JPS60126087A - Plasmid ptp18-2, ptp18-4 and ptp18-6 for promoter cloning - Google Patents

Abstract

PURPOSE:To obtain a plasmid to which the promoter DNA can be inserted easily, by creating novel pTP18-2, pTP18-4 and pTP18-6 by changing the cloning site of pTP18-1. CONSTITUTION:The plasmid pTP18-2, pTP18-4 and pTP18-6 has a molecular weight of about 4.9k-base pair, and has a beta-lactamase gene and DHFR gene devoid of transcription translation activity. Since only a specific DNA can be integrated in pTP18-1 owing to the utilization of the incision site with restriction enzyme HindIII as a cloning site, the plasmid is modified to be able to utilize the incision site with restriction enzyme SmaI in a pTP18-2, the incision site with restriction enzyme BamHI in pTP18-4, and the incision site with restriction enzyme SmaI and BamHI in pTP18-6.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides plasmid pT for promoter cloning.
P 18-2. pTP18-4 and pTPl8-6
(Hereafter, p'rp 18=2. p TP
18-4 and pTPl8-6).

The present inventors have been working on improving a plasmid containing the dihydrofolate reductase (DHFR) gene based on a plasmid isolated from E. coli, and have already been able to estimate the intensity of transcriptional translation activity. A plasmid pTP18-1 (hereinafter abbreviated as pTP18-1) suitable for this purpose has been created (Japanese Patent Application No. 58-133769).

In pTP 18-1, the restriction enzyme HindII
[Promoter DNA is ligated to the site that is cut by
It is introduced into the E. coli K12 G600 strain by the transformation method and has the characteristic that the strength of the transcriptional translation activity can be estimated using the strength of trimethoprim resistance or the amount of DHFR produced in the transformed E. coli. However, the promoter DNA insertion site, i.e.
The only cloning site is the restriction enzyme HindIII cleavage site, and for DNA fragments other than those obtained by cleavage with this enzyme, it was necessary to take great care such as connecting a HindIII linker, which is a synthetic NA. Therefore,
We conducted intensive research to create a plasmid that can improve this shortcoming without losing the characteristics of pTPl8-1 and allow for easier insertion of pTPl8-1 and motor DNA.
A new plasmid pTP 182. with a changed cloning site of p 18-1. ' pTP 18-4 and pT
They succeeded in creating Pl8-6.

p'rp 18-2 of the present invention. p'rp18-4 and pTPl8-6 have a molecular weight of approximately 4.9 kilobase pairs and lack the β-lactamase gene and transcriptional translation activity.
These plasmids contain the HFR gene and have the restriction enzyme cleavage maps shown in FIGS. 1, 2, and 3, respectively. E in Figure 1, Figure 2 and Figure 3 is restriction enzyme 1Ec
The cleavage site of oRI, P is the cleavage site of restriction enzyme Pst I, Pvl is the cleavage site of restriction enzyme Pvu I, Pv2 is the cleavage site of restriction enzyme Pvun, A is the cleavage site of restriction enzyme AvaI, H2 is the cleavage site of restriction enzyme HincII. Cutting site, T
indicates the cleavage site of restriction enzyme Taq I, respectively. Further, a means the β-lactamase gene (ampicillin resistance gene), and the arrow indicates the direction of expression. And b means a DHFR gene lacking transcriptional translation activity, and the arrow represents the direction of expression when promoter DNA is introduced.

pTP 18-2 of the present invention. The methods for creating p'rp 18-4 and p'rp 18-6 are as described in the Examples below, but they are novel plasmids. Then, they were introduced into E. coli K12 C600 strains and maintained in a stable state, pTPl, 8-2, and p'
E. coli K12C600 strains containing rp 1B-4 and p'rp 18-6, respectively, were supplied to Kikoken with FERMP-7347, FERMP-7348 and FE, respectively.
It has been deposited as RMP-7349.

The plasmid of the present invention is an improved plasmid of pTPl8-1, which has already been created by the present inventors, and pTPl8
-1 (Japanese Patent Application No. 58-133769), the method for creating each plasmid shown in the Examples and the structure of the plasmid shown in Figures 1, 2, and 3. It's obvious when you see it.

pTPl8-1. In order to utilize the restriction enzyme Hindll cleavage site as a cloning site, a special kDN
A.I.

However, in pTP 18-2, the restriction enzyme Sm was used as a cloning site.
In pTPl8-4, the aI cleavage site was cut using the restriction enzyme BamH.
I cleavage site, and in pTPl8-6, the restriction enzyme Sma
I and BamHI cleavage sites have been modified so that T')NA can bind to the elementary SmaI cleavage site.
Any NA fragment with blunt ends can be ligated using T4 DNA IJ gauze. Restriction enzymes that generate blunt NA ends include AluI, Hae'lu, HincIr, Hp
aI, PvuTI, Sma, 1. .

Bat I,' Dpn I, Hae I, Rr
Many enzymes such as u I are known, and double-stranded DNA can be made blunt-ended by treatment with nuclease BAL31 or DNA polymerase I.

On the other hand, the restriction enzyme BamHI acts on the 5'-G of double-stranded DNA.
↓GATCC-3' is recognized as 3'-αKoshima↑G 5' and cut at ↓, so 5'-G and ↑
3'-CCTAG Generates a DNA end with the 5' end sticking out. However, restriction enzymes that give a cleavage that is complementary to this region are not only BarnHI, but restriction enzymes that recognize 6 bases such as Bgl II and BclI, and restriction enzymes that recognize 4 bases such as Mbo I and Sau 3. AI and the like are known, and have the advantage that various DNA fragments can be used. In addition, in pTP]8-6, the structure of the cloning site is -BamHI-Sma
I -BamHI-, so the restriction enzyme S
It has the advantage that it can be easily excised without cutting the promoter DNA fragment introduced into the maI cleavage site with the restriction enzyme BamHI.

Figure 4 shows pTP18-1, p'rp18-2, pT
The nucleotide sequences near the cloning sites of P18-4 and p'rp 18-6 are shown.

Next, examples of the present invention will be shown.

Example 1 Preparation of pTP18-2 pTP18-1 (described in Japanese Patent Application No. 133769/1982)
Approximately 1 paddle of 1004 reaction solution IC? mM Tris-
HCl, pH 7, 4, 7mM MgClz, 7mM
2-mercaptoethanol (hereinafter referred to as 2ME),
1 unit of restriction enzyme Hi in 6 mM NaCl)
After reacting at 37°C for 2 hours using ndIII, 6
Keep at 5°C for 5 minutes to inactivate HindIII, keep in water (0-4°C), 15#A! 50 mM MgCJ!
□, 15 pH of 0.1M dithiothreitol (hereinafter D
), 15all of 5mM ATP, 7
al IMTris HC4, pH7,4 and phosphorylated adapter pH1nd III/Sma I (d
(pAGCTCCCGGG), sold by Collaborative Inc.] was added, and T4DNA!
Using approximately 0.2 units of gauze, place it in the refrigerator (4 to 1 unit).
The DNA ligation reaction was carried out at 0°C for 18 hours. Take 50 IJl from the reaction solution in which the ligation reaction was carried out, and add 40μ of water and 1 portion of a solution 10 times the concentration of reaction solution ①.
0μ! In addition, 5 units of the restriction enzyme Hindlr were used to react at 37°C for 2 hours.

This reaction was prepared using the method CM, V of Norgard et al.
, Norgard.

K, Keen, J-J-Monahan; Ge
ne, 3.299 (1978)), E.
, was introduced into coli K12 C600 strain.

The treated cells were spread on a nutrient agar medium containing 20 μg/ml ampicillin sodium and incubated at 37°C for 24 hours.
By culturing for hours, more than 100 colonies could be obtained. Choose 8 randomly from these,
Birnboim and Doly method (H, C, Bir
nboim.

L Doly: Nucleic Ac1ds Re
5earch, 7.1513-1524゜(1979
)), isolate the plasmid and use the restriction enzyme Sma I
When examining whether the plasmids were cleaved by Sma I, it was found that all plasmids had one Sma I cleavage point. Select one strain from among these bacteria and
aka and Weisblum's method (T-Tanaka.

B, Weisblum; J, Bacteriol
ogy, 121.354. (1975) ) A plasmid was therefore prepared and named pTP18-2.

p'rp18-2 with restriction enzymes EcoRI, Pst
1. PvuLPvun, Ava I, HincI
1, Taq I and Sma I, and the cleaved fragments were analyzed by agarose gel electrophoresis and polyacrylamide gel electrophoresis.
The structure of 8-2 was estimated.

Example 2 Preparation of p'rp 18-4 Approximately lag of pTP18-2 was added to 200 u/reaction solution 1 [(1
0mM Tris-HCI, pH 8.0, MgCl
2.20 mM KC#, 7mM2-ME)
Reaction was carried out at 37°C for 2 hours using Unit's restriction enzyme Sma I. The reaction was stopped by adding 200 μl of water-saturated phenol, the aqueous layer was separated, and this was
50 mM Tris-HCe at 0+++6.

Dialyzed twice against pH 7.4. After dialysis, add 2 batches of cold ethanol and dilute in dry ice/ethanol for 10 min.
The DNA was precipitated by holding for 1 minute, and the precipitate was collected by centrifugation and dried under vacuum. Dissolve this DNA in 100 pl of water, then add 15 pl of 50 mM
0.1 M DTT in MgCl2.15 pit, 1
5 pi of 5 mM ATP, 7 pl of IMT
ris-HCe, pH 7, 4 and 5ug pBam
Add HI linker (d (p CCGGATCCGG), sold by Collaborative), add 5 units of T4 DNA ligase, and incubate with Bl for 4 hours at 37°C.
An unt end induction reaction was performed. The reaction was stopped by adding 160 μl of water-saturated phenol, and the aqueous layer was diluted with 1001 nl of 50 mM Tris-
Dialyzed twice against HCl, pH 7.4. After dialysis,
DNA was precipitated by adding twice the amount of cold ethanol and keeping it in dry ice/ethanol for 10 minutes, and the precipitate was collected by centrifugation and dried under vacuum.

After dissolving this DNA in 90 μl of water, add 1 of reaction solution I.
10 μe of a solution having a composition of 00 times the amount was added, and the reaction was carried out at 30°C for 2 hours using 5 units of the restriction enzyme Bam'HI, and then the reaction was stopped by keeping at 65°C for 5 minutes. Keep this in water and add 15 pl of 50 mM MgClt,
15 #7 0. IMDTT, 5 mM in 15 all
ATP, 7 pl IMTris-H (J, pH
Add 7,4 and incubate the DNA for 18 hours in the refrigerator (4-10°C) using approximately 0.2 units of T4 DNA ligase.
A ligation reaction was performed. 20 .mu.l of this reaction solution was taken, 70 .mu.l of water and 10 all of a solution having a composition 100 times that of reaction solution 1 were added, and the mixture was reacted at 37.degree. C. for 2 hours using 5 units of restriction enzyme Sma 2. This reaction product is Norg
E. coli K12 according to the method of ard et al.
It was incorporated into C600 strain. The treated bacterial cells are 2
By spreading it on a nutrient agar medium containing 0 μl of Ambici, sodium phosphorus and culturing at 37°C for 24 hours, 100
We were able to obtain more than one colony. Eight of these were selected appropriately, and the plasmid was isolated in the same manner as described in the Example, and the restriction enzyme BamH
When we examined whether they had the I site, all of them had the B site.
It was found that it possesses one amHI cleavage site. Appropriately select one strain from among these eight plasmid-carrying bacteria,
From this, prepare a plasmid and use it as p'rp1
I named it 8-4. The structure of p'rp 18-4 (restriction enzyme cleavage map) was estimated in the same manner as described in Example 1.

The results were as shown in FIG.

Example 3 Preparation of pTP 18-6 Approximately 1 μg of p, TP 18-4 was placed in 10 μl of reaction solution I at 30°C using 1 unit of restriction enzyme BamHI.
After reacting for 2 hours, keep at 65°C for 5 minutes, and incubate with BamHI.
inactivated and kept in water, 15 all of 50 mM
MgCAx, 15 pi of 0. IMDTT, 15#
A' 5 mM ATP, 7 μl IM Tris
HCI, pH 7,4 and phosphorylated adapter pBa
mHI/Sma I (d (p GATCCCC
Approximately 5 μg of GGG) (sold by Collaborative, Inc.) was added, and a DNA ligation reaction was carried out for 18 hours using T 4 DNA ligase (approximately 0.2 units) in a refrigerator. The reactants were prepared according to Example 1, F, coli K12 C
600 to obtain more than 1004 ampicillin-resistant transformants, 16 of these were randomly selected, their plasmids were isolated, and it was examined whether they could be cleaved with SmaI. The plasmid was found to have a Sma I cleavage site. Among these, one strain was appropriately selected, a plasmid was isolated, and this was named pTP18-6. The structure of p'rp 18-6 was estimated in the same manner as described in Example 1.

The results were as shown in FIG.

[Brief explanation of the drawing]

Figure 1 shows a restriction enzyme cleavage map of pTpls-2;
Figure 2 shows 1. The restriction enzyme cleavage map of pTP18-4 and FIG. 3 are the restriction enzyme cleavage maps of p'rp 18-6.
coRI, B is BamHI, H2 is HincIl
, Sm is SmaI, P is PstI, Pvl is Pvu
I, Pv2 stands for P, vu II, A stands for Ava I and T stands for Taq I. The unit of numbers is kilobase pairs. Figure 4 shows part of the nucleotide sequence downstream of the Hind]11 site of pTP18-1 and the p'rp1 corresponding to that site.
8-2, p'rp 18-4 and p'rp 18-6-
The base sequence of Patent applicant: Director of the Agency of Industrial Science and Technology Kawa 10 Hirobe No. 1
Figure 2

Claims (1)

[Scope of Claims] (1) Has a molecular weight of 4.9 kilobase pairs, has a β-lactamase gene and a dihydrofolate reductase gene lacking transcription and translation activity, and has a restriction enzyme cleavage method shown in FIG. Plasmid p'rp for promoter cloning, which has a map and uses a restriction enzyme Sma I cleavage site as a promoter DNA cloning site.
18-2°(2) The molecular weight is 4.9 kilobase pairs, and β
- Contains a lactamase gene and a dihydrofolate reductase gene lacking transcriptional translation activity, and has the restriction enzyme cleavage map shown in Figure 2, and utilizes the restriction enzyme BamHI cleavage site as the cloning site for promoter 1) NA. Promoter cloning plasmid p'rp 18-4゜(3) has a molecular weight of 4.9 kilobase pairs, lacks the β-lactamase gene and transcriptional translation activity (has the dihydrofolate reductase gene) and has the restriction enzyme cleavage map shown in FIG.
Plasmid p' for promoter cloning, characterized in that it utilizes a restriction enzyme BamHI cleavage site and a restriction enzyme SmaI cleavage site as a cloning site for NA.
rp is -6゜(4) Plasmid pTP for promoter cloning
] E containing 8-2, 12 C600 in col 1
KK. (5) Plasmid pTP for promoter cloning
E containing 18-4, colt K12 C600
KK. (6) Plasmid pTP for promoter cloning]
E. coli K12 C600 strain containing 8-6.