present chimeric plasmids but can be ligated or in-25 serted at varying sites provided that an origin of replication or other critical plasmid controlled physiological functions are not disrupted. Those skilled in the art understand or can readily determine which sites are advantageous for ligation or insertion 30 of a particular DNA segment. The various restriction fragments of plasmids SCP2, SCP2*, pBR325, pBR322 and the like, and also the various antibiotic resistance-conferring DNA segments canprised in the present vectors, can be modified to facilitate ligation. For example, molecular linkers can be provided to some or all of the aforementioned DNA fragments. Thus, specific sites for subsequent ligation can be constructed conveniently. In addition, the origin of replication-containing restriction fragments can also be modified by adding, eliminating, or substituting certain nucleotides to alter characteristics and to provide a variety of restriction sites for ligation of DNA. Those skilled in the art understand nucleotide chemistry and the genetic code and thus which nucleotides are interchangeable and which DNA modifications are desirable for a specific purpose. The recombinant DNA cloning vectors that contain the SCP2 or SCP2* Streptomyces tra function are self transmissable and thus readily transferred during mating between transformed and non-transformed Streptomyces taxa. This is advantageous because the present vectors therefore can be transferred not only by protoplast transformation but also by conventional genetic crosses. Consequently, the vectors are useful in Streptomyces strains which are difficult to protoplast thus greatly expanding the number of hosts in which genetic manipulation and DNA cloning can be done. More importantly, DNA-libraries constructed in the present vectors can be conveniently and rapidly screened for interesting genes by conventional replica-plate mating procedures. Without the tra function, DNA * 10JUU985 203864 K 0773 -14- must be isolated from each of the thousands of clones in the library and transformed into appropriate strains to identify clones that contain desirable genes. Since there are no broadly applicable phage vectors for use 5 in Streptomyces, the present tra* vectors fulfill the general cloning and screening role analogous to that of bacteriophage X in replica-plate transduction for screening gene libraries in E coli. Desirable genes can thus be readily identified by the replica-plate 1Q mating procedure and then easily amplified by shuttling into E. coli as described in Example 19C(2) below. The vectors of the present invention are broadly applicable and are transformed into host cells of many Streptomyces taxa, particularly restrictionless ■^5 strains of economically important taxa that produce antibiotics such as aminoglycoside, macrolide, p-lactara, polyether, and glycopeptide antibiotics. Such restrictionless strains are readily selected and isolated from Streptomyces taxa by conventional procedures well 2q known in the art (Lomovskaya et al^., 1980, Microbiological Reviews 44:206). Host cells of restrictionless strains lack restriction enzymes and therefore do not cut or degrade plasmid DNA upon transformation. For purposes of the present application, host cells containing ] 25 restriction enzymes that do not cut any of the restriction sites of the present vectors are also considered restrictionless. Preferred host cells of restrictionless strains of Streptomyces taxa that produce aminoglyco-2q side antibiotics and in which the present vectors are especially useful and are transformed, include restric- 203864 11 I773A -15- tionless cells of, for example: S_. kanamyceticus, j (kanamycins) , £[. chrestomyceticus (aminosidine) , S. griseoflavus (antibiotic MA 1267), S. microspores i, (antibiotic SF-767) , S_. ribosidificus (antibiotic 5 SF733), S. flavopersicus (spectinomycin), S. spectabilis (actinospectacin), S. rimosus forma paromomycinus (paromomycins, catenulin), S. fradiae var. italicus (aminosidine) , S_. bluensis var. bluensis (bluensomycin) , S. catenulae (catenulin), S. olivoreticuli var. cellu-10 lophilus (destomycin A), S. tenebrarius (tobramycin, apramycin), S. lavendulae (neomycin), S. albogriseolus (neomycins), S. albus var. metamycinus (metamycin), S. hygroscopicus var. sagamiensis (spectinomycin), S. bikiniensis (streptomycin), S. griseus (streptomycin), 15 S. erythrochromogenes var. narutoensis (streptomycin), S^. poonensis (streptomycin) , S. galbus (streptomycin) , S. rameus (streptomycin), S. olivaceus (streptomycin), S.' mashuensis (streptomycin) , S. hygroscopicus var. limoneus (validamycins) , S_. rimofaciens (destomycins) , 20 hygroscopicus forma glebosus (glebomycin) , S. fradiae (hybrimycins,|neomycins) , S. eurocidicus (antibiotic A16316-C), S. aquaeanus (N-methyl hygromycin B), S. crystallinus (hygromycin A), S. noboritoensis (hygromycin) , S_. hygroscopicus (hygromycins) , S_. 25 atrofaciens (hygromycin), S. kasugaspinus (kasugamycins), -S. kasugaensis (kasugamycins) , S^. netropsis (antibiotic LL-AM31), S. lividus (lividomycins), S. hofunensisj (seldomycin complex), and S. canus (ribosyl paromamine). Preferred host cells of restrictionless 30 strains of Streptomyces taxa that produce macrolide antibiotics and in which the present vectors are 203364 K 5773A -16- especially useful and are transformed, include restrictionless cells of, for example: S. caelestis (antibiotic M188), S. platensis (platenomycin), S. rochei var. blolubilis (antibiotic T2636) , S. venezuelae (methymycins), S. griseofuscus (bundlin), S. narbo-nensis (josamycin, narbomycin), S. fungicidicus (antibiotic NA-181), S. griseofaciens (antibiotic PA1033A:, B) , S. roseocitreus (albocycline) , S. griseo-| brunneus /(albocycline), S. roseochromogenes (albocycline), 0 S cinerochromogenes (cineromycin B), S. albus (albo-mycetin), S. felleus (argomycin, picromycin), S. rochei (lankacidin, borrelidin), S. violaceoniger (lankacidin), S. griseus (borrelidin), S. maizeus (ingramycin) , S. albus var. coilmyceticus (colimycin) ,| 5 S. mycarofaciens (acetyl-leukomycin, espinomycin), S. hygroscopicus (turimycin, relomycin, maridomycin, tylosin, carbomycin) , S^. griseospiralis (relomycin) , S. lavendulae (aldgamycin), S. rimosus (neutramycin), S. deltae (deltamycins) , S. fungicidicus var.. espino-0 myceticus (espinomycins), S. furdicidicus (mydeca-mycin), S. eurocidicus (methymycin), S. griseolus (griseomycin) , S_. phaeochromogenesf (aiaaromycin, shinco-mycins) , S. f imbriatus (amaroinycin) , S. fasciculatusj (aiaaromycin) , S. erythreus (erythromycins) , S. anti-bioticus (oleandomycin), olivochromogenes (oleandomycin) , S_. spinichromogenes var. suragaoensis (kujimycins) , S_. kitasatoensis (leucomycin) , S. narbonensis var. josamyceticus (leucomycin A3, josamycin), S. albogriseolus (mikonomycin), S. bikiniensis (chalcomycin), S. cirratus (cirramycin), S. djakartensis (niddamycin), S. eurythermus (angolamycin), S. fradiae (tylosin, lactenocin, macrocin), S. goshikiensis (bandamycin), S. griseoflavus (acumycin), S. halstedii (carbomycin), S. tendae (carbomycin), S. macrosporeus (carbomycin), S. thermotolerans (carbomycin) , S. albireticuli (carbomycin), and S. ambofaciens (spiramycin). Preferred host cells of restrictionless strains of Streptomyces taxa that produce 0-lactam antibiotics and in which the present vectors are especially useful and are transformed, include restrictionless cells of, for example: S. lipmanii (A16884, MM4550, MM13902), S. clavuligerus (A16886B, clavulanic acid), S. lactamdurans (cephamycin C), S. griseus (cephamycin A, B), S. hygroscopicus (deacetoxy-cephalosporin C), S. wadayamensis (WS-3442-D), S. chartreusis (SF 1623) , S. heteromorphus and S. panayensis (C2801X); S. cinnamonensis, S. fimbriatus, S. halstedii, S. rochei and S. viridochromogenes (cephamycins A, B); S. cattleya (thienamycin); and S. olivaceus, S. flavo-virens, S. flavus, S. fulvoviridis, S. argenteolus, and §.• sioyaensis (MM 4550 and MM 13902) . Preferred host cells of restrictionless straips of Streptomyces taxa that produce polyether' antibiotics and in which the present vectors are especially useful and are transformed, include restrictionless cells of, for example: S. albus (A204, A28695A and B, salinomycin), S. hygroscopicus (A218, emericid, DE39 36, A120A, A28695A and B, etheromycin, dianemycin), S. griseus (grisorixin), S. conglobatus (ionomycin), S. eurocidicus var. asterocidicus (laidlo-mycin), S. lasaliensis (lasalocid), S. ribosidificus (Ionomycin), S. cacaoi var. asoensis (lysocellin), S. 2-03S64 S-5WA -18- 15 cinnamonensis (monensin), S. aureofaciens (narasin), S. gallinarius (RP 30504), S. longwoodensis (lysocellin), S. flaveolus (CP38936), S. mutabilis (S-11743A), and S. violaceoniger (nigericin). Preferred host cells of restrictionless strains of Streptomyces taxa that produce glycopeptide antibiotics and in which the present vectors are especially useful and are transformed, include restrictionless cells of, for example: S. orientalis and ^ S. haranomachiensis (vancomycin); S. candidus (A-35512, avoparcin), and S. eburosporeus (LL-AM 374). Preferred host cells of other Streptomyces restrictionless strains in which the present vectors are especially useful and can be transformed, include restrictionless cells of, for example: S. granuloruber, S. roseosporus, S. lividans, S. espinosus, and S. azureus. In addition to the representative Streptomyces host cells described above, the present vectors are also useful and can be transformed into E. coli. Thus, vectors of the present invention have wide application and are useful and can be transformed into host cells of a variety of organisms. While all the embodiments of the present invention are useful, some of the present recombinant DNA cloning vectors and transformants are preferred. Accordingly, preferred vectors are pJL114, pJL121, pJLl25, pJLl80, pJL190, pJL192, pJL195, pJL197, pJL199 and pHJL212 and preferred transformants are Streptomyces griseofuscus/pJL114, S. griseofuscus/pJL121, S. griseofuscus/pJL125, S. griseofuscus/pJLl80, S. griseofuscus/pJL190, S. griseofuscus/pJL192, S. 20 25 30 o JUL 1985! 203864 X 5773A -19- 15 20 25 griseofuscus/pJL195, S . griseofuscus/pJLl99 , S^. griseo-fuscus/pJL197, S. griseofuscus/pHJL212, E. coli K12 C600Rk-Mk-/pJL114, E. coli K12 C600Rk-Mk~/pJL121, E. coli K12 C600R]c-Mk-/pJL125/ E. coli K12 C600Rk-Mk-/pJL180, E. coli K12 CBOORj^-M^/pJLigO, E. coli K12 C600Rk"~Mk~/ pJL192, E. coli K12 C600Rk-Mk-/pJL195, E. coli K12 CeOORj^-Mj^-ZpJLigg, E. coli K12 C600Rk-Mk-/pJL197, and E. coli K12 C600Rk-Mk~/pHJL212. Moreover, of this preferred group, plasmids pJL190, pJL192, pJL195, pJL197, pJL199 and pHJL212 and transformants griseo-fuscus/pJL190, S^. griseofuscus/pJL192, S. griseo-fuscus/pJL195, S_. griseofuscus/pJL197, griseofuscus/ pJL199, S. griseofuscus/pHJL212, E. coli K12 C600Rk-Mk~/ pJL190, E. coli K12 C600Rk-Mk-/pJL192, E. coli K12 C6 00Rk-Mk-/pJL195 and E. coli K12 C6 00Rk-Mk-/pJL197, E. coli K12 C600Rk-Mk-/pJL199 and E. coli K12 C600Rk~Mk-/ pHJL212 are most preferred. Streptomyces griseofuscus is a preferred host because it does not contain an endogenous plasmid or synthesize an antibiotic. Therefore, transformants of S. griseofuscus can be screened for clones that express genes for antibiotic synthesis. The vectors of the present invention comprises origins of replication that are functional in E. coli and Streptomyces and therefore provide flexibility in the choice of hosts. Consequently, cloned DNA sequences c'an be shuttled into E. coli for construction of new plasmids, physical analysis, and for mapping of restriction sites and then shuttled back into Streptomyces for functional analysis and improvement of strains. This is particularly advantageous because mplification and manipulation of plasmids can be done 203864 X 3773A -20- faster and more conveniently in E. coli than in Streptomyces . For example, the present vectors can be amplified conventionally in E. coli K12 by growth with spectinomycin or chloramphenicol. This is not possible in the 5 Streptomyces host system. In addition, since all the plasmid vectors contain resistance markers that are expressed in E. coli K12, recombinants are easily selected. Therefore, large amounts of plasmid DNA can be isolated conveniently and in a shorter time than 2_o that required for doing similar procedures in Streptomyces. Thus, after desired recombinant DNA procedures are accomplished in the E. coli host system, the particular Streptomyces DNA can be removed, reconstructed to plasmid form (if necessary), and then transformed into 15 a Streptomyces host cell. Since the present vectors are fully selectable in Streptomyces, identification of recombinant clones can be done efficiently. The recombinant DNA cloning vectors and transformants of the present invention have broad 2Q utility and help fill the need for suitable cloning vehicles for use in Streptomyces and E. coli. Moreover, the ability of the present vectors to confer a pock phenotype or resistance to antibiotics also provides a functional means for selecting transformants. 25 This is important because of the practical necessity for determining and selecting the particular cells that have acquired vector DNA. Additional DNA segments, that lack functional tests for their presence, can also be inserted into the present vectors and then trans-3q formants containing the non-selectable DNA can be isolated by appropriate antibiotic or other phenotype 2 03 8 64 X 5773A -21- selection. Such non-selectable DNA segments can be inserted at any site, except within regions necessary for plasmid function and replication, and include genes that specify antibiotic modification enzymes and 5 regulatory genes of all types. More particularly, a non-selectable DNA segment that comprises a gene can be inserted into a plasmid such as, for example, illustrative plasmid pJLl92, at the internal BamHI restriction site of the 10 ^7.7kb EcoRI-Hindlll resistance-conferring fragment. Such an insertion inactivates the neomycin resistance gene and thus allows for the easy identification of Streptomyces transformants containing the recombinant plasmid. Thi§ is done by first selecting for M pock 3_5 morphology and, secondarily, identifying those M transformants that are not resistant to neomycin. In a similar manner, insertion of a DNA segment into illustrative plasmid pJLl80 at, for.example, the unique PstI restriction site, inactivates the ampicillin resistance 20 gene. Thus, E. coli transformants carrying this recombinant plasmid can also be identified easily by first selecting for chloramphenicol resistance and, secondarily, identifying those chloramphenicol resistant transformants that are not resistant to ampicillin. 25 Therefore, the ability to select for antibiotic resistance or other phenotypic markers in Streptomyces and E. coli allows for the efficient isolation of the extremely rare cells that contain the particular non-selectable DNA of interest. 30 The functional test for antibiotic resis tance, as described above, can also be used to identify 2 03 8 XSU-3& -22- 10 15 20 30 DNA segments that act as control elements and direct expression of an individual antibiotic resistance gene. Such segments, including but not limited to, promoters, attenuators, repressors, inducers, ribosomal binding sites, and the like, can be used to control the expression of other genes in cells of Streptomyces and E. coli. The antibiotic resistance-conferring vectors of the present invention are also useful for insuring that linked DNA segments are stably maintained in host cells over many generations. These genes or DNA fragments, covalently linked to an antibiotic resistance-conferring fragment and propagated either in Streptomyces or E. coli, are maintained by exposing the transformants to levels of antibiotic that are toxic to non-transformed cells. Therefore, transformants that lose the vector, and consequently any covalently linked DNA, cannot grow and are eliminated from the culture. Thus, the vectors of the present invention can be used to maintain any DNA sequence of interest. The cloning vectors and transformants of the present invention provide for the cloning of genes to improve yields of various products that are currently produced in Streptomyces and related cells. Examples of such products include, but are not limited to, Streptomycin, Tylosin, Cephalosporins, Actaplanin, Narasin, Monensin, Apramycin, Tobramycin, Erythromycin, and the like. The present invention also provides selectable vectors that are useful for cloning, characterizing, and reconstructing DNA sequences that code for commercially important proteins such as, for example, human insulin, human proinsulin, human growth -X-5773A -23- horraone, bovine growth hormone, glucagon, interferon, and the like; for enzymatic functions in metabolic pathways leading to commercially important processes and compounds; or for control elements that improve 5 gene expression. These desired DNA sequences include, but are not limited to, DNA that codes for enzymes that catalyze synthesis of derivatized antibiotics such as, for example, Streptomycin, Cephalosporin, Tylosin, •o Actaplanin, Narasin, Monensin, Apramycin, Tobramycin, 10 an(3 Erythromycin derivatives, or for enzymes that mediate and increase bioproduction of antibiotics or other products. The capability of inserting, stabilizing, and shuttling the aforementioned DNA segments into Strepto-15 myces and E. coli allows for easy recombinant genetic manipulation for increasing the yield and availability of antibiotics that are produced by Streptomyces. In addition, since the plasmid SCP2 or SCP2* origin of replication codes for low copy number, almost any DNA 20 sequence, including those that are lethal when expressed from a high copy number plasmid, can be readily cloned into the present vectors and shuttled between Streptomyces and E. coli. Streptomyces coelicolor A3(2) and S. coelicolor ?5 M110, as respective sources of plasmids SCP2 and SCP2*, can be cultured in a number of ways using any of several different media. Carbohydrate sources which are preferred in a culture medium include, for example, molasses, glucose, dextrin, and glycerol, and nitrogen 30 sources include, for example, soy flour, amino acid mixtures, and peptones. Nutrient inorganic salts are also incorporated and include the customary salts QJUU985i 203864 t.* £> X-W3A -24- capable of yielding sodium, potassium, ammonium, calcium, phosphate, chloride, sulfate, and like ions. As is necessary for the growth and development of other microorganisms, essential trace elements are also 5 added. Such trace elements are commonly supplied as impurities incidental to the addition of other constituents of the medium. Streptomyces coelicolor MHO and S. coelicolor A3 (2) are grown under aerobic culture conditions 10 over a relatively wide pH range of about 5 to 9 at temperatures ranging from about 15° to 40°C. For production of plasmids SCP2 and SCP2* at highest copy number, however, it is desirable to start with a culture medium at a pH of about 7.2 and maintain a 15 culture temperature of about 30°C. Culturing Streptomyces coelicolor MHO and S. coelicolor A3 (2) under the aforementioned conditions, results in a reservoir of cells from which plasmids SCP2* and SCP2 are respectively isolated conveniently by techniques well known 20 in the art. The following examples further illustrate and detail the invention disclosed herein. Both an explanation of and the actual procedures for constructing the invention are described where appropriate. 25 30 Example 1 Isolation of Plasmid SCP2* A. Culture of Streptomyces coelicolor MHO A vegetative inoculum of Streptomyces coelicolor Ml10 (NRRL 15041) was conventionally prepared by growing the strain under submerged aerobic 203864 -X-5773A -25- conditions in 50 ml. of sterilized trypticase soy broth* at 35 g./l. in deionized water. The trypticase soy broth inoculum was incubated for 48 hours at a temperature of 30°C. The 5 50 ml. culture was then homogenized, transferred to 450 ml. of sterilized YEMESG** medium, and then incubated for at least 40, but not more than 65 hours, at 30°C. The pH was not adjusted. After incubation, the Streptomyces coelicolor MllO cells were ready for 10 harvest and subsequent isolation of plasmid DNA. * Trypticase soy broth is obtained from Difco Laboratories, Detroit, Michigan. * * YEMESG comprises .3% yeast extract, .5% peptone, 15 .3% malt extract, 1% dextrose, 34% sucrose, .1% MgCl2, and .1% glycine. B. Plasmid Isolation About 10 g. (wet wgt) of Streptomyces coelicolor MllO cells- were harvested by centrifugation (10 20 minutes, 4°C., 10,000 rpm) and then about 10 ml./g. wet wgt cells of TES buffer (.01M Tris(hydroxymethyl)amino-ethane [tris], .001M EDTA, 25% sucrose, pH 8) was /added. The cells were vortexed into suspension followed by addition of 10 ml./g. wet wgt cells of .25M EDTA, pH 8 25 and then 5 ml./g. wet wgt cells of lysozyme (10 mg./ml. in TES). After the mixture was incubated at 37°C. for about 15 minutes, about 1.5 ml./g. wet wgt cells of 20% SDS (sodium lauryl sulfate (BDH Chemicals Ltd. Poole, Endland) , >was .)added. The resultant mixture was allowed 30 to stand at room temperature for 3 0 minutes, and then 5M NaCl was added to give a final concentration of 1M 2 03864 ^5773A -26- NaCl. After standing again at room temperature (15 minutes), the mixture was placed on ice for 2 hours. The lysate was centrifuged (20 minutes, 4°C., 17,500 rpm) and the supernatant was pooled and mixed with .64 5 volumes of isopropyl alcohol. The DNA precipitate was collected by centrifugation (15 minutes, 4°C., 10,000 rpm). The precipitate was air dried and then resus-pended in 1 ml./g. wet wgt cells of TE buffer (.01M Tris, .001M EDTA). Centrifugation (20 hours, 20°C., 10 50,000 rpm) using cesium chloride gradients with propidium iodide was carried out to purify the plasmid DNA. Following centrifugation, the desired plasmid SCP2* DNA band was removed and the propidium iodide extracted by conventional procedures. The CsCl-DNA 15 solution was stored at -20°C. Prior to use, the DNA was desalted by either PD10 (Bio Rad) column exchange with TE or by dialysis against TE. The DNA was precipitated with ethanol by conventional procedures and redissolved in TE. 2 0 Example 2 Construction of Plasmid pLRl A. Hindlll Digestion of Plasmid pIJ2 25 30 About 20 ill. (20 yg.) of plasmid pIJ2 DNA, disclosed in Thompson et , 1980, Nature 286:525, 5 yl. BSA(Bovine Serum albumin, 1 mg./ml.), 19 yl. water, 1 yl. of Hindlll (containing 3 New England Bio Labs units) restriction enzyme*, and 5 yl. reaction mix** were incubated at 37°C. for 2 hours. The reaction was terminated by the addition of about 50 yl. of "F , y*! X-S773A -27- 10 15 20 4M ammonium acetate and 200 yl. of 95% ethanol. The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, suspended in 20 yl. of TE buffer, and frozen at -20°C. for storage. * Restriction and other enzymes can be obtained from the following sources: New England Bio Labs., Inc. 32 Tozer Road Beverly, Massachusetts 01915 Boehringer-Mannheim Biochemicals 7941 Castleway Drive Indianapolis, Indiana 46250 Bethesda Research Laboratories (BRL) Box 6010 Rockville, Maryland 20850 Research Products Miles Laboratories, Inc. Elkhart, Indiana 46515 * * Reaction mix for Hindlll restriction enzyme was prepared with the following composition: 600mM NaCl lOOmM Tris-HCl, pH7.9 70mM MgC^ lOmM Dithiothreitol 25 B. Hindlll Digestion of Plasmid pBR322 About 8 yl. (4 yg.) of plasmid pBR322 DNA, 5 yl. reaction mix, 5 yl. BSA (1 mg./ml.), 31 yl. water, and 1 yl. of Hindlll restriction enzyme were incubated at 37°C. for 2 hours. After the reaction was 30 X--5773A -28- tenninated by incubating at 60°C. for 10 minutes, about 50 yl. of ammonium acetate and 200 yl. of 95% ethanol were added. The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, and suspended in 5 45 yl. of water. C. Ligation of Hindlll Digested Plasmids pIJ2 and pBR322 About 20 yl. of Hindlll treated plasmid pIJ2 (from Example 2A), 20 yl. of Hindlll treated plasmid 10 pBR322 (from Example 2B), 5 yl. BSA (1 mg./ml.), 1 yl. * of T4 DNA ligase , and 5 yl. ligation mix** were incubated at 16°C. for 4 hours. The reaction was terminated by the addition of about 50 yl. 4M ammonium acetate and 200 yl. of 95% ethanol. The resultant DNA 15 precipitate was washed twice in 7 0% ethanol, dried in vacuo, and suspended in TE buffer. The suspended DNA constituted the desired plasmid pLRl. * T4 DNA ligase can be obtained from the following source: New England Bio Labs., Inc. 32 Tozer Rd. Beverly, Massachusetts 01915 * ★ Ligation mix was prepared with the following composition : 500mM Tris-HCl, pH7.8 200mM Dithiothreitol lOOmM MgCl2 lOmM ATP 30 Example 3 Construction of E. coli K12 HBlOl/pLRl About 10 ml. of E. coli K12 HB101 5 cells (Bolivar et al., 1977, Gene 2:75-93) were pelleted by centrifugation and then suspended in about 10 ml. of .01M sodium chloride. Next, the cells were pelleted again, resuspended in about 10 ml. of . 03M calcium chloride, incubated on ice for 20 minutes, 10 pelleted a third time, and finally, resuspended in 1.25 ml. of .03M calcium chloride. The resultant cell suspension was competent for subsequent transformation. Plasmid pLRl in TE buffer (prepared in Example 2C) was ethanol precipitated, suspended in 15 150 yl. of 30mM calcium chloride solution, and gently mixed in a test tube with about 200 yl. of competent E. coli K12 HB101 cells. The resultant mixture was incubated on ice for about 45 minutes and then at 42°C. for about 1 minute. Next, about 3 ml. of L-broth 20 (Bertani, 1951, J. Bacteriology 62:293) containing 50 yg./ml. of ampicillin was added. The mixture was incubated with shaking at 37"C. for 1 hour and then plated on L-agar (Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbor Labs, Cold Spring Harbor, 25 New York) containing ampicillin. Surviving colonies were selected and tested for the expected phenotype R S (Amp , Tet ), and constituted the desired E. coli K12 HBlOl/pLRl transformants. 30 -30- Example 4 Construction of Plasmid pLR4 A. Partial BamHI Digestion of Plasmid pLRl 5 About 10 yl. (10 yg.) of plasmid pLRl, 5 yl. BSA (1 mg./ml.), 29 yl. water, 1 yl. of BamHI (diluted 1:4 with water) restriction enzyme, and 5 yl. reaction mix* were incubated at 37°C. for 15 minutes. The reaction was terminated by the addition of about 50 yl. of 10 4M ammonium acetate and 200 yl. of 95% ethanol. The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, and suspended in 20 yl. water. 30 * Reaction mix for BamHI restriction enzyme was pre-15 pared with the following composition: 1.5M NaCl 60mM Tris-HCl, p 7.9 60mM MgCl2 B. BamHI Digestion of Plasmid pBR322 20 The desired digestion was carried out in sub stantial accordance with the teaching of Example 2B except that BamHI restriction enzyme and reaction mix were used in place of Hindlll restriction enzyme and reaction mix. The digested plasmid pBR322 was suspended 25 in 29 yl. of water. C. Ligation of Partial BamHI Digested Plasmid pLRl and BamHI Digested Plasmid pBR322 The desired ligation was carried out in substantial accordance with the teaching of Example 2C. The resultant ligated DNA was suspended in TE buffer and constituted the desired plasmid pLR4. 2C X 5773A -31- Example 5 Construction of E. coli K12 HBl01/pLR4 The desired construction was carried out in substantial accordance with the teaching of Example 3 except that plasmid pLR4, rather than plasmid pLRl, was used for transformation. Surviving colonies were se- R lected and tested for the expected phenotype (Amp , TetS), and constituted the desired E. coli K12 HB1Q1/ pLR4 transformants. 10 Example 6 Construction of Plasmids pJL120 and pJL121 A. EcoRI Digestion of Plasmid SCP2* 15 About 150 yl. (5.7 yg.) of plasmid S.CP2* DNA, 1 pi. water, 2 yl. of EcoRI (containing 20 BRL units) restriction enzyme, and 17 yl. EcoRI reaction mix* were incubated at 37°C. for 2.5 hours. The reaction was terminated by incubation at 65°C. for 15 minutes. 20 The reaction was conventionally analyzed by agarose gel electrophoresis (AGE) to verify that restriction was complete. The restricted DNA was stored at 4°C. for subsequent use. 25 * Reaction mix for EcoRI restriction enzyme was prepared with the following composition: 500mM NaCl lOOOnuM Tris-HCl, pH7.5 lOOmM MgCl2 30 "X-5773A -32- B. EcoRI Digestion of Plasmid pBR325 The desired digestion was carried out in substantial accordance with the teaching of Example 6A except that plasmid p3R325, rather than plasmid SCP2*, was used. The resultant DNA was stored at 4°C. for subsequent use. C. Ligation of EcoRI Digested Plasmids SCP2* and pBR325 10 A 5 20 About 40 yl. of EcoRI digested plasmid SCP2* (from Example 6A), 10 yl. of EcoRI digested plasmid pBR325 (from Example 6B), 10 yl. of MgCl2 (.1M), 10 yl. of (NH4)2S04 (.1M), 10 pi.' ATP (2mil) .1 pi. of T4 DNA • ligase, and 20 yl. ligation mix* were incubated at 4°C. for 18 hours. The reaction was analyzed by AGE to verify appropriate ligation. . The suspended DNA constituted the desired ^35.8Kb plasmids pJL120 and pJL121. Recombinant plasmids of two orientations result because the plasmid pBR325 EcoRI fragment can be oriented in either direction. A restriction site map of each of plasmids pJLl20 and pJL121 was determined (after isolation as disclosed in Example 7) and is presented in Figure 2 of the accompanying drawings. *Ligation mix was prepared with.the following composition: 50mM Tris-HCl, pH 7.5 lOmM {5-mercaptoethanol ImM EDTA 50 pg./ml. BSA 30 Example 7 Construction of E. coli K12 C600Rk-Mjc-/pJL120 and E. coli K12 C600Rk-Mk-/pJL121 5 A. Preparation of Frozen Competent E. coli K12 C600Rk-Mk- Fresh overnight cultures of E. coli K12 C600Rk-Mk~ (disclosed in Chang and Cohen, 1974, Proc. Nat. Acad. Sci. 71:103 0-1034) were subcultured 1:10 in 10 fresh L-broth (disclosed in Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbor Labs, Cold Spring Harbor, New York) and grown at 37°C. for 1 hour. A total of 660 Klett Units of cells were harvested, , washed with 2.5 ml. of lOOmM NaCl, suspended in 150mM 15 CaCl2 with 10% glycerol, and incubated at room temperature for 20 minutes. The cells were harvested by centrifugation, resuspended in .5 ml. of CaC^-glycerol, chilled on ice for 3-5 minutes and frozen. The suspensions of cells were stored in liquid nitrogen until 20 use. Preservation and storage did not adversely affect the viability or frequency of transformation by covalently closed circular DNA. B. Transformation 25 30 The competent cells were thawed in an ice bath and mixed in a ratio of .1 ml. of cells to .05 ml. of DNA (10 yl. of the sample disclosed in Example 6C and 40 yl. of .1XSSC (.015M NaCl, .0015M Sodium Citrate at pH 7). The transformation mixture was chilled on ice for 20 minutes, heat shocked at 42°C. for 1 minute and chilled on ice for 10 minutes. The samples were 10 JUL 1985 r.yi then diluted with .85 ml. of L-broth, incubated at 37°C. for 1.5 hours, spread on L-agar containing ampicillin (50 yg./ml.) and tetracycline (12.5 ug./ml.) and incubated for 18 hours at 37°C. The resultant colonies were selected and tested for the expected phenotype R R S (Amp , Tet , CM ) and constituted the desired E. coli K12 C600Rk-Mk-/pJL120 and coli K12 C600Rk~Mk-/pJL121 transformants. The amplicillin and tetracycline resistant colonies were isolated according to known procedures, cultured, and then conventionally identified by restriction enzyme and AGE analysis of the constitutive plasmids. The identified transformants were then used for subsequent production and isolation of plasmids pJL120 and pJLl21 according to known procedures . Example 8 Construction of Plasmids pJL180 and pJL181 A. Sail Digestion of Plasmid SCP2* and Isolation of ^6.Okb Sail Fragment The desired digestion was carried out in substantial accordance with the teaching of Example 6 except Sail restriction enzyme and reaction mix*, rather than EcoRI restriction enzyme and reaction mix, were used. The reaction was assayed by AGE to verify completion and terminated by heating at 65°C. for 15 minutes. The resultant Sail restriction fragments were separated by AGE and then the separated fragments were located in the gel by staining with ethidium bromide and visualizing fluorescent bands with an ultraviolet 205864 X-5WA -35- light. The gel fragment containing the ^6.0kb fragment of interest was excised from the gel and electroeluted into TBE buffer (1.6% Sigma 7-9 buffer**, .093% NajEDTA, .55% boric acid). The gel-fragment in TBE buffer was 5 placed in a dialysis bag and subjected to electrophoresis at 100 V for 1 hour. The aqueous solution was collected from the dialysis bag and passed over a DEAE cellulose column*** (.5 ml. Whatman DE52) that had been equilibrated with equilibration buffer (.1M KC1, 10 mM Tris-HCl, 10 PH 7.8). The column was washed with 2.5 ml. of equilibration buffer and the DNA (about 5 yg.) was eluted with 1.5 ml. of elution buffer (1M NaCl, 10 mM Tris-HCl, pH 7.8). The eluent was adjusted to about .35M with respect to Na+ ion concentration, and then the DNA 15 was precipitated by adding 2 volumes (about 9 ml.) of 100% ethanol followed by cooling to -20dC. for 16 hours. The DNA precipitate was pelleted by centrifugation, washed with 75% ethanol, dried, and dissolved in TE buffer. Hereinafter, this conventional isolation 2o technique is referred to as AGE/DE52/electroelution. * Reaction mix for Sail restriction enzyme was prepared with the following composition: 1500 mM NaCl 80 mM Tris-HCl, pH7.5 60 mM MgCl, 2 mM EDTA **Sigma 7-9 buffer can be obtained from Sigma Chemical Company, P.O. Box 14508, St. Louis, Missouri 63178 ***DEAE cellulose (DE52) can be obtained from Whatman Inc., 9 Bridewell Place, Clifton, New Jersey 07014. &-5773A -36- 10 B. Sail Digestion of Plasmid pBR325 The desired digestion was carried out in substantial accordance with the teaching of Example 8A except that plasmid pBR325 was employed and fragments were not separated by preparative AGE/DE52/electroelution. The resultant DNA was dissolved in TE buffer and stored at 4°C. for future use. C. Ligation of Sail Digested Plasmid pBR325 and ^6.0kb Sail Fragment of Plasmid SCP2* About 1.5 yg. of the 6.Okb Sail fragment of SCP2*, prepared in Example 8A, was mixed with .5 yg. of Sail digested pBR325, prepared in Example 8B. 15 The DNA mixture was precipitated by standard ethanol precipitation and redissolved in 3 yl. of distilled water, 4 yl. of .66M ATP, 2 yl. of ligase-kinase mixture (.25M Tris*KCl, pH 7.8, 50 mM MgC^, 25 mM dithiothreitol and 25% glycerol) and 1 yl. of T4-DNA 2Q ligase (1 unit). After incubation for 1 hour at 15°C., the reaction mixture was diluted with 12 yl. of water, 2 0 yl. of .66M ATP, 8 yl. of ligase-kinase mixture and then incubated at 15°C. for 18 hours. The resultant ligated DNA was diluted 1:5 into .1XSSC 25 and constituted the desired ^12.Okb plasmids pJL18 0 and pJLl81. Recombinant plasmids of two orientations result because the plasmid pBR325 Sail fragment can be oriented in either direction. A restriction site map of each of plasmids pJL18 0 and pJL181 is presented in Figure 3 of the accompanying drawings. 30 2 03 3 X 5773A- -37- Example 9 Construction of E. coli K12 C600Rk-Mk-/pJL180 and E. coli K12 C600Rk-Mk-/pJL181 5 The desired constructions were made in sub stantial accordance with the teaching of Example 7 except that the mixture of plasmid pJLlSO and pJL181 DNA (from Example 8C), rather than plasmid pJLl2 0 and pJL121, were used. The resultant transformant colonies 10 were selected and tested for the expected phenotype R S R (Amp , Tet , CM ), and constituted the desired E. coli K12 C6 00Rk-Mk-/pJL18 0 and E. coli K12 C600Rk"Mk-/pJL181 transformants. The ampicillin and chloramphenicol resistant colonies were isolated according to known 15 ^"procedures, cultured, and then conventionally identified by restriction enzyme and AGE analysis of the constitutive plasmids. The identified transformants can then be used for subsequent production and isolation of plasmids pJL18 0 and pJL181 according to known 20 procedures. Example 10 Construction of Plasmid pJL125 A. Sail Digestion of Plasmid pJL121 and Isolation of 25 ^10.2kb Sail Fragment The desired digestion was carried out in substantial accordance with the teaching of Example 8 except that the reaction was stopped before digestion 30 was complete and except that plasmid pJLl21, rather than plasmid SCP2*, was used. The resultant Sail ,fV %y a : "v -38- restriction fragments were not separated by preparative AGE but precipitated by standard ethanol precipitation. The restriction fragments were dissolved in TE buffer and immediately ligated. B. Ligation of ^10.2]cb Sail Fragment of Plasmid pJL121 The desired ligation was carried out in substantial accordance with the teaching of Example 8C except that the Sail fragments of plasmid pJL121, rather than the Sail fragment of plasmid SCP2* and pBR325, were used. The resultant ligated DNA constituted the desired plasmid pJL125 plus 12 other plasmids that were subsequently isolated and shown to contain additional Sail restriction fragments of pJL121. Plasmid pJL125, which was conventionally isolated and contains an origin of replication from plasmid pBR325 and also the ^5.4kb origin of replication-containing EcoRI-Sall fragment of plasmid SCP2*, was dissolved in TE buffer and stored at 4°C. for future use. A restriction site map of plasmid pJL125 is presented in Figure 4 of the accompanying drawing. The restriction site map was determined with plasmid from 10 20 25 transformed E. coli K12 C600Rk-Mk- Example 11 Construction of E. coli K12 C600Rk-Mk~/pJL125 The desired construction was made in substantial accordance with the teaching of Example 7 except that plasmid pJL125, rather than plasmids pJL120 30 and pJL121, was used. The resultant colonies were -39- selected and tested for the expected phenotype (Amp , s s Tet , CM ) and constituted the desired E. coli K12 C600Rk-Mk~/pJL125 transformants. The identity of the transformants was further confirmed by AGE and restriction ^ analysis by the procedure of Eckardt, 1978, Plasmid 1:584 and by Klein et al_. , 1980, Plasmid 3:88. The transformants were then conventionally cultured for subsequent production and isolation of plasmid pJL125 according to known procedures. 10 Example 12 Construction of Plasmid pJL!90 A. EcoRI-Hindi!I Digestion of Plasmid pJL121 and Isolation of ^19.0kb EcoRI-Hindlll Fragment 15 About 200 yl. (80 yg.) of plasmid pJL121 DNA, 30 yl. BSA (1 mg./ml.), 40 yl. of Hindlll (containing 200 BRL units) restriction enzyme, and 30 yl. Hindlll reaction mix* were incubated at 37°C. for about 3 hours 2Q and then at 65°C. for 10 minutes. The 300 yl. reaction mixture was cooled to 4°C., supplemented with 110 yl. of 10X HindIII->EcoRI diluent reaction mix** and 30 yl. EcoRI restriction enzyme (containing 300 BRL units), and then incubated at 37°C. for 3 hours, then at 65°C. __ for 10 minutes followed by cooling to 4°C. The re- 2 D sultant ^19.0kb EcoRI-Hindlll restriction fragment was conventionally isolated by AGE/DE52/electroelution. The desired DNA was dissolved in TE buffer and stored at 4°C. for future use. X-5773& -40- 10 •k Hindlll reaction mix was prepared with the following composition: 60mM Tris-HCl, pH 7.5 500mM NaCl 60mM MgCl2 **HindIII->-EcoRI diluent was prepared with the following composition: 382mM Tris-HCl, pH 7.5 50mT4 NaCl 22mM MgCl2 B. EcoRI-Hindlll Digestion of Plasmid pLR4 and Isolation of ^7.7kb EcoRI-Hindlll Fragment The desired digestion and isolation was carried out in substantial accordance with the teaching of Example 12A except that plasmid pLR4, rather than plasmid pJL121, was used. The desired ^7.7kb fragment was dissolved in TE buffer and stored at 4°C. for future use. 20 25 30 C. Ligation of ^19.0kb EcoRI-Hindlll Fragment of Plasmid pJLl21 and ^7.7kb EcoRI-Hindlll Fragment of Plasmid pLR4 The desired ligation was carried out in substantial accordance with the teaching of Example 8C except that the ^19.0kb EcoRI-Hindlll fragment of plasmid pJL121 and the ^7.7 EcoRI-Hindlll fragment of plasmid pLR4, rather than the 6.0kb Sail fragment of plasmid SCP2* and Sail digested pBR325, were used. The resultant ligated DNA constituted the desired plasmid pJL190 which was then stored at 4°C. for future use. A restriction site and functional map of plasmid pJL190 is presented in Figure 4 of the accompanying drawings. The restriction site map was determined from plasmid transformed into E. coli K12 CGOOR^-M^-. Example 13 Construction of E. coli K12 C600Rk-Mk~/pJLl90 The desired construction was made in substantial accordance with the teaching of Example 7 except that plasmid pJL190, rather than plasmids pJL120 and pJL121, was used. The resultant colonies were £ selected and tested for the expected phenotype (Amp , s Tet ) and size by conventional means (as in Example 11) - and constituted the desired E. coli K12 C600Rk~Mk-/pJL190 transformants. The transformants were then conventionally cultured for subsequent production and isolation of plasmid pJL190 according to known procedures. Example 14 Isolation of Plasmid pJL192 Plasmid pJL19 2, which confers high resistance to antibiotic neomycin (10 yg./ml.), can be conventionally isolated from E. coli K12 C600Rk-Mk~/pJL192, a strain deposited and made part of the permanent stock culture collection of the Northern Regional Research Laboratory, Peoria, Illinois under the accession number 15040. The restriction site map of plasmid pJL192 appears not to be distinguishable from the plasmid pJL190 map presented in Figure 4. K-5773A -42- 10 15 20 Example 15 Construction of Plasmid pJL!95 A. EcoRI-Sall Digestion of Plasmid pJL125 and Isolation of ^5.4kb EcoRI-Sall Fragment The desired digestion and isolation was carried out in substantial accordance with the teaching of Example 12A except that plasmid pJL125 and Sail restriction enzyme and reaction mix, rather than plasmid pJL121 and Hindlll restriction enzyme and reaction mix# were used. In addition, SalI-*EcoRI diluent* was used. , The resultant 0-5.4^ EcoRI-Sall fragment was dissolved in TE buffer and stored at 4°C. for future use. * SalI->-EcoRI diluent was prepared with the following composition: 940mM Tris-HCl, pH 7.5 55mM MgCl2 B. EcoRI-Partial Sail Digestion of Plasmid pLR4 and Isolation of %7.5kb EcoRI-Partial Sail Fragment The Sail digestion was carried out in sub-25 stantial accordance with the teaching of Example 10A except plasmid pLR4, rather than pJL121, was used. Since only a partial Sail digestion was desired, the resultant mixture was incubated first at 37°C. for 15 minutes and then at 65°C. for 10 minutes. Following 30 cooling to 4°C., the resultant partial Sail V7.7kb linear fragment was conventionally isolated by AGE/ mm X 577JA -43- DE52/electroelution. The desired DNA was dissolved in TE buffer and digested with EcoRI restriction enzyme in substantial accordance with the teaching of Example 6A except that the above fragment, rather than 5 plasmid SCP2*, was used. The desired -v7.5kb EcoRI-Sall fragment (the largest possible EcoRI-Sall fragment) was isolated by AGE/DE52/electroelution, dissolved in TE buffer, and then stored at 4°C. for future use. 10 C. Ligation of ^5.4^ EcoRI-Sall Fragment of Plasmid 15 substantial accordance with the teaching of Example 8C except that the ^5.4kb EcoRI-Sall fragment of plasmid pJL125 and the "-7.5kb EcoRI-partial Sail fragment of plasmid pLR4, rather than the ^6.0kb Sail fragment of plasmid SCP2* and Sail digested pBR325, were used. The 20 resultant ligated DNA constituted the desired plasmid pJL195 and was stored at 4°C. for future use. A restriction site map of plasmid pJL195 is presented in Figure 5 of the accompanying drawings. The restriction site map was determined with plasmid isolation from 25 E. coli K12 C600Rk-Mk-. pJL125 and ^7.5kb EcoRI-Partial Sail Fragment of Plasmid pLR4 The desired ligation was carried out in 30 2 03 10 -X-57733- -44- Example 16 Construction of E. coli K12 C600Rk-Mk~/pJL195 The desired construction was made in substantial accordance with the teaching of Example 7 5 except that plasmid pJLl95, rather than plasmids pJLl20 and pJL121, was used. The resultant colonies were R S tested for the expected phenotype (Amp , Tet ) and size (as in Example 11) and constituted the desired E. coli K12 C600Rk-Mk~/pJL195 transformants. The transformants were then conventionally cultured for subsequent production and isolation of plasmid pJLl95 according to known procedures. Example 17 15 Construction of Plasmid pJL114 A. Partial BamHI Digestion of Plasmid SCP2 The desired digestion was carried out in substantial accordance with the teaching of Example 6A 20 except that plasmid SCP2 (isolated, in accordance with the teaching of Example 1, from Streptomyces coelicolor A3 (2), a strain deposited and made part of the permanent stock culture collection of the Northern Regional Research Laboratory under the accession number 15042) , 25 and BamHI restriction enzyme and reaction mix*, rather than plasmid SCP2* and EcoRI restriction enzyme and reaction mix, were used. The desired DNA was stored at 4°C. for subsequent use. * Reaction mix for BamHI restriction enzyme was prepared with the following composition: lOOOmM Tris-HCl, pH 7.4 lOOmM MgCl2 -45- B. Ligation of BamHI Digested Plasmid SCP2 and BamHI Digested Plasmid pBR322 The desired ligation was carried out in 5 substantial accordance with the teaching of Example 6C except that the BamHI digest of plasmid SCP2 (prepared in Example 17A) and BamHI-digested plasmid pBR322 (prepared in Example 4B), rather than plasmids SCP2* and pBR325, were used. The resultant DNA was stored 10 at 4°C. and constituted the desired ^34.6kb plasmid PJL114. Example 18 Construction of E. coli K12 C600Rk~Mk-/pJLll4 The desired construction was made in substantial accordance with the teaching of Example 7 except that plasmid pJLH4, rather than plasmids pJL120 and pJL121, was used. The resultant colonies were R selected and tested for the expected phenotype (Amp , 20 Tet ), and constituted the desired E. coli K12 C600Rk-Mk-/pJL114 transformants. The ampicillin resistant, tetracycline sensitive colonies were isolated according to known procedures, cultured, and then conventionally identified by restriction enzyme and 25 . agarose gel electrophoretic analysis of the constitutive plasmids. It was revealed upon analysis that the BamHI restriction enzyme had cut only one of the Bglll restriction sites of 5CP2 during the digestion described 30 in Example 17A. Since this event is rare and has not been repeated, E. coli K12 C600R, -II, -/pJL114 has been »>_ - K k £ f J ^ o\ •/('v 10 JUL 1985 m/, 3 V.S M&864 OU5TOA -46- deposited and made part of the permanent stock culture collection of the Northern Regional Research Laboratory, Peoria, Illinois under the accession number B-15039. The strain is available as a preferred source and stock reservoir of plasmid pJL114. A restriction site map of plasmid pJL114 is presented in Figure 5 of the accompanying drawings. Example 19 Construction of Streptomyces griseofuscus/pJLl20 10 15 A. Growth of Cultures for Preparation of Protoplasts A vegative innoculum was conventionally prepared by growing the strain under submerged conditions for 20 hours at 30°C. in TSB supplemented with .4% glycine. The culture was homogenized and innocu-lated at a 1/20 dilution into the same medium and then grown for 18 hours at 30°C. B. Transformation 20 Using about 20 yg. of plasmid pJLl20 DNA and 9 1X10 protoplasts of Streptomyces griseofuscus, a strain deposited and made part of the permanent stock culture collection of the American Type Culture Collection, Rockville, Maryland, from which it is avail-25 able to the public under the accession number ATCC 23916, the desired transformation was carried out in substantial accordance with the teaching of International Publication (of International Patent Application No. PCT/GB79/00095) No. W079/01169, Example 2. 30 10JUU9& 2.03864 X 5773A 47 C. Selection To assay for transformation even at low frequencies, two procedures were employed. (1) Pock-assay: Spores were harvested from the regeneration plates containing confluent lawns of regenerated protoplasts as follows. About 10 ml. of sterile distilled water werefadded to the plate and the surface of the Xo culture gently scraped with a loop to remove the spores. The resulting spore suspension was centrifuged at 20,000 rpm for 10 minutes. The supernatant was discarded and the remaining spore pellet resuspended in . .3 ml. of 20% v/v glycerol. Serial dilutions of the 15 preparation were made down to 10 by successive transfer of .1 ml. of the spore suspension to .9 ml. of 20% v/v glycerol. The spores can then be stored at -20°C. with little loss of viability. About .1 ml. aliquots of some of the dilution series (e.g. -1-2-4 • 2 0 10 / 10 ,10 ) of each of the harvested plates were then transferred to R2 medium (Hopwood and Wright, 1978, Molecular and General Genetics 162:30) plates which had sufficient spores of the Streptomyces griseofuscus strain originally used in the transfor-25 mation procedure to produce a confluent lawn. This procedure can also be carried out with the substitution of YMX agar (.5% yeast extract .5% malt extract, .1% dextrose and 2% agar). Transformants can typically be detected after 3 days' growth at 30°C. by the appearance 30 of "pocks", a property expressed by spores containing the plasmid in expressible form within the lawn. The 203-864 X S773A -48- transformants were recovered by conventionally picking spores from the centre of the "pock" to an agar plate of YMX medium (Hopwood 1967, Bacteriological Review, 31:373). (2) Back transformation to E. coli K12 C600Rk-Mk-: The spores are collected as in (1) above but are used to innoculate 50 ml. of TSB supplemented with .4% glycine. The culture is grown for 20 hours at 30°C. and the cells are harvested followed by isolation of DNA. Isolation is as disclosed in Example IB except that centrifugation with CsCl and propidium iodide is omitted. Subsequently, 50 yl. of this DNA is used to transform E. coli K12 ceOOR^-M^- as disclosed in Example 7B. Plasmids in the transformants are verified and identified by conventional means as taught in Example 11. 10 15 20 25 30 Example 20 Construction of Streptomyces griseofuscus/pJLH4, S. griseofuscus/pJLl21, S. griseofuscus/pJL125, S. griseofuscus/pJL180, and S. griseofuscus/pJL181. The desired constructions were each individually and respectively made, selected, and recovered in substantial accordance with the teaching of Example 19 except that plasmids pJL114, pJL121, pJL125, pJL180, and pJL181, rather than plasmid pJLl20, were appropriately used for the individual construction. .203864- «- 5773A -4 9- Example 21 Construction of Streptomyces griseofuscus/pJL!90 A. Trans formation 5 The desired transformation was carried out in substantial accordance with the teaching of Example 19B except that plasmid pJLl90, rather than plasmid pJL120, was used. 10 20 B. Selection The desired transformants were selected for neomycin resistance by overlaying the regenerating protoplasts with R2| medium top agar containing sufficient neomycin to bring the final plate concentration to 1 ug./ml. The resultant Streptomyces griseofuscus/ pJL190 transformants were then tested for the expected pock morphology in substantial accordance with the procedure of Example 19C(!•).' ! Example 22 Construction of Streptomyces griseofuscus/pJL!95 The desired construction was made, selected, and recovered in substantial accordance with the teaching of Example 21 except that plasmid pJL195, 25 rather than plasmid pJL190, was used. 30 203 8 64 X- 5773ft -50- Example 23 Construction of Streptomyces griseofuscus/pJL!92 The desired construction was made, selected, 5 and recovered in substantial accordance with the teaching of Example 22 except that plasmid pJL192 and, in the selection procedure, top agar containing sufficient neomycin to bring the final plate concentration to 10 yg./ml., rather than plasmid pJL195 and top agar 10 containing sufficient neomycin to bring the final plate concentration to 1 yg./ml., were used. Example 24 Construction of Streptomyces fradiae/pJL120, S. 15 fradiae/pJL114, S. fradiae/pJL121, S. fradiae/pJL125, S. fradiae/pJL!80, S. fradiae/pJL181, S. fradiae/ pJL190, S. fradiae/pJL195, and S. fradiae/pJL192 The desired constructions are individually 20 and respectively made, selected, and recovered in substantial accordance with the respective teachings of Examples 19, 20, 21, 22, and 23 except that Streptomyces fradiae, rather than S. griseofuscus, is used. In addition, the TSB medium for protoplasting and growing 25 s_. fradiae was modified and contained only .2% glycine. 30 X-5773A -51- Example 25 i Construction of Streptomyces lividans/pJL120, S. lividans/pJL114, S. liyidans/pJL121, S. lividans/ 5 pJLl25, S. lividans/pJL!80, S. lividans/pJLl81, S. lividans/pJL190, S. lividans/pJL195, and S. lividans/ PJL192 The desired constructions are individually 10 and respectively made, selected, and recovered in substantial accordance with the respective teachings of Examples 19, 20, 21, 22, and 23 except that Streptomyces lividans, rather than S. griseofuscus, is used-. In addition, the media for protoplasting and growing S. 15 lividans is as described in International Publication (of International Patent Application No. PCT/GB79/00095) No. W079/01169, Example 2. Example 26 Isolation of Plasmid pJL192 Mutant that Confers 20 — High Resistance To Antibiotic Neomycin Streptomyces griseofuscus/pJL190 was isolated as described in Example 21. Analysis of growth of colonies on nutrient agar supplemented with different 25 concentrations of neomycin revealed that S. griseofuscus/ pJL190 exhibited resistance to 1.0 yg./ml. of neomycin. S. griseofuscus was spread conventionally on nutrient agar plates supplemented with 10 yg./ml. of neomycin. A colony was discovered that exhibited growth at this 30 high level of neomycin. After repeated analysis verified that the colony exhibited the aforementioned ' •/ _ ^ ^ X-5773A -52- resistance, the colony was designated S. griseofuscus/ pJL192. The plasmid, pJL192 was shuttled into E. coli K12 C6 00Rj,-Mk- by back transformation as taught in Example 19C. The restriction site map of pJLl92 5 appears not to be distinguishable from pJL190. Example 27 Isolation of Plasmid pJL199 Mutant that Confers High Resistance to Antibiotic Neomycin 10 The desired isolation is carried out in substantial accordance with the teaching of Example 26 except that Streptomyces griseofuscus/pJL195 (prepared in Example 22), rather than S. griseofuscus/pJL190, was used. A colony that exhibited high resistance 15 to neomycin was designated S. griseofuscus/pJL199. The plasmid, pJLl99, was shuttled into E. coli K12 C6 00Rk-Mk~ by back transformation as taught in Example 19C. The restriction site map of pJLl99 appears not to be distinguishable from pJL195. 2o Those skilled in the art will recognize that plasmid pJLl99 can also be conventionally constructed by substituting the neomycin resistance-conferring fragment of plasmid pJL19 2 (prepared in Examples 15 and 27) for the pLR4-derived neomycin resistance-conferring 25 fragment of plasmid pJL195. Such a substitution thus also results in the desired'plasmid pJL199. Example 28 Construction of Plasmid pLR2 30 A. Hindlll Digestion of Plasmid pIJ6 About 20 y 1. (20 yg.) of plasmid pIJ6 DNA, disclosed in Thompson et al., 1980, Nature 286:525, -53- 5 yl. BSA(Bovine Serum albumin, 1 mg./ml.), 19 yl. water, 1 yl. of Hindlll (containing 3 New England Bio Labs units) restriction enzyme*, and 5 yl. reaction mix** were incubated at 37°C. for 2 hours. The reac- 5 tion was terminated by the addition of about 50 yl. of 4M ammonium acetate and 200 yl. of 95% ethanol. The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, suspended in 20 yl. of TE buffer, and frozen at -20°C. for storage. 10 b. Hindlll Digestion of Plasmid pBR322 20 30 About 8 yl. (4 yg.) of plasmid pBR322 DNA, 5 yl. reaction mix, 5 yl. BSA (1 mg./ml.), 31 yl. water, and 1 yl. of Hindlll restriction enzyme were 15 incubated at 37°C. for 2 hours. After the reaction was terminated by incubating at 60°C. for 10 minutes, about 50 yl. of ammonium acetate and 200 yl. of 95% ethanol were added. The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, and suspended in 45 yl. of water. C. Ligation of Hindlll Digested Plasmids pIJ6 and pBR322 About 20 yl. of Hindlll treated plasmid pIJ6 (from Example 28A), 20 yl. of Hindlll treated plasmid 25 pBR322 (from Example 2 8B), 5 yl. BSA (1 mg./ml.), 1 yl. of T4 DNA ligase , and 5 yl. ligation mix** were incubated at 16°C. for 4 hours. The reaction was terminated by the addition of about 50 yl. 4M ammonium acetate and 200 yl. of 95% ethanol. The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, and suspended in TE buffer. The suspended DNA constituted the desired plasmid pLR2. * Refer to footnotes appended to Example ** 2A on page 27 and to Example 2C on page 28. X-5773A -54- Example 29 Construction of E. coli K12 HB101/pLR2 About 10 ml. of E. coli K12 HB101 cells (Bolivar et al., 1977, Gene 2:75-93) were C , pelleted by centrifugation and then suspended in about 10 ml. of 0.01M sodium chloride. Next, the cells were pelleted again, resuspended in about 10 ml. of 0.03M calcium chloride, incubated on ice for 20 minutes, pelleted a third time, and finally, resuspended in 1.25 ml. of 0.03M calcium chloride. The resultant cell suspension was competent for subsequent transformation. Plasmid pLR2 in TE buffer (prepared in Example 28C), was ethanol precipitated, suspended in " 150 yl. of 30mM calcium chloride solution, and gently 15 mixed in a test tube with about 200 yl. of competent E. coli K12 HB101 cells. The resultant mixture was incubated on ice for about 45 minutes and then at 42°C. for about 1 minute. Next, about 3 ml. of L-broth (Bertani, 1951, J. Bacteriology 62:293) containing 20 50 yg./ml. of ampicillin were added. The mixture was incubated with shaking at 37°C. for 1 hour and then plated on L-agar (Miller, 197 2, Experiments in Molecular Genetics, Cold Spring Harbor Labs, Cold Spring Harbor, New York) containing ampicillin. Surviving colonies 2^ were selected and tested for the expected phenotype R S (Amp , Tet ), and constituted the desired E. coli K12 HB101/pLR2 transformants. 30 Representative plasmids and transformants that can be constructed in accordance with the foregoing teaching include the following listed below in Tables 1 and 2. CO o ro en ro o Table 1 Representative Plasmids Example No. Plasmid Name % Size in kb E. coli Marker Streptomyces Marker 30 pJLl22 24.4 »' R m R Amp , Tet M 31 pJL123 27.8 R R Ai^p , Tet M 32 pJL124 21.1-24.2 . R _ R Amp , Tet M 33 pJL126 9.1-10.6 . R _ R Amp , Tet M 34 pJLl76 32.5 CH^, TetR P* 35 pJL1200 35.8 D R Amp , Tet M 36 pJL1201 35.8 AmpR, TetR M 37 pJL1202 24.4 R _ R Amp , Tet M 38 PJL1203 27.8 . R R Amp , Tet M 39 pJL1204 21.1-24.2 AmpR, TetR . M 40 pJL1205 10.2 A R Amp M 41 PJL1206 9.1-10.6 AmpR, TetR M 42 pJL1706 32.5 CM^, TetR P 43 * pJL1716 22.5 CM11, TetR P PJL3176 22.5 CM1*, TetR P* U1 !< (n • J ■ J I J tt Construction SstI Deletion of pJL120 Bglll Deletion of pJL121 Partial PstI Deletion of pJL120 Partial Xorll Deletion of pJL120 ^16.6kb PstI and extraneous M.0.0kb PstI of SCP2* into PstI Site of pBR325 SCP2 EcoRI Site into pBR325 EcoRI Site Reverse Orientation of pJL1200 SstI Deletion of pJLl200 Bglll Deletion of pJL1201 Partial PstI Deletion of pJL1200 Sail Deletion of pJL1201 Partial Xorll Deletion of pJL1200 VL0.6kb PstI and extraneous VLO.Okb PstI of SCP2 into PstI Site of pBR325 ^16.6 PstI of SCP2 into PstI Site of pBR325 VL6.6 PstI of SCP2* into PstI Site of pBR325 i Ui I ro P> u> o N5 U1 M O cn Example Plasmid ^ Size No. Name in kb 45 pJL1800 12.6 E. coli Marker AmpR, CMR Table 1 continued Streptomyces Marker 46 47 PJL1801 pJL1900 12.6 25.9 Amp . B Amp R, CM11 M, Neo R 48 PJL1902 25.9 Amp M, Neo R 49 pJL1905 12.1 Amp R M, Neo R 50 pJL193 6.9 Amp Thio 51 pJL196 12.1 Amp R M, Neo R 52 pJL197 13.1 Amp M, NeoR, ThioR t-1 cn ! < ° > J u > Construction ^.Okb Sail of SCP2 into Sail Site of pBR325 Reverse Orientation of pJL1800 ^19.Okb EcoRI-Hindlll of PJL1201 and ^7.7kb EcoRI-Hindlll of pLR4 M.9.0kb EcoRI-gindlll of pJL1201 and ^7.7kb EcoRI-Hindlll of pLR4 "v5. 4kb EcoRI-Sail of pJLl205 ^ and ^7.5kb EcoRI-Partial Sail of pLR4 VL.35kb BamHI of pLR2 into BamHI of pBR328 ^5.4kb EcoRI-SalI of pJLl25 into ^7.5kb EcoRI-partial Sail of pJL192 ^lkb Bell of pJL193 into partial BamHI of pJL196, orientation such that the Clal site of the M.kb Bell fragment is proximal to the Sail site of the pBR322 fragment and such that the Sail site of the M.kb Bell site is proximal to the neomycin resistance gene CD C/J CO ON -C*. to to fo o Ln o Example Plasmid ^ Size coli No. Name in kb Marker 53 pJL1907 13.1 AmpR 54 pJL198 13.1 AmpR 55 pHJL212 13.9 AmpR 56 pHJL213 13.9 AmpR U1 Table 1 continued Streptomyces Marker M, NeoR, ThioR M, NeoR, ThioR M, NeoR, ThioR M, NeoR, ThioR Construction VLkb Bell of pJL193 into partial BamHI of pJL1905 Same as pJL197 except the orein-tation of the Bell fragment is reversed *VLkb Bell fragment of pJL193 into partial BamHI of pJLl95, orientation and insertion the same as pJL198 Same as pHJL212 except the orien tation and insertion the same as P.JL197 i Ln I X-W3A -58- Table 2 Representative Transformants 1. Streptomyces R/R1 wherein R is griseofuscus, ambofaciens, fradiae, or lividans and R1 is 5 independently pJL122, pJL123, pJL124, pJLl26, pJL176, pJL1200, pJL1201, pJLl202, pJL1203, pJL1204, pJL1205, pJL1206, pJL1706, pJL1800, PJL1801, PJL1900, pJL1902, pJL1905, pJL196, pJL197, pJL1907, pJL198, pHJL212 and pHJ213. 10 E. coli K12 R^/R^" wherein R^ is CGOOR^-M^-, 294, C600 or RV308 and R^" is independently as defined above. ^2ffi5864 15 20 25 30 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 203864 -59- WHAT WE CLAIM IS: ; 1. A recombinant DNA cloning vector comprising: a) a functional origin of replication-containing restriction fragment of plasmid SCP2 or SCP2* (as herein defined), b) a restriction fragment comprising an E. coli origin of replication, c) one or more DNA segments that confer resistance to at least one antibiotic when transformed into a cell of E. coli, said cell being sensitive to the antibiotic for which resistance is conferred, and d) one or more DNA segments that independently confer either or both of the Streptomyces tra function or resistance to at least one antibiotic when transformed into a cell of Streptomyces, said cell being sensitive to the antibiotic for which resistance is conferred. 2. The vector of Claim 1 which is a plasmid. 3. The vector of Claim 1 or 2 wherein the restriction fragment of plasmid SCP2 or SCP2* is the ^5.4kb EcoRI-Sall fragment, ^6.0kb Sail fragment, ^19kb EcoRI-Hindlll fragment, or ^31kb EcoRI fragment. 4. The vector of Claim 1, 2 or 3 wherein the E. coli origin of replication is the pBR322 origin of replication, pBR324 origin of replication, pBR325 origin of replication, pBR327 origin of replication, or pBR328 origin of replication. 203964 -60- 5. The vector of any of Claims 1 to 4 wherein the one or more DNA segments that confer resistance in E. coli are DNA segments that confer resistance to ampicillin, chloramphenicol or tetracycline . PJL180, pJLl81, pJL125, pJL190, pJL192, pJLl95, pJL199, pJLl22, pJLl23, pJLl24, pJL126, pJLl76, P-JL1200, PJL1201, pJLl202, pJL1203, pJLl204, pJL1205, pJL1206, PJL1706, pJLl800, pJLl801, pJL1900, pJL1902, pJLl905, more DNA segments that confer resistance in Streptomyces are DNA segments that confer resistance to neomycin or thiostrepton. wherein the DNA segment that confers resistance to an antibiotic in Streptomyces is the ^7.7kb EcoRI-Hindlll restriction fragment of plasmid pJLl92, the ^7.7kb EcoRI-Hindlll restriction fragment of plasmid pLR4 (as herein defined), the ^7.5kb EcoRI-partial Sail restriction fragment of plasmid pLR4, the ^1.35kb BamHI restriction fragment of plasmid pLR2 (as herein defined), or the/vikb Bell restriction fragment of plasmid pJLl93. plasmid pJLl90, pJLl92, pJL195, pJL199, pJLl900, PJL1902, pJL1905, """pJLl96, pJL197, pJL1907, PJL198, pHJL212 or pHJL213. 10. The vector of Claim 9 which is plasmid pJL192 or pJL199 that in Streptomyces confers resistance to neomycin at levels of at least 10 yg./ml. 6. The vector of Claim 5 which is plasmid pJLl96, pJLl97, pJL198 r pHJL212, pHJL213, or pJLl907. 7. The vector of Claim 5 wherein the. one or 8. The vector of any of Claims 1 to 7 9. The-vector of Claim 7 or 8 which is Z0Z864 ''' f" • ) -61- • 1—1 rH Plasmid pJLH4. 12. Plasmid p J 1j12 0 • 13. Plasmid PJL121. 14. Plasmid pJLl92. 15. Plasmid pJL197. 16. Plasmid pJL198. 17. Plasmid PHJL212. 18 . Plasmid PHJL213. 19. A transformed host cell comprising the recombinant DNA cloning vector of any of Claims 1 to 18 . 20. The host cell of Claim 19 which is Streptomyces, preferably of species lividans, griseofuscus , fradiae, or ambofaciens. 21. The host cell of Claim 19 which is E. coli K12. 22. A restriction fragment comprising the plasmid SCP2 or SCP2* "^5. 4kb EcoRI-Sall or ^.Okb Sail restriction fragment of Claim 3. 23. A restriction fragment comprising the plasmid pJLl92 ^7.7kb EcoRI-Hindlll restriction fragment of Claim 8. 24. A process for preparing a recombinant DNA cloning vector which comprises ligating a functional origin of replication-containing restriction fragment of plasmid SCP2 or SCP2* (as herein defined) and one or more DNA sequences comprising: a) a restriction fragment comprising an E. coli origin of replication, 203864 10 15 20 25 30 -62- b) one or more DNA segments that confer resistance to at least one antibiotic when transformed into a cell of E. coli, said cell being sensitive to the antibiotic for which resistance is conferred, and c) one or more DNA segments that independently confer either or both of the Streptomyces tra function or resistance to at least one antibiotic when transformed into a cell of Streptomyces, said cell being sensitive to the antibiotic for which resistance is conferred . 25. The process of Claim 24 wherein the restriction fragment of plasmid SCP2 or SCP2* is the ^5.4kb EcoRI-Sail fragment, ^6.0kb Sail fragment, ^19kb EcoRI-Hindlll fragment, or 'v-Slkb EcoRI fragment, wherein the E. coli origin of replication is the pBR322 origin of replication, pBR324 origin of replication, pBR325 origin of replication, pBR327 origin of replication, or pBR328 origin of replication, wherein the one or more DNA segments that confer resistance in E. coli are DNA segments that confer resistance to ampicillin, chloramphenicol or tetracycline, and wherein the one or more DNA segments that confer resistance in Streptomyces are DNA segments that confer resistance to neomycin or thiostrepton. 26. A method for detecting transformants comprising: 1) mixing Streptomyces cells, under transforming conditions, with a recombinant DNA cloning vector, said vector comprising 203864 X 5773A Bow Zealand -63- a) an origin of replication and P gene (as herein defined)-containing restriction fragment of plasmid SCP2 or SCP2* (as herein defined), and 5 b) a non-lethal DNA sequence cloned into the EcoRI restriction site of said P gene, and 2) growing said Streptomyces cells on a lawn of 10 an indicator Streptomyces strain and select ing colonies that show the M pock phenotype (as herein defined). 27. The method of Claim 26 wherein the restriction fragment comprising the origin of replication 15 and the P gene is of plasmid SCP2*. 28. The method of Claim 26 or 27 wherein the Streptomyces cells and indicator strain are of species lividans , griseofuscus , fradiae or ambof aciens.. 29. The method of Claim 26, 27 or 28 wherein the recombinant DNA cloning vector is plasmid pJL12 0, pJLl21, pJLl25, pJL190, pJL192, pJL195, pJL197, pJLl98, PHJL212, or pHJL213. 30. A recombinant DNA cloning vector substantially as hereinbefore described with particular reference to Examples 8, 10, 12, 15, 26, 27, 30 to 49 and 51 to 56. 31. Restriction fragments 1 substantially as hereinbefore described in Examples 8A and 12B. 20 25 30 28WWW86rn| 203964 -64- 32. A transformed host cell substantially as hereinbefore described with particular reference to Examples 9, 11, 13, 16, and 19 to 25, and Table 2. 5 33. A process for preparing a recombinant DNA cloning vector substantially as hereinbefore described with particular reference to Examples 8, 10, 12, 15, 26, 27, 30 to 49 and 51 to 56. 34. A method for detecting transformants sub-•j_q stantially as hereinbefore described with particular reference to Example 19C. ta By -His/Their authorised Agent -15 A. J. PARK & SON Per: tfoc/- 20 25 30 : T 5?'/ </div>