JPS5877897A - Cloning vector for streptomyces genus and analog germs - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Recombinant DNA Comprising a Restriction Fragment Containing a Sexual Origin and One or More DNA Fragments Conveying Antibiotic Resistance
Concerning cloning vectors and transformants of the vectors.

This antibiotic resistance transfer cloning vector is used for Streptomyces and related host cells. Until now, the development and development of recombinant DNA technology in the above microorganisms has been... This is because cloning vectors generally do not have genetic selectable markers. This has been particularly difficult since vectors are functional and can be selected in Strephbutmyas and other host bacteria. This represents a significant advance in the art.

The vectors are particularly advantageous because they are small and veratile and can be introduced into and selected for Streptomyces cells that are susceptible to the antibiotic to which resistance is to be transferred. More than half of clinically important antibiotics are produced by strains of the genus Streptomias. It would be desirable to develop cloning systems and vectors that can be applied to this group of industrially useful bacteria. The present invention is. Provide such a vector. Thus, genes can be cloned into Streptomyces to produce new antibiotics and antibiotic derivatives as well as increase the yield of known antibiotics.

This vector provides a medium for cloning DNA into a Streptomyces host cell and allows for easy selection of transformants. Because transformation occurs very infrequently, such functional tests are difficult to determine which cells among millions of cells have plasmid DNA.
It is practically necessary to determine whether you got A. This means that non-selective DNA sequences can be inserted into the vector.

This is important because, after transformation, cells containing the vector and the particular required NA sequences can be harvested by appropriate antibiotic selection methods.

Zero-order terminology is described below for purposes of the invention disclosed and described herein.

Recombinant DNA cloning vector = spontaneously replicating and includes within its scope a plasmid containing a DNA molecule to which seven or more additional DNA fragments can be added, but this strain Introduction of DNA into host cells. , Transformee - transformed recipient host cell.

Susceptible host cells = in the presence of certain antibiotics.

A host cell that cannot grow because the DNA fragment that conveys resistance to that antibiotic is not present.

Restriction fragment = A linear portion or all of a plasmid formed by the action of one or more restriction enzymes on a plasmid.

Isomers due to differences in insertion sites, = 7 of two or more possible recombinant DNA molecules created when a DNA fragment is inserted into two or more suitable sites of the recipient strain's DNA.
One.

Bam HI restriction fragment of plasmid pLR2 - Contains plasmid pIJ kb
Same as the thiostrepton resistance transfer BamHI fragment of plasmid pLR/or pLRQ 3 Qkb Ba
mHI restriction fragment=same as the 3, g kb neomycin resistance-transferring BamHI fragment contained in plasmid PIJ2 Amp R-ampicillin resistance phenotype.

TetS-tetracycline sensitive phenotype.

Th1oR=thiostrepton resistance phenotype.

NeoR-Neomycin resistance phenotype.

This recombinant DNA cloning vector contains a) a functional origin of replication (afun) of plasmid pEL'/θ3;
ctional origin of replica
b) seven or more DNA fragments that convey resistance to at least one antibiotic when introduced into a susceptible host cell capable of transformation, cell division and culture.

Bifunctional, i.e. E,
This recombinant DNA cloning vector can be used in Coli, Streptomyces and other genera.

a) a restriction fragment containing a functional origin of replication of plasmid pEL/θ3; b) one or more restriction fragments that, when transferred into susceptible host cells capable of transformation, cell division and culture, convey resistance to at least seven antibiotics. and c) a functional replicon of the E. coli plasmid and a restriction flag knot that conveys antibiotic resistance.

This vector is constructed by ligating a DNA fragment conveying resistance to seven or more antibiotics and a restriction fragment containing the origin of replication of plasmid pEL/03. Additionally, to construct a trifunctional vector, a restriction fragment containing a functional replicon of the E. coli plasmid and conveying antibiotic resistance is further ligated.

Plasmid pE constituting the replication origin-containing fragment
L/θ3 is.

Plasmid pEL10 has several restriction sites that are particularly suitable for molecular cloning of approximately 2θθkb'''C-1.
The replication origin of 3 is localized within the 1,5'kb BamHI restriction fragment, so the 1/kb BamHI
Digesting this plasmid with restriction enzymes that cut outside the region can generate nine different origin-of-replication-containing fragments. A detailed restriction site and functional map of plasmid pEL/θ3 is shown in FIG. 1 of the accompanying drawings. For purposes of this specification, all figures, including FIG. 1, are not scaled.

Plasmid pEL/θ3 was isolated from Streptomyces granular rubber (Streptomyces g.
ranulo-ruber)&A399/Q/3/pE
L103 [This strain was obtained from Peoria, Illinois.
Northern Regional Research Laboratory (Northern Region)
alResearch Laboratory) and is a permanently preserved strain].

The above strain is under deposit number NRRL/2311t9.
This is a suitable source and repository of the above plasmids and is available to the public. Streptomyces granulolaper Nah399/U/3/pEL i o3 is a strain that itself carries the plasmid pEL/03, Streptomyces granulolaper Nah399/
2 [This strain also has accession number NRRL/23. ! ``Deposited with the above-mentioned depositary under No. 9].

Although a wide variety of replication origin-containing fragments of plasmid pEL/03 can be constructed, one herein includes, for example, two fragments.
．． and the /99 kb BamHI restriction fragment. These fragments can be combined with seven or more antibiotic resistance transfer DNA fragments (herein, for example, thiostrepton resistance transfer of plasmid pLR, 2 A kbBamHI restriction fragment and neomycin resistance transfer of plasmid pLR/or plasmid pLR4'). II kbBam HI restriction fragment) to form vectors that exemplify the invention.

Plasmid PLR,2, the source of the thiostrepton resistance transfer fragment, is approximately /, f 7 kb and Hind m-treated plasmid pIJ l [Tho
mpsoneL ato, /910. Nature
216:! ;2! ; disclosed in] and Hind[l
It is constructed by ligating the treated plasmid pBR3,2,2. Plasmid pLR/, the source of the neomycin resistance transfer fragment, has a length of approximately /1. J'kb
and plasmid pI instead of plasmid pIJ
The configuration is similar, except that J2 [disclosed in Thompson eL al,;/910] is used. Since plasmids pLR,2 and pLR/ are both functional in coli.

It can be amplified and isolated by conventional methods and used for the primary operation. A similar construction yielding plasmid pLR@ is BamHI-treated plasmid pBR3.
22 and BamHI-treated plus cdo pLR/. Restriction sites and functional maps of plasmids pLR/, pLR2 and pLR4Z are shown in Figures 2 to tI of the accompanying drawings, respectively.

For convenience and ease of construction, the thiostrepton resistance transfer lkb BamHI fragment and the neomycin resistance transfer JIIkbBamHI fragment were transferred to plasmids pEL/03, g, jkb or /?
Ligate with the 9 kb BamHI fragment containing the origin of replication. The resulting recombinant DNA was self-ligated (self-linked).
igation) to produce a plasmid that exemplifies the invention. A bidirectional recombinant plasmid results from the orientation of the individual resistance-transferring DNA fragments.

Thus, 2♂kbBam of plasmid pEL/θ3
HI fragment and plasmid pLR, 2 kb
Ligation of the BamHI fragment yields the exemplary plasmids pEL7θ7 and pEL/0! 344k of plasmid pLR/or plasmid pLR;
Ligation of the b-SiniHI fragment results in an exemplary plasmid pEL/θ? and pEL//θ and ligation of both fragments together yields the exemplary plasmid PEL//3゜p
EL//Q, pEL//! ; and p
Produces EL//4. Similarly, plasmid pEL
/θ3/99 kb BamHI fragment;
Ligation of the /6 kb BamHI fragment of plus 2 pLR2 creates the exemplary plasmids pFL /θ♂ and p
EL/θg and plasmid pLR/or plasmid pLR! Ligation of the 317 kb BamHI fragment of pEL/// and pEL
/ / 2 is generated and the nine fragments are ligated to form the exemplary plasmid pEL//? :pEL//za, pEL//9
and PEL/2θ.

Various restriction fragments of plasmid pEL103 are
, 11,5'kb BamHI restriction fragment, which, if present, can be used to ligate antibiotic resistance-transferring DNA fragments. Additional restriction fragments of plasmid pEL/θ3 useful in constructing exemplary plasmids within the scope of the invention include:
Pat I, 5phl, Bgjln.

It is not limited to these. The specific antibiotic resistance-transferring DNA fragment is not limited to one location on the plasmid pEL/θ3 fragment, but can be found in a variety of locations provided that the origin of replication or other important physiological functions controlled by the plasmid are not inhibited. It can be linked or inserted at the site. If you are a person skilled in the art.

It is understood and can be easily determined which sites are suitable for ligation or insertion of particular DNA fragments.

The thiostrepton and neomycin antibiotic resistance transfer DNA fragments exemplified herein are l 4 kb and ! of plasmids pLR,2 and pLR/, respectively. 4
Although a BamHI restriction fragment of Z kb.

Those skilled in the art can construct and use other DNA fragments that convey resistance to thiostrepton and neomycin, either individually or together. Other thiostrepton resistance transfer DNA fragments of plasmid pLR2 include, for example, the /3 kb Pat I restriction fragment, and the i
The Bcl l subfragment of the lz kbBamHl restriction fragment is mentioned. Further neomycin resistance transfer DNA fragments of plasmid pLR/ include the 35 kb Pat I control restriction fragment and the larger Sst l-Kpn l restriction fragment of the 3,1 Ilkb BamHI restriction fragment.
subfragments.

Chloramphenicol, streptomycin.

hygromycin, viomycin, tylosin.

Additional DNA fragments that convey resistance to the same or different antibiotics, such as erythromycin, can be constructed and used by those skilled in the art. Erythromycin resistance transfer DNA
Examples of fragments include 1 to 2 of plasmid pIJ Ko3.
Zakbshi41. ~, 17kb! I Bgln,
~1 Okbl'Nnd m, ~Q! ;kbss
I can do it. Plasmid pIJ 4! 3 is E, Coryza 03/pxJta3 [This strain was purchased from the American T Culture Collection, Roekville, Maryland.
ype Cu1Lure Col 1ee-Lion)
It has been deposited with the University of Tokyo and is a permanently preserved bacterial strain.

The above strain is available to the public under deposit number ATCC39/J% as a suitable source and reservoir of the above plasmid.

Functional derivatives of various antibiotic resistance-transferring DNA fragments are
Adding a specific nucleotide according to the gene codon,
It can be generated by elimination or substitution. Those skilled in the art will understand that vectors within the scope of the present invention can also be generated by ligating the modified fragments described above or other antibiotic resistance transfer DNA fragments with the origin-of-replication-containing fragment of plasmid pEL/θ3. .

Further derived vectors exemplifying the invention may also be constructed6.
For example, from plasmid pEL/03 to BcJl-
When BamHI is deleted, the exemplary plasmid pFJ/
24Z and further derivatives may be generated from this plasmid. Thus, plasmid pLR/or pL
~3 of R4t. Insertion of the 'I kbBamHI neomycin resistance transfer fragment into plasmid pFJ/24' yields exemplary plasmids pFJ/Q 4' and pFJ/Gj. Plus 2 pFJ/g
! Deletion (Bejl -BamHI) results in the exemplary plus 2-do pFJ/4Z. ~2.7 kbsa7 of plasmid pIJ#3! I-BgJ
Insertion of the II erythrominein resistance transfer fragment into plasmid pFJ/2'A results in the exemplary plasmid pFJ/4L7. Similarly.

Bluff, ido pIJII3~, l Q kbsa7!
Insertion of the I erythromycin resistance transfer fragment results in exemplary plasmids pFJ/Q and pFJ/4.

The antibiotic resistance transfer derived plasmid described above is pEL/
Since it contains the θ3 replication origin, it is within the scope of the present invention.

Restriction fragments of plasmid pEL/θ3 and various antibiotic resistance transfer DNA fragments can be modified to facilitate ligation. For example, 0 molecular-shaped linking substances (link
ey) to specific plasmid pEL/03 restriction fragments or antibiotic resistance transfer DNA fragments.

Thus, specific sites for primary ligation can be conveniently constructed. Plasmid pEL/θ3 and the replication origin-containing restriction fragments of plasmid pEL/θ3 can also be modified by adding, removing, or substituting specific nucleotides to change the properties during DNA ligation and provide various restriction sites. It is possible. One of ordinary skill in the art understands nucleotide chemistry and genetic codons and can therefore understand which nucleotides can be interconverted and which DNA can be modified to be desirable for a particular purpose.

Vector of the present invention (for example, plasmid pEL/θup
EL/05, PEL/θ9, pEL/
/ 3, pEL 10IApEL/ / /,
pEL//7, pFJ72, pFJ/Guku pFJ/447, etc.) and antibiotic resistance transfer restriction fragments containing functional replicons of E. coli plasmids (e.g., plasmids pBR112, pBR32).
t/L, pBR32K, pBR32f, etc.) 17:
can be ligated to generate a new trifunctional plasmid that can be used in E. coli and Streptomyces. These constructs are specifically referred to herein as plasmid pEL 121pE.
T-/22. pFJ/! ;0 and pFJ/j/
are particularly advantageous because plasmid propagation and manipulation is faster and more convenient in E. coli than in Streptomyces. Thus, after performing the desired recombinant DNA step in the E. coli host system,
Whole plasmid or specific Streptomyces DNA
and, if necessary, reconstituted in the form of a plasmid.
can be introduced into Streptomyces or related host cells.

The recombinant DNA cloning vector of the present invention is not limited in use to one species or seven strains of the genus Streptomyces. On the contrary, the vector has a wide range of applications, including a large number of Streptomyces species, especially aminoglycosides, macrolides, β-lactams.

Particularly unrestricted strains of economically important species that produce antibiotics such as polyether and glycopeptide antibiotics (
restriction leas 5 trains). Such unrestricted shares are well known in the art and are common law.
tal,,/910.

Microbiological Reviews
Since the host cells of the genus Streptomias are easily selected and collected from the genus Streptomias by ip:, lθb), the host cells of the unrestricted strain lack restriction enzymes.

Plasmid DNA is not cut or destroyed during transformation. For purposes of this specification, host cells containing restriction enzymes that do not cut the restriction sites of the vectors of the present invention are also considered to be unlimited.

As a preferred host cell for an unrestricted strain of the genus Streptomyces that produces aminoglycoside antibiotics and can be transferred, this vector is particularly useful.

For example, there are limitless cells of the 9th order.

(Left below) S, kanam ceticus (kanamyc
ins), S, chre mutual 7 (aminosidi
ne), dan, riseoflavus (ant
ibiotic HA1267), S, m1cro
s oreus (antibiotic 5F-76
7).

S, ribosidificus (antibio
tic SF 733), S, flavo-7(s
pectinomycin), S, 7 (actin
o-spectacin), S, rimosus
forma aromam cinus (parom
omycins, catenulin), S, f
radiae var.

1talicus (aminosidine), S
, bluensis var, bluensis (
bluensomycin), S. catenul
ae (catenulin), S.

olivoreticuli var, celluyama stop dokuyuus (destomycin A), p, t
enebrarius (tobramycin, a
pramycin), S.

1 avendulae (neomycin), S.
albo riseolus (neo-mycin
s), S, albus var, 7 (meta
mycin).

s, h rosco 1cus var, 7 (
spectino-mycin), S, bikin
iensis (streptomycin), S.
Anus”! Nagi (streptomycin), S, e
r throchromo enes var.

narutoensis (streptomycin
), S, strepto-myci
n), '4. p (streptomycin),
'S-, rameus (streptomycin)
, S. olivaceus (streptomy
cin).

S, mashhuensis (streptomyc
in), S, h rosco 1cusvar,
limoneus (valshi amycins),
S, rimofaciens (destomycin
s), S, h rosco 1cus form
a7 (glebomycin), S, fradi
ae (hybrimycins, neomycins
).

S, eurocidicus (antibioti
c A16316-C), S, aquacanus
(N-methyl hygromycin B),
S, crystallinus (hygro-m
ycin A), S. noboritoensi,
s (hy(Hromycin), S.

hygroscopicus (hygromycin)
s) + S, atrophaciens (hyniro
-mycin), S. kasugaspinus
(kasugamycins), S, kasuga
-ensis (kasugamycins), S,
netropsis (wholesale tibioticLL-A
M3k), Dan, 1ividus (Lividomy
cins), S. hofuensis (seldo
mycin complex) and 0. Ustan us
(ribosylparomamine).

Suitable host cells for unrestricted strains of the genus Streptomias that produce macrolide antibiotics and for which this vector is particularly useful include, for example.

The following species of unrestricted cells may be mentioned:

S. caelestis (antibiotic
M2B5), S. platensis (plate
nomycin), s, rochei var,
volubiLis (anti-biotic T2
6:36), S. venezuelae (meth
ymycins), S.

griseofuscus (bundlin), S
, narbonesis (josa-mycin,
narbomycin), and fungicidic
us (antibioticNA-181), S,
griseofaciens (antibioti
c PA133A+ B) sS, roseocitr
eus (albocycle), S, bru
neogriseus (albocycle),
S. roseochromogenes (albo
cycle).

S, cinerochromogenes (cin
eromycin B), S, albus (alb
omycetin), and felleus (ar
gomycin, picromycin).

S, rochei (lankacidin, bo
rreiidin), S, violaceo-ni
ger (lankacidin), mutual, grise
us (borrelidin).

(turimycin, relomycin, ma
ridomycin, tylosin.

carbomycin), S, griseospi
ralis (relomycin), S.

1 avendulae (aldgamycin),,
'S, rimosus (neutramycin)
.

each other, deltae (deltamycins),
S, fungicidicus var.

espinomyceticus
cins), S,'furdicidicus(my
decaa+ycin), S, ambofacie
ns (foromacidin D).

S, eurocidicus (methymyci
n), mutually griseolus (griseomyci
n), S. flavochromogenes (
amaromycin.

shincomycins), S. fimbria
tus (amaromycin), mutual.

fasciculus (amaromycin),
S, erythreus (erythro-myc
ins), S. antibiotics (ol.
eandomycin), ! 3゜olivochrom
mogenes (oleandomycin), S
, spinichromo-genes var,
suragaoensis (kujimycins)
, S. kitasato-ensis (1euc
omycin), 3. narbonensis v.
ar, josa-myceticus (leuco
mycin A3. josamycin), S,
albo-griseolus (mikonomy
cin), S, bikiniensis (cha
lco-mycin), S. circratus (
circramycin), S, djakarten
sis (niddamycin), S, euryt
hermus (angolamycin).

S, fradiae (tylosin, 1act
enocin, macrocin), S.

goshikiensis (bandamycin)
, S. griseoflavus (acumyci
n), S, halstedii (carbomy
cin), S. tendae (carbomyci
n), S, macrosporeus (carb
omycin), S.

thermotolerans (carbomyci
n) and S. albireticuli (carbo
mycin).

Suitable host cells for unrestricted strains of the genus Streptomyces which produce β-lactam antibiotics and which the vector is particularly useful for transfecting include, for example.

The following species have unrestricted cellular power.

S. lipmanii (A1688J MM455
0. MM13902), S, clavu-1ig
erus (A16886B, clavulanic
acid), S, lactam-durans
(cephamycin C), S, griseu
s (cephamycin A, B), S, hy
griscopicus (deacetoxycep
halosporinC), S, wadayame
nsis (1/S-341+2-D), S, ch
artrewis (SF 1623), S, het
Eromorphus! and S, panayens
is(C2081X); S, cinnamonen
sis, S. fimbriatus, S.

halstedii, mutual, = tracing and S, vir
idochromogenes (cephamycin)
s A, B); S, cattleya (thie
namycin) H and S, olivaceus,
S, flavovirens, S, flavus
.

S. fulvoviridis, S. argen
teolus and S, 5ioya-ensis (M
M 4550 and MM 13902).

For example, one vector is particularly useful and can be transfected with suitable host cells of unlimited strains of the genus Streptomyces that produce polyether antibiotics.

The following species of unrestricted cells may be mentioned:

S, albus (A204. A28695A and B, salinomycin).

S, hygroscopicus (A218. e
mericid, DE3936), A12OCA2
8695A and B, etheromycin,
diamycin), S.

griseus (grisorixin), S
, congJobatus (ionomyci
n).

S, eurocidicus var, aster
ocidicus (laidlomycin).

S, 1asaliensis (lasalocid
), S. ribosidificus (lonom
ycin), S, cacao var, asoe
nsis (lysocellin).

S, cinnamonensis (monensi
n), S, aureofaciens (naras
in), S. gallinarius (RP:
30504), S, and) ng-woodensis
(lysocellin), S, flaveolu
s ((:P38936)+S, mutabilis
(S-11%3a) and S, violaceon
iger (nigericin).

Suitable host cells for unrestricted strains of the genus Streptomyces which produce glycopeptide antibiotics and which the vector is particularly useful for transfecting include unrestricted cells of the following species, for example:

S. orientalis and S. haran
omachiensis (vanco-mycin)
; S, candidus (A-35512, av
opacin) and S, eburosporeu
s (LL-AM 374).

Other suitable host cells for unrestricted strains of the genus Streptomyces that the present vectors are particularly useful for transfecting include.

For example, there are unlimited cells of the 9th order.

S, coelicolor, S, granulor
uber, S. roseosporus.

S, 1ividans, S, tenebrari
us, S, acrimycins, S.

glaucescens, S, parvilin,
S. pristinaespiralis.

S, violaceoruber, S, vina
ceus, S. espinosus.

and S, azureus.

In addition to the representative Streptomyces genus unrestricted host cells mentioned above, the nine vectors include one eg Bacillus.

Cells of unrestricted strains of Staphylococci and other genera such as the related genera of Actinomycetes, including Streptosporangium, Actinoplans, Nocardia and Micromonospora, are also useful and can be transfected. Thus, the vectors of the present invention are widely applicable, useful, and transferable to a variety of microbial host cells.

The specific contents of the present invention are all beneficial, but one recombination D
Some NA cloning vectors and transformants are preferred over others.

That is, preferred vectors include plasmids pEL103゜pEL/θj, pEL709, pEL//
3, pELlOIA, Otsu///, PEL//7. p
EL/2/, pFJ/21ApFJ/g! ,
pFJ/4Z Otsu, pFJ/4'7, p
FJ#/.

pFJ/! ; 3, pF', T/3; 3
and pFJ/AO, and the preferred transformant is Streptomyces ambofaciens/pEL
103. s, ambofascens/pEL105. S,
ambofaciens/pEL109. S, Ambofaciens/pEL//3°S, Ambofaciens/PE
L/oa, s, ambofaciens/PEL
/t t, s, ambofacienos/PEL//
7. E, Cori K /2 HB10/ /pEL/2/:
S, Ambofaciens/pFJVx+, s, Ambofaciens/pFJ/≠+, S, Ambofaciens pFJ/4'&-8, Ambofaciens/pFJ7
Da ZE, stiffness / 2 HB10 / /pFJ / 30
, E, Cori / 2HB / θ / / pFJ / 3
3, E, stiffness/2 HB10//pFJ/!
; 3 and E, coli Ktx HBtot/pFJ/l
θ. Furthermore, within the above-mentioned preferred group, the plasmids pEL7θ3, pEL/0! ; , pEL/
/3, pEL109, pFJ/2g, pFJ
/Qg and pFJ/da7 and transformants S
, ambofaciens/pEL/θ3. S, Ambofaciens/pEL/0 modifiedS, Ambofaciens/pELt/3. s, ambofaciens/pEL1
09. s, Ambofaciens pFJ/211,
Most preferred are S, ambofaciens pFJ/ψda and S, ambofaciens/pFJ i tt 7.

The recombinant DNA cloning vectors and transformants of the present invention have broad utility.

Helps meet the need for cloning media suitable for use with Streptomyces and related microorganisms. Furthermore, the ability of the vector to convey resistance to antibiotics that are toxic to untransformed host cells also provides a functional means for selecting transformants.

This is important as it is essentially necessary to determine and select the particular cells from which the vector DNA was obtained. Even in the case of a DNA fragment for which there is no functional test to confirm its presence, one can be inserted into a vector and transformants containing non-selectable DNA can be isolated by an appropriate antibiotic selection method. Such non-selectable DNA fragments can be inserted anywhere except within regions necessary for plasmid function and replication, and include, but are not limited to, genes specifying all types of antibiotic-modifying enzymes and regulatory genes. Not done.

More specifically, the non-selectable DNA fragment containing the gene is inserted into the central SaJI restriction site of the BamHI fragment carrying the resistance transfer of a plasmid, such as the exemplary plasmid pEL//3. This insertion inactivates the thiostrepton resistance gene,
Thus, transformants containing one recombinant plasmid can be easily identified. To do this, we first screen for neomycin resistance.

Next, transformants that are not resistant to thiostrepton but resistant to neomycin are identified. Similarly, if you need an NA fragment, for example! Insertion into the internal BamHI restriction site of the resistance-transferring BamHI fragment of gkb inactivates the neomycin resistance gene. Thus, the transformants carrying this recombinant plasmid also. Transformants can be easily identified by first selecting for thiostrepton resistance and then identifying transformants that are not resistant to neomycin but resistant to thiostrepton.

Therefore, by being able to select Streptomyces and related cells based on whether they are resistant to antibiotics, extremely rare cells containing the necessary specific non-selectable DNA can be efficiently collected.

As mentioned hereinabove, functional testing by antibiotic resistance is also possible. It is also used to determine the location of DNA fragments that act as regulators and regulate the expression of individual antibiotic resistance genes. Such fragments include promoters, attenuators,
regressor, inducer.

Examples include, but are not limited to, ribosome binding sites. Such fragments are used to control the expression of other genes in cells of the genus Trebtomyces and related microorganisms.

The thiostrepton and neomycin resistance transfer vectors of the invention also include. It is useful to ensure that the ligated DNA fragments are firmly retained within the host cell over many generations. Genes or DNA fragments covalently linked to thiostrepton or neomycin resistance transfer fragments and propagated in either cells of the genus Streptomyces or related microorganisms are toxic to untransformed cells. It is maintained even if the transformants are exposed to some degree of thiostrepton or neomycin. Therefore, transformants that have lost the vector, ie, the covalently linked DNA, are unable to grow and are removed from culture. Thus, the vector of the present invention is necessary.
The A sequence can be stabilized and maintained.

The cloning vectors and transformants of the present invention provide for the cloning of genes to improve the yield of a variety of products currently produced in Streptomyces and related cells. Examples of such products include, but are not limited to, streptomycin, tylosin, cephalosporins, actaplanin, nalan, monensin, apramycin, tobramycin, erythromycin, and the like. The present invention is.

Examples: Ebachitinsulin, human gloinsulin.

Commercially important proteins such as glucagon and interferon, enzymatic functions in metabolic pathways leading to commercially important steps and compounds, and DNA sequences that encode regulatory factors that improve gene expression. Cloning,
Selectivity vectors useful for characterizing and reconstitution are also provided. Examples of the above-mentioned desired DNA sequences include streptomycin, cephalosporin, tylosin, and actagranidi. Naradin, Monensin, Abramycin.

Enzymes or antibiotics that catalyze the synthesis of antibiotic derivatives, such as tobramycin and erythromycin derivatives, also increase through the by-product of other products.

Examples include, but are not limited to, DNA encoding an enzyme that causes Thus, by inserting and stably producing such modified DNA fragments, it is possible to increase the yield and utilization rate of antibiotics produced by Streptomyces and related microorganisms.

Streptomyces Granulorubber mA399/Q/
3/PEL/θ3 can be cultured in a number of ways using any of several different media. Preferably in the culture medium)
Hydrocarbon sources include, for example, molasses, glucose, dextrino and glycerol; nitrogen sources include, for example, soy.

flour (soy-41our), amino acid mixtures and heftonka. Also contains nutritional mineral salts.

Sodium, potassium, ammonia, calcium.

Commonly used salts that can yield phosphates, chlorides, sulfates and ionic substances include. Essential trace components are also added, as are necessary for the growth and development of other microorganisms. Such minor components are normally supplied as impurities along with the addition of other media components.

Streptomyces Granulorubber NaA399/Q
/3/PEL103 has a pH of approximately 5 under aerobic culture conditions.
It is grown at temperatures within a relatively wide range of ~9°C, approximately /J-H θ°C. However, plasmid pEL/0
3, it is desirable to start the culture in a medium with a pH of about 72 and maintain the culture temperature at about 30°C. Cultivation of Streptomyces granulorubber Nah399/Q/3/pEL103 under the conditions described above results in a cellular reservoir from which plasmid pEL103 can be isolated in a conventional manner using techniques well known in the art.

The following examples further illustrate the invention described herein. Explanations and practical prescription procedures constituting the present invention will be described as necessary.

(Margins below) Isolation A of plasmid pEL/03, Streptomyces granulorubber (Strep
tomyceg granuloruber) A
A 399/, 2. / 3 /I) Culture of EL103 Streptomyas j Gramello Rubber 1'a A 3
99/2. /3/pEL/03 (NRRL/
2! The inoculum at the growth stage of G. 9) was added to sterilized tributicase soy broth (trypticase soy broth)
The strain was prepared by a conventional method of growing the strain in a solution of C. aoy broth (C30tttl) dissolved in deionized water at 339/l under deep aeration culture conditions.

Inoculum in tributicase soy broth.

After 414 hours of incubation at 30°C, approximately
Tributicase soy broth sterilized with θg/ (!θ
θml) without adjusting the pH.

After approximately 2θ hours of incubation at 3θ°C, the Streptominus strain used for recovery and subsequent isolation of plasmid DNA
Granuro Rubber A A 399/2. ／ ３／
Cells of pE L/θ3 were obtained.

Jar Tributicase Soy Broth is available from Tifco Laboratories (Difc, Detroit, Michigan).

Laboratories).

B. Plasmid isolation Streptomyces granulorubber & A399/, 2
．． /3/pEL/03 cells (wet weight approximately 72f)
Centrifugation (70 minutes, tl″C, 10,0θθrpm
) TES buffer [007M tris(hydroxymethyl)aminoethane].

OθO/MEDTA, 3t1% sucrose, pH
(approximately Sθml), followed by θyu, tMEDTA
Lysozyme (approximately θ, 25 g) suspended in (/θml)
added. After incubating this mixture at 37°C for about 75 minutes, TE buffer [007M Tris.

70% Triton (Triton) dissolved in θOθ/M EDTA, I)H♂]
g/).

Incubated at 65°C for approximately 75 minutes. After centrifuging the lysate (gt min, t″C, 15θ0θrpm), the supernatant was diluted once with isoamyl alcohol.

It was extracted seven times with a 21:/solution of chloroform and isoamyl alcohol. Next, the aqueous layer (3M sodium acetate)
about θjg/) and 3 volumes of cold 93% ethanol (-
, 20"C) was added. The ethanol precipitate was rapidly placed in a dry ice-ethanol bath, and the DNA precipitate was centrifuged (for 1 minute, q"C, 10,00 rpm).
) was collected. The obtained precipitate was vacuum-dried, and STE buffer [0θ/M Tris.

00θ/M EDTA, 00/M sodium chloride] (1
/W/). Bluff, E by centrifugation (IAO time, /j″C, 33,000 rpm) using a cesium chloride gradient with ethidium bromide.
The desired plasmid pEL/
θ,,? The DNA band was taken out, and ethidium bromide was extracted by a conventional method. Isolate plasmid pEL/03 DNA by precipitating the DNA in 3 volumes of ethanol.
Dissolve in 0 times diluted TE buffer (/l/) and -,20
It was frozen and stored at C.

Example 2 Configuration A of plasmid pLR2, Kawakuni's digestion of plasmid plJ4, plasmid pIJ 6D N A (Thampson
et at,.

/91θ, Nature 21 Otsu: Disclosed in L edition] (
approximately 2θ4°, 20 pg), BSA (bovine serum albumin,
/119/Tomi 1) (J-Ill), water (/9Th), Hariretsu m (containing 3 New England Bio Labda units) restriction enzyme 'Ctpl) and reaction solution "<5ti
) was incubated at 37°C for 2 hours. The reaction is uM
It was stopped by adding ammonium acetate (approximately Jθ) and 95% ethanol (ZOOpl'). Obtained DN
The precipitate of A was washed twice with 7θ% ethanol, dried in vacuo, and suspended in TE buffer (2θ/1 l), −,26
Stored frozen at °C.

Hata restriction enzyme can be obtained from:

New England Bio Labs, In
c.

32 Tozsr Ra1d Beverly, Massachusattaθ/9
/j Boehringer Mannheim Biochemicals B*
ehr inger -Mannheim Bioch
emicals7917/ Castleway D
liveIndianapolis, Indiana
230 Bethesda Research Laboratories
Incoholated Bethssda Re5earch Laborat
oriea IncP, 0. Box, j; 77 Gaitheraburg, Marylan+d
, 207 Otsu θm Hindl [The reaction solution for restriction enzymes was prepared with the following composition.

60θmM NaCj + 700mM Tris-HCl, p) (797θrnMM
gC1゜70mM dithiothreitol B, injection of plasmid PBR3U2■ Digested plasmid P
BR,? 22 DNA (about ♂4.ge).

Reaction solution (jμ), BSA (／■／舅l, 5 ml), water (3
/ld), HindIII restriction enzyme (/m) at 37°
It was incubated for 2 hours. After stopping the reaction, incubate for 70 minutes at 60"C.

Ammonium acetate (FJ!0ttl), 9. !
; % ethanol (2θθ/ll) was added. The resulting DNA precipitate was washed twice with 7θ% ethanol and dried under vacuum.

Suspended in water (ll-jTh).

Ligation of C0■■digested plasmid pIJ,4 and pBR3 Uni/Hind■treated plasmid pIJ2 [Example 2
] (approximately 20 p!), Hindl
J-treated plasmid PBR322C obtained in Example 2B] (20pd), BSA (/w4/yg
lA;ti), 'r<I)NA ligase' (/
pl) and ligation solution''(!;p!') were incubated for 4 hours at 16 °C. 4LM ammonium acetate (approximately
The reaction was stopped by adding 9j% ethanol (2θθl) and the resulting DNA precipitate was washed twice with 70% ethanol, dried under vacuum, and suspended in TE buffer ζ. The suspended DNA was the desired plasmid PLR2.

*T II DNA') Gauze can be obtained from the following locations.

New England Bio LabB, I
nc.

32 Tozer Rd.

Beverly, Massachusetts θ/
9/j Urn Urn A freezing solution was prepared from the following composition.

! ;00mM), Lis-HCl, pH712θOmM
Dithiothreitol/00 mM MgCA'.

/QmM ATP Example 3 E, Coli (E, Co11) K / 2HB /θ/
/pLR, 2 construction frozen appropriate E, co'JK /2 HB 10/ cell opening Bolivar et al., /977,
Gene 2 Near 3; 93] (approx. 10m1)
was precipitated by centrifugation and 007M sodium chloride (
The cells were then pelleted again, resuspended in 0θ 3M calcium chloride (about 10ml), incubated on ice for 20 minutes, and pelleted three times.
Finally, it was resuspended in 003M calcium chloride (/'25111). The resulting cell suspension was suitable for subsequent transformation.

The plasmid PLFt2C (prepared in Example 2C) suspended in TE buffer was precipitated with ethanol and added to a 30mM calcium chloride solution CI! ;Od') and gently mixed with appropriate E, CO+)K/2HB/θ/ cells (approximately 20θ) in a test tube. The resulting mixture was incubated on ice for about 3 minutes and then at 2°C for about 7 minutes. Next, L-broth CB containing ampicillin lθpg/ml)
ertani, /9! ; / , J, Bacter
iology62:293) (approximately 3 yil) and incubated at 37°C/h under shaking.
L-agar containing ampicillin [Miler, /
972, Experiments in Mo1
ec-ular Genetics, Co1d
Spring Harbor Labs, ColdS
Spring Harbor, New York]. The surviving colonies were selected and the expected phenotype (
AmpR, Tetsl was tested. This transforms the desired transformant E. coli/yuHB 10//pLR-
It was 2.

Example V Construction of Plasmid pLR/ Plasmid pLR/ was constructed using plasmid pIJ2 instead of plasmid pIJ6 CTampson et al.
,, /91rO.

Nature, lzag:! ;23] was prepared in substantially the same manner as in Examples 2A-C. The desired plasmid pLR/ was suspended in TE buffer.

Example! Construction of /2HB1θ//pLR/ in E. coli The desired construction was carried out in substantially the same manner as in Example 3, except that plasmid pLR/ was used instead of plasmid pLR2. Select the surviving colonies,
Expected phenotype (AmpR.

TetS), which was found to be /2HB/θ//PLR/ in the desired transformant E, coli.

Example 6 Plasmid pLR4+! Construction A of plasmid pLR/BamH1 partially digested plasmid pLR/(approximately 10μ, /θ~), BSA(/M9
/ IIl, ! ;ti), water (29u/),
BamHI [diluted with water] restriction enzyme (/Me)
And reaction solution 1 (j4) was incubated at 37°C for 75 minutes.

4Z M ammonium acetate (approximately jθId) (193
The reaction was stopped by adding % ethanol (2θθIi).

The resulting DNA precipitate was washed twice with 70% ethanol, dried under vacuum, and suspended in water (2θ111).

A reaction solution for Hata BamHI restriction enzyme was prepared with the following composition.

73 M NaCl 6θmM triy, -HC1, pH796θrn
M MgCl 2 B, Plasmid PBR3,2 HI Digestion The desired digestion was carried out essentially the same as in Example UB, except that the BamHI restriction enzyme was used in place of the HindlI restriction enzyme. . Digested plasmid pBR322 was suspended in water (, 29i).

C. Ligation of BamHI partially digested plasmid pLR/BamHI partially digested plasmid pBR322 The desired ligation was performed essentially in the same manner as in Example 2C. The resulting ligated DNA was suspended in TE buffer. This was the desired plasmid p'da.

Example 7 E, Ko! J K/2 HB10//pLR1i (D configuration) The desired configuration is pLRq instead of plasmid PLR,2.
The procedure was essentially the same as in Example 3, except that the transformation was performed using . Surviving colonies were selected and tested for the expected phenotype (AmpR, Te t'').
10//pLR1t.

Example! A, ShuHI digestion of plasmid PLR,2 and /6k
Isolation bluff of the thiostrepton resistance transfer fragment of b, 2 t' PLR, 2 DNA (approximately 30 pg
), reaction solution Cl0pl), BSAC/Ilf/At, 1
0id'), water (co9IJl) and BamHI restriction enzyme (Gunit/Ill.

/li) was incubated at 37°C for 2 hours. Equal amounts of &M ammonium acetate and x, s volumes of 9! % ethanol and cooled at -20"C for about an hour.
A was precipitated. The DNA precipitate was collected by centrifugation, and T
E buffer (approximately jθld). desired / otkl)
The BamHI restriction fragment was isolated from the DNA suspension by gel electrophoresis in a conventional manner. Following isolation, the fragments were resuspended in TE buffer (approximately 2θId) and used for subsequent ligations.

BamH1 partial digestion of B5 plasmid pEL/θ3 Plasmid pEL/θ3 DNA (approximately 20 py”), reaction solution (10Iil'), BSA (/WIA1.10
), water (39μ) and BamHI restriction enzyme [enzyme 2
Prepared by diluting I with aqueous solution I] (/Ji) at room temperature at about 75%
Incubated for minutes. Add equal volumes of &M ammonium acetate and j volumes of 9j% ethanol.

Approx./! at -2θ℃ The DNA was precipitated by cooling for an hour. This DNA precipitate was collected by centrifugation, washed with 70% ethanol, vacuum dried, and suspended in TE buffer (approximately jθp).

C6-ligated partially digested plasmid pEL/θ3 DNA (
approximately 20 pg), plasmid pLR, 2/Okl, B
amHI restriction fragment (/θ~), coupling solution (!;t
i), BSA (/”9Zw1.3;Me), water (/θ
μ) and T4tDNA! The mixture consisting of J gauze (/Me) was incubated at about /6°C for about 9 hours. Game M
ammonium acetate (17Iil) and cold ethanol (
200 IJl) and f: (Dchi, -20"CT - approx./
The DNA was precipitated by cooling for a while. This DNA precipitate was collected by centrifugation, washed with 70% ethanol, collected again, and placed in medium P (Hopwood and Wrig
ht/97za, JoMolecular and G
eneral Genetics /Otsu, 2:30
7] (jOd) and used for secondary transformation.

Which of the possible pEL/03 restriction fragments is 1
At least two types of recombinant plasmids can be obtained by ligating 1 and 'b to the B a m HI thiostrepton resistance transfer fragment. Thus.

Plasmid pF:, when ligated to the BamHI restriction fragment of Ykb of L103, a 411 kb plasmid pratio107 and pEL/0! ; is obtained.

/99 kb Hayu II fragment (linear pELpE
Since L/03 has three BamHI restriction sites into which a thiostrepton resistance fragment can be inserted, isomers can also be created due to differences in the insertion sites of plasmids pEL/0/ and pEL10. 1lkb BmmHI
The resistance transfer fragment can be oriented in either of two directions, resulting in a bidirectional recombinant plasmid. Restriction sites and functional maps of plasmids pEL/θ7 and pEL/θS and plasmids pEL/θ7 and pEL/04Z are shown in the accompanying Figures 5 and 6, respectively.

A person skilled in the art will understand that the plasmid DEL10J(7)BayuH
It is of course understood that I partial digestion generates several additional recombinant plasmids by mixing various restriction fragments that can be ligated with each other and with seven or more resistance-transferring DNA fragments.

2. , ♂ kb replication origin phagocytosis [further plasmids containing the BamHI fragment are both functional and
Two examples of the invention are thus provided. Additional plasmids described above can be identified by restriction enzyme and gel electrophoresis analysis after transfer into a suitable host cell by conventional methods.

Construction of Example fC (approximately 2θIig) and 10' protoplasts of Streptomyces ambofascenos [the strain was purchased from Northern Regional, Peoria, Illinois].・Research Laboratory (No.
rthern Regional Research
Lnbo-1a4ory) and is a permanently preserved strain.

available to anyone under deposit number NRRL21A2θ].
replication (International Patent Application No. PCT/GB79)
/'0θθ9j) black wo7910 / / A 9
, substantially the same operation as in Example Y was carried out. Desired transformants are prepared by growing regenerated protoplasts in R2 medium upper agar containing sufficient thiostrepton to give a final concentration on the plate of jθIJ4yll.
opagar) (R2 medium is T(opwood
and Wright.

/97J', Mo1ecular and Gene
ral Genetics /6.2:3Q] to screen for resistance to Thi/ostrepton. The resulting thiostrepton-resistant colonies of Oyohii S. ambofaciens/PEL/OII were collected according to known methods, cultured, and identified by conventional methods by restriction enzyme and electrophoretic analysis of the constituent plasmids.

Example/θ A, Bam HI digestion of plasmid pLR/ and 31
Isolation of the Neomycin Resistance Transfer Fragment of kb The desired digestion and isolation are carried out in substantially the same manner as in Example A. The 3&kb BarnHr restriction fragment is suspended in TE buffer (~204) and used for the next ligation.

, B, Plasmid pEL with Bam HI partially digested neomycin resistance transfer Bam HI restriction fragment of ligated Jlkb.
103 [prepared in Example B] is connected in substantially the same manner as in Example C.

Which of the possible restriction fragments of pEL/θ3 ligates the 3 ukb neomycin resistance-transferring Hl fragment results in at least two types of recombinant plasmids. Thus.

When ligated with the 2 kb BamHI restriction fragment of plasmid pEL/03, 4. ) kb plasmids pEL/09 and pEL/10.

/95'kb BamHI fragment (linear pEL
103) to generate the 233 kb plasmids PEL/// and pEL//2. Also.

Plasmid pET,/03 has three BamHI restriction sites into which the neomycin resistance transfer fragment can be inserted, so plasmid PEL///oyohi pEL/
/2 isomers can also be created due to differences in the insertion site. 3.1l
The resistance-transferring Bam HI fragment of kb can be oriented in either of the two directions, resulting in a bidirectional recombinant plasmid. Plasmid PEL/oqoyoo:p
Er, // o even hit C plus z de pEL / /
The restriction sites and functional maps of / and pEL//, 2 are shown in Figures 7 and g of the accompanying drawings, respectively.

A person skilled in the art will understand the following as described in Example Section C.
It is of course understood that further recombinant plasmids containing ShuHI fragments containing the origin of replication can be reproduced by the method described above. These plasmids are functional and thus further exemplify the invention. Additional plasmids described above can be introduced in conventional manner and identified by restriction enzyme and gel electrophoresis analysis.

Examples // pEL/ 09, S, 7A Boff 7'ix nos/pEL
/ / 0.

The DNA obtained in Example 10 (approximately 20 ~) and Streptomyces ambofachenos (NRRLA 24'
The desired configuration was prepared using /×10 protoplasts of 20 ), published by International Publication (
International patent application number PCT/GB 79100093) AWO7910/#9. The procedure is essentially the same as in Example 2. The desired transformant is obtained by incubating the regenerated protoplasts in R2 medium upper agar containing a sufficient amount of neomidino to give a final concentration of /py11 on the plate. Rather than covering the seeds, they are screened based on neomycin resistance.

Streptomyces ambofascens obtained/p
EL109. s, Amphofascens/pEL//θ,
Neomycin-resistant colonies of S. ambofaciens/I) EL/// and S. ambofaciens/pEL//2 were collected by known methods, and the constituting plasmids were isolated by restriction enzyme and electrophoretic analysis using conventional methods. identify

Qin antibiotic neomycin is manufactured in St. Louis, Missouri.
It is available from Sigma.

Example/2 Construction of plasmids pEL//3 and pEL//4' Plasmid pEL/θ7 was isolated from Streptomyces ambofascens/pEL107C prepared in Example 9] and carried out according to the method of Example/. Partial digestion with BamHI restriction enzyme is performed in substantially the same manner as in Example B. This Ba
m H1 partial digest was subjected to substantially the same procedure as in Example C to transform plasmid pLR/ into 3. Ligation with the I/-kb neomycin resistance-transferring Bam HI fragment [prepared in Example 10A] - yields the desired plasmid. Plasmid pEL/θ7 has two BamHI restriction sites into which the neomycin resistance transfer fragment can be inserted, so plasmids pBL//3 and pEL//g
Isomers can also be created due to differences in the insertion site. 3. 'Ikb
The neomycin resistance transfer shell HI fragment can be oriented in either of the two directions, resulting in a bidirectional recombinant plasmid. The restriction sites and functional maps of plasmids pEL//3 and PEL//g are shown in FIG. 9 of the accompanying drawings.

Example/3 //'l configuration The DNA obtained in Example/2 (20IJ9) and Streptomyces ambofascens (NRRLA2g2θ
) using /×/θ protoplasts.

The desired configuration is published in International Publication (within International Patent Application No. PCT/GB79/θ009j)
&WO7910//A9. The procedure is substantially the same as in Example Y. Desired transformants are first selected for resistance to r-ostrepton and then selected for neomycin resistance using the methods described in Examples and/or Sections above. The resulting Streptomyces ambofaciens/pEL//3 Oyohi S, ambofaciens/
PEL//qnotiostrepton and neomycin resistant colonies are picked by known methods and routinely identified by restriction enzyme and electrophoretic analysis of the constituent plasmids.

Example/l Construction of plasmids pEL//j and pEL//B The desired plasmids were the same as Example/2, except that plasmid pEL/θj was used instead of plasmid pEL107 and Bam HI partial digestion was performed. It is configured using essentially the same operations as . Plasmid pEL/θj has two Bam HI restriction sites into which the neomycin resistance transfer fragment can be inserted.

Isomers can also be created due to differences in the insertion sites of plasmids pEL//j and pEL/#. The 33 kb neomycin resistance-transferring BamHI fragment can be oriented in either of two directions, resulting in a bidirectional recombinant plasmid. Plasmid PEL//3; and pEL
/ / The restriction sites and functional maps of each of A are shown in the attached figure 1.
Shown in the θ diagram.

Example/j //6 configuration Using the DNA (Y0py) obtained in Example/q.

The desired configuration is carried out by essentially the same operation as in Example 3.

Streptomyces ambofaciens obtained/
PEL//s and HiS, Ambofanoens pEL
/ / g of thiostrepton and neomycin resistant colonies are picked by known methods and identified by conventional restriction enzyme and electrophoretic analysis of the constituent plasmids.

Example/6 The desired plasmid was prepared as in Example/2 and BamHI except that plasmids pEL/θ and pEL/θ were used in place of plasmids pEL/07 and pEL/θ, respectively. Each is configured using essentially the same operations as /game. However, since plasmids pEL10 and pEL/θ contain three Ban HI restriction sites into which the neomycin resistance transfer fragment can be inserted, plasmids pEL7/7,
pEL 1har, pEL//9 and pEL 720
Isomers can also be created due to differences in the insertion site. , the neomycin resistance-transferring BamHI fragment of R#kb can be oriented in either of two directions, resulting in a bidirectional recombinant plasmid.

Plasmids PEL//7 and pEL//jrj
! Rahi+C plasmid pEL//ri and pEL/
The restriction sites and functional maps of each of the 20 are shown in Figures 1/1 and 12, respectively.

Example/7 Plasmid pF, L//7゜pE obtained in Example/l
L//J', pEL//9 and pEL/20
(7) Introduce the DNA mixture (approximately 20 tqt) into Streptomyces ambofascens by essentially the same procedure as in Example 3 and /S. Shens/pEL//7.
Thiostrepton and neomycin resistant colonies of S. ambofaciens/pEL//S. ambofaciens/pA/yq and His. ambofaciens/pEL/2θ were collected by known methods, The constituting plasmids are identified by conventional methods using restriction enzyme and electrophoretic analysis.

Example/(5' plasmid chimera pEL/2/and PEL/,
2. Construction of 2 The desired plasmid chimera is plasmid pEL/θ7
Then, the mH1 partial digest was ligated to BamHI-digested plasmid pB using substantially the same ligation method as in Example C.
Obtained by linking to R322. Plasmid PEI, 10
7 was isolated from Streptomyces ambofaciens/pEL/θ7 [prepared in Example 9] in substantially the same manner as in Example 1/2, and partially digested with BamHI in the manner described in Example 2/h. BamHI digested plasmid pBR322
Hindll [0 is replaced by BamH] as the restriction enzyme.
Prepared in substantially the same manner as Example 2B, except using I.

The desired plasmid/chimera DNA was collected by centrifugation, washed with 70% ethanol, vacuum dried, and then suspended in TE buffer.

Plasmid pEL/07 is restricted plus 2 do pBR
Since it has two HI restriction sites into which 322 can be inserted, plasmids pEL/U/ and PEL/ can be used to create isomers with different insertion sites for 22. The restriction plasmid PBR3,22 can be oriented in either of the two directions, resulting in a bidirectional recombinant plasmid. pEL
The restriction site and functional maps of each of pEL/u2, pEL/u2, and pEL/u2 are shown in Figure 73 of the accompanying drawings.

Example/9 The desired construction is carried out in substantially the same manner as in Example 3, except that plasmids pEL/, 2/ and pEL/22 are used in place of plasmid PLR, 2 for transformation. We first select the surviving colonies,
Test whether the expected phenotype (AmpR, TaL8),
The desired transformant E is prepared in a conventional manner by restriction enzyme and gel electrophoresis analysis of the constituting plasmids.

coli/, 2 HB/θ//pEL/2/ and E,
For stiffness/, 2HB 10// PEL/ 22 To L/
”?-Identification.

Structure of Example 20/22 The desired structure is planubed pEL/θ7, pEL/
Oj, pP2L/OI Oyohi pEL/04t
(1 plasmid PEL/2/and p instead of D
The procedure is substantially the same as in Example 9 except that EL722 is used for transformation. The obtained transformants were as described in the above example. Screen for thiostrepton resistance using the method described in . Colonies of the thus constructed thiostrepton-resistant Streptomyces ambofaciens/PEL/2/and S. ambofaciens/pEL/22 were collected by known methods, and the constituting plasmids were subjected to restriction enzyme and electrophoresis. Identification is by conventional analytical methods.

Example 2/Construction A of plasmid pFJ/2'l, HI (partial) and paste of plasmid pEL/0.5''
I-digested plasmid pEL/θ5 DNA (approximately 0tni), reaction solution (/θIJ, BSA (/■/m1./θH), water C39td) and Oki1 (I restriction enzyme (/m1) [enzyme 2
4 diluted with water gt, t] was incubated at room temperature for about 75 minutes. After adding an equal volume of 4ZM ammonium acetate and a volume of 9q% ethanol, -Y θ°C
The mixture was cooled for about 7 g to precipitate the DNA. The DNA precipitate was collected by centrifugation.

Wash with 70% ethanol, vacuum dry, and add TE buffer (
approximately 2θμ). Be11 reaction solution 1 (approx. 5 pl)
, BSA (/wg/m/, 10m), water (39I) and ↓1 restriction fermentation! (/m) in New England Bio Lab Unit], and the mixture was incubated for approximately 60 minutes in so”c.Equivalent volumes of Q
M ammonium acetate and j volumes of 93% ethanol were added and cooled at -20°C for about 71 hours to precipitate DNA. The DNA precipitate was collected by centrifugation, washed with 70% ethanol, vacuum dried, and then added to TE buffer (approx.
θ1.1).

A reaction solution for the 11BelI restriction enzyme was prepared with the following composition.

73θmM KCl 6θmM) Lis-HCl, pT (7glθOmM
MgCla 70mM dithiothreitol BJ binding Partially digested DNA plasmid pEL /θj (approx. 20p9), ligation solution (!; pl), BSA (/■/m
l,! ;Me), water Cl0tt! ') and Tl DNA ligase (/me) were incubated for about 1 hour at about /6°C. After adding 4M ammonium acetate (11, Od) and cold ethanol (<200td'), the mixture was diluted to θ
The DNA was precipitated by cooling at °C for approximately 7 g hours. DNA
The precipitate was collected by centrifugation.

The cells were washed with 70Qfi ethanol, collected again, suspended in P medium (!;Ot!), and used for the next transformation.

A person skilled in the art would understand that the plasmid pEL/0,! It can be appreciated that partial BamHI and BclI digestion of ; generates several additional plasmids by mixing various fragments that can be ligated with themselves or with each other. desired plasmid p
FJ/u& can be introduced into a suitable host cell together with the further plasmids described above and identified in the conventional manner by restriction enzyme and gel electrophoresis analysis.

The desired construction was carried out in essentially the same manner as in Example 1, Example 9, except that the DNA obtained in Example 2/2 was used instead of Example 2C. The resulting thiostrepton-resistant transformant colonies were collected by known methods, cultured, and identified by conventional methods by restriction enzyme and electrophoretic analysis of the constituent plasmids. The desired transformant Streptomyces ambofaciens/PFJ/2q was cultivated and used for the subsequent production and isolation of plasmid pFJ 7211 by known methods. ~ll0kb plasmid p
The restriction site map of FJ/211 is shown in Figure 1q of the attached figures.

Example 23 Construction of plasmid pFJ/&j and pFJ/&j
The procedure is essentially the same as in Example JA, except that a J/2 game is used. The resulting DNA precipitate was TE
The desired H1 digest was obtained by suspending in buffer (approximately 5 oti).

B. Concatenation 3! The neomycin resistance-transferring ShuHI restriction fragment of kb [prepared in Example IOA] was digested with BamHI in essentially the same manner as in Example/C.
It was ligated with pFJ/2'l. , 9.4 Z kb of the resistance-transferring Ban II fragment can be transferred into suitable host cells in either of two directions and identified by restriction enzyme and gel 1 pneumophoresis analysis.

Example 24 Structure of t/&5 The desired structure is the same as that of Example/&5, except that the DNA obtained in Example U3 is used as the DNA obtained in Example/θ
Perform the same operation as /.

Obtainable Streptomyces ambofachaes/p
FJ/1ill and S, ambofascens/pFJ
/ g j neomycin-resistant colonies are collected using known methods, tested for thiostrepton resistance, and identified using restriction enzyme and gel 1 pneumophoresis analysis of the constituent plasmids using conventional methods. The resulting transformant was cultured in a conventional manner, and the restriction site map of each plasmid is shown in Figure 1 of the attached figure.

R Example 2j Construction of plasmid pFJ/u, 4 The desired construction is
Digestion is partial and plasmid pEL/
The procedure is substantially the same as in Example 2/, except that plasmid pFJ/4txt is used instead of 03;. The resulting DNA precipitate is collected by centrifugation, washed with 70% ethanol, collected again, suspended in medium P (50m), and used for subsequent transformation. The restriction site map of PlusEdopFJ/G6 is shown in Figure 1j of the attached figure.
As shown in the figure.

Example 26 The desired configuration is the DNA obtained in Example 10.
The procedure was essentially the same as in Example 1, except that plasmid pFJ/4Z, 4 was used instead.

Example 2? Construction of plasmid pFJ/41nia A, E,
:l! J,! r03/pIJq3 (D culture and isolation of plasmid pr, rg3 desired (7) E, l +J, f /73/ pIJ
113 (ATCC39/36) and the subsequent isolation of plasmid pIJlt3 were both
ual For Genetic Engineeri
ng.

Advanced Bacterial Geneti
cs, Co1d Spring Harbor La
boratory, Co1d Spring Ha
rbor, New York]. The resulting pIJ Ge3 DNA is suspended in TE buffer in a conventional manner and stored by cooling at -20''C.

B, Fra'nia, E)' prJ '73 (D~
Digestion and Isolation of the 27 kb Sal I-Bgl Fragment The desired digestion was performed using plasmid pEL/03, Ba
plasmid p instead of mHI restriction enzyme and reaction solution.
Using IJ113, Di1UI restriction enzyme and reaction solution 0,
The procedure is substantially the same as in Example 1, except that ↓■ restriction enzyme and reaction solution 0 are used instead of ① restriction enzyme and reaction solution. In addition, 5all digestion was only partial. The resulting 5alJ-BBq■ fragment was fractionated and subjected to agarose gel electrophoresis (Davis, RoW, et al., /
9J'θ), Ba of plasmid pFJ 2II
m HI and partially digested plus F, )' pFJ/u4tDNA (approximately 20p
g), BS A (/11111/x1./θ
l), water (39ti), BarnHI control mWI
g(//-/)C New Innogrand ° Bio Rough
Excess amount contained] and reaction solution (/θ4) at 37
After incubating for 6θ min at °C,
The cells were incubated for 0 minutes and cooled to 9°C. Here, the Death Liu I reaction solution "Cl0pl') and the Sil■ restriction enzyme (/
ld) Ronyu-Ingranod Bio Lab Unit] was supplemented, and the mixture was incubated again at 37°C for about 10 minutes. Equal volumes of lAM ammonium acetate and 2 volumes! (7) After adding 95% 1) -, -,
The DNA was precipitated by cooling at 20"C for approximately # hours. The DNA precipitate was collected by centrifugation, washed with 70% ethanol, dried under vacuum, and diluted with TE buffer (approximately #j.

It was suspended in water.

The reaction solution for earthwork restriction enzymes was prepared with the following composition13.
M NaCl 60mM) Lis-hydrochloric acid, pH796θmM M
gC et AOmM β-mercaptoethanol/189/
A reaction solution for restriction enzymes was prepared with the following composition.

Q, l, M NaCl 700mM Tris-HCl, pH 7, g/00m
MMgCl x /θθmM β-mercaptoethanol/■7'ml
BSA D, Ligation The ˜27 kb Jilu I-1 fragment of plasmid pIJl13 was ligated into the BamHI partial Huni digest of Bluff, 6d pFJ/2′l in substantially the same manner as in Example 1rC. The resulting DNA precipitate was collected by centrifugation, washed with 70% ethanol, and collected again.
The cells were suspended in medium P (tOm/) and used for subsequent transformation. Plasmid pFJ Example 2g The desired construction was carried out in substantially the same manner as in Example 9, except that plasmid pFJ/117c (prepared in Example 27) was used instead of the DNA obtained in Example C. Ta. The desired transformants are at a concentration on the plate of 3; 0.
/JJy41 by covering the regenerated protoplasts with R2 medium upper agar containing a sufficient amount of erythromycin to screen for erythromycin resistance. The resulting strain was Etreptomyces ambofascens/pFJ/ge7. This was confirmed by conventional methods using restriction enzyme and gel electrophoresis analysis of the constituting plasmids.

Example 29 A, Partial Digestion of Plasmids pIJ&J The desired digestion was carried out using plasmids pIJ 3 and 5a instead of plasmids pLR/ and BamHI restriction enzymes and reactions.
The procedure was substantially the same as in Example t, except that the II restriction enzyme and reaction solution were used. Obtained) 1■
The partially digested fragments were fractionated and routinely isolated by agarose gel electrophoresis. The desired Gillier fragment was ~2 kb.

B, Siri 21 partial digestion of plasmid pFJ/2lt. Desired digestion of plasmid PLR/, 13ar
The procedure was substantially the same as in Example 6, except that plasmid pFJ/2u, Sal I restriction enzyme and reaction solution were used instead of I HI restriction enzyme and reaction solution. The resulting I) NA precipitate was collected by centrifugation, washed with 70% ethanol, vacuum dried, and T
It was suspended in E buffer (approximately jθ4).

~Qf kb Sal of C1 ligated plasmid pIJl/-3
Efragment was treated in substantially the same manner as in Example ♂C.

5ali partial digest of plasmid pFJ/2''. The resulting DNA precipitate was collected by centrifugation, washed with 70% ethanol, harvested again, suspended in medium PG3; was used for transformation.

~2. The 5alJ fragment of Zakb can be oriented in either of the two directions, resulting in a bidirectional recombinant plasmid. Plus 2 de pFJ/4t,? and p
FJ/l@9 can be routinely introduced into suitable host cells and identified by restriction enzyme and gel electrophoresis analysis.

Example 3 Construction of θ/g9 The desired construction was essentially the same procedure as Example 9, except that the DNA obtained in Example 29C was used instead of the DNA obtained in Example IrC. I did it.

The desired transformant is the regenerated protoplast, and the concentration on the plate is! ; The cells were screened for erythromycin resistance by covering with agar on the top of R2 medium containing a sufficient amount of erythromycin to obtain Opy/ml.

The obtained Streptomyces ambofaciens/
pFJ/11F and S, ambofaciens/p
Erythromycin-resistant colonies of F:I/119 were picked by known methods, cultured, tested for thiostrepton resistance, and identified by conventional restriction enzyme and gel electrophoresis analysis of the constituent plasmids. After culturing the desired transformant in a conventional manner, the resulting plasmid pFJ/1tJ
It is shown in Figure 1 of the diagram with f and pFJ.

Example 31 Plasmid PBTt? 22 and pFJ/4'
The desired digestion is carried out in substantially the same manner as in Example JB, except that HindI [I restriction enzyme and Bam] [restriction enzyme and reaction solution are used in place of HindI restriction enzyme and reaction solution.

BamHI-digested plasmids pBRJJJ and pF
To connect J/g 7, substantially the same operation as in Example ♂C is performed. The desired plasmid/chimeric DNA is collected by centrifugation, washed with 70% ethanol, vacuum dried, and suspended in TE buffer (sottt). The restricted plasmid PBR32,2, can be oriented in either direction, resulting in a bidirectional recombinant plasmid. Plasmid pFJ/! ;0 and pFJ/j
/ can be identified by restriction enzyme and gel electrophoresis analysis after transfecting into a suitable host cell using conventional methods.

Example 32 The desired constructs are plasmids pEL/2/ and pEL/
Plasmids PFJ/jθ and pF instead of 22
The procedure is substantially the same as in Example 19, except that J/j/ is used. Surviving colonies are first selected and tested for the expected phenotype (AmpR, Tet8) and transformed into the desired transformants E. coli/2 by conventional methods by restriction enzyme and gel electrophoresis analysis of the constituent plasmids. HB/θ//pFJ/3θ and E, coli/2
It is identified as HB/θ//PFJ/ j /.

Plasmids pFJ/jθ and pFJ/! ;
A restriction site map of each of / is shown in FIG. 76 of the attached drawings.

Example 33 The desired construct consists of plasmid pFJ/3θ [E obtained in Example 32] instead of the DNA obtained in Example 1C.

For stiffness/2 HB10//pFJ/! The procedure is substantially the same as in Example 9, except that the procedure is as follows.

Example 3 The desired construct was plasmid PFJ/3/C obtained in Example 32 instead of the DNA obtained in Example IC.

:l IJ K/2 HB10//pFJ/! The procedure was substantially the same as in Example 1, except that the conventional method was used.

Representative plasmids and transformants constructed according to the procedures described above are shown in Table/2 below.

(Margin below) Table 2 CJJ bottom margin)

[Brief explanation of drawings]

Figure 1 shows the restriction sites and functional map of plasmid pEL103. Figure 2 shows the ffi site and functional map of plasmid pLR/. Figure 3 shows the restriction sites and functional map of plasmid pLR,2. Figure 9 shows the restriction sites and functional map of plasmid pLR&. Figure 5 shows plasmid pEL
Restriction sites and functional maps of 107 and pELloj are shown. Figure 6 shows plasmids pEL101 and pELlog.
The restriction site and functional map of is shown. Figure 7 shows the restriction sites and functional maps of plasmids pEL#79 and PELllO. FIG. 1 shows the restriction sites and functional maps of pEL/// and pEL//2. Figure 9 shows plasmids pEL//3 and pEL//4L.
The restriction site and functional map of is shown. Figure 1θ shows plasmid p
EL／／! ; and the restriction site and functional map of pEL//B are shown. FIG. 11 shows the restriction sites and functional map of plasmids pEL//7 and pEL//7. Figure 72 shows restriction sites and functional maps of plasmids pEL//9 and pEL/r. Figure 73 shows plasmid pE
Restriction sites and functional maps of L/, 2/ and pEL/λ2 are shown. Figure 1g shows plasmid pFJ/2'l. pFJ/In and pFJllll! shows the restriction site map of; Figure 15 shows plasmids pFJ/116 and pF
A restriction site map of J/lt7 is shown. Figure 16 shows plasmid PFJ/4! J, pFJ/4i, pFJ/j; θ and pFJI! ; shows the restriction site map of /. Fig. 1 Fig. 2 Fig. 3 Fig. q is cold RI amHI Fig. 5 EL107 EL105 Fig. 6 pattoe Fig. 7 Ichiku 10・ Fig. g at111 P section L112 9th tA EL113 EL114 Fig. 10 ptt115 1st/ Figure plEL11? Figure 72 EL120 Figure 3 p! L121 p! L122 No. u Zuno 1 pFJI Survey No. 75 Ward FJ147 No. 16 Figure 71) Jl 1 Continuation of page 1 ■In't, C1, 3 identification code Office serial number"/
CI2 R1/29 1/365 1/44 1,/465 Priority claim ♀ August 2, 1982 3 United States (US) 3 Engineering 404082 @ Inventor Walter M. Nakatsukasa Indianapolis, IN, USA 2118 Crossman Drive Inventor: Mark A. Richardson 3021 Aft Aft, Indianapolis, Indiana, United States of America 0 Inventor: James A. Map Little Oak Lane, Indianapolis, Indiana, United States of America 7410 address

Claims (1)

Claims: (1) a) a restriction fragment containing a functional origin of replication of plasmid pEL/θ3, and b) at least one A recombinant DNA cloning vector containing one or more DNA fragments that convey resistance to antibiotics. (2) The restriction fragment of pEL/03 is
The cloning vector according to claim (1), which is a BamHI restriction fragment of fkb. (3) 15 restriction fragments of pEL/03!
The cloning vector according to claim (1), which is a 9 kb BamHI restriction fragment. (4) One or more DNA fragments are thiostrepton. Claim (1) conveying resistance to one or more of the antibiotics neomycin and erythromycin;
The cloning vector described in (2) or (3). (5) DNA that conveys resistance to thiostrepton
Cloning vector according to claim 4, wherein the fragment is a 16 kb BamHI restriction fragment of plasmid pLR,1. (6) The cloning vector according to claim (4), wherein the DNA fragment conveying resistance to neomycin is a 1Qkb BamHI restriction fragment of plasmid pLR/. (7) Plasmid pEL/0! ; or pEL
/07 The cloning vector according to claims (2) and (5). (8) Plasmid pEL/0,9 or pEL/
/0 The cloning vector according to claims (2) and (6). (9) Plasmid pEL//3, pEL,/
/ tl, , PELi1s or pEL / /
Claim (2) which is A. The cloning vector described in (5) and (6). Full plasmid pEL/θl or pEL/θ! The vectors according to claims (3) and (5). 01) Plasmid pEL///or pEL/
/2 The cloning vector according to claims (3) and (6). α plasmid pEL//7, pEL/
/Ir, pEL//9 or PEL/, 2θ. The cloning vector described in (5) and (6). α3 DNA fragment conveys resistance to thiostrepton. The cloning vector according to claim (4), which is a plasmid pFJ/2'l or pFJ/9/. The Q41 DNA fragment conveys resistance to thiostrepton and neomycin. Plasmid pFJ/da#
, pFJ/q5, pFJ/daotsu or pFJ
The cloning vector according to claim (4), which is: /32. α5 DNA fragment conveys resistance to thiostrepton and erythromycin. Plasmid pFJ/su7
, pFJ/4'za, pFJ/≠? or pFJ
The cloning vector according to claim (4), which is IS9. 00) a restriction fragment containing a functional origin of replication of plasmid pEL/03 b) a restriction fragment containing seven or more antibiotics that, when transferred into a susceptible host cell capable of transformation, cell division and culture, conveys resistance to at least seven antibiotics. A bifunctional recombinant DNA cloning vector comprising a DNA fragment and c') a restriction fragment containing a functional replicon of the E. coli plasmid and conveying antibiotic resistance. QηE, coli plasmid is plasmid pBR3,22
A cloning vector according to claim αQ. (To) Plasmid pEL/2/or pEL/
22, the cloning process described in claim αη
Vector O0 plasmid pFJ/79 or pFJ/zaO
The cloning vector 11 DNA fragment according to claim αF conveys resistance to thiostrepton. Plasmid pFJ/! ; 3 or pFJ/
A cloning vector according to claim OQ or a, which is S6. ■ pDNA fragments convey resistance to thiostrepton and neomycin. Plasmid pFJ/j,
pFJ/! ;3, pFJ/37 or pFJ/j;
The cloning vector according to claim OQ or Q7) which is a male. (a) DNA fragments convey resistance to thiostrepton and erythromycin. Plasmid pFJ/
, io, pFJ/! ;I, pFJ/1,0
, pFJ/g/pFJ/1.2, pFJ
/63, pFJ/77 or pFJ/71r
A cloning vector according to claim OQ or αη. @&) a restriction fragment containing a functional origin of replication of plasmid pEL/θ3, and b) conveying resistance to at least one antibiotic when transferred into a susceptible host cell capable of transformation, cell division and culture. A transformed host cell having a recombinant DNA cloning vector containing a DNA fragment. (c) a) Restriction fragment containing a functional origin of replication of plasmid pEL/θ3. b) seven or more DNA fragments that convey resistance to at least seven antibiotics when transferred into a susceptible host cell capable of transformation, cell division and culture; and e) a six-functional replicon of the E. coli plasmid. and a restriction fragment that conveys antibiotic resistance. (c) The transformed host cell according to claim (c) or (c), which is an unrestricted cell of the genus Streptomyces, Streptosporangium, Actinoplans, Nocardia, Micromonospora, Bacillus, or Staphylococcus. . (e) The transformed host cell according to claim (c), which is a species of the genus Streptomyces, Frasiae, Coelicolor, Ambofaciens, or Granulorubber. (b) The transformed host cell according to claim (c), which is of the unlimited Streptomyces genus, preferably Ambofaciens sp. (c) The transformed host cell according to claim (c), which is K. coli. Ligate one or more DNA fragments that convey resistance to at least seven antibiotics when transferred into a sensitive host cell with a restriction fragment containing a functional origin of replication of plasmid pEL/θ3, and use the resulting recombinant DNA to A method for producing a recombinant DNA cloning vector comprising self-ligation to produce a cloning vector. (1) The method according to claim 4, wherein the restriction fragment of pEL/θ3 is a BamHI restriction fragment of QJ'kb. (81) The restriction fragment of pEL/03 is /99
5. The method of claim 4, wherein the BamHI restriction fragment is a BamHI restriction fragment of kb. (:!2.) One or more DNAs that convey antibiotic resistance
The method of claim H, (30) or (31), wherein the fragment is a fragment that conveys resistance to one or more of thiostrepton, neomycin and erythromycin. (33) DN conveying resistance to thiostrepton
A fragment is 74kb Bam H1 of plasmid pLR2
The method according to claim (32), which is a restriction fragment. (34) The method according to claim (32), wherein the DNA fragment conveying resistance to neomycin is a 3 kb Bam HI restriction fragment of plus, mid pLR/. (85) Plasmid pLR, 2 4kb BamH
I restriction fragment and Q of plasmid pEL f 03
Plasmid pEL/Oj or pEL107 consisting of ligation with IkbBamHI restriction fragment
The method according to claims (30) and (33) for producing. (86) 3! of plasmid pLR/or pLRllt!
kbBamHI restriction fragment and plasmid pEL
The method according to claims (30) and (34) for producing the plasmid pEL/θ9 or pEL/10, which consists of ligating the lJ' kb BamHI restriction fragment of /θ3. (37) Plasmid pLR, 2 6kbBamHI
Restriction fragment and plasmid pLR/l4n
b BamHI restriction fragment and plasmid pEL
Plasmid pEL//3 consisting of ligation with the Q I kb Bam HI restriction fragment of /03
, //I1. //! or//The method described in claims (30), (33), and (34) for producing B. (38) Plasmid PLR2 i 4kb BamHI
Restriction fragment and plasmid pEL/03/9
Plasmid pEL/Og or pEL/
Claims (31) and (33) for manufacturing Or
Method described. (39) Plasmid pLR/IA kb Bam
HI restriction fragment and pEL/03/99kb
The plasmid pEL/// or pEL// consists of ligating the BamHI restriction fragment of pEL//
Claims (31) and (a4) for manufacturing 2.
Method described. (40) i 4 kb BamHI restriction fragment of plasmid PLR2 and 3. Rik
b Barn) (I//7, pEL//1.p
The method according to claims (31), (33) and (84) for producing EL//9 or pEL/20. (41) Plasmid pEL/0! BamHI partial digest and plasmid pEL10! Plasmid PFJ consisting of ligation of BcJ, l complete digest of ;
/, 2'+ Claims 1 to (32)
) method described. (42) BamHI of plasmid pFJ/2′i
Plasmid pFJ consisting of ligation of the digest and the 3#kb BamHI fragment of plasmid pLR/
/4<'4 and PFJ/4<'4 and the method according to claims (29) to (32). (48) Plasmid pFJ/4 (4' BamHI
and plasmid pFJ/4Z, consisting of self-ligating a Bat 1 partial digest! The method according to claims (29) to (32) for producing (44) 27kb Saj of plus ε do pIJ g3! I
Bgl■Fragmenoto and Grasmid pFJ / 2
'I'm part of BamHI' Sa7! The method according to claims (29) to (32) for producing plasmid pFJ/'I-7, which comprises ligating a digested product of pFJ/'I-7. (45) Plasmid pFJ/, which consists of ligating the 5a711 partial digest of plasmid pIJ≠3 and the 5ai1 partial digest of plasmid PFJ/241.
The method according to claims (29) to (32) for producing tlJ and pFJ/V9. (46) &) Functional origin-containing restriction fragment of replication of Grasmid pEL/03. b) one or more DNA fragments that, when transferred into a susceptible host cell capable of transformation, cell division and culture, convey resistance to at least seven antibiotics; and c) one functional replicon of the F4 coli plasmid. A method for producing a bifunctional recombinant DNA cloning vector comprising ligating restriction fragments containing and conveying antibiotic resistance and self-ligating the resulting recombinant DNA to form a cloning vector. (47) E. coli plasmid is plasmid pBR32
2. The method according to claim (46). (48) BamHI partial digest of plasmid pEL/07 and BamHI digested plasmid pBR322
A trifunctional plus 2-do pEL/
2/or pEL/U2 (
47) The method described. (49) Claim (4) to produce plasmid pFJ/3;0 or pFJ/3/, which consists of ligating a BamHI digest of plasmid PBR322 and a BamHI digest of plasmid pFJ/g7
6) or the method described in (47). (5o) Plasmid pEL/θ3゜(51) Plus E□do pEL/θ3 2gkbBam
HI restriction fragment. (52) Plasmid pEL/θ3/9.9kb
BamHI restriction fragment. (53) Streptomyces granulorubber mA39
9/2. The cells obtained by culturing /3NRRL/23119 at pH 3-9, /j''c-tio °C in Eitai medium containing assimilable carbon sources, nitrogen sources, and minerals under deep aeration culture conditions. The supernatant of the lysate was extracted with a solvent, and the plasmid DNA was extracted from the extract.
A method for isolating grad4epsilon pEL103, which consists of precipitating A and isolating plasmid pEL/θ3 from the precipitate.