JPS5813598A - Recombined dna cloning vector, eukaryotic and prokaryotic transformed body thereof - 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 The present invention provides hygromycin B (hygr5omic
in B) and novel recombinant DNA cloning vectors that convey Gltll resistance to both eukaryotic and prokaryotic cells. The present invention further relates to transformants of the aforementioned vectors.

The present invention describes how to extract a cloned DNA sequence that does not have a selective function in eukaryotic cells and prokaryotic cells,
This is particularly important because it solves the problem of handling, stabilization, etc. To date, the development and development of recombinant DNA technology in eukaryotic cells has been limited by the following factors: 1) the general absence of suitable loning vectors containing selective marker markers in eukaryotic cells;
3) One generation time of eukaryotic cells during culture is long.
Due to various reasons, there were delays and difficulties.

This time: a) Eukaryotic promoter.

b) susceptible to one or both of the antibiotics Igromycin B and G11lr to which resistance is transmitted;
C) the host cell is a prokaryotic A prokaryotic replicon 1 in which the replicon is functional when
A recombinant DNA cloning vector consisting of
however,! or two structural genes and associated control sequences are adjacent to eukaryotic promoters and are transcribed from a eukaryotic promoter in a eukaryotic host cell, and one gene and associated control sequences are hygromycin B and atttt, but not both, and the gene conveying resistance to G11F does not encode the enzyme phosphotransferase), but in both eukaryotic and prokaryotic cells. Both are functional and selective, so
It has been discovered that the above problems are solved by this vector. Therefore, this versatility
) DNA sequences cloned into vectors can be handled and amplified in prokaryotic cells using conventional methods, thus avoiding the inconveniences and problems of performing the manipulations in eukaryotic cells. Moreover, because the vector is both functional and selective in eukaryotic cells, the vector can be directly transferred into eukaryotic hosts without further modification and subsequently manipulated and expanded. This can be said to be not only excellent in this technical field, but also a significantly advanced technology.

The ability to clone DNA into eukaryotic host cells and the ability to easily select transformants are important for the further advancement of recombinant DNA technology. Since transformation occurs and the resulting acquisition of plasmid DNA does not occur very often, such functional tests are useful in determining which cells out of millions of cells have plasmid DNA.
Obtained an A.

It is practically necessary to determine whether This functional test is important because non-selective NA sequences may be inserted into the vector to selectively inactivate specific antibiotic resistance genes. Therefore, after transformation, cells containing the vector and the required specific DNA sequence should be selected with appropriate antibiotics to ensure that they are sensitive to hygromycin B or Gatz and capable of taking up the DNA after cell division. They are particularly useful because they can be introduced and selected in eukaryotic or prokaryotic cells. Recombined DN until today
Advances in A technology have mostly focused on the development of eukaryotic polypeptides (e.g. - proinsulin, insulin A111, insulin B chain, human growth hormone, bovine growth pormon, interferon, etc.) in prokaryotic hosts.
Somatostatin, thymosin aj, etc.) were needed.

This is important. Prokaryotic cells cannot properly attach carbohydrate moieties to eukaryotic polypeptides. Therefore, using recombinant DNA technology, post-translation (posttransljm)
i◆nally) I4-modified eukaryotic polypeptides 9 For example, glycylated polypeptides can be produced in the absence of a eukaryotic host and a suitable eukaryotic recombinant DNA cloning vector. It is impossible.

This vector meets the above needs and has 9 recombinant D
Expanding the scope and application of NA technology. Commercial production of both unmodified and post-translationally modified proteins in eukaryotic cells is therefore enhanced.

For purposes of the invention described herein, the following terms are defined below.

Structural gene - DNA sequence encoding a polypeptide Regulator - DNA sequence that directs and regulates the transcription and expression of a structural gene Eukaryotic promoter - Partially promotes and regulates the expression of a structural gene in eukaryotic cells DNA sequences that control and prepare DNA replication in prokaryotic cells Prokaryotic replicons - DNA sequences that control and prepare DNA replication in prokaryotic cells Recombinant DNA cloning vectors
Transformation, which includes, but is not limited to, a plasmid, bacteriophage, or virus into which a plasmid, bacteriophage, or virus can be added or which consists of a DNA molecule to which an NA fragment can be added - a transformation that changes the genotype and is thus stable in the recipient cell. causing hereditary changes. Introduction of DNA into Recipient Strain Host Cells Isomers due to differences in insertion sites - of the λ or more possible recombinant DNA molecules produced when a DNA fragment is inserted into one of λ or more suitable sites in the recipient DNA The present vector is constructed by carrying out DNA sequences carrying one or two different structural genes and associated control sequences conveying resistance to one or both of the antibiotics Igromycin B and G11lf by a process consisting of the following operations: Constitute by preparation.

a) Treat plasmid pKC203 with III restriction enzyme and isolate the 73 kb C1π restriction fragment that conveys resistance to both of the antibiotics mentioned above. or b) plasmid pKc203! jL! Treated with It and Siri I restriction enzymes and conveys resistance to both antibiotics mentioned above! ? Isolate the restriction fragment. or C) plasmid pKc203 with Bglii and 5ac
Gael/B treated with I restriction enzyme to convey resistance to the antibiotic hygromycin B/, j/k b
Isolate the H4 ■ restriction fragment. or d) Plasmid pKC2o3 was treated with Siri■ and Cy'RI restriction enzymes and treated with antibiotic G4! Conveying resistance to It1,
tsklxn Dangu RI/Shivo! Isolate the restriction fragment.

The DNA sequence thus obtained is combined with a eukaryotic promoter and, if necessary, another one consisting of a prokaryotic replicon.
connect two DNA sequences. DNA obtained by ligation
The sequence is closed to form a plasmid, which is then used to transfer the strands into a host cell and treat the transferred host with the antibiotic hygromycin B or the antibiotic G4tl.
5. By culturing in the presence of T and selecting the surviving cells. Stable. Ah, I have it. 6. 9 The present invention provides bacterial plasmid DNA conveying resistance to the aminoglycoside antibiotics hygromycin B and aatt to generate novel plasmids harboring and expressing resistance in hygromycin host cells, as exemplified herein. Realize. Thus, the present invention
useful for cloning into virtually any type of cell.

Furthermore, the ability of the vectors exemplified by the 0 gene to convey resistance to antibiotics that are toxic in both eukaryotic and prokaryotic cells may be due to their ability to select and stabilize the cells from which they have been acquired. Provide exams.

ξThe specific bacterial plasmid DNA sequence used during the construction exemplified here is 73 from plasmid pKcaJ.
k b W■ restriction fragment or derivative thereof. Plasmid pKC203 is approximately 1 jkb in size and i
'Hikori (E. coli) JR2,23).

It can be easily isolated from by conventional methods. strain E, coli JR,
lj is known academically [Day, ics and
d Connor.

19ri I*jkntimicrobial Agent
s and Chemotherapy */@(1):
/:9], also Maryland (Maryland)
American Type Culture Collection in Rockville
an TypeCulture Co11ection
) The restriction sites and functional map of the 6° plasmid pKc203, which has been deposited as a conserved bacterial strain and is available to anyone without any restrictions under deposit number ATCC 31912, are shown in FIG. 1 of the accompanying drawings. All figures including FIG. 1 do not have scales.

From pKc203 (F) 7j k b cD B3
i'J (restriction fragment contains the structural genes and control elements for the expression of resistance to the antibiotics hygromycin B and Ga1r. [ 5cienc
e*, 209(471163): 1lA22,
(1910) was ligated to the known plasmid plasmid p8V, tgpt. Plasmid psVj
gPt Ha approx. 9.3kb Te, PlaX'? , dopBR3
22 origins of replication and an ampicillin resistance marker, as well as a functional SVgQ early promoter (e
early promoter). Restriction sites and functional map of plasmid psVj gpt are attached in Figure 2.
Shown below. As will be obvious to those skilled in the art, plasmid p8V
j gpt can replicate in E. coli as well as in eukaryotic cells such as mammalian cells. Yet again. Bgll of plasmid psVJ'gP'
The site specifically restricted in i is the plasmid pKcJO
It can be directly ligated with the 7jkb Carp ■ restriction fragment from J. Thus, hygromycin B and G4tllj resistance transmission 7! ;kb fragment is one!
! It can be ligated to plasmid pSVjgPt treated with guI[. Since the possible λ orientations of the Ztkb fragment are equally real, ligation of the 73 kb fragment to the plasmid p8Vjgpt yields two types of plasmids, which can be combined with the plasmid p
KC,2/4 (and pKC,i!/3) The restriction sites and functional maps of plasmids pKC,2/l and pKC,2/j are shown in FIG. 3 and the diagram of the accompanying drawings, respectively.

Illustrated plasmids pKcJ/l and pKC, 21
In 5, the eukaryotic promoter contains associated regulatory elements such as hygromycin B and G g /, and the r-resistance promoter controls transcription of the resistance gene and partially controls trait expression when transferred into a eukaryotic host cell. Adjust accordingly. Furthermore, nine plasmids are present in eukaryotic hosts and undergo chromosomal integration (Chr).
During normal eukaryotic cell division, it is replicated as part of a chromosome during normal eukaryotic cell division.

Illustrated plasmids pKc21'A and pKCλl
The eukaryotic promoter mentioned here as an example in j is the svgθ early promoter [0'Hare parallel, +
/ 9g / + Proc, Nat, Acad,
Sci, USA 7za (3) 1327; Mullig
an and Berg * 19fθ, Sci, 2θ
9:1l122J, but many other true nuclear cellular promoters can also be used. Other examples of eukaryotic promoters include the 5VIIO late promoter (latepro-J, t) and the H8VITK promoter [Pellcer et al.

197g, Ce1l III: 133co, adenovirus promoter [Thummel and
Tjiin + 19f/, Ce1l, 23:♂
, 2j co, adenovirus @2 (Ad, 2) late promoter [Rolni, ck + 19♂/, Ce11
．． 241/35], polio-v (Polyoma
) ・Promoter [Co1antuoni et a
l.

/9J'O,ProcJJst,Acad,Sci,
USA 77(7): 3zaso].

Mouse Sarcoma Virus Pro'? -1-[Va
n Beveren et al, /978Na
ture, 2♂9:, 2. tf].

Yeast trp-/promoter [Stinchcomb a1. .

/979, Nature, 2J', 2:3
91. Yeast 1eu 2 promoter [Ratmkin
and Carbon t 1977 + Proc,
Nit, Aead.

Sci, USA QC2 doors 1177], yeast his3
Promoter [Broach et al, s/9
79 + Gene♂:/, 2/], yeast alcohol dehydrogenase promoter and yeast cytochrome c, promoter [Guarenteard Ptash
ne.

/ 9771 + Proc, 'Nhtyo Acad,
Sci, USA 7♂ (1 house J/99) is included, but is not limited to ξ.

Illustrated plasmid pKC, 1141 Oyohi pKC2
The construction of 1j includes a sd ■ insertion downstream of the early SVψ promoter in the gene psVj gpt, but other similar constructions containing one or more of the eukaryotic promoters listed above can also be made. for example.

Bgl containing hygromycin B and attir resistance genes ■Linear DNA fragment is plasmid P
It can be obtained from KC, 2θ3 or a derivative thereof by treating a plasmid with Bgl and [restriction enzymes] using a conventional method. This fragment can be used immediately or can be prepared using synthetic NA linkers (finker), which are known in the art.
) can also be linked to this resistance-transfer fragment. The fragment thus prepared is 9 e.g. Ad 2
Alternatively, it can be cloned under a suitable eukaryotic promoter, such as the yeast cytochrome C promoter. When linked with a prokaryotic replicon, here as an example plasmids pGD/, pGD, 2. pGDJ and pG D4! etc., although these constructs are functional in eukaryotic and prokaryotic host cells and are thus within the scope of the present invention. Furthermore, other configurations can be made that include a variety of unusual eukaryotic promoters, and it is likely that some eukaryotic promoters and configurations will be more suitable than others when using a particular host. This is well understood and self-evident to those in the industry.

The specific hygromycin B and G&/J'i4 sex genes and restriction factors are contained in the plasmid pK.
c2111. pKc21! ,pGD/,pGDco,pG
Illustrated in D3 and pGD&, this gene contains a 73;k b fragment from plasmid PKC,20J. Furthermore, inside this IC■ fragment, there is a DNA sequence encoding resistance.2. j
kb Sal I/Machi II [localized to fragment and expressed as part of the fragment. Plasmid pKC2,22
GI is useful for isolating individual genes and regulators that convey resistance to specific antibiotics. for example.

Plasmid pKC222/Jl'kbBgl■/
The Silie fragment contains the hygromycin B resistance gene and regulatory elements +/, 4jkb of EcoRI/5
The alJ fragment contains genes and regulatory elements that convey resistance to attir. Yet again.

It is believed that the genes and associated regulatory factors that convey resistance to G11lf also convey resistance to the antibiotics abramycin and tobramycin in susceptible hosts such as E. coli.

Therefore, recombinant DNA cloning vectors conveying resistance to one, more than one, or all antibiotics are constructed using the appropriate plasmid PKC,2θ3 or pKc,222D under the appropriate eukaryotic promoter.
It can be constructed by cloning the NA fragment. When supplied with a prokaryotic replicon, plasmid pGD/θ.

pGD/no, pGD12. pGD/3. pcI) 1g
and pGDi3 are mentioned herein as examples; these vectors are functional in both eukaryotic and prokaryotic hosts and are thus within the scope of this text.

It is clear that the present invention is not limited in any way by the above-mentioned additional resistance transfer ability of the Gg/male resistance gene.
7. from Kc203. The dl' fragment of tkb also contains a prokaryotic replicon and can be ligated by itself to form a plasmid. The resulting plasmid is designated pSC7θl.

This plasmid is functional in E. coli.

It is useful for producing derivative plasmids useful as starting materials. Thus, plasmid pSC70/DNA was digested with lae and ligated to form plasmid-
△ pKc257 and pKC2! ; Get 9. Plasmid pKc,2j7 is ~1 A2 kb and conveys resistance to hygromycin B, whereas plasmid pKc,2j7
KC239 is highly resistant and conveys resistance to both hygromycin B and abramycin.

When plasmid pKC, 2j7 DNA is digested with Koishi 3AI and ligated, a fairly small plasmid is produced.

This is expressed as pKc2J/. This plasmid is also □.

Conveys resistance to Igromycin B.

The derivative plasmids described above are functional in E. coli and are useful as starting materials for constructing vectors of the invention. Thus, plasmid IKC2! ;9wopSVj
gpt 2 inserts; S dove-> s i F pLO3
11A and pLO31! produces; plasmid pKc
2j7woPSVj gpt 2 insertion suldo blasmid p
L0316 and pLOJ/7; and plasmid pKc261 wopSVj'gpt double insertion sultophrasmid pLO311 and pLO3/9
occurs. All of the aforementioned PLO plasmids are functional in both prokaryotic and eukaryotic host cells and are thus within the scope of the present invention.

Additional plasmid starting materials can also be constructed. For example, a Hael [restriction fragment] containing plac from plasmid pUR222, whose construction
)-chi (Nucleic Ac1ds Re5earc
h) t9:1t01r7, (/9lf)] is inserted into plasmid pKC24/, resulting in plasmid pKcJ7j. plasmid p
KG 27! ; is ~3,6 kb and conveys resistance to hygromycin B; plasmid psVj
Plasmid pLO32θ illustrated by inserting into gpt
and pLO321. Furthermore,
Insertion of the restriction fragment containing 2μ DNA from plasmid YEp, 2& into pKC259 creates plasmid PK.
yields C2All-. II Sweet Sube XX\\Otori\pKC
Insertion of the ~25 kb gene II/5all fragment from 2j9 into appropriately cut YE p211 generates plasmid pKC273. Then, inserting the ~3.2 kb Pst fragment from plasmid pKC,26g into pBRJ22 creates plasmid pL
yields O371. Plasmid pLO-37zatopK
C, 2'7? Both are functional in yeast and E. coli and are therefore within the scope of the invention.

The construction of these and other vectors using the plasmid starting materials described above is even more clearly disclosed in Example 2S below.

Still other D that also convey resistance to either or both hygromycin B and Gg/male
It will be clear to those skilled in the art that NA sequences can be constructed or isolated, and that these sequences can be used in place of the resistance genes and regulatory elements listed as examples herein.

Furthermore, a 7jkb di-d ■ fragment or a jk
Functional derivatives of the Sal/BgU subfugment of b can be constructed by adding, removing or substituting certain nucleotides according to the gene codon. It will be appreciated by those skilled in the art that such derivatives, as well as other hygromycin B and aatr resistance conveying DNA fragments, may be used to generate vectors within the scope of the present invention.

The prokaryotic replicon exemplified here in the exemplified plasmid is the plasmid pBRJ, 2.2 replicon, although many other replicons can be used in similar constructions. Other examples of eukaryotic replicons include e pBM/replicon [Betlac
h at al, s 1976 t Fed, Pr
oc, 33:203], NR/Replicon [R
own and midcel.

19riha Nature 231e, 0ko, RK, 2 Replicon [Beringer * 197tl * J
, Gen. Microbiol, II: 11
13 sm9 replicon [kontomichalpu
at al, s 19riO9J, Bacterio
l, IO'A: 344] -p8C10 Rupriconro Cohen and Chang + / 9ri7
+ J, Bacteriol, / 32: 73
11 Co., RP/Revryn [Grinsted]
et al., /972 s J.

Bacteriol, 110:j29], Rpg
Replico: / [/ 977-Nagahari et
al, , Gene / : /Q/] + R
8F 101θ Reply: l N [Guerry et
al, , /9ri4! , J.Bacteriol.
, 117:6/9, PUB/10 Reply: ln [Grycian, et al.

1971, J. Baeteriol, 13u:
31. r co and 5 LPt, 2 rev: l :/ [
Bibb et i, Nature 214A:
This includes, but is not limited to, 326 files.

Many additional prokaryotic replicons can be constructed, and there is a general consensus that some prokaryotic replicons are more suitable than others and may be selected when using a particular host. It is well understood and obvious to those skilled in the art. for example.

RBFlolo, PUBllO and 5LPt,! The replicons are each pseudomonas
) *Bacillus.

(Bacillus lus) 1 and Streptomyces x (Strepto-myces) genus 1
is preferred, whereas the other replicons cited above are preferred for use in Nikoli.

The present invention (1>R')l- is highly transferable and functional in almost any prokaryotic and eukaryotic host cell. Necessary requirements are: /) the host cells are capable of dividing and culturing; 2) the host cells are capable of transformation; 3) the non-transformed host cells are sensitive to hygromycin B and/or G11lt and therefore die. To end up.

Only. Therefore, the 9 vectors are bacteria and fungi.

Useful for yeast, plant cells, animal cells and free-living unicellular eukaryotes.

The wealth of genetic and biochemical information regarding E. coli K12 makes it a convenient and suitable prokaryotic host cell for purposes of the present invention.

tilis), Pseudomonas
s), Agrobacterium (Agrolmeter
ium) s, Treptomyce x (8trapt
omyees) *Staphylococcus (S--pb
ylocoecus), x Treptochotca x (
8 trapto-coccus), 7 Actinomycetas and 5erratia] and blue-green algae.

Suitable eukaryotic host cells include fungi [! Irospora (NL)
Iurospora) s Cephalosporium (Ce
phalos-porium) s 7 Aspergillus, Penicillium (
Easily culturable cells derived from tissues of multicellular organisms, including but not limited to Penicillium) and yeast; animal cells.

Plant cells, including but not limited to gymnosperm cells and angiosperm cells, and free-living unicellular organisms including, but not limited to, algae and protozoa.

As noted above, the vectors of the present invention are functional in virtually any type of prokaryotic and eukaryotic host cell, 9 such as E. coli KI2BEt27, E. coli K/21.
11ri lr3m K,:lsuK/,l BB10
4A1. ? '7 XLtk-cells and Saccharomyces cerevisini
cerevisiae) to convey the desired antibiotic resistance to hygromycin B- and G1111-sensitive cells. Thus, 9 transformants of the present invention [exemplified transformants E. coli K/2 BEI27
/PKC21u, E, Near! JK/JB
EJJ7/pKC211, mouse Ltk/pKc, 2
/g, Mouse I, tk-/pKc21! , E., Cori K.
12 B'E71r3/pKc273 and S. cerevisiae/PKC273] express resistance to either or both of the above-mentioned antibiotics, and therefore appropriate screening conditions This vector is useful for propagating and maintaining the vector under the following conditions. Additionally, non-selective structural genes can be cloned into vectors and transformation and subsequent culture in mammalian or other host cells under hygromycin B or aait selection pressure can stabilize, retain and express the cloned genes. Party B can be understood as a person skilled in the art. Only host cells thus transformed can survive in the presence of igromycin B or autt, thus allowing initial identification.

The majority of host cells that can be transformed with the vectors of the invention are important because they allow eukaryotic vectors to be easily propagated and manipulated in prokaryotic host cells. This is particularly advantageous because the genetic background of prokaryotes such as E. coli is well known and conventional recombinant DNA methods can be easily applied to this system. Therefore, the present recombinant 117 DNA cloning vectors, including those containing selective or non-selective structural genes, are first manipulated and amplified in prokaryotic cells and then transferred into eukaryotic host cells for expression. , thus overcoming the serious problems associated with being exclusively restricted to eukaryotic cell systems.

While all embodiments of the present invention are useful, certain of the present recombinant DNA transformation vectors and transformants are even more preferred. Therefore, suitable vectors include plasmids pKc21upKC,2/j and P
KC23, and the preferred prokaryotic transformant is E.
, Ko+J K/J BEI27/pKc2/4!
, E. coli Kt2BE12/pKC core 3 and E. coli K t2BE713/pKC core 3.

A preferred eukaryotic transformant is the mouse Ltk-/pK
C,2/μ, mouse Ltk-/pK(,2/j and S. cerevisiae/pKC,?3).

The utility of the vectors and transformants of the present invention broadly allows recombinant DNA technology to be applied to eukaryotic systems to a greater extent and more rapidly. for example.

The ability of the present vectors, including plasmid-coated bacteriophages and viruses, to convey resistance to antibiotics that are toxic to both eukaryotic and prokaryotic cells provides a functional testing method for selecting transformants. This is important because such tests are virtually necessary to determine and select the specific cells from which vector DNA has been obtained.

Another DNA/A sequence for which there is no functional testing method for its presence can be inserted into this vector, and transformants containing non-selective DNA can then be treated with hygromycin B or G'I-/
It can be isolated by lr selection. This non-selective D
The NA sequence is 9 e.g. fibronectin (B antig
It includes, but is not limited to, genes that define post-translationally modified proteins such as @n) (both of which are also glycosylated in eukaryotic cells after translation).

More specifically, 2, 7. for example from plasmid pKC, 2θ3. tkb Sal I/Bgl I [9 containing restriction fragments e.g. exemplified plasmid pG
Rvu of plasmids such as D/4tu and pGD/j
A non-selective gene sequence can be inserted at one site. Such insertion inactivates the hygromycin B resistance gene, thus allowing easy removal of transformants containing the recombinant plasmid. This is done by first selecting G'l/f resistant transformants and then removing G4 (/f resistant transformants) that are not resistant to hygromycin B.Similarly, meaningful gene sequences are selected. For example, Xho1
When inserted into the site, it inactivates the G&/za resistance gene. Thus, transformants carrying this recombinant plasmid can also be easily identified by first screening for hygromycin B resistance and then identifying hygromycin B-resistant transformants that are not resistant to auH. can. Therefore, the ability to screen for antibiotic resistance in eukaryotic or prokaryotic transformants
Meaningful specific non-selectivity is possible.

As mentioned above, antibiotic resistance functional testing can also be used to identify DNA sequences that act as regulators and control the expression of individual antibiotic resistance genes. Such sequences [including promoters, attenuators, *repressors, inducers, bosome binding sites, etc.]
[but not limited to] i can be used to control the expression of other structural genes in both eukaryotic and prokaryotic cells.

Hygromycin B and G4t/? of the present invention Resistance transfer vectors are also useful for stabilizing various linked DNA sequences. Hygromycin B or G4 (/
Genes or fragments covalently linked to resistance genes and propagated in either eukaryotic or prokaryotic cells: genes or fragments that are hygrolated to a degree that is toxic to untransformed cells. Mycin B or Gg/
Transformants can be further retained in males. Therefore, the transformants that have lost the vector and the resulting covalently linked DNA die and are eliminated from the culture medium. Thus.

Vectors of the invention are capable of stabilizing and retaining meaningful DNA sequences.

The cloning vectors and transformants of the invention provide commercially viable production of both unmodified and post-translationally modified polypeptides in eukaryotic cells as well as in prokaryotic and eukaryotic cells. Improve the yield of various products commonly manufactured. Examples of such products are streftomycin, tyrosine, cephalosphorin.

actaplanin, biosynthetic insulin, biosynthetic human insulin, biosynthetic human proinsulin.

Includes, but is not limited to, biosynthetic interferon and biosynthetic human interferon.

The present invention also identifies and characterizes DNA sequences encoding 1) commercially important proteins, 2) enzymatic actions in metabolic pathways leading to commercially important processes and compounds, and 3) restriction factors that improve gene expression. Provides selective vectors that can be used for reconstitution. These desired DNA sequences can be used for hydrocarbon biological degradation.
ion), or antibiotic derivatives such as cephalosporins, tyrosine and actafranin derivatives, or DNA encoding enzymes that mediate and increase the production by biological methods of antibiotics and other products. It is not limited. Like this N
The ability to insert and stabilize A sequences into eukaryotic and prokaryotic cells using recombinant DNA methods is important because a eukaryotic host is required for the production of certain antibiotics and post-translationally modified proteins. is important. Moreover, the vector can be easily manipulated and amplified since it can be functional in prokaryotic cells, such as E. coli, and thus can be easily manipulated and propagated in eukaryotic cells, where such manipulation is difficult if not impossible. The problems associated with this can be resolved.

The present invention further describes the composition of new species or strains of multicellular organisms that are able to withstand relatively well the aggressive domination of antibiotics intended to eliminate parasites, pathogens, and other infectious agents within one body. provide. For example, one can insert the hygromycin B or G4t/f resistance genes of the invention individually or together into germ cells or early fetal cells to create multicellular organisms highly resistant to these antibiotics. .

5 Therefore, resistant multicellular species such as pigs, cows, and sheep are exposed to high concentrations of antibiotics, which is required to better control harmful parasites and infections that produce substances that slow growth and reduce physical strength. become more tolerable.

The following examples present embodiments of the invention in detail. Explanations and practical prescription procedures constituting the present invention will be described as necessary.

(Margin below) Plasmid pKC, 2θ3 E-1') JR,! 2! ; (ATCCN, o, 3/9
/2) according to conventional microbiological methods with the antibiotic hygromyein B (/0
ON/ml) containing TY medium (1% tryptone, 9
5% yeast extract, θS% sodium chloride, pH 7g). After culturing for 17 hours, approximately 0.03% of the culture solution
m1 to i! ;ml Eppendorf (Eppend, o
rf) Transferred to a test tube and centrifuged for about /j seconds. All operations were performed at room temperature unless otherwise noted. The obtained supernatant was aspirated using a fine-tip aspirator (fine-tipa).
lysozyme (21111/sl), jOrnM glucose.

/77mMCDTA (cyclohexane diaminetetraacetate) and p23mM) lis-hydrochloric acid (p
Freshly prepared or zozyme solution (approximately 1
00td). After incubation at 0″C for 30 minutes, alkaline SDS (sodium dodecyl sulfate) rum) #
'& (02N sodium hydroxide, 1% 5DS) (approximately 20
0 pl) was added and the tube was gently portexed and held at 0°C for 75 minutes. Next, dissolve 3M sodium acetate (approximately lsθll1) (sodium acetate (3 mol) in a very small amount of water.

(prepared by adjusting the pHIAlr/l with glacial acetic acid) was added, and the contents of the test tube were gently mixed for several seconds by inversion, during which time a DNA clot was formed.

The test tube was kept at 0°C for 6 g and then centrifuged for S minutes to obtain an almost clear supernatant. Supernatant (approximately 011
m1) to another centrifuge tube and add cold ethanol (7 ml) there.
) was added. After holding the centrifuge tube at -2θ'''C for 30 minutes, the resulting precipitate was collected by centrifugation (2 minutes), and the supernatant was removed using an aspirator. Sodium 1100K Tris-
Dissolve in hydrochloric acid (pHr) and reprecipitate by adding twice the amount of cold ethanol. After holding at 2g°C for 70 minutes, the precipitate was collected by centrifugation as described above to obtain the desired E. coli JR22! ; Plus ε-do DNA is obtained.

E, Cori JRQ2! ; Approximately 10 ml of plasmid DNA sediment
0111 07M Sodium Acetate 100! ;M Tris-
It was dissolved in hydrochloric acid (pHl') and precipitated with twice the amount of cold ethanol. The obtained plasmid DNA was collected and dissolved in water or a dilute buffer solution (approximately 4 tOJil), and then added to a CWensink Cell 3:3/
3 (19711) by essentially the same method of transfer as E, coli/2BE/r27. E,:+9/2BE
The 27th is Maryland (Whryland)
American Type Culture Collection in Rockville
icanType Culture Co11ecti
On), it has been deposited as a preserved bacterium, and anyone can obtain it from there under the number ATCC 379// without any restrictions.The obtained transformant is treated with the antibiotic hygromycin B ( TY containing 200IJg/ml)
Agar medium (1% tryptone, 05% yeast extract, 2t%
Sorted on sodium chloride, 15% agar, IIH'21). Some transformants were tested by gel electrophoresis [Rao (Rao)].
) and Rogers, /971] and other studies, it contained both large and relatively small (/3 kb) plasmids and was resistant to both ampicillin and hygromycin B antibiotics. In addition, some transformants have relatively small /, fkb
Contains only the plasmid of the antibiotic hygromycin B
and G11nt but sensitive to ampicillin.

Transformants of the latter type were transferred to TY agar containing the antibiotic hygromycin B (/IP/g/) and cultured using standard microbiological procedures.

From the obtained cells, according to the method of Example/A.

/j kb hygromylene B and 0111 as described above
! A resistance transfer plasmid was isolated. This will hereinafter be referred to as plasmid pKCJ7J. Plasmid pKC203
The presence of the resistance gene was confirmed by a series of transformation and selection analyses.

Example 2 Plasmid pKC203 DNA (approximately !I4) was treated with h±■ restriction enzymes under defined conditions according to the manufacturer's instructions. Fragments of 1tkb, jtkb and 0jkb are recovered, among which Bgll of 7jtkb
The fragment contained the desired antibiotic hygromycin B and G4t/r resistance genes and regulatory elements. This means that cells that are normally sensitive to the antibiotics hygromycin B and aatr are 7 j kb (ri!
! It was confirmed by a series of transformation and selection assays that the introduction of the LL II fragment resulted in resistance to these antibiotics.

Qin restriction enzymes and instructions are readily available from: Bethesda Research Laboratories, Inc.;
ories In6゜Box 6010 Roekvilla, Maryland 2Qlf3Q
Boehringer e Mannheim Biochemicals; Boe
hringer Mannheim Bioehemi
ca1g79'l/ CmsCm5Lle Drive
eP, 00BO! j 01/4 Indianapolis, Indiana uls2
30 New England Bio Labs, Inc.
.

2f3 Cabot Beverly, Massaehusetts O/9
/! rResearch ProductsRe5eareh Products Miles Laboratories In6゜El
le:hurt, Indiana 116! /j Example 3 Plasmids pKC2/lI- and pKC2/j and plasmid pSVjgpt were constructed as described by Mulligan and Berber.
g) [Be1ernoe, 209 ('I'1
t3): /'722 (/970)], but this plasmid has a Bgll-specific site inside the gpt gene.By cloning as shown below, the antibiotics hygromycin B and Glt/ can express the trait of the resistance gene.

Boo7 SEF psVj gptDNA (approx. 3p
g) Treated with Bgll [restriction enzyme] under conditions specified according to the manufacturer's instructions. After the enzyme was inactivated by heating at 70°C for 5 minutes, D, NA (approx./p
g) was mixed with the 7jkb Bgll fragment taken from pKC203 in a l:l ratio.
4! It was ligated using DNA ligase.

*T4! DNA ligase and instructions are readily available from: Bethesda Research Laboratories Bethasda Ra5earoh Laborat
orieaBox 6I10 Roakville Is, Maryland 201
j OThis combination is Penzink's cell 3:3/! (19
The antibiotics ampicillin and abramycin were each introduced using essentially the same method of transfer as in 1 year).
E on the TY plate containing 2/2 Belr2
It was introduced into the country. This recombinant clone was treated with ampicillin (
/ 00 ptp/ml) and the antibiotic hygromycin B (200147 Roj). Approximately half of the antibiotic hygromycin B-resistant recombinant clones contained plasmid pvcc21a (Fig. 3), and the remaining half contained plasmid pKCJ/j (Fig. D).

Plasmid DNA in the various clones mentioned above.

It was isolated according to the method of Example/A and identified by restriction enzyme analysis in a conventional manner. By this method, the newly constructed transformed clone 9K/2BE♂27/pKC2/'
I and E, sol i K/, 2 BEr, lli/
pKc2/j was removed and subsequently isolated for further use.

Example 1 Construction of Mouse Ltk-/pKC2/Da Mouse Lik- cells were incubated with Earle's salt (Earle'a 5
alt).

Non-essential amino acids [Eagle (E turtle gl.), /939.
Science, /30':It32'J, 1096 /V
The cells were cultured in a conventional manner in a medium consisting of a minimum essential medium containing newborn calf serum and G-glutamine (292 pg/dl) until they were maintained in a constant state.Mouse Ltk cultured in this way - Wiggler (W
(Pros, Nat, A
eaLBei, USMu 7-4 (J): <373(
(1979)] and plasmid pKC■, in essentially the same manner with the following exceptions.

2/44 was imported. The difference is l) Plasmid DNA
(100 to 300 mg) to precipitate of calcium phosphate (L
d) was added to each plate in the medium, and 2) the culture medium was as described above. About Hong Kong time,
After culturing at 3°C.

The medium was replaced with fresh medium and the cells were cultured for an additional 20 hours. At that time, antibiotic hygromycin B selection pressure was applied. This is done by adding the antibiotic hygromycin B (approximately 73-1000 ps) to the above medium.

This was carried out by changing the medium to a selective medium that also contained ./ll1). The concentration of antibiotic suitable for selective media is 2
00I4/Dew j. The selective medium is used after the 18th round, the first stage is carried out 2 days later, and then the transformed clones appear 2 days later.
It was replaced every three days for ~3 weeks.

Colonies were picked by hand using a pipette and grown in mass culture under constant selective pressure. The desired mouse Lik-/pKcJ/4 (transformant) can be obtained from the antibiotic hygromycin B-resistant cells cultured in this manner.

Example j Construction of Mouse Ltk-/pKc2/j Cells of mouse Ltk-/pKC-2/ are transfected with plasmids pKc, 2. It was obtained essentially in the same manner as described in Example G, except that plasmid pKC'2/j was used in place of 'l II. The thus obtained transformed mouse Ltk/pKc2
/j was grown in large scale culture.

Example 6 Plasmids pKcJOJ, pKC2/da and pK
C2/3 antibiotics hygromycin B and G'I-
The ability to convey resistance to /r was determined by testing the growth of transformed and untransformed L. coli and mouse Ltk- cells on media containing varying amounts of antibiotic. The media and culture conditions for L. coli and mouse Lik cells are essentially as described in each of Examples/A-G! ζ is the same. The test results are shown in Tables 1 and 2 below. In Table 9, #10 indicates growth, ter' indicates no growth, and 'NET' indicates no test was conducted.

Example 7 Plasmid pLG449 was described by Guarenie and Ptashne for its construction.
') description (Pro oe, Nat, Acad
, Sci, USA7<5'('l) :2/
99 (/9jr/year)], which is cytochrome C.
It has a unique site inside the gene that is restricted by BamHI. 73 taken from plasmid PKC,2,θ3
By cloning the Kb Bgl'l fragment into this region as described below, the trait of the antibiotic hygromycin B resistance gene can be expressed. , the desired insertion is facilitated because the Bgl II fragment has a GATC sequence in its j'-extension that is identical to the S'-extension of the BamHI fragment. Therefore, the Bgll fragment and the BamHI fragment can be directly linked together to form a recombinant association.

Plused pGD/and PGD, 2 and E, coli/, 2BEza27/pGD/ and E, coli/,
2 BEza27/PGD, 2 can be obtained by essentially the same procedure as in Example 3, except that plasmids pLG4&9 having a functional site are used instead of plasmid psVtgP. Bgl l
Depending on the orientation of the fragment, a λ-oriented plasmid is obtained. Plasmid pGD/ represents a recombinant plasmid in which the Sal/ restriction site is closest to the ura gene. Plasmid pGD,2 represents the plasmid in the opposite orientation.

Example 1 Yeast (Saccharomyces cerevisiae) 1% yeast extract 72% bacto-pepion by microbiological methods well known in the prior art.
') in a typical manner. To transfer the plasminide pGD/ into yeast, use hinene (
(Proe, Nat, Ac
ad, Sci, USA73:/9Z (797g)] by adding a lethal dose of N.B to the culture medium.

Example 9 Construction of S. cerevisiae/pGD2 The desired transformant can be obtained by essentially the same procedure as in the example except that plasmid PGD, 2 is used in place of plasmid pGD/. .

Example 10 Plasmid φ cap, its composition is explained by Tsurnik (
Cell 21/-:/3! ;(/91r
/year), but this plasmid is located on the map coordinates “/J:4' (EeoRI) to /l-6 (Hi
ndll) to 1 □ between the major adenoviruses and 2 (Adenoviruses).
s2; Ad2) Contains the late promoter. The antibiotic hygromycin B was produced by cloning the 7jkb Bglll fragment taken from plasmid pKC203 into the Hindn1 site of plasmid pφq.
Can express the traits of resistance genes.

To achieve the desired configuration, use the Ullrich
) etc. (Seienee /9A:/313(
A Hindll linkage was added to the 75 kb Bgl ■ fragment in essentially the same procedure as (1977)].

A conventional method is used in which the obtained modified fragment is ligated to Hindll-treated plasmid pφg using T4Z DNA ligase. Hindll restriction enzyme and TgDN
The A'J gauze enzymes are available from the companies cited in Examples λ and 3, respectively, with instructions for use. 7jkb Bgllll
After preparing the fragment and HindI[I ligation material, essentially the same procedure as in Example 3 is performed to ligate the fragment to plasmid pφg to create plasmids pGD3 and pGDg. Then E, Cori/2
Transferred to BE♂27 and became Kukori/2 BEjr27
/pGD3 and E, coli/, 2 BEza27/pG
The procedure of Example 3 is followed to prepare Du.

As explained in Examples 3 and 7, plasmids with two orientations are generated depending on the orientation of the inserted resistance transfer fragment. Plasmid pGD3 represents a recombinant plasmid in which the restriction site of the inserted antibiotic hygromycin B resistance transfer fragment (5ad) is closest to the EeoRI site of the M2 promoter. Plasmid pGD
Q represents a plasmid in the opposite direction.

过Hi nd m C(d (CCAAGCTTGG
), :] and other linking materials are collaborative
Research Incorporated Collabor
active Re5search Inc.

/31. ! ; Main 5treet, NValt
ham, MA O/2! 'l) can be easily obtained.

EXAMPLE //Construction of Mouse Ltk/pGDJ The construction of mouse Lik/pGD3 is carried out essentially as in Example t, except that plasmid pGD3 is used in place of plasmid pKC2/g.

Example 12 Construction of mouse Ltk-/pGDg To construct mouse Ltk/pGD&, plasmid p
The procedure is essentially the same as in Example D, except that GD4() is used in place of plasmid pKC2/!.

Example 13 Plasmid pKC203 (approximately 11 g) was transformed into 8al and Bgl under defined conditions according to the manufacturer's instructions.
1 restriction enzyme. Antibiotic hygromycin B
and 7 fragments of 2 JKB containing the aaiz resistance gene and regulatory elements were recovered by a commonly used method.

1111111--1) 1 - Restriction enzymes and instructions can be obtained from the sources cited in Example 2.

The composition of plasmid pKC7 (approximately spg) is described by Rao and Rodgers (Gone Rin99). Processed with.

After the enzyme was inactivated by heating at 70°C for 5 minutes.

DNA (approximately ln) to PKC20 in the ratio of / : / (2)
From 3? )L7jkb Sal I/Bgl
II 7 fragments. The fragments were ligated together using Tlt DNA ligase under conditions defined according to the manufacturer's instructions cited in Example 3.

When the obtained plasmid pKC222 was transferred into E. co9/2 by essentially the same procedure as in Example 3, it was found that resistance to the antibiotics ampicillin, hygromycin Ill, and aatr was transmitted.

Isolation of Example 11 The desired DNA fragment was isolated in essentially the same manner as in Example 3A, except that 8aal was used together with Bgl in place of Sag during the restriction digestion.

Example/j The desired DNA fragment was isolated in essentially the same manner as in Example/3, except that EeoRl was used with S<em>Turtle II rather than Bgl■ during the restriction digestion.

Example 16 The desired plasmid is an i5/kb 8aa I/Bgl II fragment taken from the plasmid KC222 and the plasmid KC203 and Tutnia j k
Essentially the same procedure as Example 3 and IO, except for substituting the Bgl fragment for the Bgl l linker and adding the Bgl l linker to the antibiotic resistance transfer fragment rather than the Hindu linker. .

Bgl l-linked substance added/! j''/kb fragment was transformed into plasmid p in essentially the same manner as in Example 3.
Insert into sVjgpLO.

As explained in Examples 3 and 7, bidirectional plasmids are generated depending on the orientation of the antibiotic resistance transfer fragment inserted. Plasmid pGDIO is a hygromycin B resistance transfer fragment inserted into the AV turtle! It represents a recombinant plasmid whose restriction site is closest to the Hind■ site of the gpt gene. Plasmid pGD// represents the plasmid in the opposite orientation.

Plasmids pGD10 and PGD// in E. coli
2 Transfer to BBr2 and compare each IJK/2BE
12ri/pGD10 and Otori'sL\XXJ 'llj
<Kokugoi X11iaxX\'i,, Performance 1. Ko+JK/2
BEt27/pGD//' was produced using essentially the same procedure as in Example 3.

Example 17 Construction of Mouse Ltk/pGD#) The desired construction was performed using plasmid pKC2 during the transformation process.
The procedure is essentially the same as in Example 1, except that plasmid pGD10 is used instead of /41. The thus obtained mouse Lck7/pGD/θ can be grown in large scale culture.

Example/r Construction of mouse Ltk-/pGD//
The procedure is the same as in Example 1 except that plasmid pGD// is used instead of /4'. The thus obtained mouse Lik-/pGD// can be grown in large scale culture.

Example 19 The desired plasmid consisted of a 743 kb (Q EaoRI/Sall fragment taken from plasmid pKC222 in place of the 7! kb Bgll-- fragment taken from plasmid pKC203 and a Bgllj ligation in place of the Hind ■ ligation material. Except for adding substances to antibiotic resistance transfer fragments.

The procedure is essentially the same as in Example 3 and lθ. B
7 fragments of gjkb were added to the plasmid pS in essentially the same manner as in Example 3.
Insert into V j g p *.

As explained in Example/4, bidirectional plasmids are generated. Plaid PGD/2 represents a recombinant plasmid in which the Pa&1 restriction site of the antibiotic a4Iit resistance transfer fragment is closest to the Hind 111 site of the gPt gene. Grasmid pGD/3 represents the plasmid in the reverse orientation.

Plasmids pGD/2 and PGD/l into E. coli/
2 Transferred to BE/27 and respectively E and Cori/2BE
t27/pGD/2 and E. coli/2 BBF27
/pGD/'J was generated using essentially the same procedure as in Example 3, except that Gμ/male was used instead of the antibiotic hygromycin B when selecting transformants. . ' Example θ Construction of Mouse Ltk-/pGD/2 The desired construction was performed using Grasmid pxc in the transformation process.
The procedure was essentially the same as in Example q, except that plasmid PGD/,2 was used instead of 27g, and autt was used instead of the antibiotic hygromycin B when selecting transformants. Let's do it. The thus obtained mouse Ltk-/pGD/J can be grown in large scale culture.

Example 21 Construction of Mouse Ltk-/pGD/J The desired construction is to construct the plasmid pKc during the transformation process.
Essentially the same procedure as in Example 1, except for using plasmid PGD/3 instead of J/4 (and using atir instead of the antibiotic hygromycin B when selecting transformants) The thus obtained mouse Lt k7/p GD/3 can be grown in large scale culture.

Example 22 From plasmid pKc203! 75 kl) was isolated according to the method described in Example 13. A molecular linker was added according to the procedure of Example 10, except that a Bgll linker was used in place of the Hind■ linker.

The resulting fragment is inserted into plasmid p8Vjgpt in essentially the same manner as in Example 3 to form the desired plasmid.

As described in Example 16, bidirectional plasmids are generated. Plasmid pGD/Q has the Ava I restriction site of the antibiotic resistance transfer fragment connected to the H of the gpt gene.
Represents the recombinant plasmid closest to the indl1 site. Plasmid GD/j represents the plasmid in the opposite orientation.

Plasmid pGD/4 (and pGD/j into E, coli/2 B) J! Transferred to 7 and respectively E, Cori/2
BE127/pGD/4! and L Cori KI2BE12
Essentially the same operations as in Example 3 were performed to generate pGD/3.

Example 23 The desired construct is the plasmid pKC2 during the transformation process.
The procedure was essentially the same as in Example 1 except that plasmid pGD71 was used instead of /u. The thus obtained mouse Ltk/pGD/& +i can be grown in the large volume medium.

Example 21 Construction of Mouse Lik/pGD15 The desired construction
The procedure is essentially the same as in Example 1, except that plasmid pGD/j is used instead of /'t. The thus obtained mouse Ltk/pGD/11. can be grown in large scale culture.

Example 2j Hula 7. jpKC203 (isolated in Example 1. Approximately jp1
．． jItg) in TE buffer (10mM Tris-HCl.

pu, rθ, /mM EDTA), ,DTT('100
mM dithiothreitol, jp), BSA (bovine serum albumin; /θθOW/ml, !ip!), water (
26ti').

The cells were cultured in reaction solution (Sμ) at 37°C for about 7 hours.

After incubating at 70"C for 5 minutes to stop the reaction, the mixed reaction solution was cooled on ice, extracted with phenol and a 21t:/mixture of chloroform and isoamyl alcohol, and precipitated with ethanol. The obtained Bgl ■The restriction fragment was dissolved in jmM Na1l (jp!) and ligated.The ligation was carried out using Bgllll-restricted DNA (/
ld) in water (approximately 31 td), 10 mM), TP (
! ;ti').

Ligation solution (jpri and Tl DNA ligase [~io'
New England Bio Lab Un
The reaction was carried out by reacting with its ') ) (/4) at 16@C for about 16 hours. The reaction was stopped by incubating at 70"C for 5 minutes.

After cooling on ice, the resulting ligation mixture was transformed on TY plates containing the antibiotic hygromycin B (200*/*l) using the Beto Zinc transformation method (/97'l-year) essentially Using the same method B, transfer to E and Cori/2 BEf27. Some transformants were tested by gel electrophoresis [Rao (Rao)].
Contains the desired ~73 kbO plasmid as previously shown by tests by J.D.') and Rogers et al./97g] and other studies. Such a transformant is herein referred to as E.

coli /2 BE127/psc7θ/, which was selected and treated with the antibiotic hygromycin B (2θOpg
/ml) and cultured using commonly used microbiological techniques. Plasmid pSC7θl is isolated from the obtained cells in the same manner as in Example/A. The presence of antibiotic hygromycin B and G4Z/male resistance genes in plasmid psc70/ was further confirmed by a series of transformation 9 selections and restriction enzyme analysis. The restriction sites and functional map of plasmid p8c70/ are shown in Figure 6 of the attached figure.

The reaction solution (/OX) for the Hata Bgll restriction enzyme was prepared from the following components: 600 mM NaCl 700 mM) Lis-HCl, pH 74! / 0 () inM Mg CI J Qinbo coupling solution was prepared from the following ingredients:! ;00mM Tris-HCl,
Kure 2g 200mM dithiothreitol/00mM Mg CI! Example 26 The desired plasmid is plasmid pKc103.

Plasmid ps instead of Bgll restriction enzyme and reaction mixture
It was produced in essentially the same manner as in Example 2j, except that c7θ/, Haall restriction enzyme, and reaction solution 0 were used. Plasmid psc7θl has / more than one Hael
Hael digestion as it contains restriction sites.

Subsequent ligations generate various plasmids.

The resulting ligated material contains the desired 1-Igromycin B
Resistance transfer ~442kb plasmid pKC2, f7 contains hygromycin B, G4 (/J' and abramycin resistance transfer ~θkb plasmid pKCJ-9, which was essentially transformed into the transformation process of Example 2j. In the same way, E, Cori / 2 BE 7 Za 3 [Illinois (Il.
Northern Regional Research Laboratory (Nor.linois) Peoria 7
thern Re'onal Re5earch L
aboratory)t with deposit number B-/! The strain was deposited at θ2θ and is a permanently preserved strain]. Desired transformants were selected and transplanted onto TY agar medium containing the antibiotic hygromycin B (20θR/II/) and cultured separately using commonly used microbiological techniques. Transformants containing pKC2j7 were easily isolated by a conventional method of screening for hygromycin B resistance, and transformants containing plasmid pKC2j9 were isolated by screening for abramycin and hygromycin B resistance. . These transformants are herein referred to as E, coIJK/2BE.
7za3/pKC2! ;7 and E, Cori/2 BE7
It is expressed as jr3/pKC, 2,5-9, but is it? Plasmids pKC2j7 and pKC259 were isolated from each 77 in the same manner as in Example. The presence of antibiotic resistance genes in each plasmid indicates that a series of transformations is required.

Further elucidation was achieved by selection and restriction enzyme analysis. The restriction sites and functional maps of each of plasmids PK(-2j7 and pKC2!;9 are shown in the accompanying figures, Figures 6 and 7, respectively.

A reaction solution (10X) for the Hael Hael restriction enzyme was prepared from the following components: 477m, M) Lis-HCl, pH 71t t OmM MgC1a Example 27 The desired plasmid was plasmid psc70/.

μ--■ Plasmid PKC than restriction enzyme and reaction solution
2! ;7, Sau,? Essentially the same procedure as in Example 2j except for using the A'I restriction enzyme and reaction solution.
．． It is ~3.2 kb and conveys resistance to hygromycin B.

The resulting ligated material was transformed into E, Co! in essentially the same transformation process as in Example 2j. Transfer to J K72 BF, 773. The transformant is E. coli/2BE7g.
,? /pK(-24/) is selected and plasmid pKc26/ is isolated using the same method as in Example/A. The presence of the antibiotic hygromycin B resistance gene in plasmid pKC2AI is a result of a series of traits. This was further confirmed by conversion 9 selection and DNA sequence analysis.The restriction site and functional map of plasmid pKc26/ is shown in Figure 7 of the accompanying drawings.

A reaction solution (10X) for the 5auJAI restriction enzyme was prepared from the following components: SOθmM NaCl tOmM Tris-HCl, pHZ, j! ; OmM
Mget.

Example 2g Plasmid pKC,24/ (approximately jμ,!; isolated in pyYC Example 27) was dissolved in TE buffer, DTT (jμ).

BS AC/ 00011g/ml, jp! ),
Water (2!;p!').

Hael restriction enzyme (3Id, !; U=t) and /θ
The cells were cultured in X reaction solution (3; pl') at 37°C for about 1 hour.

After incubating at 70"C for 5 minutes to stop the reaction, the reaction mixture was cooled on ice, extracted with each of phenol and 214:/mixed Fi of chloroform and isoamyl alcohol, and precipitated with ethanol. The obtained The Haell control fragment was dissolved in 3mM NaCl (31d').

B, μL111 [including ends ~394! nt plae
Isolation of fragments of plasmid PUR222 (approximately !pl, !;py)
C By essentially the same operation as in Example 1, E, CO! JK/2
BE/#, isolated from g/pUR222] in Example 2♂A, except that the reaction mixture was incubated at 37° C. for approximately 2 hours to ensure complete, but not partial, digestion.
Haell digestion in TE buffer in essentially the same manner as above. The obtained Hae ■ restriction fragment was added to 5mMN
Dissolved in aC1(j J ).

E. coli /2 BE//66/pUR222 is a strain that has been deposited and permanently stored at the Northern Regional Research Laboratory in Peoria, Illinois, and the plasmid is listed under deposit number B-#θ23. pU
A suitable source and storage material for R222 is available to anyone.

CO ligation and transformation plasmid pKc24/, plasmid pUB222H
a and ■ restriction fragments (approximately 4 IId each) (Example 2
7 and B respectively], water (3/〆).

/ Q mM ATP (j 141 ), coupling solution (
! pl) and Tl DNA ligase [~lO to England Bio Laboratories Units] (/
PI') was reacted at 73°C for about 16 hours. After incubating at 70°C for 5 minutes to stop the reaction, the resulting ligated material was cooled on ice. The DNA is E, i Suni/2 BE1017
/“:11 (Northern Regional, Peoria, Illinois)
Example 2j has been deposited with the Research Laboratory under deposit number B-/302/ and is a permanently stored strain.
was transferred using essentially the same transformation process as . desired ~, i
, transformants containing only 4 kb plasmid pKC273 were transformed into E, co! JK/2BE10111/pKC27
! It was expressed as The transformants were selected, transplanted, cultured, and the plasmids were subsequently isolated. ~39 including plae
The presence of 7 un* fragments and the detailed structure of plasmid pKc27j were determined by conventional methods using gel electrophoresis and restriction enzyme analysis.

Since plasmid pKcJ4/ has three Ha/■ restriction sites, a mixture of various Hael fragments in which 9 partial Ha@I digestions occur is produced.

Therefore, by the method exemplified above, plasmid pK
C! Isomers arise due to differences in the various insertion sites of 73 and plasmid pKc27j. Approximately 39 dunt pla
Fragments containing e can be linked in either of two directions, so there are two directions.

A recombinant plasmid is generated. It will be appreciated by those skilled in the art that plasmid isomers of various orientations and the resulting transformants can be readily isolated and identified by conventional methods. Plasmid pKC27! The restriction site and functional map of is shown in Figure 1 of the accompanying drawings.

Example 29 The desired digestion uses plasmid pKC2j instead of plasmid pUR222, μael restriction enzyme and reaction mixture. , E
The procedure was essentially the same as in Example 21, except that coRI restriction enzyme and reaction solution 5 were used. The resulting KooRI restriction fragment was digested with jmMNaC1 (j l
d) IC dissolved.

*EaoRI restriction enzyme reaction solution (10X) was prepared from the following components.

j 00 mM NaCl 100 θmM) Lis-hydrochloric acid, pH 77j Q mM
MgCI J Plasmid YEp for primary λμ DNA isolation, 34! The RI-digested plasmid YEp2u of E. coli was isolated from E. coli /2BE//39/YEp2μ by essentially the same procedure as in Example 1. E. coli /2BE//39fYEp,Zda is a strain that has been deposited at the Northern Regional Research Laboratory in Peoria, Illinois and is a permanently preserved strain. It is publicly available under deposit number B-/J'022 as a suitable source and storage material for the above-mentioned plasmids. The desired EeoRI digestion of plasmids YEp, Z was performed essentially in the same manner as in Example 29A, and the resulting EeoRI restriction fragment was
Dissolved in W NaCl (j Id,).

CO ligation and transformation The KeoRI-digested plasmid pKc2j9 and plasmid YlpJ (prepared in Example 9A and B, respectively) were ligated and subsequently transferred into E. coli/2 BE7r3 using essentially the same procedure as in Example 21rC. did. desired ~
7. A transformant containing only the lkb hygromycin B, abramycin and Gll/jr resistance transfer plasmid is designated E. coli/2 BE713/pKc244'. The transformants are selected, transplanted, and cultured in a conventional manner, and then the plasmid is isolated.

co-μDNA can be linked in either direction, thus
It is clear to those skilled in the art that the above-described method produces both the previous plasmid pKC26μ and its reverse orientation. Various plasmids and the resulting transformants can be easily isolated and removed by conventional methods. The restriction sites and functional map of plasmid pKC-244' are shown in Figure 1 of the accompanying drawings.

Example 30 Desired digestion is plus E! f', pUR222 and Ha・m restriction enzyme and plasmid instead of reaction solution,
Essentially the same N as in Example 21rB except for using KC2!9, Bgl IIl restriction enzyme and reaction solution.
Dissolve in aC1.

The desired digestion was carried out using plasmid PUR2J 2 , Hae
plasmid pSvj′ instead of m restriction enzyme and reaction solution.
The procedure was essentially the same as in Example 2FB, except that gpt, Bglm restriction enzymes and reaction solutions were used. The obtained Bgl [digested material is j mM NaCl
dissolve in

C0 ligation and transformation The desired ligation is plasmid pKc26/ and pUR2
Bgl l digested plasmid pKC2 instead of 22
j9) tJ: psVjgpt + use rokoj:,,
The procedure was essentially the same as in Example 2rC except for the following.

The resulting ligated material was transformed into the antibiotic hygromycin B using essentially the same transformation method as Penzink (797g).
TY play including (20ri IIg/sl), 13□7
．． ni7yuBE7r3 +c$ioh. A6 transformants were subjected to conventional gel electrophoresis (Rao and Rogers, /97
1 year) and other tests, the desired plasmid PLO,? /4' or desired plasmid p
LO3/! Contains only; When these transformants are selected, transplanted, and cultured.

/2BK7♂3/pL0 to the desired transformant mouse
31μ and E, Furini/2 [7za3/pLO3/3
Configure. These transformants are then subjected to isolation of the desired plasmid pLO3/4 (and pLO3/j).

The DNA 7 fragments are ligated in either direction, resulting in a bidirectional recombinant plasmid. It will be apparent to those skilled in the art that the desired plasmid can be easily identified and removed using conventional techniques such as restriction enzyme and electrophoretic analysis. Plasmids pLO3/II- and pLO3/
! The restriction sites and functional maps of each are shown in FIG. 9 of the accompanying drawings.

Example 31 Construction of mouse Ltk-/pLOJ/4' The desired construction is:
Plasmid pLOJ/ than plasmid pKC2/II
The procedure is essentially the same as in Example 1, except that a damascene is used. The thus obtained transformed mouse Ltk
/pLO3/II is grown in large scale culture.

Example 32 Mouse Ltk/pLo,? / j configuration The desired configuration is plasmid pKc2/4! plasmid pLO instead of
Except using 3/j.

The operation is essentially the same as in Example D. The thus obtained transformed mouse Ltk/pLOJ/j is grown in large scale culture.

Example 33 The desired anti-inflammatory drug was obtained using plasmid pKC2!7 instead of plasmid pUR222/Heel restriction enzyme and reaction solution.
The procedure is essentially the same as in Example 2♂B, except that the Sacl restriction enzyme and reaction solution are used.

To add the Bglll linkage substance to the Sac digested plus epsilon pKc2s7 end, use the method of Saint John's (St.
, /! ;2:3/7 (191/year)].

The reaction solution for Hajime Sac I restriction enzyme (/θX) is prepared from the following components.

1 = 00 mM NaCl 1θQmM) Lis-HCl, pH 74</i>00 m
M MgCI J Series Bg l n C (d (CAGATCTG))
and other linking materials are readily available from: Collaborative Research, Inc.
nc.

/36! Main Street Waltham MA 02/! ;44'B, Ligation and Transformation □""1 Linear plasmid pKc237 having Bgl fl ends is ligated into Bgll plasmid pKc237 by essentially the same procedure as in Example 30C. The ligated plasmid is transferred into E. coli/2 BE7f3 using the method also disclosed in Example 30C. The obtained transformant was infected with co'J K/2BE7r3/
pLO3/& and E, :+! JK/2 BE7r
Isolate the desired plasmids pLO3/O and pLO3/7 from 3/PLO3/7. As explained in Example 30C, depending on the orientation of the resistance transfer fragment inserted, a bidirectional plasmid is generated. These plasmids and the resulting transformants can be easily identified and isolated by conventional methods. Plasmid pLO3/l
, pLO3/7, and pLO3/7 are shown in FIG. 9 of the accompanying drawings.

Example 31 Each of the desired constructions is performed essentially the same as in Example 3, except that either plasmid 4'i t6 or pLO3/7 is used rather than plasmid pKC2/'l. The thus obtained transformed mouse Ltk
/pLO3/7 are each grown in large scale culture.

Example 3j The desired construction except that plasmid PKC2t/ is used instead of plasmid pKC,257.

The procedure is essentially the same as in Example 33. The resulting transformant E, coli/2BF2za3/pLO3/I
Isolate the desired plasmids pLO37g and pLOJ/9 from r and E. coli/2 BE7r3/pLO3/9. As explained in Example 30C, two orientations of the plasmid result, depending on the orientation of the resistance transfer fragment inserted. These plasmids and the resulting transformants can be easily identified and isolated by conventional methods. The restriction sites and functional maps of each of plasmids pLO3/r and pLO3/9 are shown in FIG. 9 of the accompanying drawings.
゛Example 36 Mouse Ltk/pLOJ/r Oyohimouse Ltk/desired constructs are respectively plasmid pKC2/
'I- instead of plasmid pLO3/t or pLO
The procedure is essentially the same as in Example 1 except that either 3/9 is used. The thus obtained transformant mouse Ltk/pLO3/zato mouse Ltk
/pLO3/9 are each grown in mass culture.

Example 37 The desired configuration is plasmid pKC27! instead of plasmid pKC,17! Except using ;

The procedure is essentially the same as in Example 33. Obtained.

Transformant E, coli/2 BE104t//pL
O320 and E, Cori K12 BEIO Hiroshi//pLO
32/ to the desired plus (de pLO32θ and p
Isolate LO32/. As explained in Example 30C, two orientations of the plasmid result, depending on the orientation of the resistance transfer fragment inserted. These plasmids and the resulting transformants can be easily identified and isolated using conventional methods. Plasmids pLO320 and pLO
The restriction sites and functional maps of each of 32 and 32 are shown in FIG. 9 of the accompanying drawings.

Example 3F Each of the desired constructions is performed essentially the same as in Example 1, except that either plasmid pLo 320 or pLOj21 is used in place of plasmid pKC2'/l. The thus obtained transformant mouse L
t km/p LO320 and mouse Ltk/pL
O32/ are each grown in large scale cultures.

Example 39 Plasmids pYEp, Zll (approx.
/le1. ! ;pl), water (2!;td), B
amHI restriction enzyme (jμ, j Units) and /θX reaction solution (jp!') was incubated at 37°C for 1 hour and then at 70°C for 5 minutes.B a mHI-digested D
NA was water-cooled, precipitated with ethanol, and 10XSall
Buffer solution (reaction solution (3; pl), DTT (!; ttl)
, BSA (/θθθda/ml, !; ti') and 5al) restriction enzymes (jp!, !; unit) were dissolved in water (30p!). 37% of the obtained mixture
℃ for 7 hours, then incubated at 7θ°C for 5 minutes, and finally cooled to t°C. The mixture was then extracted with each of phenol and a 24c:/mixture of chloroform and isoamyl alcohol.

Finally, it was precipitated with ethanol. The obtained precipitate contains the desired linear DNA, and j mNacl (
! 27500mM NaCl, tOmM) Lis-HCl, pT (79tOmM) and stored at l''C for subsequent use.
Mg CI J The desired linear DNA was obtained by essentially the same procedure as in Example J9A, except that plasmid pKC2,3-9 was used instead of plasmid pYEp2'l. The obtained precipitate contains the desired linear DNA, and j m
It was dissolved in M NaCl (!; pl') and stored in &'C for subsequent use.

C0 ligation and transformation The desired ligation and E, co') transfer into K/2 BE7r3 was performed by ligating the DNA fragments prepared in Example 39h and B over the Hae fragments taken from plasmids pUR, 222 and pKc26/. The procedure was essentially the same as in Example 2♂C, except for the subsequent transformation. Some of the transformants obtained were as shown by conventional methods by selection, gel electrophoresis (Rao and Rogers, 1997) and other tests.

Contained the desired plasmid. This transformant was transformed into E.
, Coli/2 BE7r3/pKC273 and table eagle.

After selection, transplantation, and culture, plasmid pKC273
was isolated. The restriction sites and functional map of plasmid pKC273 are shown in Figure 70 of the attached figure.

Example 110 Yeast constituting Satucharomyces cerevisiae/pKC273 (Satucharomyces cerevisiae) was grown in /% yeast extract 72% Bacto peptone/1% glucose using conventionally known microbiological methods. Ta. Plasmid pKC273 was transferred into yeast using the method of Hinen et al. (Proc.

Nat, Acad, Sci, USA 7j:
/929 (/97g)]. Transformants were selected by their ability to grow on minimal medium lacking uracil (ura+phenotype). ura+
Type transformants were subsequently treated with hygromycin B (200 η
/II/) was tested for its ability to grow on a complex medium supplemented with. This amount of drug is lethal to non-transformed cells. Transformed somatic cells Saccharomyces cerevisiae/pKc23 grow in these media, but untransformed Saccharomyces I cerevisiae dies.

Example g/ The desired digestion is performed using plasmids PUR222 and Han
Plasmid pBRJ2. instead of restriction enzyme and reaction solution.
2. The procedure was essentially the same as in Example 21''B, except for using the PsL I restriction enzyme and reaction solution 1. The obtained Pst I digest was dissolved in tmMNmCl (
Dissolve in j me).

- Reaction solution for Pstl restriction enzyme (/OX') was prepared from the following components: 500 mM NaCI 40 mM) Lis-HCl, puZu is OmM Mg CI J The desired digestion was performed using plasmid pxc, 26 g instead of plasmid pBRJ2J. Except using .

The procedure was essentially the same as in Example g/A. The resulting Pstl digest was added to j mM NaCl (in ld)
It was dissolved.

C0 ligation and transformation For the desired ligation and transfer into E, coli K/2 BE7t3, ligate the DNA fragment prepared in run 45F/A and B instead of the Haa fragment taken from plasmids pUR222 and pKc2t/ The procedure is essentially the same as in Example 2/rC, except for the subsequent transformation. Some of the transformants were subjected to antibiotic selection, gel electrophoresis [Rao et al.
Contains the desired plasmid, as shown by conventional methods in the following tests: This transformant was selected and transplanted into E.coli/2BE713/pLO37r.

Following cultivation, plasmid pLO371r is isolated.

Two orientations of the plasmid result, depending on the orientation of the resistance transfer fragment inserted as described in Example 30C. Therefore, in addition to plasmid pL037F, a plasmid with the opposite orientation can also be obtained by the above method. Those skilled in the art can easily identify and isolate these plasmids and the resulting transformants using conventional methods.

The restriction sites and functional map of plasmid pLO37♂ are shown in Figure 1θ of the attached figure.

Example D2 Construction of Saccharomyces cerevisiae/pLO77♂ For the desired transformation, plasmid pKc27J was replaced by G.
11n! Plasmid pL O371 containing resistance gene
The procedure is essentially the same as in Example 1O, except that . Transformants are isolated by conventional methods of screening recipient yeast cells for the presence of plasmid DNA. The desired transformant S. cerevisiae/pLO371 is thus easily removed and isolated.

[Brief explanation of the drawing]

Figure 1 shows the restriction sites and functional map of plasmid pKC203. Figure 2 shows the restriction sites and functional map of plasmid psVjgpt. Figure 3 shows the restriction sites and functional map of plasmid pKcJ/l. Figure 4 shows plasmid pKC
Restriction sites and functional maps of 2/; are shown. Figure 5 shows the restriction sites and functional map of plasmid pKC222. Figure 6 shows the restriction sites and functional map of plasmids psc70/ and pKC2j7. Figure 7 shows the restriction sites and functional maps of plasmids pKc2j9 and pKc2tl. Figure 1 shows plasmid pKC
! 7j and pKC244! The restriction sites and functional map of . Figure 9 shows plasmids pLO3/'A to pLO32.
The restriction sites and functional map of / are shown. Figure 10 shows the restriction sites and functional map of plasmids pLO37t and PKC,2ri3. However, in FIGS. 2, 3, and 1, the PY Tag includes a polyoma sequence or a 5vao sequence. Fig. 1 Fig. 2 Fig. 3 Fig. q Fig. hoI Fig. 5 Fig. 6 Fig. 7 Fig. 7 Zuhoko ■ Ha@ll Fig. , C1,3 identification symbol Office reference number/
CI2 R1/44 746 1/465 766 1/745 1/80 1/91 0 Inventor Ramachandra N. Rao 6818 Tuber Road, Indianapolis, Indiana, United States of America

Claims (1)

[Claims] /, a) Eukaryotic promoter. b) When transferred into host cells that are sensitive to one or both of the antibiotics hygromycin B and G g/male and are amenable to transformation, cell division and cultivation. Convey resistance to one or both of the above antibiotics
or a recombinant D consisting of two different structural genes and associated control sequences and C) a prokaryotic replicon in which the replicon is functional when the above-mentioned host cell is a prokaryotic cell.
NA cloning vectors (provided that one or two structural genes and associated control sequences are flanked by and transcribed from a eukaryotic promoter in a eukaryotic host cell, and one The gene and associated regulatory sequences shall convey resistance to either hygromycin B or G411r, but not both, and shall not code for the gene enzyme phosphotransferase conveying resistance to a4ctl. ). 2 prokaryotic replico'Ap B RJ 2J replicon. pM B / = NR / −RKJ s RK 6
*pSC/0/-RP/, RP4'
. selected from the group consisting of R8FIQIQ, PUBIIO and SLP/2, with an independent eukaryotic promoter 8VI1. Q early promoter, SV@o late promoter, H8VITK promoter, adenovirus promoter, AdJ promoter -, polyoma promoter, mouse sarcoma virus promoter* yeast trp -/promoter. The claim is a plasmid selected from the group consisting of yeast 1euJ promoter, yeast his3 promoter and yeast cytochrome C promoter! Vectors listed. 3 pla xz de pKc JOJ (D7Jkb) Bgl'
The vector according to claim 1 or 2, which contains a restriction fragment. The vector according to claim 3, wherein the bamboo prokaryotic replicon is pB, Rj22 replicon, and the eukaryotic promoter is the SVgQ early promoter. j The vector according to claim 3, wherein the prokaryotic replicon is pBRJ22 replicon, the eukaryotic promoter is Ad 2 promoter, and the eukaryotic promoter is yeast cytochrome C. 2 genes and related control sequences are hygromycin. 2.7jkb of Bgl from plasmid pKC,203.
The vector according to claim 1 or λ, which is a plasmid containing the ll:/αsl restriction fragment. and the vector according to claim 1 or 2, which is a plasmid in which the number of said structural genes is limited to one.
□ A vector according to claim 1, in which the RI gene and associated control sequences convey resistance to hygromycin B, and which contains an alpha rie/cho 1 restriction fragment of /, 5-/k b from plasmid pKC222. . The IQ gene and associated regulatory sequences convey resistance to atiit and plasmid PKC222 (7)
/, 63 kb ノα30 RBl / Sal l
Claims containing restriction fragments? Vectors listed. Il, plasmid pKC, 2/4! , pKC,2/j
, PGD/. PGD, 2, PGDJ, pGD&, pGD
lo, pGD//, pGD/2. pGD/J, p
The vector according to claim 1 or 2, which is a plasmid selected from the group consisting of GI)lg and pGD/j. 12, plasmid pLO371, pKc27j, pLO
3111PLO31! ;, pLO311,, pLo3
/7. pLO311, pL. 319, pL0320J-3 and pLO321. 1′:,・. /j, a) Eukaryotic □ promoter. b) Antibiotics hygromycin B and G11l/if
when introduced into host cells that are susceptible to one or both of the following and are amenable to transformation, cell division, and culture. Convey resistance to one or both of the above antibiotics!
or a recombinant D consisting of two different structural genes and associated control sequences and C) a prokaryotic replicon in which the replicon is functional when the above-mentioned host cell is a prokaryotic cell.
NA cloning vector (provided that 1 or 2 structural genes and associated control sequences are transcribed from a eukaryotic promoter in a eukaryotic host cell, flanking the X eukaryotic promoter, and The gene and associated control sequences shall convey resistance to either hygromycin B or Gsull, but not both, and the gene conveying resistance to aair shall not encode the enzyme phosphotransferase). A transformed host cell containing. The transformed host cell according to claim 13, which is an llA prokaryotic cell. 1.5: The transformed host cell according to claim 13, which is a eukaryotic cell. /4. E, Cori, E, Cori K12. E, for stiffness/, 2B
g! 27. The transformed host cell of claim 11 selected from the group consisting of Bacillus, Bacillus subtilis, Pseudomonas, Agrobacterium, Streptomyces, Staphylococcus and Streptococcus. 17 Neurospora, -1? A transformed host cell according to claim 1j selected from the group consisting of Pharosporium, Aspergillus≦Penicillium and yeast. A transformed host cell according to claim is selected from the group consisting of 11 plant cells, 5 amphibian cells and mammalian cells. From lz plasmid FPKC203 (7)? : jkb 屡■ restriction fragment, plasmid pKC203 to -2,7J-k b 詑2x/and alJ restriction? Ragment. i,5tkb from plasmid pKC222/
III restriction fragment and plasmid pKC222
A restriction fragment selected from the group consisting of /, 6jkb EcoRI/SidI restriction fragments+. 20 plasmid PKC, 2θ3. PK C2,1,l
, pKC23'7. pKC,259, pKc2AI, pKc26e,
pKC27! ; and pSC701. 2/, plasmid PKC203, PKC, 2co2,
pKc, 237. PKC, 2! ;9, pKc261, pKC21
, pKC273 and psc701. 22, E-Cori, E, Cori Ki2+E, Cori /, 2
BEza27. Bacillus, Bacillus subtilis, Pseudomonas, Agrobacterium, Streptomyces,
22. The transformed host cell of claim 21 selected from the group consisting of Staphylococcus and Streptococcus. 23, &) Eukaryotic promoter. b) sensitive to one or both of the antibiotics hygromycin B and GIAII, and transformed into cells with one or two different structural genes and associated control sequences conveying resistance to one or both of the above antibiotics; and C) as described above. Recombinant D consisting of a prokaryotic replicon, where the replicon is functional when the host cell is a prokaryotic cell.
NA vector (provided that one or two structural genes and associated control sequences are adjacent to a eukaryotic promoter and have been transcribed from a eukaryotic promoter in a eukaryotic host cell, and one gene and The relevant control sequences are true (shall convey resistance to either Igromycin B and Gltlf, but not both; and the gene conveying resistance to G11lf shall not encode the enzyme phosphotransferase). Nuclear cell: A method for producing a post-translationally modified polypeptide by transfecting it into Ayan cells and culturing the resulting cells.