JPS6237958B2 - - Google Patents

Description

Detailed Description of the Invention The present invention is a plasmid for detecting a DNA fragment having promoter activity based on the expression of trimethoprim resistance, and relates to a plasmid vector having an ampicillin resistance gene as a genetic marker and a method for producing the same. be.

In recent years, with the development of molecular biology and genetic engineering, methods for producing useful substances using recombinant DNA techniques have been attracting attention. Based on the results of molecular biology to date, gene information expression is determined by DNA
The stage in which Messenger RNA (hereinafter abbreviated as mRNA) is produced (transcription stage) and mRNA
is read and regulated during the two stages of translation, which produces proteins.In addition to the DNA sequence that encodes the primary structure of the protein, genes also contain promoters, which are responsible for initiating transcription and translation.
The existence of DNA sequences has been revealed. recently,
Using genetic engineering techniques, extremely useful substances such as somatostatin, insulin, and interferon are being produced using microorganisms. It has become clear that simply introducing DNA obtained by transcription into a plasmid does not initiate the production of useful polypeptides; a promoter is required to read the introduced foreign DNA. Furthermore, promoters have varying degrees of activity, and it has come to be considered that the expression of the introduced heterologous DNA, that is, the production of the target polypeptide, depends on the strength of the promoter activity. When producing useful polypeptides in a foreign organism using genetic engineering methods, it may be possible to add a strong promoter to increase the production amount, but the gene product of the introduced foreign DNA may be produced in large quantities. It is expected that the host bacteria will be adversely affected by this, and such cases have actually been revealed. Therefore, it is necessary to find a promoter that is convenient for efficiently expressing the introduced foreign DNA without causing any harm to the host bacteria, and to use it to produce the gene product of the foreign DNA. be done.

In view of these circumstances, the present inventors have keenly realized that it is necessary to extract promoters of various strengths that exist within the genes of host bacteria, and to find promoters suitable for expressing specific foreign DNA. I started to do that. For this purpose, promoter detection is essential. Therefore, as a result of extensive research into promoter detection methods, we discovered that a plasmid for promoter detection can be obtained by modifying a plasmid vector that incorporates the trimethoprim resistance gene that the present inventors had already developed. It was completed.

That is, the present invention provides promoter activity
After removing the specific DNA of a plasmid vector that expresses trimethoprim resistance by introducing a DNA fragment into a specific restriction enzyme site, and a plasmid vector that incorporates a trimethoprim resistance gene developed by the present inventors, The present invention provides a method for producing a plasmid vector for promoter detection by ligating the cleaved sequences.

The host bacterium used in the present invention is Escherichia coli, but any host in which the plasmid can be autonomously replicated, such as a near green bacterium of Escherichia coli, can be used.
The following describes E. coli.

Trimethoprim is a strong competitive inhibitor of dihydrofolate reductase (hereinafter abbreviated as DHFR), and by inhibiting DHFR, the supply of tetrahydrofolate, which is essential for amino acid and nucleic acid synthesis reactions, is stopped. From this, trimethoprim is
It is used as an antibacterial agent, but in order to impart resistance to trimethoprim in the host, as the inventors have already revealed (patent application, 56-143520), the DHFR gene must be incorporated into a multicopy plasmid. This can be achieved by introducing this gene into the cell, increasing the amount of this gene in the cell, and producing a large amount of DHFR. The DHFR gene is
It consists of a DNA sequence that encodes the amino acid sequence of the protein to be produced, and a promoter sequence that allows this code to be read. Therefore, the latter, i.e., by removing the promoter sequence, the DHFR gene no longer produces DHFR, but the former, i.e., it encodes DHFR itself.
have other promoter activities before the DNA sequence
When you connect the DNA, this regenerated gene produces DHFR
began to produce. Therefore, in order to detect promoter activity, a plasmid containing the DHFR gene is modified to remove the DHFR gene promoter sequence and encode DHFR itself.
A specific restriction enzyme site may be formed in front of the DNA sequence to facilitate connection of other DNA. The plasmid thus formed becomes capable of producing DHFR by connecting a promoter sequence, and thus can confer trimethoprim resistance to the host. Therefore, a plasmid into which a promoter sequence has been introduced can only be obtained from host bacteria that grows in a medium containing trimethoprim, and a host bacterium that has a plasmid into which DNA without a promoter sequence has been grown can grow in a medium containing trimethoprim. The promoter can be easily detected because it cannot be grown directly. However, until now, a promoter detection plasmid vector using trimethoprim resistance as a marker has not yet been developed.

Next, embodiments of the present invention will be described.

The plasmid vector pTP5-3 was already prepared by the present inventor for the purpose of improving the plasmid vector pTP30-5 (described in Japanese Patent Application No. 143520/1982), and the manufacturing process thereof is shown in Reference Example.
pTP5-3 is a plasmid vector containing genes that confer resistance to three types of drugs, trimethoprim, tetracycline, and ampicillin in the host, and is kept stable in E. coli.
E. coli containing 3 was sent to the Microtech Institute as FERM P-8648.
It has been deposited as.

First, plasmid vector pTP5-3 is cut with Sal and then religated again using T4 DNA ligase to create a plasmid DNA consisting of approximately 4300 base pairs. Sal, pTP5
-3 is cleaved at three locations to form three DNA fragments of approximately 4300, 1200, and 600 base pairs. Approximately of this
The 4300 base pair DNA fragment contains an ampicillin resistance gene, a gene involved in plasmid replication and
Contains the DHFR gene that lacks a small portion of the promoter. So, you get about 4300
Base pair plasmid (referred to as pTP6-6)
confers ampicillin resistance to the host, but cannot confer trimethoprim resistance. However, since this plasmid contains most of the promoter of the DHFR gene, even if a DNA fragment without promoter activity is introduced,
Since there is a possibility of producing DHFR, pTP6-
Most of the promoter is removed by cutting 6 with Sal and then applying exonuclease BAL31, an enzyme that specifically acts on double-stranded DNA to degrade nucleic acids from both ends. At this time, the action of exonuclease depends on the amount of enzyme, temperature, and time for action. The inventor has determined that the final concentration is 2 units/
At 400μ enzyme amount and 25℃ reaction conditions, approx.
They confirmed that the enzymatic reaction progresses at a rate of 120-180 base pairs/min, and are planning to delete the promoter sequence based on this result. According to this condition,
In order to preserve the sequence encoding dihydrofolate reductase present approximately 100 base pairs downstream of the Sal cleavage sequence of pTP6-6, it is necessary to stop the reaction within 33 seconds (100/180 minutes). The resulting linear
Both ends of DNA are blunt ended. Furthermore, heterogeneous
Circular DNA is created by adding a DNA sequence that can be cut with a specific restriction enzyme to make it easier to introduce DNA.
and obtain the desired plasmid vector.

Plasmid pTP6-6 is kept stable in E. coli, and E. coli containing pTP6-6 is transferred to FIKEN.
Deposited as FERM P-8647. The creation of a site that is cleaved by a specific restriction enzyme, for example, a site that is cleaved by Hind,
DNA with a sequence d (pCAAGCTTG), called a linker, is ligated to the blunt end of linear DNA obtained by exonuclease BAL31 using T4 DNA ligase, and then cut again with Hind, resulting in both ends of the DNA. It becomes the adhesive end.
When both ends are ligated with T4DNA ligase, a circular DNA is obtained, and the site ligated with T4DNA ligase is
A sequence is formed that is cleaved by Hind.
Whether or not the plasmid vector obtained in this way is a promoter detection vector is
This can be verified as shown below.
That is, pBR322 was cut with Hinc, and further,
Attach the Hind linker to the cutting point and Hind again
Cut with. This and the plasmid obtained above cut with Hind are ligated with T4 DNA ligase and incorporated into the host. If the host acquires trimethoprim resistance through this operation, the promoter detected in the plasmid used will be determined to be a promoter-detecting plasmid vector. {Hinc of pBR322 and
At least one of the fragments obtained by cleavage with Hind has a complete promoter structure. [JGSutcliffe, Cold Spring Harbor
Symbosium, 43, 77 (1979)]} The plasmid vector of the present invention can also be obtained by the following method.

That is, pTP6-6 is cut with Sal and a Hind linker is attached to the linear DNA obtained by treating with exonuclease BAL31. This and
The chromosomal DNA of E. coli is mixed, cut with Hind, and ligated with T4 DNA ligase to create various plasmid DNAs, which are then incorporated into E. coli.
Escherichia coli that has become resistant to trimethoprim is isolated, and a plasmid is isolated from it. This plasmid
The DNA is cleaved with Hind, ligated again with T4 DNA ligase, and incorporated into E. coli to isolate a strain that is resistant to ampicillin but not resistant to trimethoprim. When a plasmid is isolated from this strain, a plasmid vector for detecting the desired promoter can be obtained.

The Hind cleavage site of the plasmid vector obtained in this way is the so-called adapter.
Synthetic DNA, called DNA, can be used to convert sites that are cut by other restriction enzymes. Therefore, the specific restriction enzyme site in the present invention is
It is obvious that it is not limited to the Hind site.

Next, the present invention will be explained in more detail with reference to Examples.

In addition, isolation of plasmids from bacterial cells in Examples was performed using the method of Tanaka and Weisblum {T.
Tanaka, B. Weisbum; J. Bacteriology, 121,
354, (1975)}, the uptake of DNA into the host is
Norgard et al.'s method {MV Norgard, K. Keem, J.J.
Monahan; Gene, 3, 279, (1979)}.

Examples Plasmid vector pTP5-3 is a plasmid vector that confers resistance to three types of drugs, trimethoprim, tetracycline, and ampicillin in the host, and was developed by the inventors. Figure 1 shows a restriction enzyme cleavage map of pTP5-3. About 0.3μg of pTP5-3 was added to 75μ of reaction solution {6mM MgCl ₂ , 0.2mM ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA) and 150mM NaCl.
in 8mM Tris-HCl buffer (PH7.6) containing
Reaction was carried out at 37°C for 2 hours using 5 units of restriction enzyme Sal. Furthermore, after inactivating the restriction enzyme Sal by keeping it at 65°C for 5 minutes, keep it on ice.
15μ of 50mM MgCl ₂ , 15μ of 0.1M dithiothreitol (hereinafter abbreviated as DTT) 15μ of 5mM
ATP, 7μ of 1M Tris-HCl buffer (PH7.4),
23μ of water and 0.2 units of T4 DNA ligase were added, and the mixture was allowed to react at 4°C for 18 hours to ligate the DNA ends to perform a recircularization reaction. This reaction product was prepared according to the method of Norgard et al.
It was incorporated into Escherichia coli K-12 C600 strain. The treated cells were sown on a nutrient agar medium containing 20 μg/ml ampicillin sodium (medium consisting of 2 g/glucose, 1 g/K ₂ HPO ₄ , 5 g/polypeptone, 5 g/yeast extract, and 15 g/agar) and allowed to grow. Approximately 1000 bacterial cells were obtained. Randomly select 100 of these bacteria,
When the cells were transferred onto a nutrient agar medium containing 2 μg/ml trimethoprim and 20 μg/ml ampicillin sodium to examine whether they exhibited resistance to trimethoprim, 72 cells were unable to grow on this medium. A plasmid was isolated from one such bacterial cell, that is, a bacterial cell that was resistant to ampicillin and sensitive to trimethoprim. The size of the obtained plasmid is approximately 4300 base pairs, and the restriction enzyme
It was cleaved at one site each by Sal, EcoR and Pst, but not by Hind and BamH. This plasmid is pTP6-6
It was named. Figure 2 shows a restriction enzyme cleavage map of pTP6-6.

Next, about 5μg of pTP6-6 was added to 400μ of reaction solution {6mM Mgcl ₂ , 0.2mM EDTA, and 150mM
Using 10 units of the restriction enzyme Sal in 8mM Tris-HCl buffer (PH7.6) containing Nacl,
After reacting for an hour, add 8μ of 1M Tris-HCl buffer (PH8.0), 4μ of 1M MgCl ₂ , 5μ of 1M
CaCl ₂ , 8μ 5M NaCl and 1μ 0.25M
EDTA was added, kept at 25°C, and treated with 2 units of exonuclease BAL31 for 20 seconds. The exonuclease reaction was stopped by adding 400μ of water-saturated phenol, separated into an aqueous layer and a phenol layer by centrifugation at 3000 rpm.
Take the aqueous layer and mix it with 50mM containing 50mM NaCl.
Dialyzed against Tris-HCl buffer (PH7.4). Dialysate
40μ of 3M CH ₃ COONa was added to 400μ of the DNA solution, and 1ml of ethanol was added, and the mixture was kept at -20°C overnight to precipitate the DNA. Collect the precipitate by centrifugation at 12,000 rpm for 15 minutes, and
It was dissolved in 50mM Tris-HCl buffer (PH7.4).
Add to this 15μ of 50mM MgCl ₂ , 15μ of 0.1M
DTT, 15μ of 5mM ATP, 7μ of 1M Tris
-HCl buffer (PH7.4), 23μ water, 7μ 3.5
μM Hind nucleotide linker {d
(pCAAGCTTG)} and 10 units of T4 DNA ligase were added and allowed to react at 4°C for 18 hours. this
It was called DNA mixture.

Take 20μ of the DNA mixture, add 48μ of water, and add 7μ of 0.6M NaCl, 70mM 2-mercaptoethanol, and 70mM _MgCl2 .
Add 70mM Tris-HCl buffer (PH7.4) and incubate at 37°C.
5 units of Hind was added to this and allowed to react for 2 hours. This reaction solution was heat-treated at 65℃ for 5 minutes to inactivate Hind, and then 15μ of 50mM
_MgCl2 , 15μ 0.1M DTT, 15μ 5mM
ATP, 7μ of 1M Tris-HCl buffer (PH7.4),
23μ of water and 2 units of T4 DNA ligase were added and reacted at 4°C for 18 hours. The DNA solution treated in this manner was introduced into Escherichia coli K-12 C600 strain. 20μg/bacterium treated with this treatment
Approximately 500 growing bacterial cells were obtained by seeding on a nutrient agar medium containing ml of ampicillin sodium. From these bacterial cells, 24 plasmids were appropriately selected and isolated.
When cut with Hind, three types of plasmids were found.
Severed by Hind. The three plasmids were pTP7-2, pTP7-8 and pTP7-16, respectively.
It was named.

Saito and Miura method (H.Saito, K.Miura;
Biochim, Biophys. Acta, 72, 619 (1963)} from Escherichia coli K-12 C600 strain.
Approximately 1 μg of DNA and pTP7-2 (or pTP7-
8 or pTP7-16) Approximately 1μg of 75μ reaction solution {7mM Tris-HCl buffer (PH7.4), 7mM
MgCl ₂ , 7mM 2-methylcaptoethanol,
Digestion was performed using 5 units of Hind in 60mM NaCl at 37°C for 1 hour. Furthermore, after inactivating Hind by keeping it at 65°C for 5 minutes, keep it on ice.
15μ of 50mM _MgCl2 , 15μ of DTT, 15μ
5mM ATP, 7μ of 1M Tris-HCl buffer (PH 7.4), 23μ of water and 1 unit of T4 DNA ligase were added and reacted at 4°C for 18 hours. This reaction solution was introduced into Escherichia coli K-12 C600 strain. When this treated strain was plated on a nutrient agar medium containing 20 μg/ml ampicillin sodium and 2 μg/ml trimethoprim, pTP7
When using -2 and pTP7-8, growing bacterial cells were obtained, but not with pTP7-16.

When the molecular sizes of pTP7-2 and pTP7-8 were compared by agarose gel electrophoresis, pTP7-8 was found to be smaller. As already stated herein
It is clear that both pTP7-2 and pTP7-8 can be used as promoter detection plasmid vectors.

Both pTP7-2 and pTP7-8 are other than Hind.
It was cleaved at one site each by EcoR, Pst, and Pvu, but not by BamH and Sal. The restriction enzyme cleavage map of pTP7-8 is shown in FIG. pTP7-8 is maintained in a stable state in E. coli, and the E. coli containing pTP7-8 has been deposited with the Institute of Fine Technology as FERM P-8646.

Reference example TP5-3 production method 1 μg of pTP30-5 and 1 unit of restriction enzyme
After cutting with BstE, 2 units of exonuclease BAL31 was applied at 25°C for 30 seconds.
Pst located near the BstE site of pTP30-5
The part was deleted. The deleted DNA is converted into T4-DNA
The Escherichia
coli K12C600 strain according to the method of Norgard et al. The treated bacterial cells were plated on a nutrient agar medium containing 20 μg/ml ampicillin sodium, 10 μg/ml tetracycline hydrochloride, and 5 μg/ml trimethoprim to obtain 50 or more growing bacterial cells.
From these, we randomly selected six plasmids, isolated them, examined the cleavage pattern with the restriction enzyme Pst, and selected those that were cleaved at only one site.Of the six plasmids examined, five had a Pst site at one site. I had one, and the other one had two. One of the five was randomly selected and named pTP5-3.

[Brief explanation of the drawing]

Figure 1 is a restriction enzyme cleavage map of pTP5-3, Figure 2 is a restriction enzyme cleavage map of pTP6-6, and Figure 3 is a restriction enzyme cleavage map of pTP7-8. represents a restriction enzyme,
E is EcoR, H is Hind, B is BamH, S is
Sal and P indicate Pst. Furthermore, the units of numbers are kilobases, and although the circular DNA structure is shown as a straight line for convenience, both ends are originally the same site.

Claims (1)

[Scope of Claims] 1. Provides ampicillin resistance to the host, has an E. coli dihydrofolate reductase gene lacking a promoter, and inserts DNA with promoter activity upstream of the sequence encoding dihydrofolate reductase. 1. A plasmid vector for promoter detection, characterized in that it has a specific restriction enzyme site for the purpose of the present invention and that it imparts trimethoprim resistance to a host by inserting a DNA fragment having promoter activity into the specific restriction enzyme site. 2. The size of the plasmid is approximately 4.3 kilobase pairs, and the cleavage site is a specific restriction enzyme site for inserting a DNA fragment with promoter activity;
The plasmid vector pTP7-8 for promoter detection according to claim 1, which has the restriction enzyme cleavage map shown in the figure below. [Table] 3 From a plasmid vector incorporating the E. coli dihydrofolate reductase gene, its promoter activity is deleted by cutting with a specific restriction enzyme and exonuclease, and then the promoter activity is deleted immediately before the sequence encoding dihydrofolate reductase. 1. A method for producing a plasmid vector for promoter detection, which comprises adding a DNA sequence that is cleaved by a specific restriction enzyme to a plasmid vector.