GB2118947A - Cloning vectors - Google Patents

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

SPECIFICATION Cloning vectors The present invention relates to selectable novel recombinant DNA cloning vectors comprising a 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 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 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 Streptomyces 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 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 inserted into the vectors and, upon transformation, 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 disclosed and claimed herein, the following terms are as defined below.

Plasmid pLR 1 or pLR4 3.4 kb BamHI Restriction Fragment -- the same 3.4 kb BamHI neomycin resistance-conferring fragment contained in plasmid plJ2.

AmpR -- the ampicillin resistant phenotype.

Amp5 -the ampicillin sensitive phenotype.

TetR - the tetracycline resistant phenotype.

Tet5 - the tetracycline sensitive phenotype.

CMR - the chloramphenicol resistant phenotype.

CM5 - the chloramphenicol sensitive phenotype.

NeoR - the neomycin resistant phenotype.

Neo5 - the neomycin sensitive phenotype.

ThioR - the thiostrepton resistant phenotype.

Thio5 - the thiostrepton sensitive phenotype.

NeoR, Neon, ThioR, and Thios refer only to results of tests in Streptomyces as used in this disclosure. AmpR, Amps, TetR, Tets, CmR and Cms refer only to results of tests in E. coli as used in this disclosure.

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*, 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.

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: 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 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.

The invention further comprises transformants 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 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 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 best constructed by ligating an origin of replicationcontaining and Streptomyces tra function-conferring restriction fragment of plasmid SCP2 or SCP2* into an E. collorigin of replication-containing and antibiotic resistance-conferring restriction fragment of an E. coli plasmid. Plasmids SCP2 and SCP2*, from which origins of replication are constructed, are each 31 kb 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 pock is distinguishable from that of plasmid SCP2, in other ways plasmids SCP2 and SCP2* are virtually identical.

Since the present disclosure teaches that the Stremptomyces tra function and the origin of replication of plasmids SCP2 and SCP2* are within their respective --5.4 kb EcoRI-Sall restriction fragments, a variety of different origin of replication-containing and Streptomyces tra functionconferring fragments can be generated. This is accomplished by digestion with restriction enzymes that cut outside the 5.4 kb EcoRI-Sall region. A detailed restriction site map of plasmid 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 coellcoior A3(2) and Streptomyces coelicolor M1 10 strains deposited and made part of the permanent stock culture collection of the Northern Regional Research Laboratory, Peoria, Illinois. Streptomyces coelicolorA3(2) is available to the public as a preferred source and stock reservoir of plasmid SCP2 under the accession number 1 5042. Streptomyces coellcoior M1 1 O is available to the public as a preferred source and stock reservoir of plasmid SCP2* under the accession number 1 5041.

Many tra function-conferring and origin of replication-containing restriction fragments of plasmids SCP2 and SCP2* can be constructed. Those specifically exemplified, for illustrative purposes, include the 5.4 kb EcoRI-Sall, the -6.0 kb Sail, the 19 kb EcoRI-Hindlll, and the --31 kb EcoRI restriction fragments of plasmid SCP2* and the -31 kb Bglll restriction fragment of plasmid SCP2. The aforementioned plasmid SCP2* and SCP2 fragments were respectively ligated to an origin of replication-containing and antibiotic resistance-conferring fragment of E. coli plasmids pBR325 and pBR322.Those skilled in the art will recognize that although not required, it is convenient for both the 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 31 kb EcoRI fragment of plasmid SCP2* and the 6 kb 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* -6.0 kb Sall fragment and the 6 kb Sall fragment of pBR325 results in the illustrative plasmids pJLl 80 and pJL181; ligation of the SCP2* 5.4 kb EcoRI Sa/l fragment and the 4.8 kb EcoRI-Sall fragment of pBR325 results in the illustrative plasmid pJL125; and ligation of the SCP2 Bath1 digest and the 4.4 kb BamHI fragment of plasmid pBR322 results in the illustrative plasmid pJLl 14.

All of the aforementioned vectors are readily selectable in each of E. coil and Streptomyces. For example in E. coli, plasmids pJL120 and pJL121 confer ampicillin and tetracycline resistance; plasmids pJLl 80 and pJL181 confer ampicillin and chloramphenicol resistance; and plasmids pJL125 and pJL1 14 confer only ampicillin resistance. Therefore, the vectors are conventionally selectable in the E. coli host system by adding the appropriate antibiotic to the culture medium.

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 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 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 pJLl 20, pJL121, and pJL125 code for M phenotype, that plasmids pJL1 80 and pJL181 code for P* phenotype, and that plasmid pJL1 14 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 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, specifically exemplified for illustrative purposes by plasmids pJL1 90 and pJL1 95, express an additional selectable phenotype. Plasmid pJLl 90 was constructed by ligating the neomycin resistance-conferring or7.7 kb EcoRI-Hindlil fragment of plasmid pLR4 to the 19 kb EcoRI-Hindlil fragment of plasmid pJL121. Plasmid pJL195 was constructed by ligating the pLR4 7.5 kb EcoRI-partial Sall fragment to the 5.4 kb EcoRI-Sall fragment of plasmid pJL125. The latter pJL125 plasmid comprises the largest (5.4 kb) EcoRI-Sall fragment of plasmid pSCP2* and was constructed by Sail deletion of plasmid pJL1 21. Illustrative plasmids pJL1 90 and pJL1 95, in addition to neomycin resistance, also express the M phenotype as discussed above.

Plasmid pLR4, the source of the neomycin resistance conferring fragments, is 7.7 kb and is constructed by ligating BamHI-treated plasmids pBR322 and pLR1. Plasmid pLR1 is -14.8 kb and is constructed by ligating Hindlll-treated plasmid plJ2, disclosed in Thompson et al., 1980, Nature 286:525, to Hindill-treated plasmid pBR322. As is readily apparent to those skilled in the art, both plasmids pLR4 and pLR1 contain the same neomycin resistance gene and thus either plasmid can be used for constructing the aforementioned pJL neomycin resistant vectors.

An additional neomycin resistance-conferring plasmid, designated as pJL1 92, was isolated as a spontaneous mutant of plasmid pJL190 resident in Streptomyces grlseofuscus. Plasmid pJLl 92 specifies resistance to elevated levels of neomycin and therefore comprises a novel neomycin resistance gene which is distinguishable from the resistance gene comprising plasmids pJLl 90, pJLl 95, plJ2, pLR4, and pLR1. In a similar manner, an additional neomycin resistance-conferring plasmid, designated as pJLl 99, was isolated as a spontaneous mutant of plasmid pJL195.Those skilled in the art will recognize that the novel neomycin resistance gene of plasmid pJLl 92 or pJL199 can be readily excised and ligated to other vectors. The gene allows for improved and more efficient selection of transformants. As in the case of plasmids pJLl 90 and pJLl 95, transformants of plasmids pJL1 92 and pJL1 99 express the M phenotype when plated on an appropriate indicator strain.

Plasmid pJLl 92 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. It is available to the public as a stock reservoir and preferred source of plasmid pJL192 under the accession number B-l 5040.

A DNA segment that confers resistance to antibiotic thiostrepton, exemplified by the -1.35 kb 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 -18.7 kb and is constructed by ligating Hindlll treated plasmid plJ6, disclosed in Thompson et al., 1980, Nature 286:525, to Hindill treated plasmid pBR322. Plasmid pLR2 is functional in E. coli and therefore can be amplified and isolated conveniently for subsequent manipulation.

For convenience and ease of construction, the thiostrepton resistance conferring 1.35 kb BamHI fragment of plasmid pLR2 was ligated into the BamHI restriction site of plasmid pBR328 to form plasmid pJL1 93. The 1 kb BciI restriction fragment of pJL193 contains the thiostrepton resistanceconferring 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 replication contained in their respective 5.4 kb EcoRI-Sall restriction fragments is present. Such additional plasmid SCP2 and SCP2* restriction fragments include, but are not limited to, the 6 kb Sall, -1 5 kb Pstl, 23 kb Bill, N1 5 kb BamHI, -14 kb EcoRI-Pstl, 13 kb EcoRI-BamHI, and -1 5 kb 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 restriction 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 use of either plasmid pBR322 or pBR325 as exemplified in several pJL constructions.

Although the neomycin and thiostrepton antibiotic resistance-conferring DNA segments exemplified herein are respectively the 7.7 kb EcoRI-Hindlll and the 7.5 kb EcoRI-partial Sall fragments of plasmid pLR4 and the pLR2 -1.35 BamHI and the pJL193 1 kb Bcll 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 pLR1 include, for example, the 3.4 kb BamHI restriction fragment, the 3.5 kb Pstl restriction fragment, and the larger of the Sstl-Kpnl subfragments of the 3.4 kb BamHI restriction fragment. Other thiostrepton resistance-conferring segments include, for example, the 13 kb Pstl fragment of plasmid pLR2. Still other DNA segments conferring resistance to the same or to different antibiotics such as, for example, hygromycin, viamycin, 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 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 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 plasmids that are within the scope of the present invention. Therefore, an antibiotic resistance-conferring 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 limited to the use of tra alone or in combination with an antibiotic resistanceconferring 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 inserted 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 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 comprising 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 nontransformed Streptomyces taxa. This is advantageous because the present vectors therefore can be transformed 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 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 in Streptomyces, the present tra+ vectors fulfill the general cloning and screening role analogous to that of bacteriophage A in replica-plate transduction for screening gene libraries in E coli. Desirable genes can thus be readily identified by the replica-plate mating procedure and then easily amplified by shuttling into E. coli as described in Example 20C below.

The vectors of the present invention are broadly applicable and are transformed into host cells of many Streptomyces taxa, particularly restrictionless strains of economically important taxa that produce antibiotics such as aminoglycoside, macrolide, p-lactam, polyether, and glycopeptide antibiotics. Such restrictionless strains are readily selected and isolated from Streptomyces taxa by conventional procedures well known in the art (Lomovskaya et ai., 1980, Microbiological Reviews 44:206). Host cells of restriction less strains lack restriction enzymes and therefore do not cut or degrade plasmid DNA upon transformation. For purposes of the present application, host cells containing restriction enzymes that do not cut any of the restriction sites of the present vectors are also considered restriction less.

Preferred host cells of restrictionless strains of Streptomyces taxa that produce aminoglycoside antibiotics and in which the present vectors are especially useful and are transformed, include restrictionless cells of, for example: S. kana-myceticus (kanamycins), S. chrestomyceticus (aminosidine), S. griseoflavus (antibiotic MA 1267), S. microsporeus (antibiotic SF-767), S.

ribosidificus (antibiotic SF733), S. flavopersicus (spectinomycin), S. spectabilis (actinospectacin), S.

rimosus forma paromomycinus (paramomycins, catenulin), S. fradlae var. italicus (aminosidine), S.

bluensis var. bluensis (bluensomycin), S. catenulae (catenulin), S. oilvoretlcullvar. cellulophilus (destomycin A), S. tenebrarius (tobramycin, apramycin), S. Ia van duiae (neomycin), S. albogriseolus (neomycins), S. albus var. metamycinus (metamycin), S. hygroscopicus var. sagamiensis (spectinomycin), S, bikiniensis (streptomycin), S. griseus (streptomycin), S. erythrochromogenes var.

narutoensls (streptomycin), S. poolensis (streptomycin), S. galbus (streptomycin), S. rameus (streptomycin), S. olivaceus (streptomycin), S. mashuensis (streptomycin), S. hygroscopicus var.

limoneus (validamycins), S. rimofaciens (destomycins), S. hygroscopicus forma glebosus (glebomycin), S. fradiae (hybrimycins neomycins), S. eurocidicus (antibiotic Al 6316-C), S. aquacanus (N-methyl hygromycin B), S. crystallinus (hygromycin A), S. noboritoensis (hygromycin), S. hygroscopicus (hygromycins), S. atrofaciens (hygromycin), S. kasugaspinus (kasugamycins), S. kasugaensis (kasugamycins), S. netropsis (antibiotic LL-AM31), S. lividus (lividomycins), S. hofuensis (seldomycin complex), and S. canus (ribosyl paromamine).

Preferred host cells of restriction less strains of Streptomyces taxa that produce macrolide antibiotics and in which the present vectors are especially useful and are transformed, include restrictionless cells of, for example: S. caelestis (antibiotic M188), S. platensis (platenomycin), S.

rochelvar. voiubllis (antiblotic T2636), S. venezuelae (methymycins), S. griseofuscus (bundlin), S.

narbonensis (josamycin, narbomycin), S. fungicidicus (antibiotic NA-181), S. griseofaciens (antibiotic PAl 33A, B), S. roseocitreus (albocycline), S. bruneogriseus (albocycline), S. roseochromogenes (albocycline), S. cinerochromogenes (cineromycin B), S. albus (albomycetin), S. felleus (argomycin, picromycin), S. rochei (lankacidin, borrelidin), S. violaceoniger (lankacidin), S. griseus (borrelidin), S.

maizeus (ingramycin), S. albus var. coilmyceticus (coieimycin), 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. espinomyceticus (espinomycins), S. furdicidicus (mydecamycin), S.

eurocidicus (methymycin), S. griseolus (griseomycin), S. flavochromogenes (amaromycin, shincomycins), S. fimbriatus (amaromycin), S. fascisulus (amaromycin), S. erythreus (erythromycins), S. antibioticus (oleandomycin), S, 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), 5. 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 p-lactam antibiotics and in which the present vectors are especially useful and are transformed, include restrictionless cells of, for example: S. lipmanii (Al 6884, MM4550, MM 13902), S. clavuligerus (Al 6886B, clavulanic acid), S. Iactamdurans (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 (C2081X); S. cinnamonensis, S. fimbriatus, S. halstedii, S. rochei and S. viridochromogenes (cephamycins A, B); S. cattleya (thienamycin); and S. olivaceus, S.

flavovirens, S. flavus, S. fulvoviridis, S. argenteolus, and S. sioyaensis (MM 4550 and MM 13902)..

Preferred host cells of restrictionless strains 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, DE3936), A120A, A28695A and B, etheromycin, dianemycin), S. griseus (grisorixin), S. conglobatus (ionomycin), S. eurocidicus var. asterocidicus (laidlomycin), S. lasaliensis (lasalocid), S.

ribosidificus (lonomycin), S. cacaoi var. asoensis (lysocellin), S. cinnamonensis (monensin), S.

aureofaciens (narasin), S. gallinarius (RP 30504), S. longwoodensis (lysocellin), S. flaveolus (CP38936), S. mutabilis (S-1 1743a), 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, avaparcin), 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 transfromed 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,pJL125, pJL180, pJL190, pJL192, pJL195, pJL197, pJL199 and pHJL212 and preferred transformants are Streptomyces griseofuscus/pJL1 14, S. griseofuscus/pJL121 , S.

grlseofuscus/pJLl 25, S. griseofuscus/pJL 180, S. grlseofuscus/pJLl 90, S. grlseofuscuslpjLl 92, S.

grlseofuscus/pJL1 95, S. grlseofuscus/pJLl 99, S. grlseofuscus/pJL1 97, S. griseofuscus/pHJL2 12, E.

coli K 12 C6OORk-Mk-pJL 114,E, coli K12 C6OORk-Mk-/pJL121,E. coli K12 C6OORk-Mk/-pJL125,E.

coli K12 C6OOPk-Mk-/pJL180, E. coli K12 C600Rk- Mk-/pJL190, E. coli K12 C6OORk-Mk-/pJL192, E.

coli K1 2 C6OORk-Mk-/pJL195, E. coli Kl 2 C6OORk-Mk-/pJL199, E. coli Kl 2 C6OORk-Mk-/pHL197, and E. coli K12 C6OORk-Mk-/pHJL212. Moreover, of this preferred group, plasmids pJL190, pJL192, pJLl 95, pJL1 97, pJL1 99 and pJL , and transformants S. griseofuscus/pJL190, S.

grlseofuscus/pJLl 92, S. grlseofuscus/pJL1 95, S. griseofuscs/pJL197. S. griseofuscus/pJL1 99, S.

griseofuscus/pHJL212.E. coli K12 C6OORk-Mk-/pJL190,E. coli K12 C6OORk-Mk-/pJL192,E. coli K12 C6OORk-Mk-/pJL1 195 and E. coli Kl 2 C6OORk-Mk-/pJL197, E. coli K1 2 C6OORk-Mk-/pJL199 and E. coli K12 C6OORk-M,-pHJL212 are most preferred. Streptomyces griseofuscus is a preferred host because it does not contain an endogenous plasmid or synthesize an antibiotic. Therefore, transform ants 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 can be shuttled into E. colt 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 amplification and manipulation of plasmids can be done faster and more conveniently in E. colithan 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 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 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 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 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. 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 transformants containing the non-selectable DNA can be isolated by appropriate antibiotic or other phenotype selection.Such non-selectable DNA segments can be inserted at any site, except within regions necessary for piasmid function and replication, and include genes that specify antibiotic modification enzymes and regulator 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 pJLl 92, at the internal BamHI restriction site of the 7.7 kb 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. This is done by first selecting for M pock 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 pJL180 at, for example, the unique Pstl restriction site, inactivates the ampicillin resistance 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.

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 nonselectable DNA of interest.

The functional test for antibiotic resistance, as described above, can also be used to identify 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 hormone, 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 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, Actaplanin, Narasin, Monensin, Apramycin, Tobramycin, and 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 Streptomyces 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 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 Ml 10, 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 sources include, for example, soy flour, amino acid mixtures, and peptones.

Nutrient inorganic salts are also incorporated and include the customary salts capable of yielding sodium, potassium, ammonia, calcium, phosphate, chloride, sulfate, and like ions. As is necessary for the growth and development of other microorganisms, essential trace elements are also added. Such trace elements are commonly supplied as impurities incidental to the addition of other constituents of the medium.

Streptomyces coelicolor Ml 10 and S. coelicolor A3(2) are grown under aerobic culture conditions over a relatively wide pH range of about 5 to 9 at temperatures ranging from about 1 50 to 400C. 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 culture temperature of about 300C.

Culturing Streptomyces coelicolor M 10 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 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.

Example 1 Isolation of plasmid SCP2* A. Culture of Streptomyces coelicolor M110 A vegetative inoculum of Streptomyces coelicolor Ml 10 (NRRL 1 5041) was conventionally prepared by growing the strain under submerged aerobic 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 300 C. The 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 coellcoior Ml 10 cells were ready for harvest and subsequent isolation of plasmid DNA.

*Trypticase soy broth is obtained from Difco Laboratories, Detroit, Michigan.

**YEMESG comprises .3% yeast extract, .5% peptone, .3% malt extract, 1% dextrose, 34% sucrose, .1% MgCl2, and.l% glycine.

B. Plasmid isolation About 10 g (wet wgt) of Streptomyces coelicolor Ml 10 cells were harvested by centrifugation (10 minutes, 40C, 10,000 rpm) and then about 10 ml/g wet wgt cells of TES buffer (.01 M Tris(hydroxymethyl)aminoethane [tris], .001 M EDTA, 25% sucrose, pH 8) was added. The cells were vortexed into suspension followed by addition of 10 ml/g wet wgt cells of .25 M EDTA, pH 8 and then 5 ml/g wet wgt cells of lysozyme (10 mg/ml in TES). After the mixture was incubated at 370C for about 15 minutes, about 1.5 ml/g wet wgt cells of 20% SDS (sodium lauryl sulfate (BDH Chemicals Ltd.

Poole, England), was added. The resultant mixture was allowed to stand at room temperature for 30 minutes, and then 5 M NaCI was added to give a final concentration of 1 M NaCI. 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 volumes of isopropyl alcohol. The DNA precipitate was collected by centrifugation (15 minutes, 40C, 10,000 rpm). The precipitate was air dried and then resuspended in 1 ml/g wet wgt cells of TE buffer (.01 M Tris, .001 M EDTA). Centrifugation (20 hours, 2O0C, 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 CsCI-DNA solution was stored at-20 C. Prior to use, the DNA was desaited by either Pud 10 (Bio Rad) column exchange with TE or by dialysis against TE. The DNA was precipitated with ethanol by conventional procedures and redissolved in TE.

Example 2 Construction of plasmid pLR1 A. Hindlll digestion of plasmid plJ2 About 20 yl (20 ,ug) of plasmid plJ2 DNA, disclosed in Thompson et al., 1980; Nature 286:525, 5 ul BSA (Bovine Serum albumin, 1 mg/ml), 19 u1 water, 1 u1 of Hindlll (containing 3 New England Bio Labs units) restriction enzyme*, and 5 yI reaction mix** were incubated at 370C for 2 hours. The reaction was terminated by the addition of about 50 l of 4 M ammonium acetate and 200 41 of 95% ethanol.

The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, suspended in 20 l of TE buffer, and frozen at -2O0C 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 Laboraties, Inc.

Elkhart, Indiana 46515 **Reaction mix for Hindlll restriction enzyme was prepared with the following composition: 600 mM NaCI 100 mM Tris-HCI, pH 7.9 70 mM MgCl2 10 mM Dithiothreitol B. Hindlll digestion of plasmid pBR322 About 8 l (4 g) of plasmid pBR322 DNA, 5 jul reaction mix, 5 ,u1 BSA (1 mg/ml), 31 yI water, and 1,ul of Hindlll restriction enzyme were incubated at 37 OC for 2 hours. After the reaction was terminated by incubating at 60 C for 10 minutes, about 50 l of ammonium acetate and 200 l of 95% ethanol were added. The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, and suspended in 45 l of water.

C. Ligation of Hindlll digested plasmids plJ2 and pBR322 About 20 Ul of Hindlll treated plasmid plJ2 (from Example 2A), 201 of Hindlll treated plasmid pBR322 (from Example 2B), 5 U1 BSA (1 mg/ml), 1 l of T4 DNA ligase*, and 5 l ligation mix** were incubated at 1 60C for 4 hours. The reaction was terminated by the addition of about 50 ,u1 4 M ammonium acetate and 200 l 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 pLR1.

*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: 500 mM Tris-HCI, pH 7.8 200 mM Dithiothreitol 100mM MgCl2 10 mM ATP Example Construction of E. coli K12 HBI OI /pLRI About 10 ml of frozen competent E. coli K12 HB101 cells (Bolivaretal., 1977, Gene 2:75-93) were pelleted by centrifugation and then suspended in about 10 ml of .01 M sodium chloride. Next, the cells were pelleted again, resuspended in about 10 ml of .03 M calcium chloride, incubated on ice for 20 minutes, pelleted a third time, and finally, resuspended in 1.25 ml of .03 M calcium chloride. The resultant cell suspension was competent for subsequent transformation.

Plasmid pLR in TE buffer (prepared in Example 2C) was ethanol precipitated, suspended in 150 l of 30 mM calcium chloride solution, and gently mixed in a test tube with about 200 ,u1 of competent E. coli K12 HB101 cells. The resultant mixture was incubated on ice for about 45 minutes and then at 420C for about 1 minute. Next, about 3 ml of L-broth (Bertani, 1951, J. Bacteriology 62:293) containing 50,ug/ml of ampicillin was added. The mixture was incubated with shaking at 370C for 1 hour and then plated on L-agar (Miller, 1 972, Experiments in Molecular Genetics, Cold Spring Harbor Labs, Cold Spring Harbor, New York) containing ampicillin. Surviving colonies were selected and tested for the expected phenotype (AmpR, Tets), and constituted the desired E. coli K12 HB1 01/pLR1 transformants.

Example 4 Construction of plasmid pLR4 A. Partial BamHI digestion of plasmid pLR1 About lOul (10 g) of plasmid pLR1, 5 Ul BSA (1 mg/ml), 29 u1 water, 1 ul of BamHI (diluted 1:4 with water) restriction enzyme, and 5 ul reaction mix* were incubated at 370C for 15 minutes. The reaction was terminated by the addition of about 50 U1 of 4 M ammonium acetate and 200 U1 of 95% ethanol. The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, and suspended in 20 ,ul water.

*Reaction mix for BamHI restriction enzyme was prepared with the following composition: 1.5 M NaCI 60 mM Tris-HCI, pH 7.9 60 mM MgCl2 B. BamHI digestion of plasmid pBR322 The desired digestion was carried out in substantial 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 in 29 ul of water.

C. Ligation of partial BamHI digested plasmid pLR1 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.

Example 5 Construction of E. coli K12 HBI Ol /pLR4 The desired construction was carried out in substantial accordance with the teaching of Example 3 except that plasmid pLR4, rather than plasmid pLR1, was used for transformation. Surviving colonies were selected and tested for the expected phenotype (AmpR, Tets), and constituted the desired E. coli K12 HBl 01/pLR4 transformants.

Example 6 Construction of plasmids pJL120 and pJLI 21 A. EcoRI digestion of plasmid SCP2 About 150 l (5.7 ug) of plasmid SCP2* DNA, 1 ml water, 2 u1 of EcoRI (containing 20 BRL units) restriction enzyme, and 17 l EcoRI reaction mix* were incubated at 370C for 2.5 hours. The reaction was terminated by incubation at 650C for 15 minutes. The reaction was conventionally analyzed by agarose gel electrophoresis (AGE) to verify that restriction was complete. The restricted DNA was stored at 40C for subsequent use.

*Reaction mix for EcoRI restriction enzyme was prepared with the following composition: 500 mM NaCI 1000 mM Tris-HCI, pH 7.5 100mM MgCl2 B. EcoRI digestion of plasmid pBR325 The desired digestion was carried out in substantial accordance with the teaching of Example 6A except that plasmid pBR325, rather than plasmid SCP2*, was used. The resultant DNA was stored at 40C for subsequent use.

C. Ligation of EcoRI digested plasmids SCP2* and pBR325 About 40 l of EcoRI digested plasmid SCP2* (from Example 6A), 10 l of EcoRI digested plasmid pBR325 (from Example 6B), 10 l of MgCI2 (.1 M), 10 l of (NH4)2SO4 (.1 M), 10 l ATP (2 mM) .1 l of T4 DNA ligase, and 20 ul ligation mix* were incubated at 40C for 18 hours. The reaction was analyzed by AGE to verify appropriate ligation. The suspended DNA constituted the desired 35.8 kb 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 pJL120 and pJL12 1 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: 50 mM Tris-HCI, pH 7.5 10 mM ss-mercaptoethanol 1 mM EDTA 50 mg/ml BSA Example 7 Construction of E. coli K12 C600Rk-Mk-/pJL120 and E. coli K12 C600Rk-Mk-/pJL121 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. 71:1030-1034) were subcultured 1:10 in fresh L-broth (disclosed in Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbor Labs., Cold Spring Harbor, New York) and grown at 370C for 1 hour. A total of 660 Klett Units of cells were harvested, washed with 2.5 ml of 100 mM NaCI2, suspended in 1 50 mM CaCI2 with 10% glycerol, and incubated at room temperature for 20 minutes. The cells were harvested by centrifugation, resuspended in .5 ml of CaCl2-glycerol, chilled on ice for 3-5 minutes and frozen. The suspensions of cells were stored in liquid nitrogen until use. Preservation and storage did not adversely affect the viability or frequency of transformation by covalently closed circular DNA.

B. Transformation 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 ssl of the sample disclosed in Example 6C and 40 Ml of .1 XSSC (.01 5 M NaCI, .0015 M Sodium Citrate at pH 7). The transformation mixture was chilled on ice for 20 minutes, heat shocked at 420C for 1 minute and chilled on ice for 10 minutes. The samples were then diluted with .85 ml of Lbroth, incubated at 370C for 1.5 hours, spread on L-agar containing ampicillin (50 yg/ml) and tetracycline (12.5 yg/ml) and incubated for 18 hours at 370C.The resultant colonies were selected and tested for the expected phenotype (AmpR, TetR, CM9), and constituted the desired E. coli K12 C600Rk- Mk-/pJL120 and E. coli K12 C600Rk-Mk-/pJL121 transformants. The ampicillin 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 pJL121 according to known procedures.

Example 8 Construction- of plasmids pJL180 and pJL181 A. Sail digestion of plasmid SCP2* and isolation of 6.0 kb San fragment The desired digestion was carried out in substantial accordance with the teaching of Example 6 except Sall 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 650C for 1 5 minutes. The resultant Sall 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 light.The gel fragment containing the 6.0 kb fragment of interest was excised from the gel and electroeluted into TBE buffer (1.6% Sigma 7-9 buffer**, .093% Na2EDTA, .55% boric acid). The gel-fragment in TBE buffer was 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 (.1 M KCI, 10 mM Tris-HCI, pH 7.8). The column was washed with 2.5 ml of equilibration buffer and the DNA (about 5 ug) was eluted with 1.5 ml of elution buffer (1 M NaCI, 10 mM Tris-HCI, pH 7.8).The eluent was adjusted to about .35 M with respect to Na+ ion concentration, and then the DNA was precipitated by adding 2 volumes (about 9 ml) of 100% ethanol followed by cooling to -200C for 16 hours. The DNA precipitate was pelleted by centrifugation, washed with 75% ethanol, dried, and dissolved in TE buffer. Hereinafter, this conventional isolation technique is referred to as AGE/DE52/electroelution.

*Reaction mix for Sall restriction enzyme was prepared with the following composition: 1500 mM NaCI 80 mM Tris-HCI, pH 7.5 60 mM MgCl2 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.

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 40C for future use.

C. Ligation of Sail digested plasmid pBR325 and 6.0 kb Sail fragment of plasmid SCP2* About 1.5 g of the 6.0 kb Sail fragment of SCP2*, prepared in Example 8A, was mixed with .5 g of Sa# digested pBR325, prepared in Example 8B. The DNA mixture was precipitated by stard ethanol precipitation and redissolved in 3 l of distilled water, 4 l of 66 M ATP, 2 l of ligasekinase mixture (.25 M Tris HCI, pH 7.8, 50 mM MgCl2, 25 mM dithiothreitol and 25% glycerol) and 1 l of T4-DNA ligase (1 unit). After inculbation four at 15 C, the reaction mixture was diluted with 12 l of water, 20 l of 66 M ATP, 8 l of ligase-kinase mixture and then incubated at 15 C for 18 hours.The resultant ligated DNA was diluted 1:5 into.1 XSSC and constituted the desired #12.0 kb plasmids pJL1 80 and pJL181.

Recombinant plasmids of two orientations result because the plasmid pBR325 Sa# fragment can be oriented in either direction. A restriction site map of each of plasmids pJL1 80 and pJL181 is presented in Figure 3 of the accompanying drawings.

Example 9 Construction of E. coli K12 C6OORk-Mk-/pJL-180 and E. coli K12 C600Rk-Mk-/pJL181 The desired constructions were made in substantial accordance with the teaching of Example 7 except that the mixture of plasmid pJL1 80 and pJL181 DNA (from Example 8C), rather than plasmid pJL120 and pJL121, were used. The resultant transformant colonies were selected and tested for the expected phenotype (AmpR, Tets, CMR), and constituted the desired E. coli K12 C600Rk-Mk-/pJL180 and E. coli K12 C600Rk-Mk-/pJL181 transformants. The ampicillin and chloramphenicol 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 can then be used for subsequent production and isolation of plasmids pJL1 80 and pJL181 according to known procedures.

Example 10 Construction of plasmid pJL125 A. Sail digestion of plasmid pJL121 and isolation of #10.2 kb 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 was complete and except that plasmid pJL1 21, rather than plasmid SCP2*, was used. The resultant Sall 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 kb Sail fragment of plasmid pJLI 21 The desired ligation was carried out in substantial accordance with the teaching of Example 8C except that the Sall fragments of plasmid pJL121, rather than the Sall 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 5.4 kb origin of replication-containing EcoRI-Sall fragment of plasmid SCP2*, was dissolved in TE buffer and stored at 40C 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 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 and pJL121, was used. The resultant colonies were selected and tested for the expected phenotype (AmpR, Tets, CMs) and constituted the desired E. coli K12 C6OORk-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.

Example 12 Construction of plasmid pJL1 90 A. EcoRI-Hindlll digestion of plasmid pJLI 21 and isolation of 19.0 kb EcoRI-Hindlil fragment About 200 all (80 ug) of plasmid pJL121 DNA, 3OuI BSA (1 mg/ml),40,ui of Hindlll (containing 200 BRL units) restriction enzyme, and 30 yI Hindlll reaction mix# were incubated at 37 OC for about 3 hours and then at 650C for 10 minutes.The 300 ul reaction mixture was cooled to 40C, supplemented with 110 l of lOx Hindlll#EcoR1 diluent reaction mix** and 30 iul EcoRI restriction enzyme (containing 300 BRL units), and then incubated at 370C for 3 hours, then at 650C for 10 minutes followed by cooling to 40C. The resultant #19.0 kb EcoRI-Hindill restriction fragment was conventionally isolated by AGE/DE52/electroelution. The desired DNA was dissolved in TE buffer and stored at 40C for future use.

*Hindlil reaction mix was prepared with the following composition: 60 mM Tris-HCI, pH 7.5 500 mM NaCI 60 mM MgCl2 **HindIII#EcoR1 diluent was prepared with the following composition: 382 mM Tris-HCI, pH 7.5 50 mM NaCI 22 mM MgCl2 B. EcoRI-Hindlll digestion of plasmid pLR4 and isolation of 7.7 kb EcoR1-HindIII fragment The desired digestion and isolation was carried out in substantial accordance with the teaching of Example 1 2A except that plasmid pLR4, rather than plasmid pJL1 21, was used. The desired 7.7 kb fragment was dissolved in TE buffer and stored at 40C for future use.

C. Ligation of 19.0 kb EcoRI-HindIII fragment of plasmid pJL121 and 7.7 kb EcoRI-HindIII fragment of plasmid pLR4 The desired ligation was carried out in substantial accordance with the teaching of Example 8C except that the 19.0 kb EcoRI-Hindlll fragment of plasmid pJL121 and the 7.7 EcoRI-Hindlll fragment of plasmid pLR4, rather than the 6.0 kb Sa# fragment of plasmid SCP2* and Sall digested pBR325, were used. The resultant ligated DNA constituted the desired plasmid pJL1 90 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. coliK12 C6O0Rk-Mk-.

Example 13 Construction of E. coli K12 C600Rk-Mk-/pJL190 The desired construction was made in substantial accordance with the teaching of Example 7 except that plasmid pJL1 90, rather than plasmids pJL1 20 and pJL12 1, was used. The resultant colonies were selected and tested for the expected phenotype (AmpR, Tets) and size by conventional means (as in Example 11) and constituted the desired E. coli K12 C6OORk-Mk-Mk-/pJL190 transformants.

The transformants were then conventionally cultured for subsequent production and isolation of plasmid pJLl 90 according to known procedures.

Example 14 Isolation of plasmid pJL192 Plasmid pJL1 92, which confers high resistance to antibiotic neomycin (10 g/ml), can be conventionally isolated from E. coli K12 C6OORk-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 1 5040. The restriction site map of plasmid pJL192 appears not to be distinguishable from the plasmid pJL190 map presented in Figure 4.

Example 15 Construction of plasmid pJL195 A. EcoRI-Sa# digestion of plasmid pJL125 and isolation of 5.4 kb EcoRI-Sa# fragment The desired digestion and isolation was carried out in substantial accordance with the teaching of Example 1 2A except that plasm id pJL125 and Sa/l restriction enzyme and reaction mix, rather than plasmid pJL121 and Hindlll restriction enzyme and reaction mix, were used. In addition, SalloEcoRI diluent* was used. The resultant 5.4 kb EcoRI-Sall fragment was dissolved in TE buffer and stored at 4 C for future use.

*Sa/leEcoRI diluent was prepared with the following composition: 940 mM Tris-HCI, pH 7.5 55 mM MgCI2 B. EcoRI-partial Sail digestion of plasmid pLR4 and isolation of 7.5 kb EcoRI-partial San fragment The Sa# digestion was carried out in substantial accordance with the teaching of Example 1 2A except plasmid pLR4, rather than pJL121, was used. Since only a partial Sa/l digestion was desired, the resultant mixture was incubated first at 370C for 15 minutes and then at 650C for 10 minutes.

Following cooling to 40C, the resultant partial Sall 7.7 kb linear fragment was conventionally isolated by AGE/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 the Sa# fragment of SCP2*, was used. The desired 7.5 kb EcoRI-Sall fragment (the largest possible EcoRI-Sall fragment) was isolated by AGE/DE52/electroelution, dissolved in TE buffer, and then stored at 40C for future use.

C. Ligation of -5.4 kb EcoRI-San fragment of plasmid pJL125 and 7.5 kb EcoRI-partial Sail fragment of plasmid pLR4 The desired ligation was carried out in substantial accordance with the teaching of Example 8C except that the 5.4 kb EcoRI-Sall fragment of plasmid pJL125 and the 7.5 kb EcoRI-partial Sall fragment of plasmid pLR4, rather than the 6.0 kb Sall fragment of-plasmid SCP2* and Sall digested pBR325, were used. The resultant ligated DNA constituted the desired plasmid pJL195 and was stored at 40C for future use. A restriction site map of plasmid pJLl 95 is presented in Figure 5 of the accompanying drawings.The restriction site map was determined with plasmid isolation from E. coli K12 C600Rk-Mk-.

Example 16 Construction of E. coli K12 C600Rk-Mk-/pJL195 The desired construction was made in substantial accordance with the teaching of Example 7 except that plasmid pJLl 95, rather than plasmids pJLl 20 and pJL121 , was used. The resultant colonies were tested for the expected phenotype (AmpR, Tets) and size (as in Example 1 1 ) and constituted the desired E. coli K12 C600Rk-Mk-/pJL195 transformants. The transformants were then conventionally cultured for subsequent production and isolation of plasmid pJL195 according to known procedures.

Example 17 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 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 1 5042), 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 40C for subsequent use.

*Reaction mix for BamHI restriction enzyme was prepared with the following composition: 1000 mM Tris-HCI, pH 7.4 100 mM MAC12 B. Ligation of BamHI digested plasmid SCP2 and BamHI digested plasmid pBR322 The desired ligation was carried put in substantial accordance with the teaching of Example 6C except that the BamHI digest of plasmid SCP2 (prepared in Example 1 7A) and BamHI-digested plasmid pBR322 (prepared in Example 4B), rather than plasmids SCP2* and pBR325, were used. The resultant DNA was stored at 40C and constituted the desired 34.6 kb plasmid pJL1 14.

Example 18 Construction of E. coli K12 C600Rk-Mk-/pJL114 The desired construction as made in substantial accordance with the teaching of Example 7 except that plasmid pJL1 14, rather than plasmids pJL120 and pJL1 21 , was used. The resultant colonies were selected and tested for the expected phenotype (AmpR, Tets), and constituted the desired E. coli K12 C6OORk-Mk-pJL1 14 transformants. The ampicillin resistant, tetracycline sensitive colonies were isolated according to known procedures, cultured, and then conventionally identified by restriction enzyme and 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 SCP2 during the digestion described in Example 1 7A. Since this event is rare and has not been repeated, E. coli K12 C600Rk-Mk-/pJL1 14 has been 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 pJL1 14. A restriction site map of plasmid pJL1 14 is presented in Figure 5 of the accompanying drawings.

Example 19 Construction of Streptomyces grlseofuscus/pJLI 20 A. Growth of cultures for preparation of protoplasts A vegetive innoculum was conventionally prepared by growing the strain under submerged conditions for 20 hours at 300C in TSB supplemented with .4% glycine. The culture was homogenized and innoculated at a 1/20 dilution into the same medium and then grown for 18 hours at 300C.

B. Transformation Using about 20 jug of plasmid pJL120 DNA and 1 x109 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 available 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/BG79/00095) No. W079/01169, Example 2.

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 was added to the plate and the surface of the 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 preparation were made down to 10-5 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 allquots of some of the dilution series (e.g. 10-1, 10-2, 10-4) 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 transformation 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 300C by the appearance of "pocks", a property expressed by spores containing the plasmid in expressible form within the lawn.The 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 3O0C and the cells are harvested followed by isolation of DNA. Isolation is as disclosed in Example 1 B except that centrifugation with CsCI and propidium iodide is omitted. Subsequently, iul of this DNA is used to transform E. coli K12 C600Rk Mk- as disclosed in Example 7B. Plasmids in the transformants are verified and identified by conventional means as taught in Example 11.

Example 20 Construction of Streptomyces griseofuscus/pJL114,S. griseofus/JL121,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 20 except that plasmids pJLl 14, pJL121, pJL125, pJL1 80, and pJL1 81, rather than plasmid pJLl 20, were appropriately used for the individual construction.

Example 21 Construction of Streptomyces griseofuscus/pJL190 A. Transformation The desired transformation was carried out in substantial accordance with the teaching of Example 19B except that plasmid pJL190, rather than plasmid pJL120, was used.

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 jug/ml. The resultant Streptomyces griseofuscus/pJL190 transformants were then tested for the expected pock morphology in substantial accordance with the procedure of Example 20B.

Example 22 Construction of Streptomyces griseofuscus/pJL1 95 The desired construction was made, selected, and recovered in substantial accordance with the teaching of Example 21 except that plasmid pJL195, rather than plasmid pJL1 90, was used.

Example 23 Construction of Streptomyces griseofuscus/pJL192 The desired construction was made, selected, 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 Mg/ml, rather than plasmid pJL195 and top agar containing sufficient neomycin to bring the final plate concentration to 1 yg/ml, were used.

Example 24 Construction of Streptomyces fradiae/pJL120, S. fradiae/pJL114,S. fradiae/pJL121,S.

fradiae/pJL125,S. fradiae/pJL180, S. fradiae/pJL181,S. fradiae/pJL190,S. fradiae/pJL195, and S. fradiae/pJL192 The desired constructions are individually and respectively made, selected, and recovered in substantial accordance with the respective teachings of Examples 1 9, 20, 21, 22, and 23 except that Streptomyces fradiae, rather than S. griseofuscus, is used. In addition, the TSB medium for protoplasting and growing S. fradiae was modified and contained only .2% glycine.

Example 25 Construction of Streptomyces lividans/pJL120,S. lividans/pJL114,S. lividans/pJL121,2.

lividans/pJL125,S. lividans/pJL180,S. ,lividans/pJL181,S. lividans/pJL190,S. lividans/pJL195, and S. lividans/pJL192 The desired constructions are individually 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. lividans is as described in International Publication (of International Patent Application No.

PCT/BG79/00095) No. W079/01169, Example 2.

Example 26 Isolation of plasmid pJL192 mutant that confers high resistance to antibiotic neomycin Streptomyces grlseofuscus/pJL1 90 was isolated as described in Example 21. Analysis of growth of colonies on nutrient agar supplemented with different concentrations of neomycin revealed that S.

griseofuscus/pJL190 exhibited resistance to 1.0 g/ml of neomycin. S. griseofuscus was spread conventionally on nutrlent agar plates supplemented with 10 g/ml of neomycin. A colony was discovered that exhibited growth at this high level of neomycin. After repeated analysis verified that the colony exhibited the aforementioned resistance, the colony was designated S. grlseofuscus/pJL1 92.

The polasmid, pJL192 was shuttled into E. coli K12 C600Rk-Mk- by back transformation as taught in Example 19C. The restriction site map of pJL192 appears not to be distinguishable from pJL1 90.

Example 27 Isolation of plasmid pJL199 mutant that confers high resistance to antibiotic neomycin The desired isolation is carried out in substantial accordance with the teaching of Example 26 except that Streptomyces griseofuscus/pJL195 95 (prepared in Example 22), rather than S.

grlseofuscus/pJL1 90, was used. A colony that exhibited high resistance to neomycin was designated S. grlseofuscus/pJL1 99. The plasmid, pJL1 99, was shuttled into E. coli K12 C6OORk-Mk- by back transformation as taught in Example 19C. The restriction site map of pJL199 appears not to be distinguishable from pJL1 95.

Those skilled in the art will recognize that plasmid pJL199 can also be conventionally constructed by substituting the neomycin resistance-conferring fragment of plasmid pJL192 (prepared in Examples 1 5 and 27) for the pLR4-derived neomycin resistance-conferring fragment of plasmid pJL1 95. Such a substitution thus also results in the desired plasmid pJL1 99.

Example 28 Construction of plasmid pLR2 A. Hindlll digestion of plasmid plJ6 About 20 ul (20 ug) of plasmid plJ6 DNA, disclosed in Thompson et ai., 1980, Nature 286:525, 5 l BSA (Bovine Serum albumin, 1mg/ml), 19 l water, 1 l of HindIII (containing 3 New England Bio Labs units) restriction enzyme*, and 5 l reaction mix** were incubated at 37 C for 2 hours. The reaction was terminated by the addition of about 50 L of 4 M ammonium acetate and 200 L of 95% ethanol. The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, suspended in 20 L of TE buffer, and frozen at -200C for storage.

B. HindIII digestion of plasmid pBR322 About 8 ul (4 ug) of plasmid pBR322 DNA, 5 ul reaction mix, 5 ul BSA (1 mg/ml), 31 ul water, and 1 ul of Hindlll restriction enzyme were incubated at 37 OC for 2 hours. After the reaction was terminated by incubating at 600C for 10 minutes, about 50 l of ammonium acetate and 200 ,zl of 95% ethanol were added. The resultant DNA precipitate was washed twice in 70% ethanol, dried in vacuo, and suspended in 45 l of water.

C. Ligation of HindIII digested pklasmids pJL6 and pBR322 About 20 l of HindIII treated plasmid pJL6 (from Example 2A), 20 l of HindIII treated plasmid pBR322 (from Example 2B), 5 l BSA (1 mg/ml), 1 l of T4 DNA ligation mix** were incubated at 1 60C for 4 hours. The reaction was terminated by the addition of about 50 ul 4 M 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.

Example 29 Construction of E. coli K12 HB101/pLR2 About 10 ml of frozen competent E. coli K12 HB101 cells (Bolivar et al., 1977, Gene 2:75-93) were pelleted by centrifugation and then suspended in about 10 ml of 0.01 M sodium chloride. Next, the cells were pelleted again, resuspended in about 10 ml of 0.03 M calcium chloride, incubated on ice for 20 minutes, pelleted a third time, and finally, resuspended in 1.25 ml of 0.03 M calcium chloride.

The resultant cell suspension was competent for subsequent transformation.

Plasmid pLR2 in TE buffer (prepared in Example 2C) was ethanol precipitated, suspended in 150 L of 30 mM calcium chloride solution, and gently mixed in a test tube with about 200 l of competent E. coli K12 HB101 cells. The resultant mixture was incubated on ice for about 45 minutes and then at 420C for about 1 minute. Next, about 3 ml of L-broth (Bertani, 1951, J. Bacteriology 62:293) containing 50 g/ml of amplicilin 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 Genetlcs, Cold Spring Harbor Labs, Cold Spring Harbor, New York) containing ampicillin. Surviving colonies were selected and tested for the expected phenotype (AmpR, Tets), and constituted the desired E. coli K12 HB101/pLR2 transformants.

Representative plasmids and transformants that can be constructed in accordance with the foregoing teaching include the following listed below in Tables 1 and 2.

Table 1 Representative plasmids Example Plasmid #Size E.coli Streptomyces Construction No. Name in kb marker marker 30 pJL122 24.4 AmpR,TetR M SstI Deletion of pJL120 31 pJL123 27.8 AmpR,TetR M Bg#II Deletion of pJL121 32 pJL124 21.1-24.2 AmpR,TetR M Partial Pastl Deletion of pJL120 33 pJL126 9.1-10.6 AmpR,TetR M Partial Xorll Deletion of pJL120 34 pJL176 32.5 CMR,TetR P* #16.6 kb Pstl and extraveous #10.0kb Pstl of SCP2* into Pstl Site of pBR325 35 pJL1200 35.8 AmpR,TetR M SCP2 Ecol Site into pBR325 EcoRI Site 36 pJL1201 35.8 AmpR,TetR M Reverse Orientation of pJL1200 37 pJL1202 24.4 AmpR,TetR M Sstl Deletion of pJL1200 38 pJL1203 27.8 AmpR,TetR M Bg#II Deletion of pJL1201 39 pJL1204 21.1-24.2 AmpR,TetR M Partial Pstl Deletion of pJL1200 40 pJL1205 10.2 AmpR,

M Sa# Deletion of pJL1201 41 pJL1206 9.1-10.6 AmpR,TetR M Partial Xorll Deletion of pJL1200 42 pJL1706 32.5 CMR,TetR P #10.6 kb Pstl and extraneous 43 pJL1716 22.5 CMR,TetR P #10.0 kb Pstl of SCP2 into Pstl Site of pBR325 44 pJL3176 22.5 CMR,TetR P* #16.6 Pstl of SCP2* into Pstl Site of pBR325 45 pJL1800 12.6 AmpR,CMR P #6.0 kb Sa# of SCP2 into Sa# Site of pBR325 46 pJL1801 12.6 AmpR,CMR P Reverse Orientation of pJL1800 47 pJL1900 25.9 AmpR M,NeoR #19.0 kb EcoRI-HindIII of pJL1201 and #7.7 kb EcoRI-HindIII of pLR4 Table 1 (contd.) Representative plasmids Example Plasmid #Size E.coli Streptomyces Construction No.Name in kb marker marker 48 pJL1902 25.9 AmpR M,NeoR #19,0 kb EcoRI-HindIII of pJL1201 and#7.7 kb EcoRI-HindIII of pLR4 49 pJL1905 12.1 AmpR M,NeoR #5.4 kb EcoRI-Sa# of pJL1205 and #7.5 kb EcoRI-Partial Sa# of pPR4 50 pJL193 6.9 AmpR ThioR #1.35 kb BamHI of pLR 2 into BamHI of pBR328 51 pJL196 12.1 AmpR M,NeoR #5.4 kb EcoRI-Sa# of pJL125 into #7.5 kb EcoRI-partial Sa# of pJL192 52 pJL197 13.1 AmpR M,NeoR,ThioR #1 kb Bc# of pJL 193 into partial BamHI of pJL 196, onentation such that the Clal site of the #1 kb Bc# fragment is proximal to the Sa# site of the pBR322 fragment and such that the Sa# site of the #1 kb Bc# site is proximal to the neomycin resistance gene 53 pJL1907 13.1 AmpR M,NeoR,ThioR #1 kb Bc# of pJL193 into partial BamHI of pJL 1905 54 pJL198 13.1 AmpR M,NeoR,ThioR Same as pJL197 except the orienta tion of the Bc# fragment is reversed 55 pHJL212 13.9 AmpR M,NeoR,ThioR #1 kb Bc# fragment of pJL193 into partial BamHI of pJL195, orientation and insertion the same as pJL198 56 pHJL213 13.9 AmpR M,NeoR,ThioR Same as pHJL212 except the orienta tion and insertion the same as pJL197 Table 2 Representative transformants 1. Streptomyces R/pR wherein Ris griseofuscus, ambofaciens, fradiae, or lividans and R is independentyl pJL122, pJL123, pJL124, pJL126, pJL176, pJL1200, pJL1201, pJL1202, pJL1203, pJL1204, pJL1205, pJL1206, pJL1706, pJL1800, pJL1801, pJL1900, pJL1902, pJL1905, pJL196, pJL197, pJL1907, pJL198, pHJL212 and pHJ213.

2. E. coli K12 R/pR wherein R is C6OORk-Mk-294, C6OO or RV308 and R is independently as defined above.

Claims (34)

Claims

1. A recombinant DNA cloning vector 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.

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 #5.4 kb EcoRI-Sa# fragment, #6.0 kb Sa# fragment, #19 kb EcoRI-HindIII fragment, or #31 kb 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.

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.

6. The vector of any of Claims 1 to 5, wherein the one or more DNA segments that confer resistance in Streptomyces are DNA segments that confer resistance to neomycin or thiostrepton.

7. The vector of Claim 6 which is plasmid pJL1 80, pJL181, pJL1 25, pJL1 90, pJL1 92, pJL1 95, pJL199, pJL122, pJL123, pJL124, pJL126, pJL176, pJL1200, pJL1201, pJL1020, pJL1203, pJL1204, pJL1205, pJL1206, pJL1706, pJL1800, pJL1801, pJL1900, pJL1902, pJL1905, pJL193, pJL1 96, pJL1 97, pJL1 98, pHJL212, pHJL21 3, or pJL1 907.

8. The vector of any of Claims 1 to 7, wherein the DNA segment that confers resistance to an antibiotic is the #7.7 kb EcooRI-HindIII restriction fragment of plasmid plasmid pJL192, the #7.7 kb EcoRI HindIII restriction fragment of plasmid pLR4, the #7.5 kb EcoRI-partial Sa# restriction fragment of plasmid pLR4, the #1.35 kb BamHI restriction fragment of plasmid pLR2, or the #1 kb Bc# restriction fragment of plasmid pJL193.

9. The vector of Claim 8 which is a plasmid pJL1 90 or pJL195 high resistance mutant that in Streptomyces confers resistance to neomycin at levels of at least 10 g/ml.

10. Plasmid pJL1 14.

11. Plasmid pJL1 20.

12. Plasmid pJL1 21.

13. Plasmid pJL192.

14. Plasmid pJL193.

15. Plasmid pJL1 97.

16. Plasmid pJL1 98.

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* #5.4 kb EcoRI-Sa# or #6.0 kb Sa# restriction fragment of Claim 3.

23. A restriction fragment comprising the plasmid pJL1 92 #7.7 kb EcoRI-Hindlil 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* and one or more DNA sequences comprising: 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. coll, 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 5.4 kb EcoRI-Sail fragment, ""6.0 kb Sail fragment, ""19 kb EcoRI-Hindill fragment, or ""31 kb EcoRI fragment and wherein the E. collorigin 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, and 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 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 2) growing said Streptomyces cells on a lawn of an indicator Streptomyces strain and selecting colonies that show the M pock phenotype.

27. The method of Claim 26, wherein the restriction fragment comprising the origin of replication and the P gene is of plasmid SCP2*.

28. The method of Claim 26 or 27, wherein the Streptomyces cells and indicator strain are preferably of species lividans, griseofuscus, fradiae or ambofaciens.

29. The method of Claim 26, 27 or 28, wherein the recombinant DNA cloning vector is plasmid pJL1 20, pJL1 21, pJL1 25, pJL1 90, pJL1 92, pJL1 95, pJL1 97, pJL1 98, pHJL21 2, or pHJL21 3.

30. A recombinant DNA cloning vector substantially as hereinbefore described with particular reference to Examples 8, 10, 12, 15, 26, 27 and 30 to 56.

31. A restriction fragment substantially as hereinbefore described with particular reference to Examples 8a and 12b.

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.

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 and 30 to 56.

34. A method for detecting transformants substantially as hereinbefore described with particular reference to Example 19C.