JPH0211233B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a plasmid vector for cloning a terminator, which is a gene involved in termination of transcription, namely, terminator cloning plasmid pTP19-1 (hereinafter referred to as pTP19
-1. ). Gene expression generally means that the information stored in DNA is produced as a protein, but in the gene expression process, the information in DNA is first transferred to RNA, and then the RNA is It consists of a two-step process: reading and producing proteins. Although the former is called the transcription stage and the latter the translation stage, it has been revealed that regulation of microbial gene expression is mainly carried out at the transcription stage. Transcription is carried out by RNA polymerase
This is a reaction in which RNA is synthesized using the template as a template.
When we investigate this process, we find that RNA polymerase recognizes a site called a promoter on DNA and creates DNA.
The process of initiation of transcription, which binds to DNA
The process of transcription succession in which RNA polymerase synthesizes RNA according to template DNA by an enzymatic reaction, and
The transcription termination process can be divided into three stages: RNA polymerase recognizes a specific site called a terminator on DNA, stops RNA synthesis, and RNA polymerase leaves the DNA. With recent advances in genetic engineering technology, it is now possible to produce large quantities of the products of specific genes. When the above transcription mechanism is utilized and the transcription initiation reaction is accelerated, specifically by using a strong promoter, a large amount of RNA is produced, and as a result, the target protein is produced. This means that it can be done. Generally, such operations are performed on genes cloned onto plasmids, but when a specific gene on a plasmid is expressed under the control of a strong promoter, sometimes not only the target gene but also its It is known that it also affects the expression of downstream genes, and that the increase in downstream gene products has a negative impact on the survival of the host microorganism, making it impossible to stably produce the product of the target gene. In order to solve this problem, it is considered necessary to ensure the termination of transcription of the target gene and to prevent the expression of downstream genes from being affected. However, to date, research on transcription termination has not made much progress. For this reason, there is a need to develop a technique for efficiently extracting a gene involved in termination of transcription, that is, a terminator. In view of this background, the present inventors conducted extensive research and detected a promoter, a gene involved in the initiation of transcription using the already created dihydrofolate reductase (hereinafter referred to as DHFR) gene. Plasmid vector pTP18-4 for
[Microtechnical Research Institute No. 7348 (see Japanese Patent Application Laid-open No. 126087, 1987)]
By recombining this, we have created a plasmid vector that can efficiently isolate terminators, that is, a plasmid for terminator cloning. The principle of terminator cloning of the present invention is to introduce some kind of promoter upstream of the DHFR gene lacking a promoter, and to create a structure with a specific restriction enzyme site between the introduced promoter and the DHFR gene lacking the promoter. The fusion gene is transcribed from the introduced promoter,
The DHFR gene is transcribed, allowing large amounts of DHFR to be produced. However, when a DNA fragment containing a terminator is introduced into a specific restriction enzyme site between the introduced promoter and the promoterless DHFR gene, transcription from the upstream promoter terminates at the newly introduced terminator. , transcription of the downstream DHFR gene stops occurring and DHFR cannot be produced.
On the other hand, if a DNA fragment that does not contain a terminator or a signal that weakens transcription is introduced into a specific restriction enzyme site, transcription from the upstream promoter will be interrupted by the newly introduced DNA fragment.
Termination does not occur on the DNA fragment, so downstream
The DHFR gene is transcribed, and DHFR is produced in large amounts. In this way, DHFR production can be suppressed only when terminator DNA is introduced, so if there is no DNA insertion,
It can also be clearly distinguished from cases in which a DNA fragment that does not contain a terminator or a transcription attenuation signal is introduced. By the way, as the present inventors have already clarified (Japanese Patent Application No. 143520/1982), the bacterial cells that produce a large amount of DHFR are trimethoprim (hereinafter abbreviated as Tp).
It is possible to develop resistance to these drugs. In addition, the strength of the resistance is determined by the DHFR in the bacterial cells.
content dependent (Iwakura et al; J.
Biochemistry, vol93, pp927 (1983)). Utilizing this property, it is possible to easily select bacterial cells in which terminators have been cloned.
That is, the bacterial cells containing the terminator cloning plasmid of the present invention have a concentration of 7 μg/ml.
Although it can grow in a medium containing Tp, a bacterial cell containing a plasmid in which a terminator has been cloned into this plasmid can no longer grow in a medium containing 7 μg/ml of Tp. Since the terminator cloning plasmid of the present invention has a gene conferring ampicillin resistance as a selection marker, the bacterial cells into which the plasmid has been introduced are obtained as colonies on an agar medium containing ampicillin. About the colony
If a colony is examined to see if it exhibits Tp resistance, a colony that does not exhibit Tp resistance is isolated, and a plasmid is isolated from that bacterial cell, the terminator has been incorporated into that plasmid. Plasmid pTP19-1 for terminator cloning of the present invention (hereinafter abbreviated as pTP19-1)
can confer ampicillin resistance and Tp resistance to the host, has the structure shown in the restriction enzyme cleavage map in Figure 1, and is obtained by in vitro DNA recombination with a size of approximately 5.3 kilobase pairs. This is a new plasmid. And pTP19-1 is
Escherichia coli introduced into Escherichia coli K12C600 strain and maintained in a stable state, containing pTP19-1.
The coli K12C600 strain has been deposited with FERMP-7476 at FEI. The pTP19-1 of the present invention was obtained by introducing the tetracycline resistance gene promoter of the plasmid vector pBR322 into the promoter cloning plasmid pTP18-4 (Japanese Patent Application No. 58-234051), which the present inventors had already created. can be created. As described in Examples 1 and 2, pTP19-1 contains BamH and pTP18-4.
One of the two DNA fragments obtained by Pst cleavage is approximately 4.2 kilobase pairs in size, and the other is approximately 1.1 of the two DNA fragments obtained by BamH and Pst cleavage of plasmid vector pBR322.
It has a structure in which kilobase pair DNA fragments are linked by complementarity at restriction enzyme cleavage sites. Therefore,
pTP19-1 produced according to Example 1,
Among the two DNA fragments obtained by BamH and Pst cleavage of pTP18-4, a DNA fragment with a size of approximately 4.2 kilobase pairs and a plasmid vector
It is obtained by combining two DNA fragments of approximately 1.1 kilobase pairs of DNA fragments obtained by BamH and Pst digestion of pBR322; conversely, pTP19 -1 is
Among the two DNA fragments obtained by BamH and Pst cleavage of pTP18-4, a DNA fragment with a size of approximately 4.2 kilobase pairs and a plasmid vector
Of the two DNA fragments obtained by BamH and Pst digestion of pBR322, approximately 1.1 kilobase pair
It can be obtained by joining DNA fragments through complementarity of restriction enzyme cleavage sites. 2 pieces
Joining of DNA fragments can be performed using T4 DNA ligase. Various types of conjugates may be formed by binding, but only one in which the above two DNAs are properly combined and takes the structure of pTP19-1 can confer resistance to ampicillin and Tp to E. coli. pTP19-1 can be obtained by isolating a recombinant plasmid from Escherichia coli transformed to be resistant to ampicillin and Tp. The only restriction enzyme present in pTP19-1
The BamH site is connected to the introduced promoter.
DHFR lacking the promoter present in pTP18-4
It exists between genes. Escherichia coli K12C600 strain containing pTP19-1 contained 7 μg/ml
It can grow in a medium containing Tp and produces about 0.24 units/mg protein of DHFR, which is about 24 times the content of Escherichia coli K12C600 strain that does not contain a plasmid. Terminator cloning can be performed by normal cloning, using restriction enzymes.
DNA fragment cut by BamH and pTP19
-1 using T4 DNA ligase to create a fusion plasmid, and this
coli K12C600 strain by the usual transformation method, for example, by treating cells in the logarithmic growth phase with CaCl ₂ at low temperature to create cells with the ability to take in DNA, and then introducing the cells with ampicillin. By spreading the plasmid on an agar medium containing the plasmid and culturing it, the bacterial cells that have taken up the plasmid can be isolated as colonies. Save the resulting colony and pick a portion of it with a toothpick to 7 μm.
Transfer to an agar medium containing g/ml of Tp and continue culturing.
Check for growth, and after 24-48 hours, isolate saved colonies corresponding to those that do not yield colonies. As mentioned above, the bacterial cells isolated in this way have DNA containing terminators.
It should contain a fusion plasmid containing the fragment. By isolating a plasmid from this bacterial cell and cleaving it with the restriction enzyme BamH, a DNA fragment containing a terminator can be detected and isolated. In pTP19-1 of the present invention, the restriction enzyme BamH site is used as a cloning site, but according to the method shown by the present inventors (Japanese Patent Application No. 58-234051), the restriction enzyme BamH
Obviously, it is not limited to. In addition, the method for creating a plasmid shown in the present invention,
Those skilled in the art can easily implement the present invention, such as methods for introducing plasmids, based on this specification. Next, examples of the present invention will be shown. Example 1 Preparation of pTP19-1. Approximately 1 μg of pTP18-4 and approximately 1 μg of pBR322 in 100μ
reaction solution (7mM Tris-HCl, PH7.4,
MgCl ₂ , 7mM 2-mercaptoethanol (hereinafter referred to as
Shown as 2ME. ) 6mM NaCl) using 2 units of restriction enzyme BamHI and 2 units of restriction enzyme Pst at 30°C for 2 hours.
Add 300μ cold ethanol to stop the reaction.
After leaving this in dry ice and ethanol for 10 minutes, it was centrifuged at 12,000 rpm for 10 minutes.
The DNA was collected as a precipitate and the supernatant was discarded. After sufficiently removing ethanol in a vacuum desiccator,
100μ of ligase reaction solution (70mM Tvis-
HCl, PH7.4, 7mM MgCl ₂ , 10mM dithiothreitol (hereinafter referred to as DTT), 0.5mM
About 0.5 units of
Add T4 DNA ligase and refrigerate (approximately 4 to 10 minutes).
The DNA ligation reaction was carried out for 18 hours at 10°C.
This reaction product was prepared using the method of Norgard et al. (MV
Norgard, K. Keem, JJ Nonahan; Gene,
vol3, pp299 (1978)), Escherichia
coli K12C600 strain. The treated bacterial cells were treated with 50 μg/ml ampicillin sodium,
and 7 μg/ml of Tp (an agar medium containing 2 g of glucose, 1 g of K ₂ HPO ₄ , 5 g of yeast extract, 5 g of polypeptone, and 15 g of agar) at 37°C. By culturing for 24 hours, we were able to obtain over 500 colonies. 1 from these colonies
Piece selection, Tanaka and Weisblum's method (T.
Tanaka, B. Weisblum; J. Bacteriology,
A plasmid was prepared according to Vol 121, pp 354 (1975) and named pTP19-1. Example 2 Structure of pTP19-1. In order to determine the restriction enzyme cleavage map of pTP19-1 obtained in Example 1, cleavage with the restriction enzymes shown in the table was performed, and this was analyzed by agarose gel electrohydrometry. Using the DNA fragments obtained by this method as molecular size markers, the molecular sizes of the DNA fragments obtained by each cleavage were determined, and the results shown in the table below were obtained.

【table】

[Table] From the results shown in this table, the restriction enzyme cleavage map shown in FIG. 1 was determined. Example 3 Cloning of terminator using pTP19-1. Approximately 3 μg of Escherichia coli K12 chromosomal DNA and
Approximately 1μg of pTP19-1 was added to 100μ reaction solution for 5 minutes.
using the unit restriction enzyme BamH at 30°C.
The cleavage reaction was allowed to occur for 2 hours. After that, 300μ of cold ethanol was added to stop the reaction, and this was left in dry ice and ethanol for 10 minutes.
By centrifuging at 12,000 rpm for 10 minutes,
Collect the DNA as a precipitate and discard the supernatant. After sufficiently removing ethanol in a vacuum desiccator,
The DNA precipitate was dissolved in 100μ of a ligase reaction solution, approximately 0.5 units of T4 DNA ligase was added thereto, and the DNA ligation reaction was carried out in a refrigerator for 18 hours. This reaction product was introduced into Esoheririchia coli K12C600 strain according to the method of Norgard et al. The treated bacterial cells were spread on a nutrient agar medium containing 50 μg/ml ampicillin sodium and cultured at 37° C. for 24 hours to obtain more than 500 colonies. 100 colonies were randomly selected from these colonies, transferred one by one using a toothpick onto a nutrient agar medium containing 50 μg/ml ampicillin sodium and 7 μg/ml Tp, and cultured at 37°C for 24 hours. , 88 pieces are
It could not grow on medium containing Tp. We selected 2 plants that grew on Tp medium and 8 plants that failed to grow, and examined the production amount of DHFR.
The results shown in the table were obtained.

[Table] Strain number 11 is a strain containing pTP19-1, and strain number 12 is a strain that does not contain any plasmid. In addition, the plasmids possessed by strains 1 to 10 were
Birnboim and Doly's method (HC Birnboim, J.
Doly；Nucleic Acids Research, vol7, pp1513
(1979)), and their sizes were examined by agarose gel electroflow method, and it was found that all of the plasmids were larger than approximately 5.3 kilobase pairs. The change in Tp sensitivity in strains 3 to 10 was due to the DNA inserted into the restriction enzyme BamH site of pTP19-1.
This is due to the presence of a transcriptional attenuation signal, which causes DHFR to decrease from 0.0787 in strain 9 to 0.0787 in strain 5.
It was shown that the value was 0.0094, which is 1/3 to 1/24 of the value of 0.240 for the strain containing pTP19-1.

[Brief explanation of drawings]

Figure 1 is a cleavage map of pTP19-1 using restriction enzymes, where the symbols in the figure represent restriction enzymes and E is
EcoR, B is BamH, H is Hind, P is Pst
, A represents Ava, and Pv represents Pvu. Furthermore, the unit of numbers is kilobase pairs.

Claims (1)

[Claims] 1. Consisting of approximately 5.3 kilobase pairs, capable of imparting ampicillin resistance and trimethoprim resistance to the host, and having the restriction enzyme cleavage map shown in the figure below, the ability to impart trimethoprim resistance is reduced. A terminator cloning plasmid that has the characteristic of determining whether or not a terminator exists in the inserted DNA based on whether or not the terminator exists in the inserted DNA
pTP19-1 (in the figure below, E is EcoR, B is
BamH, H is Hind, P is Pst, A is Ava
, Pv indicates Pvu. Also, the unit of numbers is kilobase pairs). 2 Plasmid for promoter cloning
Among the two DNA fragments obtained by BamH and Pst cleavage of pTP18-4, a DNA fragment with a size of approximately 4.2 kilobase pairs and a plasmid vector
A terminator cloning plasmid characterized by joining a DNA fragment approximately 1.1 kilobase pairs in size from two DNA fragments obtained by BamH and Pst cleavage of pBR322.
Method for producing pTP19-1. 3 Plasmid for terminator cloning
Escherichia coli containing pTP19-1
K12C600 shares. 4 Plasmid for terminator cloning
A recombinant plasmid obtained by inserting a heterologous DNA containing a transcriptional attenuation signal into the BamH site of pTP19-1 was introduced into a host strain, Escherichia coli, and transformed into ampicillin-resistant.
A transformed strain of Escherichia coli that does not exhibit trimethoprim resistance is isolated, and a heterologous strain inserted from a recombinant plasmid contained in the isolated transformed strain.
A method for separating DNA fragments containing a transcription attenuation signal, the method comprising separating DNA.