CA1207685A - Chimeric cloning vectors for use in streptomyces and e. coli - Google Patents

Abstract

Abstract The present invention discloses chimeric plasmid SCP2 and SCP2* cloning vectors that are useful in Streptomyces and E. coli. The invention further discloses transformants and a method for detecting transformants of the aforementioned vectors.

Description

~ZS3'~6~S

Chimeric Cloning Vectors For Use In Streptomyces And E. coli The present invention relates to selectable novel recombinant DNA cloning vectors comprising a functional origin of replication-containing restriction fragment of plasmid SCP2 or SCP~* and a functional origin of replication-containing and antibiotic re-sistance-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 : 15 _. coli. Since over hal of the clinically importan-t 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-myces both for increasing the yields oE known anti-b.iotics as well as for the production of new anti biotics and antibiotic derivatives~
The method of the present invention provides for the convenient selection of transformantsO Since transformation is a very low ~requency event, such a runctional test is a practical necessity for deter-mining which cell(s), of among the millions of cel~s, has ac~ ired the plasmid DWA. This is important because DNA sequences that are non~selectable can be inserted into the vectors and, upon transformation, X-5773A -~

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 ~re as defined below.
Plasmid pLRl or pLR4 3.4kb ~amHI Res~riction Fragment - the same 3.4kb BamHI neomycin resistance-conferring fragment contained in plasmid pIJ2.
AmpR - the ampicillin resistant pheno~yp~
pS _ the ampicillin sensitive phenotyp~.
Tet - the tetracycline resistant phenotype.
TetS - the tetracycline sensitive phenotype.
CMR - the chloramphenicol resistant phenotype.
CMS - the chloramphenicol sensitive phenotype.
Neo ~ the neomycin resistant phenotype.
Neo - the neomycin sensitive phenotype.
ThioR - the thiostrepton resistant phenotype.
ThioS - the thiostrepto,n sensitive phenotype.
Neo , NeoS, Thio , and ThioS refer only to results of tests in Streptomyces as used in this dis-`closure. AmpR, AmpS, TetR, TetS, CmR and CmS refer only to results of tests in ~. coli as used in this disclosure.
~5 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*, 3~76~

X-5773A -3~

b) a restriction fragment comprising an Eo coli origin of replication, c) one or more DNA segments that confer rasis-tance to at least one antibiotic when trans-formed into a cell of E. coli, said cell being sensi~ive 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 restric-tion fragmen-t of plasmid SCP2 or SCP2* and one or more DN~ sequences comprising:
a) a restriction fragment comprising an E~ coli origin of replication, b) one or more DNA segments that confer resis-tance to at least one antibio*ic when trans-formed 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 ; 30 antibiotic when transformed into a cell of i2~3t,lt;~5 S~reptomyces, said cell being sensitive to the antibiotic for which resistance is con-~erred.
The invention further comprises trans~ormants ~nd a method for detecting transformants of the afore-mentioned vectors comprising:
l) mixing Streptomyces cells, under transform ing conditions, with a recombinant DNA clon-ing vector, said vector comprising a) an origin of replication and P gene~
containing restriction ragment of plasmid SCP2 or SCP2*, and b) a non-lethal DNA sequence cloned into the EcoRI restriction sit.e of said P
~ gene, and

2) growing said Streptomyces cells on a lawn of an indicator Streptomyces strain and select-ing colonies that show the M pock phenotype~
The vectors of the present invention are 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 ragment of an Eo coli plasmid. Plasmids SCP2 and SCP2*, from which origins of replication are constructed, are each ~31kb and show similar restriction patternsO 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 iden tical.

t~6~3~

X-5773A 4a-In the drawings appended to this specification:
Figure 1 is a detailed restriction site map of plasmids SCP2 and SCP2*~
Figure 2 is a restriction site map of each of 5 plasmids pJL120 and pJL121;
Figure 3 is a restriction site map of each of plasmids pJL180 and pJL181;
Figure 4 is a restriction site map of plasmid pJL125 and is also a restriction site and functional map 10 of plasmid pJLl90; and Figure 5 is a restriction site map of plasmids pJL195 and pJL114.

.
~ .
~ ,,`, s X-5773A _5 Since the present disclosure teaches that the Streptomyces tra function and the origin of repli--cation of plasmids SCP2 and SCP2* are within their respective ~5.4kb EcoRI-SalI restriction fragments, a 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-SalI region. A detailed restriction site map of plasmid SCP2* (and thus also pla~mid SCP2) is presented in Figure 1 of ~he accompanying drawings.
Plasmids SCP2 and SCP2* can be conventionally isolated respectively rom the Streptomyces coelicolor A3(2) and Stxeptomyces coelicolor MllO strains deposited and made part of the permanent stock culture collection of the Northern Regional Research Laboratory, Peoria, Illinois. Streptomyces coelicolor A312) is avai'able to the public as a preferred source and stock reservoir of plasmid SCP2 under the accession number 150420 Streptomyces coelicolor MllO is available to the public as a preferred source and stock reservoir of plasmid SCP2* under the accession number 15041.
Many ra 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 ~5.4kb EcoRI-SalI, the ~6.Okb SalI, the ~19kb EcoRI~
, .
HindIII, and the ~31kb EcoRI restric-tion fragmen~s o plasmid SCP2* and the ~31kb BglII restriction fragment of plasmid SCP2. The aforementioned plasmid SCP2* and SCP2 fragments were respectively ligated to an oxigin ~2~'7~S

of replication-containing and antibiotic resistance-conerring 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 construc-tion, the ~31kb EcoRI fragment of plasmid SCP2* and the ~6kb EcoRI fragment of plasmid pBR325 were ligated to form illustrative plasmids pJL120 and pJL121. Re-combinant plasmids of two orientations result because the fragments can be ligated in either direction.
Similarly, ligation of the SCP2* ~6.Okh SalI Eragment 15 and the 36kb SalI fragment of pBR325 results in the illustrative plasmids pJL180 and pJL181; ligation of the SCP2* ~5.4kb EcoRI-SalI fragment and the ~4.8kb EcoRI-SalI fragment of pBR325 results in the illus-trative plasmid pJL125; and ligation of the SCP2 BamHI
20 digest and the ~4.4kb BamHI fragment of plasmid pBR322 results in the illustrative plasmid pJL114.
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 ampicillin and tetracycline resistance; plasmids pJL180 and pJL181 conEer ampicillin and chloramphenicol re-sistance; and plasmids pJL125 and pJL114 confer only ampicillin resistance. Therefore, the vectors are conventionally selectable in the Eo coli host system by adding the appropriate antibiotic to the culture medium~

76~

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 StreptGmyces sex factors.
Three distinct 'pock' morphologies are associated with transformants, when plated on an appropxiate 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 J15 EcoRI restriction site of SCP2 or SCP~*. 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 ~y 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-con~aining restriction fragment of plasmid SCP2 or SCP2*, and b) a non-lethal DNA sequence cloned into the EcoRI restrictioll site of said P gene, and ~2(~6~3S

X-5773A ~8-2) growing said Streptomyces cells on a lawn of an indicator Streptomyces strain and select~
ing 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 pJL120, pJL121, and pJL125 code for M phenotype, that plasmids pJL180 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 ~resent 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, speci-ically exemplified for illustrative purposes by plasmidspJL190 and pJL195, express an additional selectable phenotype. Plasmid pJL190 was constructed by ligating the neomycin resistance-conferring ~7.7kb EcoRI-HindIII
fragment of plasmid pLR4 to the ~19kb EcoRI-HindIII
2S fragment of plasmid pJL121. Plasmid pJL195 was con-structed by ligating the pLR4 ~7.5kb EcoRI-partlal SalI
fragment to the ~5.4kb EcoRI-SalI fragment of plasmid pJL125. The latter pJI,125 plasmid comprises the largest (5.4kb) EcoRI-SalI fragment of plasmid pSCP2*
and was constructed by SalI deletion of plasmid pJL121.
Illustrative plasmi~s pJL190 and pJL195, in addition to neomycin resista~ce, also express the M phenotype as discussed above.

7~5 X-5773A _g_ 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 HlndIII-treated plasmid pIJ2, disclosed in Thompson et al., 1980, Nature 286.525, to HindIII-treated plasmid pBR322. As is readily apparent to those skilled in the art, both plasmïds pLR4 and pLRl contain the same neomycin resistance gene and thus either plasmid can be used for constructing the afore-mentioned pJL neomycin resistant vectors.
An additional neomycin resistance-conferring plasmid, designated as pJL192, was isolated as a spontaneous mutant of plasmid pJLl90 resident in Streptomyces ~riseofuscus. Plasmid pJLl92 specifies resistance to elevated levels of neomycin and therefore comprises a novel neomycin resistance gene which is distinguishable Erom the resistance gene comprising plasmids pJLl90, pJLl95, pIJ2, pLR4, and pLRl. In a similar manner, an additional neomycin resistance;
conferring plasmid, designated as pJLl99, was isolated as a spontaneous mutant of plasmid pJL195. Those skilled in the art will recognize that the novel neomycin resistance gene of plasmid pJLl92 or pJL19g can be readily excised and ligated to other vectors.
The gene allows for improved and more efficient selec-tion of transformants. As in the case of plasmids pJL190 and pJLl95, transformants of plasmids pJLl92 and pJL199 express the M phenotype when plated on an appropriate indicator strain.

~Z()'7~5 Plasmid pJL192 can be conventionally isolated from E coli K12 C600~ -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-15040.
A DN~ 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 ~he thiostrepton resistance conferrin~ fragment, is ~1807kb and is constructed by ligating HindIII treated plasmid pIJ6, disclosed in Thompson et al., 1980, Nature 286:525, to HindIII treated plasmid pBR322. Plasmid pLR2 is functional in E. coli and there-Eore can be amplified and isolated conveniently for subsequent manipulation.
For convenience and ease of construction, the thiostrepton resistance conferring ~1035kb BamHI
frasment of plasmid pLR2 was ligated into the Bam~lI
restriction site of plasmid pBR328 to form plasmid pJI,193. The ~lkb ~clI restriction fragment of pJL193 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 or purposes of constructing the present invention provided that the origin of repli-cation contained in their respective ~5.4kb EcoRI~SalI
restriction fragments is present. Such additional plasmid SCP2 and SCP2* restriction fragments include, but are not limited to, the ~6kb SalI, ~15kb PstI, ~23kb BglII, ~15kb BamHI, ~14kb EcoRI-PstI, ~13kb EcoRI--BamHI, and ~15kb PstI-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-striction fragment of an E. coli plasmidO Such E.
coli plasmids include, for example, plasmids pBR322, pBR324, pBR325, pBR327, pBR328 and the like. There~
fore, 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 anti-biotic resistance-conferring DNA segments exemplified herein are respectively the ~7.7kb EcoRI-HindIII and the ~7.5kb EcoRI-partial SalI fragments of plasmid 20 pLR4 and the pLR2 ~1.35 BamHI and the pJL193 ~lkb BclI
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 example, the ~3.4kb BamHI restriction fragment, the ~3.5kb PstI restriction fragment, and the larg~-r of the SstI-KpnI subfragments of the ~3.4kb BamHI restriction fragment. Other thiostrepton resistance~conferr:ing segments include, for ~xample, the ~13kb PstI fragment of plasmid pLR20 Still other DNA segments conferring resistance to the same or to different antibiotics such ~l2~:~'7~3S

X-;7 73A --12--as, for example, hygromycin, viomycin, tylosin, erythro-mycin and the like, can also be constructed and used by those skilled in the art. In addition, various func-tional derivatives of the above described antibiotic resistance-conferring DNA segments can be constructed by adding, eliminaking, 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 anti-biotic resistance-conferring DNA segment, an E. coli origin of replication-containing restriction fragment, and also an origin of replication-containing restric-tion fragment of plasmids SCP2 or SCP2*, ~esults in plasmids that are within the scope of the present invention. Therefore, an antibiotic resistance~con-ferring DNA seyment 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 DN~ segment is not limited to a single ~osition on the present chimeric plasmids but can be ligated or in-2~ serted at varying sites provided that an origin of replication or other critical plasmid controlled physiological functions ~re not disrupted. Those skilled in the art understand or can readily determlne which sites are advantageous for ligation or ins~rtion of a particular DNA segment.

.~ .

The various restriction fragments of plasmids SCP2, SCP2*~ pBR325, pBR322 and the like, and also the various antibiotic resistance conerring DNA segmen~s comprising the present vectors, can be modified to facilitate ligation. For example, molecular linkexs 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 modiied by adding, eliminating, or substituting certain nucleotides to alter charac-teristics and to provide a variety of restriction sites for ligation of DNA. Those skilled in the art under-stand 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 durlng mating between transformed and non-transformed Strepto-myces taxa~ This is advantageous because the present vectors therefore can be transformed not only by proto-plast transformation but also by conventional genetic cros es. 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 proceduresO Without the tra function~ DNA

7~

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 fox 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 OL many Streptomyces taxa, particularly restrictionless strains of economically important taxa that produce antibiotics such as aminoglycoside, macroiide, ~-lactam, polyether, and glycopeptide antibiotics. Such re-strictionless strains are readily selected and isolated from Streptomyces taxa by conventional procedures well -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 restriction enzymes that do not cut any of the restric-tion ites of the present vectors are also considered restrictionless.
Preferred host cells of restrictionless strains of Streptomyces taxa that produce aminoglyco-side anti~iotics and in which the present vectors areespecially useful and are transformed, include restric-s X-;773A -15-tionless cells of, for example: S. kanamyceticus (kanamycins), S. chrestomyceti ~s (aminosidine), S.
griseoflavus (antibiotic MA 1267), S. microspor us (antibiotic SF-767), S. ribosidificus (antibiotic SF733), S. flavopersicus (spectinomycin), S. spectabilis (actinospectacin), S. rimosus fo.rma paromomycinus (paromomycins, catenulin), S. fradiae var. italicus (aminosidine), S. bluensis var. bluensis (bluensomycin), S. catenulae (catenulin), S. olivoreticuli var. cellu-lophilu~ (destomycin A), S. tenebrarius (tobramycin, apramycin), S. lavendulae (neomycin), S. albogriseolus (neomycins), S. albus var. m ~ (metamycin), S.
hyaroscopicus var. sagamiensis (spectinomycin), S.
bikiniensis (streptomycin), S. griseus (streptomycin), l; S. erythrochromogenes var. narutoensis (streptomycin), S. poolensis (streptomycin), S. galbus (streptomyci.n), S. rameus (streptomycin), S~ olivaceus (streptomycin), . .
S. mashuensis (streptomycin), S. hygroscopicus ~ar.
limoneus (validamycins), SO rimofaciens (destomycins), S. hygroscopicus forma glebosus (glebomycin), S.
fradiae (hybrimycins neomycins), _. eurocidicus (antibiotic Al6316-C), S. aquacanus (N methyl hygromycin B), S. crystallinus (hygromycin A), S. noboritoensis (hygromycin)~ S. hygroscopicus (hygromycins), S.
2~ atrofaciens (hygromycin), S. kasugaspinus (kasugamycins), S. kasugaensis (kasugamycins), S. netropsis (antibiotic _ LL-A~31), S. lividus (lividomycins), _. hofuensis (seldomycin complex), and _. canus (ribosyl paromamine).
Preferred host cells of restric~ionless strains of Streptomyces taxa that produc~ macrolide antibiotics and in which the present vectors are ., .
~' d 37~

especially useful and are transformed, include restric-tionless c911s of, for example: S. caelestis (anti-biotic M188), S. platensis (platenomycin), S. rochei var. volubilis (antibiotic T2636), S. venezuelae (methymycins), S. griseofuscus (bundlin), S. narbo-nensis ~josamycin, narbomycin), S. fungicidicus (antibiotic NA-181), S. griseofaciens (antibiotic PA133A, B), S. roseocitreus (albocycline), S. bruneo-grlseus (albocycline), S. roseochromogenes (albocycline~, S cinerochromogenes (cineromycin B), S. albus (albo-.
mycetin), S. felleus (argomycin, picromycin), SO
rochei (lankacidin, borrelidin), S. violaceoniger (lankacidin), S. griseus (borrelidin), S. maizeus (ingramycin), S. albus var. coilmyceticus (coleimycin), S. mycarofaciens (acetyl-leukomycin, espinomycin), S.
hygroscopic_ (turimycin, relomycin, maridomycin, tylosin, carbomycin), S. griseospiralis (relomycin), S.
lavendulae (aldgamycin), S. rimosus (neutramycin), S.
deltae (deltamycins), S. fungicidicus var. espino-myceticus (espinomycins), S. ~urdicidicus (mydeca-~ . .. _ . . .. .
mycin), S. eurocidicus ~methymycin), SO g eolus(griseomycin), S. flavochromogenes (amaromycin, shinco-mycins), S. fimbriatus (amaromycin), S. fasciculus (amaromycin), S. _ ~ (erythromycins), S. anti-bioticus (oleandomycin), S. olivochromogenes (oleando-mycin), S. spinichromo~enes var. suragaoensis (kujimycins), S. kitasatoensis (leucomycin), S. narbonensis var.
josamyceticu~ (leucomycin A3, josamycin), S. albogr~seolus (mikonomycin), SO bikiniensis (chalcomycin), S. irratus (cirramycin), S. djakartensis (niddamycin), S~ eurythermus (angolamycin), S. fradiae (tylosin~ lactenocin, macrocin), ~-5773A -17-S. goshikiensis (bandamycin), S. griseoflavus (acumycin), S. halstedii (carbomycin), S. ~endae (carbomycin) J S.
macrosporeus (carbomycin), S. thermotolerans (carbo-_ mycin), S. albireticuli (carbomycin), and S. ambofaciens (spiramycin).
Preerred host cells of restrictionlessstrains of Streptomyces taxa that produce ~-lactam anti~iotics and in which the present vectors are espec.ially useful and are transformed, include re-strictionless cells of, for example: S. lipmanii(A16884, MM4550, ~M13902), S. clavuligerus (A16886B, clavulanic acid), S. lactamdurans (cephamycin C), S.
griseus (cephamycin A, B)~ S. h groscopicus (deacetoxy-cephalosporin C), S. wadayamensis (WS-3442-D), S.
chartreusis (SF 1623), S. heteromorphus and S. panayens1s ~C2081X); S. cinnamonensis, S. f.imbriatus, S. halstedii, S. rochei and S. viridochromogenes (cephamycins A, B);
_~
S. cattleya (thienamycin); and S. ol _aceus, S. flavo-virens, S. ~lavus, 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 vectoxs are especially useful and are transformed, include re-strictionless cells of, for example: S. albus (A204,A28695A and ~, salinomycin~, S. hygroscopicus (A218, emericid, DE3936) , A120A, A28695A and B, etheromycin, dianemycin), S. griseus (grisorixin~, S. onglobatus (ionomycin), S. eurocidicus var~ asterocidicus ~laidlo-mycin), _. lasallensis (lasalocid), S. ribosidificus (lonomycin), S. cacaoi var. asoensis (lysocellin), S.

~LZ~9'~

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 useul and are transformed, include re-strictionless cells of, for example: S. orientalis and S. haranomachiensis (vancomycin); S. candidus ~A-35512, avoparcin), and . eburosporeus (LL-AM 374).
Pre-ferred 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. ros~osporus, S. lividans, S. e.spinosus, and S.
azure _.
In addition to the representative _repto-myces 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 2~ invention are useful, some of the present recombinant DNA cloning vectors and transformants are preferredO
Accordingly~ preferred vectors are pJL114, ~JL121, pJL125, pJL180, pJLl90, pJL192, pJL195, pJL197, pJLl99 and pHJL212 and preerred transformants are Strepto~
myces griseofuscus/pJL114, S. griseofu cus/pJL121, S.
griseofuscus/pJL125, S. ~ /pJL180, S.
griseofuscus/pJLl90, S. griseofuscus/pJL192, S.

Z1)7~

X-5773A . 19-griseofuscus/pJL195, S.griseofuscus/pJLl99, S. griseo-fuscus/pJL197, S. griseofuscusJpHJL212, E coli K12 C600Rk-M~-/pJL114, E. coli K12 C600Rk-Mk-jpJL121, E.
coli K12 C600Rk Mk-tpJL125, E. coli K12 C600Rk Mk-/pJL180, E. coli K12 C600R M -/pJLlgO r E . coli K12 C600R -M -/
- k k - - k k pJL192, E. coli K12 C600Rk-Mk-/pJL195, E. coli X12 C600Rk-Mk-/pJL139, E. coli X12 C600Rk-Mk~/pJL197, and E. coli K12 C600Rk-Mk-/pHJL212. Moreover, of this preferred group, plasmids pJLl90, pJL192, pJL195, pJL197, pJLl99 and pJL and transfor~.ants S. griseo=
fuscus/pJLl90, S. griseofuscus/pJL192, S. griseo-fuscus/pJL195, S. griseofuscus/pJL197, S. griseofuscus/
pJLl99, S. griseofuscus/pHJL21?, E~ coli K12 C600Rk-Mk-/
pJLl90, E. coli X12 C600Rk-Mk-/pJL192, E. coli K12 C600Rk-Mk-/pJ~95 and E. coli K12 C600Rk-Mk-/pJL197, E.
coli K12 C600Rk-Mk-/pJLl99 and E. coli K12 C600Rk-Mk-/
pHJL212 are most preferred~ Streptomyces 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.
Tne vectors of the present invention com-prises origins of replication that are functional in E.
~5 coli and Streptomyces and therefore provide flexib.ility : in the choice of hosts. Consequently, cloned DNA
sequences can be shuttled into E. coli for construction of new plasmids, physical analysis, and for mapping of restriction sites and then shuttled back into Strepto-myces for functional analysis and improvement of strains. This is particularly advantageous because . amplification and man.ipulation of plasmids can.be done ' ~

~2~

X-5773A ~20-faster and more conveniently in E. coli than in Strepto-myces. For example, the present vectors can be amplified conventionally in E. coli K12 by growth with spectino-mycin or chloramphenicolr 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, recombinant~ are easily selected. Therefore, large amoun~s o~ plasmid DN~ can be isolated conveniently and in a shorter time than that required for doing similar procedures in ~
myces. Thus, after desired recombinant DNA procedures are accomplished in the E. coli host system, the partic-ular Strep omyces 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. More-over, the ability of the present vectors to con~er a pock phanotype 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 prPsent vectors and then trans-formants 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 plasmid function and replication, and include genes that specify antibiotic modification enzymes and 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 pJL192, at the internal BamHI restriction site of the ~7.7kb EcoRI-HindIII 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 ~or M pock morphology and, secondarily, identifying those ~ trans formants that are not resistant to neomycin. In a similar manner, insertion of a DNA segment into illus~
trative plasmid pJLl80 at, for example, the unique PstI
restriction site, inactivates the ampicillin resistance gene. Thus, E. coli transformants carrying this re-combinant 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 re-sistance 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.
The functional test for antibiotic resis-tance, as described above, can also be used to identify :~2~'7~

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 ex-pression of other genes in cells of Streptomyces and _.
coli.
The antibiotic resistance-conferring vectors of the prese~t invention are also useful for .insuring that linked DNA segments are stably maintained in host cells over many generations. These genes or DNA fxag-ments, covalently linked to an antibiotic resis~ance conferring fragment and propagated either in Strepto~
myces or E. coli, are maintained by exposing the trans-_ formants to levels of antibiotic that are toxic tonon-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 oE 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. Exampl~s of such products include, but are not limited to, Streptomycin, Tylosin, Cephalosporins~ Actaplaninl Nara~in, ~onensin, Apramycin, ~obramycin, Erythromycin, and the like. The present invention also provides selectable vectors that are useful for cloning, charac-terizing, and reconstructing DNA sequences that codefor comm~rcially important proteins such as, for exam-ple, human insulin, human proinsulin, human growth ~Z~t~6 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 suoh as, for example, Streptomycin, Cephalosporin, Tylosin, Actaplanin, Narasin, Monensin, Apramycln, 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 ~
myces and E. coli allows for easy recombinant genetic manipulation for increasing the yield and availabili~y 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 ex-pressed from a high copy number plasmid, can be readily cloned into the present vectors and shuttled between Streptomyces and E. coli _ .
Strep~omyces coe color A3(2) and S. coelicolor MllO, 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 prefexred 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 1L2~3"7~i~1S

capable of yielding sodium/ potassium, ammonium, calcium, phosphate, chloride, sulfate, and like ions. ~s i~
necessary for the growth and development of other microorganisms, essential trace elem~nts are also added. Such trace elements are commonly supplied as impurities incidental to the addition of other con-stituents of the medium.
Streptomyces coelicolor MllO and S. coeli~
color A3(2) are grown under aerobic culture conditions over a relatively wide pH range of about 5 to 9 at temperatures ranging from about 15 to 40C. 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 30C. Culturing Stxepto-myces coelicolor M110 and S. coelicolor A3(2) under the aforementioned conditions, results in a reservoir of cells from which plasmids SCP2 and SCP2* are respec-tively isolated conveniently by techniques well known in the art.
The following examples further illustrate and detail the invention disclosed herein. Both an ex-- planation of and the actual procedures for constructing the invention are descxibed where appropriate.
Example 1 - Isolation of Plasmid SCP2*
. .
A. Culture of Streptomyces coelicolor MllO
_ A vegetative inoculum of S~reptomyces coelicolor MllO (NRRL 15041) was conventionally pre-pared by growing the strain under submerged aerobic . .

X-5773~ -25-conditions in 50 ml. of sterilized ~Trypticase~l soy broth* at 35 q./l. in deionized water.
The 'Trypticase'l soy broth inoculum was incu-bated for 48 hours at a ~emperature of 30C. The 50 ml. culture was then homogenized, transferred to 450 ml. of sterilized YEMESG** medium, and then in-cubated for at least 40, but not more than 65 hours, at 30C. The pH was not adjusted. After incubation, the Streptomyces coelicolor M110 cells were ready for -harvest and subsequent isolation of plasmid DMAo *'Trypticase'1 so~- ~roth is obtained from Difco Laboratories, Detroit, Michigan.
YE~ESG comprises .3~ yeast extract, .5% peptone, .3% malt extract, 1% dextrose, 34~ sucrose, .1% MgCl2, and .1% glycine.
B. Plasmid Isolation -About 10 g. (wet wgt) of Streptomyces coeli-color MllO cells were harvested by centrifugation (lO
2~ minutes, 4C., 10~000 rpm) and then about lO ml./g. wet wgt cells of TES buffer (oOlM Tris(hydroxymethyl)amino-ethane [tris], .OOlM EDTA, 25% sucrose, p~ 8) were added.
The cells were vortexed into suspension followed by addition of 10 ml./g. wet wgt cells of .25M EDTA, pH 8 2~ and then 5 ml./g. wet wgt cells of lysozyme (lO mg./ml.
in TES). After the mixture was incubated at 37C. for about 15 minutes, about 105 ml./g~ wet wgt cells o~ 20 - SDS (sodium lauryl sulfate (BDH Chemicals Ltd. Poole, England), were added. The resultant mixture was allowed to stand at room temperature for 30 minutes, and then 5M NaCl was added to give a final concentration of lM

1. Trademark for dehydrated peptone prePared from i ~ casein by pancreatic digestion.

)'7~

NaCl. After standing again at room temperature (15minutes), the mixture was placed on ice for 2 hours.
The lysate was centrifuged (20 minutes, 4C., 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, 4C., 10,000 rpm). The precipitate was air dried and then resus pended in 1 ml./g. wet wgt cells of TE buffer (.OlM
` Tris, .OOlM EDTA). Centrifugation (20 hours, 20Co 10 50,000 rpm) using cesium chloride gradients with propidium iodide was carried out to purify the plasmid DNA. Following centrifugation, the desir~d plasmid SCP2* DNA band was removed and the propidium iodide extracted by conventional procedures. The CsCl DNA
solution was stored at -20C. Prior ~o use, the DNA
was desalted by either P~lO~(Bio Rad)'column exchange with TE or by dialysis against TE. The D~A was precip--itated with ethanol by conventional procedures and redissolved in TE.
~3 Example 2 Cons~ruction of Plasmid pLRl A. EindIII Digestion o~ Plasmid pIJ2 ?5 Abvut 20 ~1. (20 ~g.) of plasmid pIJ2 DNA, disclosed in Thompson et al., 1980, Nature 286:525, 5 ~1. BSA(Bovine Serum albumin, 1 mg./mlO), 19 ~1.
water, 1 ~1. of HindIII (containing 3 New England Bio Labs units) restriction enzyme*, and 5 ~1. reaction mix** were incubated at 37C. for 2 hours. The reac tion was terminated by the addition of ahout 50 ~1~ of 2. Trademark i~ ?

s X-5773A -27~

4M ammonium acetate and 200 ~1. of 95% ethanolO The resultant DNA precipitate was washed twice in 70~
ethanol, dried in acuo, suspended in ao ~1. of TE
buffer, and frozen at -20C. 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, Maxyland 20850 Research Products Miles Laboratories, Inc.
Elkhart, Indiana 46515 Reaction mix for HindIII restriction enæyme was pre pared with the following composition:
60OmM NaCl lOOmM Tris-EICl, pH7.9 70mM MgCl~
lO~M Dithiothreitol B. HindIII Digestion of Plasmid pBR322 .. . . ...
About 8 ~1. (4 ~g.) of plasmid pBR322 DNA, 5 ~1 reaction mix, 5 ~1. BSA (1 mg./ml.), 31 ~1.
water, and 1 ~1. of H _ III restriction enzyme were incubated at 7C. for 2 hours. After the reaction was

3~

~L~24~7~

X-5773A ~28-terrninated by incubating at 60C. for 10 minutes, about 50 ~1. of ammonium acetate and 200 ~1. of 95% ethanol were added. The resultant DNA precipitate was washed twice in 70~ ethanol, dried ln vacuo, and suspended in 45 ~1. of water.
C. Ligation of HindIII Digested Plasmids pIJ2 and pBR322 .
About 20 ~1. of HindIII treated plasmid pIJ2 (from Example 2A), 20 ~1. of HindIII treated plasmid pBR322 (from Example 2B), 5 ~l o BSA (1 mg./ml.), 1 ~1.
of T4 DNA ligase , and 5 ~1. ligation mix** were incubated at 16C. for 4 hours. The reaction was ter-minated by the addition of about 50 ~1. 4M ammonium acetate and 200 ~1. of 95gO ethanol. The resultant DNA
precipitate was washed twice in 70% ethanol, dried ln vacuo, and suspended in TE buffer. The suspended D~A
constituted the desired plasmid pLRl.
*
T4 DNA ligase can be obtained from the following source:
New England Bio Labs., Inc.
32 Tozer ~d.
Beverly, Massachusetts 01915 **
Ligation mix was prepared with the following composi-tion:
500mM Tris-HCl, pH7.8 200~ Dithiothreitol lO~)mM MgC12 lOm2~ ATP

o~

X-5773~ -29-Example 3 Construction of E. coli K12 HBlOlJpLRl About 10 ml. of 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 .OlM sodium chloride~ Next, the cells were pelleted again, resuspended in about 10 ml. o .03M
calcium chloride, incubated on ice for 20 minutes, pelleted a third time, and finall~, resuspended in 1025 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 ~1. of 30mM calciu~ chloride solution, and gently mixed in a test tube with about 200 ~1. o competent E.
coli K12 HB101 cells. The resultant mixture was in cubated on ice for about 45 minutes and then at 42C.
for about 1 minute. Next, about 3 ml. of L-broth 20 (Bertani, 1951, J. Bacteriology 62:2g3) containing 50 ~g./ml. of ampicillin was added. The mixture was incubated with shaking at 37C. for 1 hour and then plated on L-agar (Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbor Labs, Cold Spring Harbor, New York) containing ampicillin. Surviving coloni s were selected and tested for the expected phenotype (AmpR, TetS), and constituted the desired E coli K12 HB101/pLRl transformants.

~3'7~
,, X~5773A -30-Example 4 _ ,_ Construction of Plasmid pLR4 A. Partial BamHI Digestion of Plasmid pLRl About 10 ~1. (10 ~g.) of plasmid pLRl, 5 ~1. BSA
(1 mg./ml.), 29 ~1. water, 1 ~1. of BamHI ~diluted 1:4 with water) restriction enzyme, and 5 ~1. reaction mix* were incubated at 37C. for 15 minutes. The reac~
tion was terminated by the addition of about 50 ~lo of 10 4M ammonium acetate and 200 ~1. of 95~ ethanol. The resultant DNA precipitate was washed twice in 70%
ethanol, dried in vacuo, and suspended in 20 ~1. water.
*
Reaction mix for BamHI restriction enzyme was pre-pared with t~e following composition:
1.5M NaCl H
60mM Tris-HCl, p 7.9 6OmM MgC12 B. BamHI Digestion of Plasmid pBR322 ; 20 The desired digestion was carried out in su~-stantial accordance with the teaching of Example 2B
except that BamHI restriction enzyme and reaction mix were used in place of ~indIII restriction enzyme and reaction mix. The digested plasmid pBR322 was suspended 2 5 in 2 9 ~1 . o f water.
C. Ligation of Partial BamHI Diyest~d Plasmid pLRl and BamHI Di~ested Plasmid pBR322 .. . _ . . . _ The desired ligation was carried out in substantial accordance with the teaching of Example 2C. The resul-tant ligated DNA was suspended in TE buffer and consti-tuted the desired plasmid pLR4.

~Z~3~6~3~

X--5'773A -31-Example 5 Construction of E. coli Xl2 HB101/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-lected and tested for the expected phenotype (AmpR, Tet ), and constituted the desired E. coli ~12 HBlOl/
pLR4 transformants.
Example 6 Construction of Plasmids pJLl20 and pJL121 A. EcoRI Digestion of Plasmid SCP2*
lS About lSO ~l. (5.7 ~g.) of plasmid SCP2* D~A, l~l. water~ 2 ~l. of EcoRI (containlng 20 BRL units) restriction enzyme, and 17 ~l. EcoRI reaction mix* were incubated at 37C~ for 2.5 hours. The reaction was ; terminated by incubation at 65C. for 15 minu~es~
The reaction was conventionally analyzed by agarose gel electrophoresis (AGE) to verify that restriction was complete. The restricted DNA was stored at 4C. for subsequent use.
.
_ ~ *
Reaction mix for EcoRI restriction enzyme was - prepared with the following composition:
500~ NaCl 1000~`~1 Tris-HCl, pH7.5 lOOm,~ MgC12 -.

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 4~C. for subsequent use.
C. Ligation of EcoRI Digested Plasmids SCP2* and pBR325 About 40 ~1. of EcoRI digested plasmid SCP2*
(from Example 6A), 10 ~1. of EcoRI digested plasmid -pBR325 ~'rom Example 6B), 10 ~1. of MgC12 (.lM), 10 ~1.
of ~NH4)2SO4 (.lM), 10 ~1. ATP (2~'1) .1 ~1. of T4 DNA
ligase, and 20 ~1. ligation mix* were incubated at 4C.
for 18 hours. The reaction was analyzed by AGE to verify appropriate ligation. The suspended DNA con-stituted the desired ~35.8kb plasmids pJL120 and pJL121.
; Recombinant plasmids of two orientations result because the plasmid pBR325 Eco~I fragment can be oriented in either direction. A restriction site map of each of plasmids pJL120 and pJL121 was determined (after isolation as disclosed in Example 7) and is presented in Figure 2 of the accompanying drawingsO

~5 Ligation mix was prepared with the following composition:
50~ Tris-HC1~ pH 7.5 lOmM ~-mercaptoethanol lmM EDTA
50 ~g./ml. BSA

Example 7 Construction of E. coli K12 C600 ~ -Mk-/pJL120 and E. coli K12 C600Rk-Mk-/pJ~121 A. Preparation of Frozen Competent E. coli K12 C600Rk-Mk-- -- --- Fresh overnight cultures of E~ coli K12 C600Rk-Mk- (disclo~ed 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 37C. for 1 hour.
A total of 660 Klett Units of cells wera harvested, washed with 2.5 ml. of lOO~M NaCl, suspended in 150mM
is CaC12 with 10% glycerol, and incubated at room tem-perature for 20 minutes. The cells were harvested by centrifugation, resuspended in .5 ml. of CaC12-glyc rol, chilled on ice for 3-5 minutes and frozen. The sus-pensions of cells were stored in liquid nitrogen until use. Preservation and storage did not adversely a~ect the viability or frequency of transformation by co~a-lently 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 O05 ml.
of DNA (10 ~1. of the sample disclosed in Example 6C
and 40 ~1. of .lXSSC (.015M NaCl, ~0015M Sodium Citrate at pH 7). The transformation mixture was chilled on 30 ice for 20 minutes, heat shocked at 42C. for 1 minute and chilled on ice for 10 minutesD The samples were :~p ~

X~5773A -34-then diluted with .85 ml. of L broth, incubated at 37C.
for 1.5 hours, spread on L-agar containing ampicillin (50 ~g./ml.) and tetracycline (12.5 ~g./ml~) and incubated for 18 hours at 37C. The resultan~ colonies were selected and tested for the expected phenotype (Amp , Tet , CM ) and constituted the desired E. coli K12 C600Rk-Mk-/pJL120 and E~ coli K12 C600Rk-Mk-/pJL121 transformants. The ampicillin and tetracycline re-sistant colonies were isolated according to known procedures, cultured, and then conventionally iden~
tified by restriction enzyme and AGE analysis of the constitutive plasmids. The identified transformants were then used for subsequent production and isolation of plasmids pJL~20 and pJL121 acc~rding to known pro-cedures.
Example 8 Construction of Plasmids pJL180 and pJL]al A. SalI Digestion of Plasmid SCP2* and Isolation of ... _ . _ . . . .
2~ ~. okb S eI r~9m-nt The desired digestion was carried out in substantial accordance with the teaching of Example 6 except SalI restriction en7yme and reaction mix*, rather than EcoRI restriction enzyme and reaction mix, were used. The reaction was assayed by AGE ~o verify completion and terminated by heatin~ at 65Co for 15 minutes. The resultant SalI restriction fragments were separated by AGE and ~hen the separa~ed fragments werP
located in tAe gel by staining with ethidium bromide and visualizing fluorescent bands wi~h an ultraviolet ~IL2(17~S

light. The gel fragment containing the ~6.Okb fragment of interest was excised from the gel and electroeluted in.o TBE buffer (1.6~'Sigma 7~9'~uffer**, .093~ Na~EDTA, .55~ boric acid). The gel-fragment in ~BE 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 baa and passed over a DEAE cellulose colum~*** (.5 ml.'h~atman DE;2~ that had been e~uilibrated with equilibration buffer (.lM KCl, 10 ~ Tris HCl, pH 7.8). The column was washed with 2.5 ml. of equili-bration buffer and the DNA (about 5 ~g.) was eluted with 1.5 ml. of elution buffer (lM 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
was precipitated by adding 2 volumes (about 9 ml.) of 100~ ethanol followed by cooling to -20C. for 16 hours. The DNA precipitate was pelleted by cen~ri-fugation, washed with 75% ethanol, dried, and dissolved in TE buffer. Hereinafter, this conventional isolation 2~ technique is referred to as AGE~DE52~electroelution~

Reaction mix for SalI restriction enzyme was prepared with the following composition:
1500 m~`~l NaCl ~; 80 ~ Tris-HCl, pH7.5 60 ~ ~gC12 ~ ~ 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 What~an Inc., 9 Bridewell Place, Clifton, New Jersey 07014.

S c.~ 3. Trademark

4. Trademark ~ z~t7~ ~ ~

B. SalI Digestion of Plasmid pBR325 TAe desired digestion was caxried out in sub-stantial 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 ~NA was dissolved in TE buffer and stored at 4C. for future use.
C. Ligation of SalI Digested Plasmid pBR325 and ~6.Okb SalI Fragment of Plasmid SCP2*
.
About 1.5 ~g. of the 6.Okb SalI fragment of SCP2*, prepared in Example 8A, was mixed with .5 ~g.
of SalI digested pBR325, prepared in Example 8B.
The DNA mixture was precipitated by standard ethanol precipita-tion and redissolved in 3 ~1. of distilled water, 4 ~lo of .66M ATP, 2 ~1. of ligase-kinase mixture (.25M Tris-HCl, pH 7.8, 50 mM MgC12, 25 mM
dithiothreitol and 25~ glycerol) and 1 ~1. of T4-DNA
ligase (1 unit). After incubation for 1 hour at 15C., the reac-tion mixture was diluted with 12 ~1. of waterl 20 ~1. of .66M ATP, 8 ~1. of ligase-kinase mixture and then incubated at 15C. for 18 hours. The resultant ligated DNA was diluted 1:5 into .lXSSC
and constituted th~ desired ~12.Okb plasmids pJI,180 and pJL181.
Recombinant plasmids of two orientations result because the plasmid pBR325 SalI fragment can be oriented in either direction. A restriction site map of each of plasmids pJL180 and pJL181 is presented in Figure 3 of the accompany~ng drawings.

. , .

:3L20~

X-5773A -37~

Example 9 Construction of E. coli K12 C600Rk-Mk-/pJL180 and s;
.
E~ coli X12 C6ooRk-Mk-/pJLl8l The desired constructions were made in sub-stantial accordance with the teaching of Example 7 except that the mixture of plasmid pJL180 and pJLl81 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 Kl2 C600Rk-l~k-/pJLl80 and E. coli Kl2 C600Rk-M~/pJLl8l transformants. The ampicillin and chloramphenicol resistant colonies were isolated according to known procedures, cultured, and then con~entionally iden-tified by restriction enzyme and.AGE analysis of theconstitutive plasmids. The identified transforman~s can then be used for subsequent production and iso-lation of plasmids pJLl80 and pJLl81 according to known procedures.
Example lQ
Construction of Plasmid pJLl25 A. SalI Digestion of Plasmid pJLl21 and Isolation of ~10.2kb SalI Fragment The desired dige~tion was carried out insubstantial accordance with the teaching of Example 8 except that the reaction was stopped before digestion was complete and except that plasmid pJLl21, rather than plas~id SCP2*, was used. The resultant SalI

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 ~10.2kb SalI Fragment oE Plasmid pJL121 . . . _ The desired ligation was carried out in substantial accordance with the teaching of Example 8C
except that the SalI fragments of plasmid pJL121, rather than the SalI fragment of plasmid SCP2* and pBR325, were used. The resultant ligated DNA con-stituted the desired plasmid pJL125 plus 12 other plasmids that were subsequently isolated and shown to contain additional SalI restriction fragmen-ts o~
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-SalI fragment of plasmid SCP2*, was dissolved in TE buffer and stored at 4C. for future use. A restriction site map of plasmid p3L125 is presented in Figure 4 of the accompanying drawing. The restriction site map was determined with plasmid from transformed E. coli R12 C600Rk-Mk-.
Example 11 Construction of E. coli K12 C600R~-Mk-/pJL125 _ _ The desired construction was made in sub-stantial accordance with the teaching of Example 7 except that plasmid pJL125, rather than plasmid~ pJL120 and pJL121, was used. The resultant colonies were ~20'~ 5 ~-5773A -39-selected and tested for the expected phenotype (AmpR, TetS, CMS) 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.
Example 12 Construction of Plasmid pJLl90 A. EcoRI-HindIII Digestion of Plasmid pJL121 and Isolation of ~l9.Okb EcoRI-HindIII Fragment About 200 ~1. (80 ~g.) of plasmid pJL121 VNA, 30 ~1. BSA (1 mg./ml.), 40 ~1. of HindIII (containing 200 BRL units) restriction enzyme, and 30 ~1. HindIII
reaction mix* wera incubated at 37C. for ahout 3 hours and then at 65C~ for 10 minutes. The 300 ~lo reaction ; mixture was cooled to 4C., supplemented with 110 ~1.
of lOX HindIII~EcoRI diluent reaction mix** and 30 ~1.
EcoRI restriction enzyme (containing 300 BRL units), and then incubated at 37C. for 3 hours, then at ~5C.
for 10 minutes followed by cooling to 4C. Tha re-sultant ~l9.Okb EcoRI-HindIII restriction fragment was conventionally isolated by AGE/DE52/electroelution.
The desired DNA was dissolvad in TE buffer and stored at 4C. for future use.

7~

X-5773A ~40 -~indIII reaction mix was prepared with the following COmpOSitlOn:
60m.`~ Tris-HCl, pH 7.5 500~ NaCl 60~ MgCl2 **H_ndIII~EcoRI diluent was prepared with the following composition:
382mM Tris-HCl, pH 7.5 50~1 NaCl 22mM MgCl2 B. EcoRI-HindIII Digestion of Plasmid pLR4 and Isolation of ~7.7kb Eco~I-HindIII Fragment . _ .
The desired digestion and isolation was carried out in substantial accordance with the -teaching of Example 12A ~xcept that plasmid pLR4, rather than plasmid pJLl21, was used. The desired ~7.7kb f.ragment was dissolved in TE buffer and stored at 4C. for future use.
C. Ligation of ~l9.Okb EcoRI-HlndIII Fragment of . ~
Plasmid pJLl2l and ~7.7kb EcoRI-HindIII Fragment of Plasmid pLR4-The desired ligation was carried out in substantial accordance with the teaching of Example 3C
except that the ~l9.Okb EcoRI-HindIII fragment of plasmid pJLl2l and the ~707 EcoRI-HindIII fragment of plasmid pI.R4, rather than the 6.Okb SalI fragment of plasmid SCP2* and SalI digested pBR325, were used. The resultant ligated DNA constituted the desired plasmid ~2~'7~

pJLl90 which was then stored at 4C. for future use. A
restriction site and ~unctional map of plasmid pJLl90 is presented in Figure 4 of the accompanying drawings.
The restriction site map was determined from plasmid transformed into E. coli K12 C600R ~
- k k Example 13 Construction of E. coli K12 C600~k-~qk-~pJL190 The desired construction was made in sub-stantial accordance with the teaching of Example 7 except that plasmid pJLl90, rather than plasmids pJL120 and pJL121, was used. The resultant colonies were selected and tested for the expected phenotype ~AmpR, TetS) and size by conventional means (as in Example 11) 15 and constituted the desired E. coli K12 C600Rk~Mk-/pJL190 transformants. The transformants were then conventionally cultured for sub5equent production and isolation o plasmid pJLl90 according to known procedures.
Example 14 Isolation of Plasmid pJL192 . . . _ _ . . _ _ Plasmid pJL192, which confers high resistance to antibiotic neomycin (10 ~g./ml.), can be conven-tionally isolated from E. coli K12 C690Rk~Mk-/pJL192, a strain deposited and made part o~ the permanent stock culture collection of the Northern Regional Research Laboratory, Peoria, Illinois under the accession number 15040. The restxiction site map of plasmid pJL192 appears not to be distinguishable from the plasmid pJLl90 map presented in Figure 4O

~ZO'76~

X-5773~ -42-Example 15 Construction of Plasmid pJL195 -A. EcoRI-SalI Digestion of Plasmid pJL125 and Isola~ion of ~5.4kb EcoRI-SalI Fragment , .
The desired digestion and isolation was carried out in substantial accordance with the teaching of Example 12A except that plasmid pJL125 and SalI
restriction enzyme and reaction mix, rather than plasmid pJL121 and HindIII restriction enzyme and reaction mix, were used. In addition, SalI~EcoRI
diluent* was used. The resultant ~5.4kb EcoRI-SalI
fragment was dissolved in TE buffer and stored at 4C.
for future use SalI~EcoRI diluent was prepared w.ith the followiny composition:
940~1 Tris-HCl, pH 7.5 55m~ gC12 . EcoRI-Partial SalI Digestion of Plasmid pLR4 and Isolation of ~7.5kb EcoRI-Partial SalI Fragmen~
The SalI digestion was carried out in su~-2~ stantial accordance with the teaching of Example lOA
except plasmid pLR4, rather than pJL121, was used~
Since only a partial SalI digestion was desired, the : resultant mixture was incubated first at 37C. for 15 minutes and then at 6;C. for 10 minutes. Following cooling to 4C., the resultant partial SalI ~7.7kb linear ~ragment was conventionally isolated by AGE/

~LZ~'7~

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 r rather than plasmid SCP2*, was used. The desired ~,7.5kb E RI-SalI fragment (the largest possible EcoRI~SalI
fragment) was isolated by AGE/DE52/electroelution, dissolved in TE buffer, and th~n stored at 4C. ~or future use.
13 C. Ligation of ~5.4kb EcoRI-SalI Fragment of Plasmid pJL125 and ~7.5kb EcoRI-Partial SalI Fragment of Plasmid pLR4 The desired ligation was carried out i.n substantial accordance with the teaching of Example 8C
except that the ~5.4kb EcoRI-SalI fragment of plasmid pJL125 and the ~7.Skb EcoRI-partial S I fragment of ; plasmid pLR4, ratner than the ~6.Okb SalI ~ragment of plasmid SCP2* and SalI digested pBR325~ were us2d~ The 23 resultant ligated DNA constituted the desired plasmid pJL195 and was stored at 4C. for future use. A re-striction 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--~k-.

,., 6~

~Y-5773A -44-Example 16 Construction of E. coli ~12 C600Rk-Mk-/pJL195 Th~ desired construction was made in sub-stantial accordance with the teaching of Example 7 S except that plasmid pJL195, rather than plasmids pJL120 and pJL121, was used. The resultant colonies were tested for the expected phenotype (AmpR, TetS) and size (as in Example 11) and constituted the desired E.
coli K12 C600Rk Mk-/pJL195 transformants. The trans-formants were then conventionally cultured for sub-sequen-t 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
; 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 per-manent stock culture collection of the Northern Regional Research Laboratory under the accession number 1$042), and BamHI restriction enzyme and reaction mix*, rather ~han plasmid SCP2* and EcoRI restriction enzyme and reaction mix, were used. The desired DNA was stored at 4C. for subsequent use~

... .
*
Reaction mix for BamHI restriction enzyme was prepared with the following composition:
lOOO~M Tris~HCl, pH 7O4 lOOm-~ MgC12 ~ ~2~

B. Llgation of Bam}iI Digested Plasmld SCP2 and BamHI Digested Plasmid pBR322 The desired liga~ion was carried out in substantial accordance with the teaching of Example 6C
e~cept that the BamHI digest of plasmid SCP2 ~prepared in Example 17A) and BamHI-digested plasmid pBR322 (prepared in Exampl~ 4B5, rather ~han plasmids SCP2*
and pBR325, were used. The resultant DNA was stored at 4C. and constituted the desired ~34.6kb plasmid pJL114.
Example 18 Construction of E. coli K12 C600Rk-Mk-/pJL114 .
The desired cons~ruction was made in sub-s~antial accordance with the teachin~ of Example 7 except that plasmid pJL114, rather than plasmids pJL120 and pJL121, was used. The resultant colonies were selected and tested for the expected phenotype (AmpR, 2~ TetS), and constituted the desired ~. coli K12 C600Rk-~1k-/pJL114 ~ransformants. The ampicillin re~
sistant, tetracycline sensitive colonies were isolated according to known procedures, cultured, and then conventionally identified by restriction enzyme and agarose gel electrophoretic analysis of the consti-tutive plasmids.
It was revealed upon analysis that the BamHI
restriction enzyme had cut only one of the ~glII re-striction sites of SCP2 during the digestion described in ~xample 17A. Since this event is raxe and has not been repeated, E. coli K12 C600Rk~ pJL114 has been ~)76~3~

deposited and made part of the permanent stock cultur~
collection of the Northern Regional Research Laboratory, Peoria, Illinois under the aGcession number B-15039. The strain i5 available as a preferred source and stock

5 reservoir of plasmid pJL114. A restriction site map of plasmid pJL114 is presented in Figure 5 of the accom-panying drawings.
Example 19 Construction of Streptomyces griseofuscus~pJL120 1 0 - e A. Growth of Cultures for Preparation of Protoplasts .
A vegetative inoculum was conventionally prepared by growing ~he s_rain under submerged con-ditions for 20 hours at 30C. in TSB supplemen-ted with .4% glycine. The culture was homogenized and inocu-lated at a 1/20 dilution into the same medium and then grown for 18 hours at 30C.
B. Transformation Using ~bout 20 llg. of plasmid pJL120 DNA and lX109 protoplasts. of Strept_myces grlseofuscus, a strain deposited and made part of the permanent stock culture collection of the American Type Culture Col-lection, Rockville, Maryland, from which it is avail-able to the public under the accession number ATCC
23916, the desired transformation was carried out in substantial accordance with the teaching o:E International Publication (of International Patent Application No.
PCT/BG79/00095) No. W079/01169, Example 2.

'~ ~ Sb ~L)7~

X~5773A -47-C. Selection To assay for transformation even a~ low frequencies, two procedures were employed~
(1) Pock-assay:
Spores were harvested from the regeneration plates containing confluent lawns of regenerated proto-plasts as follows. About 10 ml. of sterile distilled water were added to the plate and the surface of the : 10 culture gently scraped with a loop to remo~e the spores.
The resulting spore suspension was centrifuged at 20,000 rpm for 10 minutes. The supernatant was dis-carded and the remaining spore pellet resuspended in .3 ml~ of 20% v/v glycerol. Serial dilutions of the prepar,ation 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 -20C. with little loss of viability. About .1 ml. aliquots 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 : ~riseofuscus strain originally used in the transfor-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~ ayarl. Transformants can typically be - detected after 3 days' growth at 30C. by the appearance of "pocks", a property expressed by spores containing the plasmid in expressible form within the lawnO The ~ .
., ~ . ~

12~3~76~i X~5773A -48 transformants were recovered by conventionally picking spores frorn the centre of the "pock" to an agar plate of YMX medium (Hopwood 1967, Bacter.iological Review, 31:373).
(2) Back transformation to E. coli K12 C600 k k The spores are collected as in (1) above but are used to inoculate 50 ml. of TSB supplemented with 1 .4% glycine. The culture is grown for 20 hours at 30C. and the cells are harvested followed by isola~ion of DNA~ Isolation is as disclosed in Example lB except that centrifugation with CsC1 and pxopidium iodide is omitted. Subsequently, 50 ~1. of this DNA is used to 15 transform E,coli K12 C600Rk~Mk- as disclosed in Example 7B. Plasmids in the trans~ormants are verified and identified by conventional means as taught in Example 11.
Example 20 Construction of Streptomyces _riseofuscus/pJL114, , S. qriseofuScus/pJL121, S. griseofuscus/pJL125, -S. griseofuscus/pJL180, and S. ~riseofuscus~pJL181~
The desired constructions were each indi-vidually 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 pJL120, were appro-priately used for the individual construction.

:~;

6~5 X-5773A _49_ Example ~1 Construction of Streptomyces griseofuscus/pJL190 A. Transformation The desired transformation was carried out in substantial accordance with the teaching of Example l9B
except that plasmid pJLl90, rather than plasmid pJL120, was used.
B. Selection lQ
The desired transformants were selected for neomycin resistance by overlaying the regenerating protoplasts w.ith R2 medium top agar containing suf-ficient neomycin to bring the final plate conce~tration to 1 ~g./ml. The resultant Streptomyces riseofuscus/
pJLl90 transformants were then tested for the expected ; pock morphology in suhstantial accordance with the procedure of Example 2OB.
Example 22 2~ Construction of Streptomyces griseofuscus~pJLlgS
The desired construction was made, selected, and recovered in substantial accordance with the teaching of Example 21 except that ~lasmid pJL195, xather than plasmid pJLl90, was used~
. .
~`

, ~
~ ..J

7 f;~5 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 5uf-ficient neomycin to bring the final plate concentration to 10 ~g./ml., rather than plasmid pJL195 and top agar containing sufficient neomycin to bring the final plate concentration to 1 ~g./ml., were used.
Example 24 Construction of Streptomyces fradiae~pJL120, S.
radiae/pJL114, S. fradiae/pJL121, S. fradiae/pJL125, S. fradiae/pJL180, S. fradiae/pJL181, S. ~radiae/
_ pJLl90, S. fradiae/pJL195 r and S. _radlae~pJL192 The desired construc-tions are individually ~ and raspectively made, selected, and recovered in substantial accordance with the respective teachings of Examples 19, 20, 21, 22, and ~3 except that S-treptomyces fradiae, rather than S. ~ , is used. In addition, the TSB medium or protoplasting and growing 5. fradiae was modified and contained only .2% glycine.

~Z~'7~

X-$773A -51-Example 25 Constructi~n of Streptomyces lividans/pJL120, S.
. _ _ lividans/pJL114, S. lividans/pJL121, S. lividans/
pJL125, S. lividans/pJL180, S. lividans/pJL181, SO lividans/pJLl90, S. lividans/~JL195, 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 Strepto-myces 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. WO79/01169, Example 2.
Example 26 Isolation of Plasmid pJL192 Mutant that Confers High Resistance To Antib otic Neomycin Streptomyces griseofuscus/pJL190 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/
pJLl90 exhibited resistance to 1.0 ~g./ml. of neomycin.
S. griseofuscus was spread conventionally on nutrient 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 S

~-5773A -52-resistance, the colony was designated S. griseofuscus/
pJL192. The plasmid, pJL192 was shuttled into E. coli K12 C600Rk-Mk- by back transformation as taught in Example l9C. The restriction site map of pJL192 appears not to be distinguishable from pJLl90.
Example 27 Isolation of Plasmid pJLl99 Mutant tha~ 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 ~prepared in Example 22), rather than S. griseofuscus/pJLl90, was used. A colony that exhibited high resistance to neomycin was designated Sn griseouscus/pJLl99.
The plasmid, pJLl99, was shuttled into E. coll K12 C600Rk-Mk- by back transformation as ~aught in Example l9C. The restriction site map of pJLl99 appears not to be distinguishable from pJL195.
Those skilled in the art will recognize that plasmid pJLl99 can also be conventionally constructed by substitu-ting the neomycin resistance-conferring fragment of plasmid pJL192 (prepared in Examples 15 and 27) for the pLR4-derived neomycin resistance-conferring fragment of plasmid pJLlg5. Such a substitution thus also results in the desired plasmid pJLl99.
Example 28 Construction of Plasmid pLR2 ~.~
A. HindIII Digestion of Plasmid pIJ6 About 20 ~1. (20 ~g.) of plasmid pIJ6 DNA, disclosed in Thompson et al., lq80, Nature 286:525, - `
7~

5 ~l. BSA(Bovine Serum albumin, l mg./ml.), l9 ~l.wa~er, l ~l. of indIII (containing 3 New ~ngland Bio Labs units) restriction enzyme*, and 5 ~l. reaction mix** were incubated at 37C. for 2 hours. The reac-tion was terminated by the addition of about 50 ~l. of4~1 a~monium acetate and 200 ~l. of 95% ethanol. The resultant DNA precipita-te was washed twice in 70~
ethanol, dried _ vacuo, suspended in 20 ~l. of TE
buffex, and frozen at -20C. for storage.
lC B. HindIII D~sestion of Plasmid pBR322 About 8 ~l. (4 ~g.) of plasmid pBR322 DNA, 5 ~l. reaction mix, 5 ~l. BSA (l mg./ml.), 31 ~l.
water, and 1 ~l. of HindIII restriction enzyme were incubated at 37C. for 2 hours. After the reaction was i terminated by incubating at 60C. for lO minutes, about 50 ~1. of ammonium acetate and 200 ~l. of ~5~ e~hanol were added. The resultant DNA precipitate was washed twice in 70% ethanol, dried ln vacuo, and suspended in 45 ~l. of water.
C. Ligation of HindIII Digested Plasmids pIJ6 and pBR322 A~out ~0 ~l. of HindIII treated plasm:id pIJ6 (from ~xample 28A), 20 ~l. of HindIII treated plasmid pBR322 (from Example 28B), 5 ~l. BSA (lmg./ml.), l ~l.
of T4 DNA ligase , and 5 ~l. ligation mix** were incubated at 16C. for 4 hours. The reaction was ~er-minated by the addition of about 50 ~l. 4M 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. ~he suspended DNA
constituted the desired plasmid pLR2.

* See the footnotes on pages 27 and 28 of the ,- ** specification 3'~

X~5773A -54-Example_23 Construction of E. coll K12 HB101/pLR2 ~ .
About 10 ml. of E. coli K12 HB101 cells (Bolivar et al., 1977, Gene 2:75-93) wexe S pelleted by centrifug~tion and then suspended in about 10 ml. of O~OlM 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 10 1.25 ml. of 0.03.~1 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 ~1. of 30m~ calcium chloride solution, and gently mixed in a test tube with about 200 ~1. of competent E.
coli R12 HB101 c~lls. The resultant mixture was in~
cubated on ice for about 45 minutes and then at 42C.
for about 1 minute. Next, about 3 ml. of L-broth (~ertani, 1951, J. Bacteriology 62:293) containing 23 50 ~g./ml. of ampicillin were added. The mixture was incubated with shaking at 37C. ~or 1 hour and then plated on L-agar (Miller, 1~72, Experiments in Molecular Gene~ics, Cold Spring Harbor Labs, Cold Spring Harbor, New York) containing ampicillin. Surviving colonies were selected and tested for the expected phenotype (Amp , Tet ), and constituted the desired Eo coli K12 ~B101/pLR2 transformants.
Representative plasmids and transfoxmants that can be constructed in accordance with the fore-going teaching include the ollowing listed below in Tables 1 and 2.
, , ;~

'7~

~;-57731~ -S5-J~
~ ~ U~
O D. H O
o c~l o ~ ~1 o ~1 0 C~ O ~ ~

~: ~1 0 U~ 1 0 0 o~ 4~ ? H ~ ~ 0 ~ o~ 0 1:: ~ O
u c~ d 1 r c.) ~ O t~ U7 O ~ ~10 ~-1 a ~ ~~ "'X P~ ~ ~ ~ X P~
u c~1 a)~ o c o ~~ ~ ~ c~
W ~ C) ~ 0 _I
O O~ O~ O ~ ~ ~ ~ ~n ~ u~
1: 0 H 0 ~ ~ C 'C ~ ~H t~a O Cb 1 C1-l H H H C`l U~ C~ O rl H O1--1 H H ~H O ~H O ~1 ~r~ .LI ~ ~-IJ.l P ~ ) JJ ~ 1 ~J J- O O O
X P ~ O ~1a) ,c~ X P~ ~:4 ~) H ~ H I Q~
o ~ ~ R 0 R
H H ~ ~ ~ H ~`1 CJ H H ~ H '~ ~D H ~ H ~D
~1 ~ ~ l ~1 . J.) ~_1 ~ t-l O O ~ ~ JJ ~ J.J
u~ ~ ~ ~ ~I cO C~ o ~0 ~ ~ ~ ~1 ~I c~ ~ ~ ~ u~
W ~ P~ U~p: P~ U~ 4 rl1 .~ ta 15 ~ ~ ~ :~ X ~ X ~
~ ~ ~ X
~ C'~
Ul C~
P.
2 ~ x O ~ ~ E~
C~

2::~ `:r oo ~ O U~ o N 4! , C~l r~. 1 0 1 ~ ~`J
c~

'CJ ~ ~ ~ C> o o 0 :z: ~ ~ '~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~'~ ~ ~ ~ ~ 7 O O ~I c~ 0 _I ~ c Z ~ ~ ~ ~ ~~ ~ ~ ~ ~ ~ ~ ~ ~P

... .

V~

' 1 H H ~1 0 _~ U Pq H I ~q C
O 1~ O ,~ Cl C~
O ~ ''a H _1 ~1 .:C U~ nl a: ~ e ~ ~ ~ ~ E
~r ~ ~ O~ v3 ~ O
Co ~ 14 ~ Cl~ 'd C ~ ~ ~1 1~1 .LI O t~
~ e ~ o o c.,~ ~ u IJ ~ O 1~ U 1--i t.l ~ h 11.1 J l 1 C 1--l H O t~ ~ O ~ P- h ~ ~
~1 LI ~ 1 ,a'1:1 ~0 P~ o a) ~
e v C :~: 1~1 I H 1~ `I V
Ct~ ~~ r~ ~rl ~ O X t~l ol ~ ~ o PC ~
O ~ Ot'lU~ ~ Cl~ al E c c~ o e ~ c'~ ~ I u ~ ~I ~ ~ ~ c). c~ w ~ ~
~ o ~ ol~o1~P: ~ ~~ D ~ 1 o -'I Ul~ w C~ u D ~ D O t~ U O J~ r U~
tO~ O ~ O ~,D 1~ ~ l .L) Q~ O .LI ~ ~-- ~ ~1 o e~ 7 C O ~15 ~ ~
. ~ ~ f . ~ ta ~O ~J31 ~ W U~ C ~ --~ ~ ~ C ~ _I
~2 C o 2 ~ o 2 ~ ~ 2 cq 2 ~ CL 2 ~ ~

.~
~ ~ ;Y; Ct~
~ E a~ CO~~ o a~
~, o v Z æ z ~ æ æ
o .. ~ ~ CL~ s tJ ~ r X,~., ~ ~ ~ '' ~ u~

~o ~ C~
U
~ ¦ ~ ~ L E E ~3 E

N ~: ~~
u~, 5.-1 ~c~l ~~1 ~1 _I
. ~

C ~O ~ U~
J O O O O O
E E ~
~ Z ~2 ~ ~ ~ ,_1 , 1 ~ _1 C s ~ I
E o ~ 0 7~ zI
~; I

` ,;, '7~5 . o o o ~d O ~ ,~
o ~1 ~ .,, ~ e c~ ~ ~ O ~ ~
:~ ~ o H J, ~ U~ ~ O
:~ ~
O
~ ~ o l:q ~ O ~ I ~
~ ,~ H t~
o ~ 1 ~ 3 ~
H ,C~ ~I H ~ h c~l o ~a ~
H O 3 ~
t~ R
c' ~ u~ G t~
O O O o E~
V
.,., ~ a~ ~
R O ~ Z Z Z æ
o ~ ~
u a~) æ x x æ x .
E~ .
~0 ... ..
~, ~
O Y
S~ ~ ~ ~ ~

2 5 ~ ~ ~ c;~
N . `~
~rl O 00 ~1 ~ Z

a~ I
~ .
e o X ~ u~
;

Table 2 Representative Transformants 1. Streptomyces R/pR wherein R is griseofuscus, _ ambofaciens, fradiae, or lividans and RL is - -independently pJL122, pJL123, pJL124, pJL126 pJL176, pJL1200, pJL1201, pJI.1202, pJL1203, pJL1204, pJL1205, pJL1206, pJL1706, pJL1800, pJL1801, pJLl900, pJL1902, pJL1905, pJL196, pJL197, pJL1907, pJL198, pHJL212 and pHJ213.
2. E. coli K12 R /pRl wherein R2 is C600Rk-Mk-, 294, C600 or RV308 and R is independently as defined above.

~ 25 :

Claims (24)

The embodiments of the invention for which an exclusive property or privilege is claimed are defined as follows:

1. A recombinant DNA cloning vector com-prising:
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 resis-tance to at least one antibiotic when trans-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 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 con-ferred.

2. The vector of Claim 1 which is a plasmid.

3. The vector of Claim 2 wherein the restric-tion fragment of plasmid SCP2 or SCP2* is the ~5.4kb EcoRI-SalI fragment, ~6.0kb SalI fragment, ~19kb EcoRI-HindIII fragment, or ~31kb EcoRI fragment.

4. The vector of Claim 2 wherein the E. coli origin of replication is the pBR322 origin of replica-tion, pBR324 origin of replication, pBR325 origin of replication, pBR327 origin of replication, or pBR328 origin of replication.

5. The vector of Claim 1 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 Claim 5 wherein the one or more DNA segments that confer resistance in Strepto-myces are DNA segments that confer resistance to neo-mycin or thiostrepton.

7. The vector of Claim 2 which is plasmid pJL120, pJL121, pJL180, pJL181, pJL125, pJL190, pJL192, pJL195, pJL199, pJL114, pJL122, pJL123, pJL124, pJL126, pJL176, pJL1200, pJL1201, pJL1202, pJL1203, pJL1204, pJL1205, pJL1206, pJL1706, pJL1800, pJL1801, pJL1900, pJL1902, pJL1905, pJL193, pJL196, pJL197, pJL198, pHJL212, pHJL213, or pJL1907.

8. The vector of Claim 2 wherein the DNA
segment that confers resistance to an antibiotic is the ~7.7kb EcoRI-HindIII restriction fragment of plasmid pJL192, the ~7.7kb EcoRI-HindIII restriction fragment of plasmid pLR4, the ~7.5kb EcoRI-partial SalI restric-tion fragment of plasmid pLR4, the ~1.35kb BamHI re-striction fragment of plasmid pLR2, or the ~1kb BclI
restriction fragment of plasmid pJL193.

9. The vector of Claim 8 which is a plasmid pJL190 or pJL195 high resistance mutant that in Strepto-myces confers resistance to neomycin at levels of at least 10 µg./ml.

10. Plasmid pJL 121.

11. Plasmid pJL125.

12. Plasmid pJL192.

13. Plasmid pJL193.

14. Plasmid pJL197.

15. Plasmid pJL198.

16. Plasmid pHJL212.

17. Plasmid pHJL213.

18. A transformed host cell comprising the recombinant DNA cloning vector of Claim 2.

19. The host cell of Claim 18 which is Streptomyces, of species lividans, griseo-fuscus, fradiae, or ambofaciens.

20. The host cell of Claim 18 which is E.
coli K12.

21. A restriction fragment comprising the plasmid SCP2 or SCP2* ~5.4kb EcoRI-SalI or ~6.0kb SalI
restriction fragment of Claim 1.

22. A restriction fragment comprising the plasmid pJL192 ~7.7kb EcoRI-HindIII restriction frag-ment of Claim 8.

23. 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 resis-tance to at least one antibiotic when trans-formed 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 con-ferred.

24. The process of Claim 23 wherein the restriction fragment of plasmid SCP2 or SCP2* is the ~5.4kb EcoRI-SalI fragment, ~6.0kb SalI fragment, ~19kb EcoRI-HindIII fragment, or ~31kb EcoRI fragment and wherein the E. coli origin of replication is the pBR322 origin of replication, pBR324 origin of replica-tion, pBR325 origin of replication, pBR327 origin of replication, or pBR328 origin of replication, and wherein the one or more DNA segments that confer re-sistance in E. coli are DNA segments that confer resistance to ampicillin, chloramphenicol or tetra-cycline and wherein the one or more DNA segments that confer resistance in Streptomyces are DNA segments that confer resistance to neomycin or thiostrepton.