JPS6043384A - Hybrid plasmid with marker - 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 This invention is a continuation-in-part of US Patent Application Serial No. 462,731, filed January 31, 1983.

As is well known, whether prokaryotes (for example, Escherichia coli) or eukaryotes (for example, yeast),
The frequency of transformation of cells by plasmids is relatively low. Therefore, it is desirable to have within the genome of the plasmid undergoing transformation certain characteristics that can be used to identify transformed cells. Such distinguishing characteristics, known in the art as markers, include specific nucleotide chains, chemical resistance, such as antibiotic resistance, or the ability to synthesize specific growth factors, such as amino acids. Untransformed host cells originally lack the ability to synthesize.

The present invention relates to a plasmid-carried transformation marker suitable for indicating transformation in both prokaryotic and eukaryotic transformants. Furthermore, the present invention relates to creating a special insertion site in this plasmid E for the deoxyribonucleic acid (DNA) chain to be replicated within the eukaryotic or prokaryotic transformant. More specifically, among them, methotrexate-resistant dihydrofolate reductase (MtXr
D) A new plasmid with the nucleotide chain code for TFR) is synthesized. This nucleotide chain is ligated into a doubly digested plasmid vector, yEp13. This vector carries markers for ampicillin and tetracycline resistance with initial selectivity and genomic regions of Saccharomyces cerevisiae and E. coli origin. Specifically, the insertion position of the nucleotide chain code for MtXrDHFR is adjacent to the yeast portion of the plasmid vector at one end, and is a plasmid vector that is known to contain a replicative region of eukaryotic origin. L of the part of the bacterial part which is located below part of the yeast part of the plus (23) at the other end and adjacent to the bacterial part of the mid, which is known to contain a replicative region of prokaryotic origin. It's on the other side. In other words, an additional nucleotide chain, which is known to contain the promoter, can also be inserted below the replicative region of eukaryotic origin, creating another new plasmid. . The plasmid was cloned in E. coli sp. LE392. Plasmid MT cloned in E. coli sp. LE392
X4 has been deposited as ATCC 39276. Plasmid MT cloned in E. coli sp. LE392
X5 has been deposited as ATCC 39275. Plasmid M cloned in large intestine #4 species LE392
TX4/4269 has been deposited as ATCC 39395. MTX5/4269C was cloned in E. coli sp. LE392 and has been deposited as ATCC 39394.

The plasmid has been cloned into E. coli sp. LE392 (24) and deposited at the American Type Culture Collection Center, Rockville, Maryland, USA. The plasmids have also been deposited as required by the patent laws of each country in which a copy of the subject application is filed. The deposit of the L plasmid is itself made to the Assignee 5tauffe.
No governmental action under any patent or invention title granted to Chemreal Company shall constitute, without prejudice, a license to exploit the subject invention.

If we also describe the process by which the cholera plasmid is synthesized, it can be summarized as follows: Ba
The yEp plasmid is thoroughly and specifically digested using mHI and Hlnd m restriction endonuclease (hereinafter referred to as restrictase). The nucleotide linkage code for MtXrDHFR was isolated from plasmid pEE364 and joined with an octanucleotide linker.
Ru. Thus, a variety of new plasmids are generated in which the nucleotide chain code for MtXrDHFR is spliced into the prepared yEp13 fragment. Among them, MtXrDH
MTX4 and MTX5, which are hybrid plasmids with ER nucleotide chain codes, are also included. here 1
"Hybrid" means that it includes DNA from more than one source and can replicate in both eukaryotic and prokaryotic cells.

The synthesis of a plasmid containing an additional nucleotide chain known to contain a plant promoter is briefly described as follows: The MTX4 plasmid was
Digest specifically with lnd nT restrictase. A nucleotide chain having approximately 480 base pairs (hereinafter referred to as 480
- base pair fragment) is excised from plasmid pR4269 using Hlndm and BamHI restrictase. This nucleotide chain is joined with an octanucleotide linker, and then MTX4 or MTX
5 to create a hybrid plasmid. This plasmid contains both the nucleotide linkage code for MtXrDHER and a 480-base pair fragment with a plant promoter.

The aim of the present invention is to synthesize a hybrid plasmid having a marker nucleotide linkage therein.

Another object of the present invention is to synthesize a hybrid plasmid having therein the nucleotide chain code for dihydrofolate reductase that is resistant to mentrexate.

The present invention further aims to synthesize a plasmid having therein a nucleotide chain code for a eukaryotic promoter and a nucleotide chain code for a marker.

Yet another object of the invention is to synthesize a plasmid having therein a nucleotide linkage code for a plant promoter and a nucleotide linkage code for a marker.

The present invention further aims to synthesize plasmids that increase methotrexate resistance in eukaryotes and prokaryotes transformed by the plasmids (27).

Yet another object of the present invention is to synthesize a plasmid that produces methotrexate-resistant dihydrofolate reductase in prokaryotes and prokaryotes containing the plasmid.

These and other objects of the present invention will be understood by those skilled in the art from the description of the missing drawings.

Example 1 The examples shown below are merely illustrative and are not intended to impose any limitations. It will be apparent to those skilled in the art that various steps described below may be taken without departing from the essence of the invention as described. For example, the strength of the buffer solution, centrifugation speed and time, extraction operations, precipitation operations, washing, etc. can be changed without changing the essence of the described invention.
It can be changed within that range. Similarly, the sources of nucleotide molecules, enzymes, and the like may be varied without departing from the essence of the invention described herein.

The yEp plasmid was obtained from V. Williamson, University of Washington, Seattle, WA. yEp13 was reported by Broch, Strathern and Hicks in Qene, Vol. 8, 121-133 (1979). The yEp plasmid is a hybrid species, and has a yeast DNA region derived from π, Einjiru 1, and Gyen, which are found in eukaryotic microorganisms. Inside the yeast DNA region, there is a replication domain of eukaryotic origin. be.

yEp13 is also a prokaryotic microorganism, Escherichia coli.
It also contains a bacterial DNA region derived from Rlchia coll. Inside the bacterial DNA region is a replicative region of prokaryotic origin. )'EP 13 has early selectivity markers for ampicillin and tetracycline resistance and LEU2 of the leucine biosynthetic pathway.

The yEp13 plasmid was isolated from E. coli by the Birnboimu cesium chloride method. Birnboim,
H, C., Tori, J.

(1979), Nucleic Ac1d Re5e
arch, 7 volumes.

1513-1523゜60 mmol (mM) sodium chloride, 7mM TRl5, 7mM magnesium chloride in about 2 milliliters (-) at 37℃, pH
7.5, approximately 50 micrograms (μf) of yEp13
About 150 units of Hl nd ■ restrictase (Be
thesda Re5earch Lab-orato
(hereinafter abbreviated as BI'LI).

After approximately 3 hours under these conditions, the digestion mixture was heated to 65° C. for 3 minutes and then poured onto ice to inactivate the Hindl'l'[reductase. Sodium chloride, β-mercaptoethanol and the first Bam restrictase (BR
L) was added to the mixture described in L, and finally 100 m of each
M, 10 mM and 150 units. The mixture was kept at 37° C. for 3 hours, then immediately heated at 65° C. for 3 minutes, poured into ice F, and rapidly cooled.

The digested plasmid DNA was then precipitated in a final concentration of 0.3 molar acetic acid and 25 volumes of 95% ethanol. After suspending the concentrated DNA twice in ethanol, the pellet was diluted with pH 8.0 containing 1mM EDTA.
80 microliters of 10mM TRl5-chloride (
μt).

Ram RI/Hlnd l' [digested with] rE
p13 produced a 1o, 3 kilobase fragment and a 0.4 kilobase fragment. This was determined by electroelution using 40 mM TRIS, 1 mM EDTA, pH adjusted to 8.2 with glacial acetic acid, and eluting with a 0.8 thiagarose horizontal gel and applying 3 o volts for 115 hours. . The 10.3 kilobase fragment obtained in this way is 0
．． In the presence of IJium (5 moles of chloride), phenol/
It was extracted twice with a mixed solvent of chloroform (1:lu/u) and further extracted twice with ether. This 10.3 kilobase fragment was precipitated with ethanol and then, after an ethanol wash, 400 at of TRl5EDTA at pH 8.0.
Redissolved in buffer. The redissolved belene (31) was reprecipitated with ethanol, washed, and finally added with 50 μt of 10 mM TRl5-chloride buffer adjusted to pH 8.0.
resuspended in.

The thus obtained yEpta vector has one sticky ' Bam HI end and one sticky ' Bam HI end (I
The 10.3 kilobase plasmid with nd m ends was of suitable purity for cloning the DNA fragment containing the nucleotide linkage code for the MtxrDHFR gene therein.

Looking at Figure 3, which is a depiction of the restriction map of yEp13, we see that the first Hlnd [EcoR located clockwise from the position]
■The location and the second location clockwise from the L EcoRI location.
The region between the first Hindm position and the BamHI position, including the Hlnd If position, was removed from the original 7Ep13 plasmid by the method described in E to create a linear 10.3 kilobase plasmid. You should understand.

As a result of this excision, the 10.3 kilobase fragment has lost the tetracycline resistance function in the region of the Bam HT(32) position of the original yEp13 plasmid. Further digestion by H1ndl'l restrictase results in a beta with an insert flanking the yeast portion of the 10.3 kilobase fragment. 10.3
The kilobase fragment also contains a gene coding for ampicillin resistance from Escherichia coli and a LEU2 gene coding for an enzyme in the leucine biosynthetic pathway from S. cerevisiae (hereinafter referred to as the genus Saccharomyces). .

E. coli carrier of plasmid ppE364 containing prokaryotic Mtx'DHFR nucleotide linkage was obtained from Wellcome Re5earchLabo, North Carolina.
It was taken from MFling by ratorles.

Fling, M.E., and Elwell.
L, P. (1980).

Journal of Bacteriology,
Volume 141, 779-785゜Plasmid is Birn
boim-cesium chloride was prepared by this notation,
Large (33) coliform bacteria cells containing pFE364 were treated with the methotrexate derivative trimethoprim (Sigma) instead of ampicillin 100 μf/-.
The difference is that the growth was performed in the presence of 10 μf/−. The pFE364 plasmid obtained with the following recipe is MtxrDHFR.
It was reserved for the isolation of nucleotide chain codes.

pFE364 plasmid 100 micrograms (pf)
TRl8-chloride 20mM, pH 7.0° Magnesium chloride 7mM, sodium chloride 100mM, β
- 150μ of a solution consisting of 2mM mercaptoethanol (Sigma) in 16 units of BamH at 37°C for about 16 hours.
I (BRL) and 36 units of EcoRI (Blopab
a) Digested with v strictase. Transfer the remaining plasmid to 1.2 inch horizontal agarose gel E130 volt 1
Electroelution for 6 hours and 150 volts for 3-4 hours yielded 2.5 kilobases of Bam HI/Eeo.
The Ri fragment (containing the MtxrDHFR gene) was isolated. The electroelution buffer was the same as described above. Bam
HI/E co RI MtxrDHFR(q
a) The fragments were collected, extracted twice with phenol, once with chloroform, and precipitated with isopropyl alcohol and ethanol. Approximately 10 μt of final DNA precipitate was added to 10 mM TRl.
Resuspended in 100 μM of 5 chloride, pH 8.0, EDTAo, 1 mM buffer. This same method was used for each batch. BamHI/EcoRI Mtx DH
All three batches of FR fragments were precipitated with ethanol and combined with Has m and Alu as described below.
Digested with I.

Approximately 30μ2 of the Bam HI/EeoRI fragment was suspended in 80μt of a solution containing 50mM TRl5 chloride, pH 7.5, 5mM magnesium chloride, and 0.5mM dithiothreitol, and stock Hae restrictase ( 5 μt containing 45 units (BRL) were added. The mixture was kept at 37°C for 2 hours, then the stock Al
5μ containing 40 units of u I restrictase (BRL)
t and 10 μt of 50 mM sulfur chloride were added.

The digestion mixture was further aged at 37°C for 2 hours.

(35) A 370 base pair Hae III/Alu I DN with the nucleotide chain code for Mtx DIIFR in it.
Fragment A is 150 volts with 2 thiagarose horizontal gels;
It was isolated by electroelution for 2 hours. Elution buffer is L
Same as described above.

The Hae III/Alu I fragment was purified by extraction three times with phenol and twice with chloroform, and precipitated twice with 0.3 molar acetic acid/ethanol.

The generated solution contains a 7.5 μm DNA fragment with the nucleotide chain code for MtxrDHFR, and the entire content)
JL with 50 pL of 10 mM TRl5-chloride buffer,
It contained pH 8.0 and EDTA O, 1mM.

Prior to splicing with the 370 base pair DNA containing the nucleotide chain code for M t x rD HF R,
The terminal phosphate group was regenerated into a linker molecule. A suitable octanucleotide linker (Co11aborati
(manufactured by Re8earch) 5 microlog (36) rum was treated with TRl5-chloride 70mM, pH 7°5. Magnesium chloride 10mM, dithiothreitol (Sigma
m) 5 mM, "p-adenosine triphosphate
) (ATP) A total volume of 10μ containing 50μt was suspended in it. The mixture was heated to 70° C. for 2 minutes before 5.5 units of T4 polynucleotide kinase (BRL) were added. 3
After 30 minutes at 0°C, the mixture was supplemented with 5.5 units of kinase enzyme and 1mM unlabeled ATP and left for an additional 30 minutes. After inactivation by heating to 65℃ for 10 minutes, −2
Stored at 0°C. It was confirmed that the phosphorylation linker was self-linking and that phosphorylation was effectively carried out. Phosphorylation of the linkers was visualized in 7-thiacrylamide gel E using conventional autoradiographic techniques, revealing the formation of large ladders of self-linking linkers. Hlnd m and bam
Redigestion of the self-linking linker using HI under appropriate conditions regenerated the linker monomer (37).

Mtx in it before connecting with regeneration linker.
Since the DNA fragment with the nucleotide chain code for rDHFR has unpaired terminal bases, it was treated with DNA polymerase I in the presence of deoxynucleotidase to complete the base pairing. A purified DNA fragment of approximately 1μ containing the nucleotide chain code for MtxrDHFR in it.
V, 2 units of DNA polymerase I (BRL),
10mM β-mercaptoethanol, 60mM TR
l5-chloride.

Magnesium chloride at pH 7, 5, 8mM, and 0, 2
m M deoxyadenosine triphosphate, deoxycytidine triphosphate, thioxythymidine triphosphate, and deoxyguanosine triphosphate (P, L).

Biochemicals) in a total volume of 20 pL for 60 minutes at 16°C. Then 1 at 65℃
After heating for 0 minutes, the reaction was inactivated by pouring onto ice. The DNA fragment with the MtxrDT (FR nucleotide (38) linkage code in it) was now ready for blunt-end ligation to BamHI and Hlndm octanucleotide linkers.

- John β-mercaptoethanol [M t x rDHF
Added to the R mixture to give a final concentration of 10mM. 6 about 1
μm St p-phosphorylated BamHI and Hln
d ■ A linker was also added. Mt x rDHFR1μ
2. Linker-1T4DNA ligase 200 units, TRl
5 chloride 66mM, pH 7.5, magnesium chloride 6
．． Plant endoligation was performed in 40 μt of a solution containing 6 mM, ATPo, 25 mM and dithiothreitol 10 mM at 20° C. for 20 hours. The ATP mixture was heated to inactivate and then sodium chloride was added to a final concentration of 60 mM. The ligation mixture (39) was digested with 6 units of ndl' (restriction enzyme) for 285 hours at 37°C. To the above mixture was directly added 8-enzyme BamHI restrictase, 2mM β-mercaptoethanol and sufficient sodium chloride. The final sodium chloride concentration was 100mM. Digestion was then carried out at 37°C for 2.5 hours. It was precipitated with ethanol, concentrated, and purified to remove the linker by electroeluting on a 2% agarose horizontal gel h150 volts for about 1.5 hours using the buffer described above. The electroeluted fragments were extracted with phenol, further extracted with ether, precipitated with ethanol, and washed. The DNA fragment with the nucleotide chain code for MtxrDHFR in it thus has one or more terminal Bam
Bam HI / Hl n
The cells were cloned into the dm-digested yEp13 vector.

(40) D containing the nucleotide chain code for MtxrDHFR
Splicing of the NA[i piece into the 3'gp13 vector was performed using 1.8 μp% 1l of 41fiBamHI and Hlndll digested yEp13 in the form of a linear 10.3 kb fragment.
p-phosphorylated BamHI and/or H1nd
nl linker, in which MtxrDH
fW DNA0.0 with nucleotide chain code for FR
6μg 1 bovine serum albumin (BRL) 21μ, 9SA
TP (Sigma) 1mM, TRI S-chloride 30mM5 pH 7,5, magnesium chloride 10mM
, 50 mM sodium chloride, and 10 mM β-mercazotoethanol were added together in a total volume of 30 μl solution. Heat the mixture at 37 °C for 2 minutes, then 20
0 units of T4 DNA Suga-ze was added. Joining was carried out at 4°C for 24 hours. The resulting mixture contained various free DNA species as well as incomplete and complete redaction products.

E. coli strain L with MtxrDHFR tethering products
E392 was irradiated at 600 nanometers with an optical density of 0.6 and cultured at 37° C. in 15° L-broth in an incubator rotating at 20 Orpm.

The cell solution was cooled on ice for 5 minutes, separated in a tabletop centrifuge at approximately 5,000 rpm, and the pellet was resuspended in 10 mM magnesium ijfate. Cells were washed with magnesium sulfate, centrifuged, and then resuspended in 2.5 mg of chilled 50 mM calcium chloride.

Cells were kept in calcium chloride/ice for approximately 30 minutes, then centrifuged again and resuspended in 1 rnl of 50 mM calcium chloride. One hundred microliters of suspended E. coli cells were placed in a sterile 1.5 ml Eppendorf tube and added to 30 μl of the previously prepared r-john mixture. Transformation was performed at 0°C for 45 minutes. The cells were then resuspended in 2-L-broth and incubated for approximately 1 hour at 37°C, after which the culture was centrifuged and the cell pellet was resuspended in 10mM magnesium sulfate. .

Approximately 100 μl of this solution was then incubated on an L-agar plate containing 100 μy/− of ampicillin (Sigma). Incubate the plate for 3
After 16 hours at 7°C, 17 ampicillin-resistant colonies were hatched.

Mtx DH
The presence of the FR-containing DNA fragment insertion site was confirmed. Namely, calculation of plasmid size by agarose dal electrophoresis, BamHI using acrylamide gel electrophoresis.
Evaluation of the /H1ndul restriction enzyme digestion and extraction of total cellular DNA from transformants of the original DNA fragment (43) labeled with 3-P-labeled MtxD)IFR. This includes hybridization into the plot. MTX2, MTX respectively
3. Plasmids called MTX4 and MTX5 were isolated from the above transformants by the following method. Each transformant cell was separately incubated at 37°C and 25° rpm in 50°C of L-broth containing 100μi/at of ampicillin until the cells reached an optical density of 1.1-1.3 (incubated for 2 hours). Next, 15 milligrams of spectinomycin (UpJohn) was added. After an additional 15 h of growth under the same conditions, 5orvall SS
-340- Sterilized eyes with tar? Cells were obtained by centrifugation in a linolobilene screw tube. Cell pellet was treated with glucose 5mM, EDTAIOmM, TRl5
- Chloride buffer 25mM.

pH 8.0, and egg yolk lysozyme, Gradee (Si
gma) was resuspended in solution 2- containing 21 da/-. The cells were kept at 0° C. for 30 minutes and then mixed well with solution 4- containing 0.2N sodium hydroxide and 1% sodium decyl sulfate (44) rum to obtain a clear lysate. After 5 minutes at 0°C, the lysate was treated with 3 molar sodium acetate to pH 4.8, mixed well and incubated at 0°C for 1 hour. Cell debris was pelleted by centrifugation. Supernatant ffi containing plasmid DNA, 8
Transfer ml to a new test tube and add 20Wle of water-cooled ethanol.
The DNA was then precipitated at -35°C for 20 minutes.

Terminate the Grasmid DNA by centrifugation and add TRI S-chloride 50mM, pH 8, lithium chloride 0.2MSEDT.
Resuspended in Bath 2- containing 20 mM A and 0.2 sodium tidodecyl sulfate. Approximately 1:1 DNA
Extracted with an equal volume of v/v chloroform-phenol mixture. The phase-containing glasmid was separated from the phenol by centrifugation. The approximately 1.6-mL DNA solution was transferred to a new test sample, precipitated with ethanol, resuspended in 0.5 mm of 0.3M sodium acetate in distilled water, and reprecipitated with ethanol. The DNA pellet thus obtained was dissolved in 10 mol TRl5-chloride, pi8.0.0,2g, and 201t9/-1(Na
treated with se. After the third ethanol precipitation, Grasmid DNA was resuspended in 50 μl of distilled water. 0.7% of each 1 microliter of plasmid prepared by the above method
Treated on an agarose horizontal barrel with standard cilasmids of known size at 25 volts for 18 hours. The MTX plasmids were each approximately 10.7 kilobases in size.

Each MTXf lasmid also has approximately 0.3
5 μl of a plasmid suspension containing μl of cilasmid was electroeluted to obtain a nucleic acid-free grasmid and characterized. Each Grasmid was then treated with 4 units of BamHI and 8 units of Hln in exactly the same manner as described above.
89mMTRl5-chlori
Fragment size was analyzed using 8.3.2.5mM EDTA, 89mM phosphate buffer, 4% acrylamide, 150% alcohol for 3 hours. Motsu and small cilasmid fragments obtained from MTX noranumid were compared to standards of known size from 0.37 to 0.38, respectively.
It was on the order of kilobases. The largest fragment of each MTX plus band is 3mJTRIS-chloride, pi
s, 2, 1mMgDTA, in 5mM sodium acetate, on 1% agarose roll, 50 delts, 9 hours of treatment, was approximately 1O55 to 10.7 kilobases.

These fragments were digested with BamHI/Hindl) rEp.
Mtx DHFR was cloned in 13.
The size was consistent with that expected for a DNA fragment with a P chain code.

Characterization by Hybridization Studies DNA from each of the 17 E. coli transformants was isolated from lap-labeled Mtx DHF by nick translation using a technique called “colony hybridization.”
It was hybridized into a 370 base pair DNA #FfT piece with a nucleotide chain code containing R in it. These techniques are (
47) Both are generally known to those familiar with the industry. (5
outer XE, (1975), Mo1ac,
Biol.

98, 503-517; Gruenstein,
M, and Hognesg, D, S, (197
5), Proc, Nacl, Aead.

Set, vol. 72, pp3961-3965: Rlg
by.

P., M., Drickmann, M., Rod.
as, C, and Bug, P, J.

(1977), Mo1. Blol, vol. 113, p.
, 237° hybridization test using Kodak x-OMAT
This was done by applying automatic radiography to AR-5 film. 14 transformants including 2.3.4 and 5 l) matching the original MTX DHFR insert
It contained A.

Expression of MTX3 and MTX5.8 and MtxDHF
A total of 5 E. coli transformants, 3 lacking the R insert, were 2
L- containing 5μg/ml trimethoprim (81gma)
Growth on agar medium was poor even after 15 days at room temperature. Growth of these transformants was sparse and uneven. E. coli LE392 without MTX-containing (48) M glasmid showed the same appearance.

Twelve E. coli bacteria containing MTX2 and MTX4 grew densely on L-agar containing trimethnorim under the same conditions. This is Mtx DHFR in MTX2 and MTX4
indicates partial or complete expression.

Further analysis of the -oriented cilasmids MTX2, MTX 3, MTX 4 and MTX5 by digestion with 5AU3AI and BamHI revealed that the MtxDHFR insert was oriented within the plasmids of MTX2 and MTX4.
x Transcription of the DHFR insertion site is performed by Hindll of cilasmid.
! region to the BamHI region, whereas transcription of the Mtx DHFR inserts in MTX3 and MTX5 proceeds in opposite directions within the vector from the BamHI site to the BamHI region.
nd site. This orientation is clearly illustrated in FIGS. 1 and 2. As can be seen from FIGS. 1 and 2, the nucleotide chain code for 11C1Mtx DHFR has the Sau 3 A I 1llJ end position off-center within the nucleotide chain. Analysis of the digested fragments was performed using BamHI and Sau 3A I to determine the orientation of the Mtx DHFR nucleotide chains within the cilasmid.

Methods for digesting and analyzing single pieces of cilasmid are well known to those skilled in the art and are briefly summarized below. Each cilasmid was digested with BamHI. Terminal phosphates were removed by alkaline phosphatase digestion followed by phenol extraction to remove alkaline phosphatase. After removing residual phenol by repeated ether extraction, the digested cilasmid DNA was precipitated with ethanol-acetate and redissolved in distilled water. Radiolabeling of the terminal phosphates of individual cilasmids was performed using T4 polynucleotide kinase (BRL) and NRp-labeled ATP in the appropriate kinase buffer, followed by the addition of non-labeled TP in portions. After labeling, the mixture was extracted with phenol then ether and precipitated with sodium acetate-ethanol (as above). Each glasmid with a 52p-labeled terminal phosphate group was then extracted with S
The cells were treated with au 3 A restrictase (formerly olabs) for a sufficient period of time until they were completely digested. Digested plasmid DNA fragments are precipitated with ethanol, concentrated, and treated with 6% acrylamide in an appropriate buffer for sufficient time to remove the transferred DNA.
The fragments were separated. BamHI/5au3AI fragment is Ko
Visualization was performed by automated 2-photography on dak Control DNA fragments of standard size were treated in the same way as Bam HI
/5au3A lDNA Alli piece size was calculated.

10 μy of yeast pearl DBy-746 I'M
Transformation was performed with TX2, MTX3, MTX4 and MTX5 (all of which have the Mtx DHFR gene). (Yeast pearl DBY-746 was sent to Mortimer University, R.C., Parkley, California.
). Yeast cells grown in 50-7 EPD broth to a final optical density of 600 nanometers between 0.6 and 0.7 were washed once with sterile 1 molar sorbitol solution of 10- and collected by centrifugation. The cell pellet was then resuspended in 4-sorbitol plus 10 μl β-mercaptoethanol (Sigma) for 15 min at 30°C. After another sorbitol wash, 0,5η Somolyar-1! h60,000 enzyme (Kirin) at 30°C, 6
The cells were treated for 0 minutes to remove cell walls. Spheroglosto yeast cells, which had lost their cell walls and became spherical, were incubated in a 50 ml capped polypropylene tube without stirring, and the highest yield was obtained. Spheroplasts were washed with 10 d sorbitol and incubated at 4°C for 12 (51
) obtained by centrifugation at 1500 rpm for minutes.

Residual molyase was further removed by washing with 20-ml sorbitol. The spheroplasts were then incubated for 45 min in 67% YEPD broth containing 1 molar (M) sorbitol, followed by a sorbitol wash, followed by a further 0.8-1 M sorbitol, 10 mM TRl5 buffer, pi 7.5.10 mM calcium chloride.
The cells were resuspended in solution and transformed.

The yeast transformation mixture contains approximately 3-5 μg of each plasmid DNA.
1 Yeast Spheroplus) 0.1 td and 10 m M
T R I S % p) I 7.5. Contained sterile 40 polyethylene glycol 4000 (aqueous) ltnld solution in 10 mM calcium chloride. Grasmid DNA and spheroplasts were preincubated for 15 min at room temperature, followed by 1.5 m
Transformations were carried out in Eppend-orf tubes for at least 1 hour on ice. The transformation mixture of approximately 0.1-(52) mol sorbitol, 0.67 t yeast nitrogen base without amino acids (Difao), 2 t glucose (Bake
r), 3% agar (Difco), 2011 adenine sulfate, uracil, histidine, arginine, tritophane and methionine, 30IIl&/J tyrosine, isoleucine, and above links, 50■/l phenylalanine, 11001R/l It was diluted with recycled bare substrate 10- consisting of glutamic and aspartic acids, 150119/l of valine, 200~//l of threonine and 3751149/l of serine. All nucleotides and amino acids were purchased from Slgma Chemical. The regeneration suspension was mixed and poured onto agar containing all of the above ingredients except sorbitol.

Transformants became visible after 4 days at 30°C.

Transformants of yeast 1 were collected from regeneration grate and maintained on minimal medium without leucine. Yeast transformants selected for their ability to grow without leucine confirmed the presence of the Grasmid LEU2 gene. The Emoreda yeast transformants thus obtained were further tested for mentreguisate resistance.

To test for methotrexate resistance, a strain suitable for growth of yeast species, but lacking leucine, may be used.
A minimal medium containing mM methoxate was prepared. Y
3m different yeast transformants carrying either Ep13, MTX4 or MTX57°2 smids were grown on this medium for a sufficient time to confirm growth. YEp13
The growth of MTX4- and MTX4-containing yeast transformants was clearly inhibited, but the growth of MTX5-containing yeast transformants did not show the same inhibition situation.

Example 2 Preparation and preparation of MTX4 vector DNA (55) The MTX4 plasmid was prepared using Birnboirn as described above.
-Cesium chloride prepared by this notation.

Approximately 15 μg of MTX4 was dissolved in 7 m M T RI S-chloride, I) I (7,5,7mM magnesium chloride, 6
in 50 μl solution of 0 mM sodium chloride at 37°C.
for 3.5 hours H amount n d ll restrictase (B
lolmbg) Digested with 5 units. To this reaction mixture was added 1 microliter of 2MTRl5-chloride buffer, p118.0 to a final concentration of 40mM, and calf alkaline phosphatase (Boeringe
1 μg containing 3.3 units of R Mannheim) was added. The mixture was incubated at 65° C. for 20 minutes, extracted once with phenol previously maintained at pH 7.Q with TRl5-sodium chloride-EDTA, and twice with chloroform. DNA was dissolved in 95% ice-cold ethanol and 0.3M sodium acetate at -35°C.
, twice over 45-60 minutes. DNA was resuspended in distilled water.

(56) One microliter of the MTX4 preparation obtained above was treated with 200 units of T4 DNA IJ gauze (formerly o1mbm) in a total volume of 20 μl of an appropriate buffered salt solution for 2 h at room temperature to prepare the redundant. I checked. Finally, all phosphatase-treated MTX4 remained in linear form when analyzed by agarose gel electrodialysis with appropriate standards.

However, almost all of the untreated MTX4 preparations converted to a more gradual transition species, indicating that effective redaction had taken place.

The final MTX4 vector is 11μ! resuspended in distilled water. In this regard, the 10.7 kilobase vector is Hl
It was a linear molecule that had been cleaved with ndlll, had unpaired bases at both ends, and the terminal phosphate group had been removed, making it impossible to form a cellulose r-ion.

Preparation of a 480-base pair fragment using the Hlnd Ill linker The 480-base pair fragment was originally injected with Agrobacterium tumefaciens tumefaciens norasmite A 6 (pTi
This is part of A6). To generate a 480-base pair fragment, pTiA6 is digested with <<ndli>IJ<tb>ase. A 2.75 kb fragment with HIndIII ends is isolated by agarose gel electrodialysis. 2.75kb
The fragments are electroeluted, concentrated by ethanol precipitation, and resuspended in distilled water. A fixed amount of 2.75kb DNA fragment KSa
u3AI restrictase digestion and Sau3A
Supplement the salts necessary for I restrictase itself and allow sufficient time for complete digestion. The digestion mixture of the 2.75 kb fragment contains many fragments, only 2s of which have Sau a A I and old ndll ends. One of these 24-fold fragments is 480
- It is a base pair fragment.

Digesting pBR327 with BamHI and Hlndll yields pR4269. When saponified, one Bam
HI “sticky” end and one Hl ndll “sticky”
A linear DNA fragment with an end can be changed. This fragment was combined with the previously obtained 2,75 kb fragment Sau3 in an appropriate ligation buffer with T4 ligase and ATP.
Incubate with A I digestion mixture. BamHI“
Because the sticky” end is compatible with the 5au3A1 end,
The two fragments with the 5au3AI end and the old ndl end were ligated together into BamHI/HlndIII digested pB R327 and covalently closed (CCC
) Generates a DNA plasmid. Of the two CCCf lasmids, a 480-base pair fragment joined together was
It is called R4269.

Techniques well known by experts in the industry (see: BRL
User Manual, M 13 Cloning/
Dideoxy” Continuation Manual 1980 BRLINc,,
Galtharburg%Maryland 207
Serial analysis of the 480-base pair fragment using 60) yielded the following sequence: where J is C or G, A is adenine, T is thymine, C is cytosine, and G is guanine.

(59) The 480 base pair fragment was excised from E. coli plasmid pR4269. 100 microliters of pR4269 containing 25 μm of plasmid DNA, as found in the Birnbo 1m cesium chloride method described above, was mixed with 10 units of Hlndl [restrictase and 150 μl I
Digestion was performed in d II buffer salt at 37° C. for approximately 11 hours.

After adding sodium chloride and TRl5-chloride at pH 7.0 to final concentrations of 100mM and 20mM, respectively, 8 units of B,am HI restrictase were added to the digestive fluid. After reacting at 37°C for 9 hours, B
Added 5 units of indm and 4 units of Bam HI. 3
After 12 hours at 7°C, an additional 5 units of Hind were added to the digestive fluid.
1 and 4 units of Bam HT and allowed to digest for 3 hours at 37°C. The 480 base pair fragment was electroeluted from a 0.71 agarose gel at 125 volts for 2 hours in a suitable washing solution. The 480 base pair fragment was purified by extraction three times with phenol and three times with chloroform, and precipitated twice with ethanol (aO) 2 under the same conditions used in Example 1 (J: described). The purified 480 base pair fragment thus obtained was resuspended in 50 μt of distilled water at a concentration of approximately 0.2 μt/mt.

The 480-base pair fragment had one Hlnd II' cut end and one Bam HI cut end. 4
To compensate for the terminal unpaired bases of the 80-base pair fragment, approximately 3μ
The 480-base pair fragment of
) lyphosphate, and deoxyguanosine-5'-
Triphosphate (P, L, Biochemica
lg) of 20mM β-mercaptoethanol, 60mM
In a total reaction volume of about 22μ containing MTR18-chloride, pH 7.5, and 8mM magnesium chloride, about 10
Unit of Klenow fragment enzyme (BRL.

(large subunit of E. coli DNA polymerase I). After 60 minutes at 16°C, the mixture was inactivated by heating to 65°C for 10-12 minutes.

Prior to joining the 480-base pair fragment to the Hlnd If linker, the terminal phosphate group was regenerated on the linker molecule using the same method used in Example 1. However, in precedent! ! Although p-labeled ATP was used, this time unlabeled ATP was used instead. To join the 480-base pair fragments, add 22 μt of polymerase mixture containing approximately 3 μt of the 480-base pair fragment to the linker.
was added to 10 μt of the Hlnd m linker mixture at a molar ratio of 100:1 to the fragment. 66mMT in this solution
Rl5-chloride, p)i7.5.66mM magnesium chloride, 10mM dithiothreitol, 0.6At 1
0mM ATP.

1 μt of 0.2 mM β-mercaptoethanol and 1
4 consisting of 00 units of T4DN A IJ gauze
A solution of μt was added to bring the total to 40 μt. mix 20
Incubated for 9 hours at ~22°C, supplemented with 100 units of glue gauze and incubated for another 40 hours.

480-(63
) The base pair fragments immediately represent the ligation mixture seen in L,
Add sodium chloride directly to the ligation solution and adjust the volume to 50 μt with distilled water, then add 10 units of HlndI.
Obtained by treating with II restrictase enzyme at 37°C for 4 hours.

The 480-base pair fragment with the H1ndm' sticky end group was separated from the free linker by precipitation with ethanol for 15 min at 0°C in the presence of 100 mM sodium chloride and 10 mM spermine (Sigma) for 10 min. Collect the pellet by centrifugation at 200mMTRl5-
Chloride, p) was resuspended in 5μ of 17.5.7 mM magnesium chloride, 1 molar sodium chloride and 2mM β-mercaptoethanol.

This solution was diluted with 45 μt of distilled water and reprecipitated twice with 5 mM spermine. 480-base pair fragment at 150mM
TRlS-chloride, pH 8,0° was resuspended in 8 μt of ligase buffer containing 50 mM magnesium chloride, 256 mM sodium chloride, 50 mM β-mercaptoethanol and 250 μ2/− of bovine serum albumin (64) (BRL).

The thus obtained 480-base pair fragment with Hlnd III linkers at both ends was treated with ligase ligase in 1mM ATP, and a multimer was generated from the fragment, which was
m Digestion resulted in a monomer volume. Bam HI restrictase also converted multimers into monomers, but this was because the terminal Bam HI site of the fragment was ligated to Hind m
This shows that it was reconfigured into Lincoln.

Hln in the same manner as used for the 480-base pair fragment.
Approximately 5 μt of MTX4 plasmid, which had been previously digested with d lll restriction enzyme, was added to both ends of the MTX4 plasmid.
■ A linker was also added to 0.4μ2 of the 9480-base pair fragment. 30mM TRl5-chloride, pH 8, 10m
M magnesium chloride, 10mM β-mercaptoethanol, 5onf/- bovine serum albumin, 1mM A
Total volume 4 containing T P (65) and 200 units of 74 DNA ligase
Ligation was performed at 0 μt.

After 2 hours at 20°C, the mixture was heated to 65°C for 1o min.
It was inactivated. Thirty microliters of the ligation mixture was used to transform E. coli cells.

Characteristic U of E. coli cells M t x rD of E. coli is added to E. coli of bacterial species LE392.
For transformation with HFR ligation, the plasmid thus obtained was transformed in substantially the same manner as described in Example 1. However, the mixture used for transformation contained M
The difference is that it includes TX4.

Two hundred ampicillin-resistant colonies obtained by clonal analysis transformation were treated with a 480-base pair fragment ( 66) Cleaning was performed, except that an l1lp-labeled 480-base pair fragment generated by nick translation was used.

3! Eleven colonies containing DNA hybridized to p-labeled 480-base pair fragments were analyzed for insert copy number and orientation. Plasmid DNA was obtained from Blrnbolm at
L-broth plus 100 μm/
-1,5 grown in ampicillin (Sigma)
It was isolated from a 1 nt cell. Under the amended law,
Plasmid DNA from the clear E clear solution was precipitated with ethanol and 10mM TRl5. Instead of resuspending in 100 microliters, pH 8, and then purifying with a cesium chloride gradient, the cells were treated with 100 μm/− glue bonuclease (Sigma) at 37° C. for 30 minutes. To calculate the plasmid size, 25 μt of the plasmid solution was used directly and applied to a 0.8 thiagarose gel.

Nine clones had plasmid solutions larger than 10.7 kb. Each (67) plasmid from nine clones was ethanol precipitated and then suspended in 10 microliters of deionized water and digested with BamHI restriction enzyme. Following Bam HI digestion for 3 hours at 37°C, half of each plasmid sample (volume f*) was precipitated with ethanol, dissolved in 44 μt of deionized water, and then incubated in appropriate buffered salts for 3 hours at 37°C. Treated with 6 units of pstI restrictase (Biolalls). Ethanol precipitates of Bam HT and pstI digests of each plasmid sample were treated in 1.0 thiagarose at 75 volts for 5 hours. Vector 7Ep for cloning
The 13 digests yielded fragments of known length and number, which provided a standard for molecular size and confirmed the complete progress of the digestion. Lambda DNA predigested with Bind ■ and pBR327 plasmid DNA predigested with Hlnf I served as standards for fragment size.

The orientation of the insert could be determined by mapping the specific rearrangement am HI (68) sites occurring at specific ends of the 480-base pair fragment in the following manner.

The Bam 'sticky' end of the 480-base pair fragment is 5'GA
It has chain sequences such as TCC3'-. This ant sticky l
The end becomes a functional Ram restriction site and the chain sequence 5'CAAG
When linked to a Hindm linker with CTTG3'-, etc., it has a chain sequence such as 5'GGATCC3'-, which gives 5'CAAGCTTGGATCC3', etc. H1
The 3'-terminal guanosine residue of the ndTII linker is Ba
It serves as the 5'-initial guanosine residue of the mHI sticky end, thereby reconstituting the BamII restriction site. Localization of the tandem H1nd m/Bam HI sites with respect to the second pat) restriction site of the vector was used to orient the insert.

Analysis of MX4/4269 Plasmid A plasmid called MTX4/4269-5, obtained by joining a 480-base pair fragment to the MTX4 plasmid, was digested with pst ① and Bam HI restrictase, and the fragment was digested as described in 1.F. It was developed on a 0.0 thiagarose gel. It was expanded into five fragments of the following approximate sizes: 4.6kb, 3.5kb, 0.9kb,
2.2kb, and 0゜38kb 0Figure 4 is MTX
Restriction map for the fragment obtained from 4/4269-5.

The 2.2 kb fragment is a 480-base pair fragment and vector DNA.
It consists of The 2.2k b fragment was reconstituted as described above by Ban HI digestion of the am HI site and vector D.
It was generated by pstI digestion of one of the N A p st ■ sites. The reconstructed amHI site has a 0.38 mtxrDHFR nucleotide linkage within it.
Located next to the kb fragment.

Plasmid MTX4/4269-2 to MTX4/42
It was treated in the same manner as No. 69-5. Five fragments were extracted with the following sizes: 4.6kb, 3.5kb, 1.6kb.
5kb, and 0.8kb0. Figure 5 is a restriction map showing these results. The 1.65kb fragment is vector DN
The result was a pat digestion of one of the ApstI sites and a Bam HI digestion of the adjacent rearranged am HI site.

A 0.8 kb fragment with a 480-base pair fragment and Mt in it.
It consists of a DNA fragment with the nucleotide chain code for xrDHFR.

MT another plasmid MTX4/4269-13
Digested in the same manner as X4/4269-2, 4.6 kb.

3.5kb, 1.65kb, 0.9kb, 0.8
kb, and 6 fragments of 0.45 kb. Figure 6 is a restricted area showing these results. The 0.45 kb fragment contains at least two 480-base pair fragments inserted in tandem in the same direction as the single 480-base pair fragment of MTX/4269-2.
- suggests the presence of base pair fragments.

MT another plasmid MTX4/4269-1
Digested in the same way as X4/4269-13, 4,6kb
, 3.5kb, 0.9kb, 2.2kb, 0.
3F! kb, and 6 fragments of 0.45 kb. Figure 7 is a restriction map showing these results. 1.5kb
The fragment was inserted in tandem in the opposite direction to the tandemly inserted 480-base pair fragment of MTX/4269-13.
This suggests the presence of an 80-base pair fragment.

(71) MTX/4269-5 plasmid MTX4/4269-5 of yeast DBV-746. MTX4. The yeast DB)'-746 was transformed using the same method as described in Example 1, except that MTX5 and yEp13 were used.

Plasmid DN from 50 ml of yeast spheroplasts
Af: Extracted using the modified method of Birnboim et al. Yeast was grown in advance in a minimal medium that does not contain leucine, stored at 5°C for 20 hours, and then dried.
Spheroplasts were obtained by modifying the previously described method. Wash cells with 1M sorbitol, 25mM EDTA, 50mM
Resuspended in 1/20 volume of M dithiothreitol, pH 8.0, for 10 minutes at 30°C. In vitro cells
Wash with 1M sorbitol, 0.1M sodium citrate, pH 5, 0, 10mM EDTA and 5 pf/ml Modiase 60,000 (K
lrln) (72) for 20 min at 30°C. The mixture was placed in a 5orvall SA-600 rotor and centrifuged at 15,000 rpm for 30 minutes at 0°C to obtain a clear liquid. The DNA was precipitated with ethanol and then incubated with buffer plus 40 μf/r for 30 min at 37°C.
n1. Resuspended in RNA5e T (Sigma). It was further precipitated with ethanol, and the portion containing DNA was extracted three times with phenol and once with chloroform/isopropyl alcohol (24=1), and reprecipitated with ethanol.

Plasmid DNA content extracted from yeast spheroplasts was run on a 0.7 thiagarose gel L125 volt.
The mixture was treated for 5 hours and then transferred to a nitrocellulose filter sheet for 5-outhern hybridization. yeast dna
The filtrate was subjected to the dextran sulfate formamide method (W
ahl at al,, P, N, A, S, USA
(1979) 76:3693-368F) in separate experiments, p-radiolabeled pBH327,MT
Hybridization was performed on specimens of X5° and Mtx DHFR nucleotide chains. Radiolabeled specimen is Example 1
This was seen with the Nick translation method as described in .

yEpta, MTX4. MTX5 and MTX4/4
A total of four plasmids of 269-8 were detected. Analysis of the relative migration of the plasmids to HindIIT-digested lambda DNA used as a standard shows that no changes in plasmid size were detected in yeast cells.

As expected, excluding y E p 13 (MTX4.MT
X5 and MTX4/4269-5 are radiolabeled Mt
It was detected by hybridization with xrDHFR specimen.

Radioactive labeling of plasmid extracted from transformed yeast M
Hybridization with TX5 had the same results. Additionally, all four plasmid contents contained a highly mobile species that was highly hybridized to the MTX5 sample, leading to the inference that this was an endogenous 2 micron portion of the yEp13 plasmid.

Plasmid MTX4. MTX5. MTX4/4269-
Yeast cells transformed with one of 5 or 7Ep13 were processed into spheroplasts as described above. Spheroplasts were digested in a buffer containing inhibitors of nuclease activity. The digestion buffer was 50mM TRl5. pH7
,5,50mM NacL, 10mM EDTA, 50m
M β-mercaptoethanol, 1% SDs and 1 thidiethylpyrocarbohydrate (DEPC). The mixture was extracted with phenol and treated with DNA5e to obtain a pure sample of RNA.

Total yeast cell RNA was isolated from each transformed yeast, denatured, processed on a formaldehyde-containing agarose gel, and then transferred to a nitrocellulose filter. Deoxtadeoxynucleotide chain code for M t x rDHFR with p 5) Oxidized. Three MtxrDHFR clones, namely MTX4. MTX5 and MTX4/4269-5
Complete hybridization to RNA was observed.

Very little hybridization between the sample and yEp13 or yeast liposome RNA was observed.

The gel pattern of RNA from four transformants was also 8tp.
- Hybridized with a sample of labeled 480-base pair fragment DNA. Complete hybridization occurred in MTX/4269-5.
It was only. Furthermore, the discontinuous band shows MTX4/426 strongly hybridized to the 31p-labeled 480-base pair sample.
9-5 RNA gel, indicating that at least a portion of the 480-base pair fragment is transcribed into yeast.

Experiments to determine the strand characteristics of the transfer revealed that 480-
A considerable amount of transcription in the area is correct! It can be seen that it comes from the strands.

(76) Preparation of MTX5 Vector DNA
Prepared by m-cesium chloride method. MTX5 plasmid D
Approximately 18.6 microliters of NA was mixed with a solution containing 2e units of Bam HI (BRL) in 200 microliters of a suitable salt.
Digestion was performed at 7°C for 16 hours. Complete digestion was indicated by the appearance of a single 11.2 kb band in the 0.8 thiagarose gel. Digested M T X 5
DNA was electroeluted and precipitated twice with ethanol for 25 min.
resuspended in μt of distilled water.

Approximately 15 μt (1012 μt) of resuspended digested MTX5
F) 30mM TRl5-chloride, pH 7,8
Terminal phosphates were removed by treatment with 3.3 units of calf intestine alkaline phosphatase at 65°C for 15 minutes.

The mixture was extracted and precipitated as described in section 7 for MTX4. Untreated vector gave rise to viable species under standard ligation conditions, although phosphatase-treated MTX5 remained linear as analyzed by agarose gel electrodialysis. The linear MTX5 preparation was resuspended in 15 μt of distilled water.

Preparation of 480-base pair fragment with Bam HI linker A 3 μm 480-base pair fragment was added to K as previously described.
Complete double helixization was achieved using lenow fragment enzyme.

Bam HI octanucleotide linker (Co11a
(borative Research) approximately 5 μ2 was phosphorylated using unlabeled ATP.

A phosphorylated Bam HI linker was ligated to the 480-base pair fragment as described for MTX4. However, ligation is at room temperature (20℃~22℃).
) for 16 hours, and then heated to 65°C for 10 minutes to inactivate.

Double helix 480-base pair fragment and phosphorylated Bam
35 .mu.t of the mixture containing the HI linker was added to a final concentration of 100 ml of sodium chloride in 100 mM XBa units, and then distilled water was added to bring the total volume to 50 .mu.t. 3
480- with Bam HI end after treatment at 7°C for 40 hours.
A base pair insert was obtained. Spermine precipitation method (Hoop
ea.

B, C, and W, R, McClure, 1
981. Nucl Ac1d Rika.

9, 5493-5504) to remove the free linker. The purified 480-base pair fragment with Bam HI ends was resuspended in 6 μt of ligase buffer salt. Treatment of a fixed amount of the fragment preparation with T41J gauze and ATP resulted in multimers. 13am HI converted multimers to monomers, but Hlndm did not. From this, 480
It can be seen that the terminal H1nd m site of the 1 base pair fragment is not reconstructed by the linker.

2.5 μF of the Bqm HI series ligated 480-base pair insert was ligated to 5 μt of MTx vector as described above in a solution of 30 μF of ligase buffer salt, 1.0 mM ATP, and 200 units of T4 ligase.

(79) Transformation of E. coli 200 p of antagonistic LE392 E. coli cells were grown using 15 microliters each of the ligation mixture described in E.
Transformation of L. Transformants were selected on L-agar E containing 100 μV/− ampicillin.

Analysis of MTX5/42697 Clones Plasmids from nine ampicillin-resistant E. coli colonies were isolated using the Birnbo im method as described above. The orientation of the 480-base pair fragment is the pv of the vector.
ul' (a single internal p of the 480-base pair fragment with respect to the site
v u ll' [determined qualitatively by mapping restriction sites.

The pvu [site of the 480-base pair fragment is located 150 base pairs from the 'Bam end' of the 480-base pair fragment. Therefore, one orientation of the 480-base pair fragment is 480-base pair in the opposite orientation. Approximately 30 from the base pair fragment pvu■ fragment
It was expected to yield a 0-base pair large pvull fragment. Hind l' MTX5/4269 plasmid
[6 units of pvul' in mild (80) buffered salt for 3 hours at 37°C] and then separated on a 1% agarose gel. The three plasmids yielded digested fragments of approximately 9.2 kb and 1.9 kb, whereas the 480-base pair fragment was
9A (FIG. 8) shows the orientation.

Six clones had fragments of approximately 9.2 kb and 1.6 kb, which were 480-base pair fragments of MTX5/
4269C (FIG. 9) shows that it is oriented. All DNA from 6 colonies is 8t
It showed homology to the p-marked 480-base pair fragment.

It will be readily apparent to those skilled in the art from what has already been explained and shown by examples that the plasmid of No. L has a more general use. For example, although methotrexate-resistant dihydrofolate reductase was used as a marker chain in the present invention, other DNA fragments with nucleotide chains or chain codes for Nozomi's gene products can also be used.

Therefore, one chain of markers other than the MTX'DHFR nucleotide chain described above can be used depending on the characteristics of the host to be transformed and the needs of the user. For example, DNA chains can be used that are resistant to substances that are toxic to the host. Such linkages are well known to those skilled in the art and include antibiotic resistance, heavy metal resistance, and many more. Such markers may be considered within the scope of this invention. Additionally, although a chain of early selectivity markers for ampicillin, tetracycline resistance and the yEp code for LEU2 have been used, other early selectivity markers may be used if available on other hybrid plasmids.

It is also clear from the foregoing that DNA fragments carrying the nucleotide linkage code for the desired gene product other than the marker property may optionally be used together with or in place of the marker nucleotide linkage. It is. In the former case, a property that is not easily selectable, such as enhanced amino acid productivity, may be included in the plasmid along with or next to the marker nucleotide linkage, and selection of transformed hosts may be performed using the marker. Good. In the latter case, it is preferable to place the DNA fragment with the nucleotide linkages for the desired gene product in a plasmid rather than in a marker.

One of the particular advantages of the vector systems described so far is the ability to shuttle between prokaryotic and eukaryotic hosts. That is, a vector with the desired nucleotide linkage is assembled in vitro, allowed to transform and replicate in a prokaryotic host, and is rapidly transformed into a prokaryotic host due to the short generation cycle of prokaryotic organs. Multiply. Vectors propagated in this manner can then be recovered at high copy numbers from prokaryotic hosts and used to transform eukaryotic hosts in vitro.

Therefore, by propagating a vector using a prokaryotic host and then using the propagated vector to transform a eukaryotic host, large numbers of eukaryotic transformants can be produced in a short period of time. becomes possible.

As is well known to those skilled in the art, it is also clear that the nucleotide chain containing the plant promoter can be replaced with promoters of other eukaryotic or prokaryotic origin or function. Here, the term "electronic function" refers to promoters that are not derived from eukaryotic cells, but which act as promoters for RNA replication within eukaryotic cells. An example of this is the promoter derived from the TI plasmid of Agrobacterium tumefaciens.These plasmids directly synthesize various RNAs using the replication apparatus of the host plant cell.

(83)

[Brief explanation of the drawing]

Figure 1 is a schematic restriction map of the MTX4 plasmid, which contains the nucleotide chain code for MtxrDHFR; Figure 2 is a schematic restriction map of MTX5, a variant of MTX4, which also contains the nucleotide chain code for MtxrDHFR. It has a chain code. Figure 3 is a schematic restriction map of yEp13; Figure 4 is a schematic restriction map of MTX4/4269-5; Figure 5 is a schematic restriction map of MTX4/4269-2; The figure is a schematic restriction map of MTX4/4269-13; Figure 7 is a schematic restriction map of MTX4/4269-1; Figure 8 is a limited restriction map of MTX5/4269A; and Figure 9 is a limited (84) limit map of MTX5/4269C. Agent Patent Attorney Hideaki Kuwahara Continuation of page 1 ■Int, C1,' Identification code Office serial number Priority claim @1983*January 3 [phase] United States (US) @4
62731■ Inventor John Antony United States of America Force Howard Beachview-262 483- Lifornia 94044 Pacifi force, #2 (0 shots) Procedural amendment (method) Showa GQ ΔR-3, Director of the Japan Patent Office M Fatsuo Wakasugi 1. Indication of the case Patent Application No. 14542/1983 3. Relationship between the person making the amendment and Case I. Applicant Bekki

Claims (1)

[Claims] 1. Transforming at least one prokaryotic and eukaryotic host having at least one insertion site for at least one DNA fragment and at least one initial selectable marker. Recombinant plasmid suitable for. 2. The plasmid according to claim 1, further comprising at least one DNA ligated to the insertion site. 3. A plasmid according to claim 2, wherein at least one of said DNA fragments carries at least one nucleotide linkage code for the desired gene product. 4. The plasmid according to claim 3, wherein the desired gene product is a marker. 5. The plasmid according to claim 4, wherein the marker is mentrexate-resistant dihydrofolate reductase. 6. The plasmid according to claim 2, further comprising a nucleotide chain having a promoter code as a part thereof near at least one of the nucleotide chains. 7. The plasmid according to claim 6, wherein the nucleotide chain having a promoter code as a part thereof has the following basic chain. 8. The plasmid according to claim 6, wherein the promoter functions as a eukaryotic promoter. 9. The promoter is a eukaryotic promoter;
The plasmid according to claim 6. 10. The plasmid according to claim 6, wherein the promoter is a plant promoter. 11. The plasmid according to claim 5, further comprising a nucleotide chain having a promoter code in a part thereof near at least one nucleotide chain code for methotrexate-resistant dihydrofolate reductase. 12. The plasmid according to claim 11, wherein the nucleotide chain having a promoter code in a part thereof has the following basic chain. 13. The plasmid according to claim 11, wherein the promoter functions as a eukaryotic promoter. 14. The plasmid according to claim 11, wherein the promoter is a eukaryotic promoter. 15. The plasmid according to claim 11, wherein the promoter is a plant promoter. 16. The recombinant plasmid according to claim 1, consisting of (a) a first DNA region containing a eukaryotic replica and (b) a second DNA region containing a prokaryotic replica. 17. The insertion site is located between the first DNA region and the second DNA region.
17. The plasmid according to claim 16, which is located between the plasmid region and the plasmid region. 18, and at least one tethered to the insertion site.
18. The plasmid according to claim 17, characterized by a DNA fragment of . 19. 19. The plasmid of claim 18, wherein at least one r)NA fragment consists of at least one nucleotide linkage for the desired gene product. 20. The plasmid of claim 19, wherein the desired gene product is a marker. 21. The plasmid according to claim 20, wherein the marker is methotrexate-resistant dihydrofolate reductase. 22. The plasmid according to claim 18, further comprising at least one null and nucleotide linkage having a promoter code as a part thereof near the nucleotide chain. 23. The plasmid according to claim 22, wherein the nucleotide chain having a promoter code in a part thereof has the following basic chain: 24. The plasmid according to claim 22, wherein the promoter functions as a promoter for eukaryotes. The plasmid according to item 22. 25. The plasmid of claim 22, wherein the promoter is a eukaryotic promoter. 26. The plasmid according to claim 22, wherein the promoter is a plant promoter. 27. The plasmid according to claim 21, further comprising a nucleotide chain having a code for a promoter as part of the nucleotide chain in the vicinity of at least one nucleotide chain. Claim 2, wherein the nucleotide chain, a part of which is a code for a promoter, has the following basic chain:
The plasmid according to claim 1: 29, wherein the promoter functions as a eukaryotic promoter.
The plasmid described in Section 1. 30. The plasmid of claim 21, wherein the promoter is a eukaryotic promoter. 31. The plasmid according to claim 21, wherein the promoter is a plant promoter. 32, the first DNA region is of eukaryotic origin, and the second
Claim 1, wherein the DNA region is of prokaryotic origin
The plasmid described in Section 6. 33. The insertion site is located between the first DNA region and the second DNA region.
33. The plasmid according to claim 32, which is located between the plasmid and the region. 34. The plasmid of claim 33, further comprising at least one DNA fragment ligated to the insertion site. 35. at least one said DNA fragment consists of at least one nucleotide linkage for the desired gene product;
The plasmid according to claim 34. 36. The plasmid of claim 35, wherein the desired gene product is a marker. 37. The plasmid of claim 36, wherein the marker is methotrexate-resistant dihydrofolate reductase. 38. The plasmid according to claim 34, further comprising a nucleotide chain having a promoter code as a part thereof near at least one of the nucleotide chains. 39. The plasmid according to claim 38, wherein the nucleotide chain having a promoter code as part thereof has the following basic chain: (11) (12) 40. The promoter is a eukaryotic promoter. 39. A plasmid according to claim 38, which functions as a plasmid. 41. The plasmid according to claim 38, wherein the fromotor is a eukaryotic promoter. , 42. 39. The plasmid of claim 38, wherein the promoter is a plant promoter. 43. Further, a patent claim characterized by a nucleotide chain having a promoter code as a part thereof near at least one of the nucleotide chains (a plasmid according to item 37 in the rNa box). 44. The plasmid having a promoter code as a part thereof. 44. The plasmid of claim 43, wherein the nucleotide chain has the following basic chain: 45. The plasmid of claim 43, wherein the promoter functions as a eukaryotic promoter. 46. 47. The plasmid of claim 43, wherein the promoter is a eukaryotic promoter. 47. The plasmid of claim 43, wherein the promoter is a plant promoter. 48. The plasmid of claim 43, wherein the promoter is a plant promoter. The plasmid according to claim 32, wherein the plasmid belongs to the genus Yeast, and the prokaryote is a bacterium. 49. The insertion site includes the first DNA region and the 21)N
The plasmid according to claim 48, which is located between the A regions. 50. The plasmid of claim 49, wherein said plasmid is digested with Bam HI and Hlnd m to generate said insertion site. 51. The plasmid according to claim 50, further comprising at least one DNA fragment ligated to the insertion site. 52. The plasmid of claim 51, wherein at least one said DNA fragment consists of at least one nucleotide chain code for the desired product. 53. The plasmid of claim 52, wherein the desired product is a marker. 54. The plasmid of claim 53, wherein the marker is methotrexate resistant dihydrofolate reductase. 55. The plasmid according to claim 51, further comprising a nucleotide chain having a promoter code as a part thereof in the vicinity of at least one nucleotide chain. 56. The plasmid according to claim 55, wherein the nucleotide chain having a promoter code in a part thereof has the following basic chain: (16) 57. The promoter functions as a eukaryotic promoter. , the plasmid according to claim 55. 58. The plasmid according to claim 55, wherein the promoter is a eukaryotic promoter. 59. The plasmid of claim 55, wherein the promoter is a plant promoter. 60. The plasmid according to claim 54, further comprising a nucleotide chain having a promoter code as part of the nucleotide chain in the vicinity of at least one nucleotide chain. 61. The plasmid according to claim 54, wherein the nucleotide chain having a promoter code in a part thereof has the following basic chain. (19) 62. The plasmid according to claim 54, wherein the promoter functions as a eukaryotic promoter. 63. The plasmid of claim 54, wherein the promoter is a eukaryotic promoter. 64. The plasmid according to claim 54, wherein the promoter is a plant promoter. 65, Plasmid MTX4/4269 A TCC39
395°66, plasmid MTX5/4269CATC
C39394゜(20)