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

Plasmid ptp18-2, ptp18-4 and ptp18-6 for promoter cloning

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Publication number
JPS60126087A
JPS60126087A JP58234051A JP23405183A JPS60126087A JP S60126087 A JPS60126087 A JP S60126087A JP 58234051 A JP58234051 A JP 58234051A JP 23405183 A JP23405183 A JP 23405183A JP S60126087 A JPS60126087 A JP S60126087A
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JP
Japan
Prior art keywords
ptp18
restriction enzyme
plasmid
site
cloning
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
JP58234051A
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Japanese (ja)
Other versions
JPS6335236B2 (en
Inventor
Masahiro Iwakura
正寛 巖倉
Tsukasa Sakai
坂井 士
Keishiro Tsuda
津田 圭四郎
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National Institute of Advanced Industrial Science and Technology AIST
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Agency of Industrial Science and Technology
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Priority to JP58234051A priority Critical patent/JPS60126087A/en
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Granted legal-status Critical Current

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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70Vectors or expression systems specially adapted for E. coli

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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

【発明の詳細な説明】 本発明は、プロモータ・クローニング用プラスミドpT
P 18−2. pTP 18−4及びpTPl8−6
 (以下、それぞれp’rp 18=2. p TP 
18−4及びpTPl8−6と略す)に関するものであ
る。
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).

本発明者らは、E、coliから分離したプラスミドを
基本にして、ジヒドロ葉酸還元酵素(以上DHFRと略
す)遺伝子を持つプラスミドの改良を行っており、すで
に、転写翻訳活性の強度を推定するのに好適なプラスミ
ドpTP18−1(以下pTP18−1と略す)を創成
している(特願昭58−133769号)。
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).

p TP 18−1においては、制限酵素HindII
[によって切断される部位にプロモータDNAを結合し
、組換えプラスミドを作成し、これを宿主菌体であるE
、 coli K12 G600株にトランスホーメシ
ョン法により導入し、形質転換したE、 coliのト
リメトプリム耐性の強さ、もしくは、DHFR生産量を
用いて転写翻訳活性の強度を推定できるという特徴を有
していたが、プロモータDNAの挿入部位、すなわち、
クローニング部位が制限酵素HindIII・切断部位
のみであり、この酵素の切断によって得られるDNA断
片以外は、合成りNAであるHindIIIリンカ−を
接続するなどのくふうをこらす必要があった。そこで、
pTPl8−1の特徴を失なわせずに、この欠点を改良
しより容易にプ占、モータDNAの挿入を行うことがで
きるプラスミドを創成するべく鋭意研究を行い、p’r
p 18−1のクローニング部位を変更した新たなプラ
スミドpTP 182.’ pTP 18−4及びpT
Pl8−6を創成することに成功したのである。
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.

本発明のp’rp 18−2. p’rp 18−4及
びpTPl8−6は、分子量が約4.9キロ塩基対であ
り、β−ラクタマーゼ遺伝子及び転写翻訳活性を欠<D
HFR遺伝子を有し、それぞれ、第1図、第2図及び第
3図に示される制限酵素開裂地図を有するプラスミドで
ある。第1図、第2図及び第3図のEは制限酵素1Ec
oRIの切断部位、Pは制限酵素Pst Iの切断部位
、Pvlは制限酵素Pvu Iの切断部位、Pv2は制
限酵素Pvu nの切断部位、Aは制限酵素AvaIの
切断部位、H2は制限酵素HincIIの切断部位、T
は制限酵素Taq Iの切断部位をそれぞれ示している
。また、aはβ−ラクタマーゼ遺伝子(アンピシリン耐
性遺伝子)を意味し、その矢印は発現方向を現わしてい
る。そして、bは転写翻訳活性を欠<DHFR遺伝子を
意味し、その矢印はプロモータDNAが導入された場合
の発現方向を現わしている。
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.

本発明のp TP 18−2. p’rp 18−4及
びp’rp 18−6の創成方法は後述の実施例に述べ
る通りであるが、それらは新規なプラスミドである。そ
して、それぞれE、 coli K12 C600株に
導入されて安定状態に保たれ、pTPl、8−2、p’
rp 1B−4及びp’rp 18−6をそれぞれ含有
するE、coli K12C600株は、各々機工研に
FERMP−7347、FERMP−7348及びFE
RMP−7349として寄託されている。
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.

本発明のプラスミドは、すでに本発明者らが創成してい
るpTPl8−1の改良プラスミドであり、pTPl8
−1の特長(特願昭58−133769号)をすべて有
していることは実施例に示した各プラスミドの創成方法
及び第1図、第2図及び第3図に示したプラスミドの構
造をみれば明らかである。
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.で(床、クローニング部位として制限
酵素Hindll切断部位を利用するため特別なkDN
AI。
pTPl8-1. In order to utilize the restriction enzyme Hindll cleavage site as a cloning site, a special kDN
A.I.

か組み込めないという難点を有していたので、クローニ
ング部位として、p TP 18−2では制限酵素Sm
aI切断部位を、pTPl8−4では制限酵素BamH
I切断部位を、またpTPl8−6では制限酵素Sma
I及びBamHI切断部位を利用できるように変化させ
たものであ素SmaI切断部位に結合できるT’)NA
断片としては、平滑な末端を有するものならばどんなり
NA断片でも、T4DNAIJガーゼを用いて結合する
ことができる。平滑なりNA末端を生成する制限酵素と
しては、AluI、Hae’lu、HincIr、Hp
a I 、PvuTI、Sma、1.。
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
u Iなど多くの酵素が知られている上に、ヌクレアー
ゼBAL31とか、DNAポリメラーゼIで処理するこ
とにより二本鎖DNAを平滑末端にすることができる。
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.

一方、制限酵素BamHIは、二本鎖DNAの5′−G
↓GATCC−3’を3′−α工嶋↑G 5’ 認識し、↓ の所で切断するため、5′−G と、↑ 
3’−CCTAG 5′末端がつき出た形のDNA末端を生じる。ところが
、この部分と相補性を示すような切断を与える制限酵素
は、BarnHIばかりでなく、6塩基認識の制限酵素
では、Bgl II及びBclIが、4塩基認識の制限
酵素では、Mbo I 、 Sau 3 A Iなどが
知られており、種々のDNA断片を利用することができ
るという利点を有する。また、pTP]8−6において
は、クローニング部位の構造が−BamHI −Sma
 I −BamHI−となっているために、制限酵素S
ma I切断部位に導入したプロモータDNA断片を制
限酵素BamHIで切断す不ことにより容易に切り出す
ことができるという特長を有している。
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.

第4図は、pTP18−1、p’rp 18−2、pT
P18−4及びp’rp 18−6のクローニング部位
近傍の塩基配列を示している。
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.

次に本発明の実施例を示す。Next, examples of the present invention will be shown.

実施例1 pTP18−2の作製 pTP18−1(特願昭58−133769号に記載)
約1櫂を1004の反応液I C? mM Tris−
HCl、 pH7,4,7mM MgC1z、 7mM
2−メルカプトエタノール(以下、2MEと示す) 、
6 mM NaC1)中で、1ユニツトの制限酵素Hi
ndIIIを用いて、37℃ 2時間反応させた後、6
5℃5分間保ち、HindIIIを失活させ、水中に保
ち(0〜4°C)、15#A!の50 mMMgCJ!
□、15 pHの0.1Mジチオトレイト」ル(以下D
TTと示す) 、 15allの5 mM ATP、7
alのIMTris HC4、pH7,4及びリン酸化
アダプターpH1nd III/ Sma I (d 
(p AGCTCCCGGG) 、コラボレイティブ社
で販売しているもの〕を、約5μg加え、T4DNA!
jガーゼ約0.2ユニツトを用いて、冷蔵庫中(4〜1
0°C)で、18時間DNAの連結反応を行わせた。連
結反応を行った反応液から、50IJlを取り、これに
40μノの水及び反応液■の10倍濃度の組成の液を1
0μ!加え、5ユニツトの制限酵素Hindlr[を用
いて、37℃、2時間反応させた。
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.

この反応物を、Norgardらの方法CM 、 V 
、 Norgard。
This reaction was prepared using the method CM, V of Norgard et al.
, Norgard.

K、 Keen、 J−J−Monahan ; Ge
ne 、 3.299 (1978) )に従って、E
、 coli K12 C600株に取り込ませた。
K, Keen, J-J-Monahan; Ge
ne, 3.299 (1978)), E.
, was introduced into coli K12 C600 strain.

この処理をした菌体を20μg/mlのアンピシリンナ
トリウムを含む栄養寒天培地上に広げ、37°Cで24
時間培養することにより、100個以上のコロニーを得
ることができた。これらのうちから、適当に8個選び、
Birnboim及びDolyの方法(H,C,Bir
nboim。
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
))に従ってプラスミドを分離し、制限酵素Sma I
によって切断を受けるかを調べたところ、すべてのプラ
スミドがSma Iの切断点を1ケ所有することが判明
した。これらの菌体のうちから適当に1株選び、Tan
akaとWeisblumの方法(T−Tanaka。
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) )Iこ従
ってプラスミドの調製を行い、これをpTP18−2と
名ずけた。
B, Weisblum; J, Bacteriol
ogy, 121.354. (1975) ) A plasmid was therefore prepared and named pTP18-2.

p’rp 18−2を制限酵素EcoRI 、 Pst
 1. PvuLPvu n、Ava I、HincI
l、Taq I及びSma Iで切断し、アガロースゲ
ル電気泳動及びポリアクリルアミドゲル電気泳動により
切断フラグメントを分析し、第1図に示すp’rp 1
8−2の構造を推定した。
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.

実施例2 p’rp 18−4の作製 pTP18−2約lagを200u/の反応液1[(1
0mMTris −HCI 、 pH8,0、MgCl
2.20 mM KC#、7mM2−ME) 中で、2
ユニツトの制限酵素Sma Iを用いて、37°C2時
間反応させた。反応は、200μlの水飽和フェノール
を加えることにより停止し、水層を分離し、これを10
0+++6の50 mM Tris−HCe。
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.

pH7,4に対して2回透析した。透析後、2製置の冷
エタノ→しを加え、ドライアイス・エタノール中に10
分間保つことによりDNAを沈澱させ、遠心分離により
沈澱を回収し、これを真空下に乾燥した。このDNAを
100plの水に溶かした後、15plの50 mM 
MgCl2.15 pitの0.1 M DTT、 1
5 piの5 mM ATP、 7 plのI M T
ris −HCe、 pH7,4及び5ugのpBam
HIリンカ−(d (p CCGGATCCGG)、コ
ラボレイティブ社で販売〕を加え、さらに5ユニツトの
T4 DNA リガーゼを加え、37℃で4時間、Bl
unt end Iigation反応を行わした。反
応は、160μlの 水飽和フェノールを加え停止させ
、水層を1001nlの50 mM Tr i s −
HCl、 pH7,4に対して2回透析した。透析後、
2倍量の冷エタノールを加え、ドライアイス・エタノー
ル中に10分間保つことIこよりDNAを沈澱させ、遠
心分離により沈澱を回収し、これを真空下に乾燥した。
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.

このDNAを90μlの水に溶かした後、反応液Iの1
00倍量の組成の液を10μe加え、5ユニツトの制限
酵素Bam’HIを用いて、30°C2時間反応させた
後、65℃5分間保つことにより反応を停止した。これ
を水中に保ち、15 plの50 mM MgC1t、
15 #7の0.IMDTT、15 allの5 mM
 A T P、7plのIMTris−H(J、 pH
7,4を加え、T4 DNAリガーゼ約0.2ユニツト
を用いて、冷蔵庫中(4〜10°C)で18時間DNA
の連結反応を行わせた。この反応液から20μlを取り
、70μlの水及び反応液■の100倍量の組成の液を
10 all加え、5ユニツトの制限酵素Sma■を用
いて、37℃2時間反応させた。この反応物をNorg
ardらの方法に従って、E、 col i K 12
 C600株に取り込ませた。この処理をした菌体を2
0μlのアンビシ、リンナトリウムを含む栄養寒天培地
上に広げ、37℃24時間培養することにより、100
個以上のコロニーを得ることができた。これらのうちか
ら適当に8個選び、実施例工に述べである方法と同様に
して、プラスミドを分離し、それが、制限酵素BamH
I部位を有するかについて検討したところ、すべてがB
amHI切断部位を一ケ所有することが判明した。これ
ら8個のプラスミド保持菌のうちから適当に1株選び、
これからプラスミドの調製を行い、これをp’rp 1
8−4と名ずけた。実施例1に述べである方法と同様に
してp’rp 18−4の構造(制限酵素による切断地
図)を推定した。
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.

結果は、第2図に示す通りであった。The results were as shown in FIG.

実施例3 pTP 18−6の作製 p、TP 18−4約1μgを10μlの反応液I中で
、■ユニットの制限酵素BamHIを用いて、30°C
2時間反応させた後、65°C5分間保ち、BamHI
を失活させ、水中に保ち、15 allの50 mM 
MgCAx 、15 piの0.IMDTT、15 #
A’の5 mM ATP、7 μllのIM Tris
 HCI、pH7,4及びリン酸化アダプターp Ba
mHI / Sma I (d (p GATCCCC
GGG)、コラボレイティブ社で販売〕を約5μg加え
、T 4 DNAリガーゼ、約0.2ユニツトを用いて
冷蔵庫中で、18時間DNAの連結反応を行わせた。反
応物を実施例1に従い、F、 col i K12 C
600に取り込ませ、100四以上のアンピシリン耐性
トランスホーマントを得、これらのうち、適当に16個
選び、そのプラスミドを分離し、SmaIによって切断
されるか否かを調べたところ、6株由来のプラスミドが
、Sma I切断部位を有することが判明した。これら
のうち、1株を適当に選び、プラスミドを分離し、これ
をpTP18−6と名ずけた。実施例1に述べである方
法と同様にしてp’rp 18−6の構造を推定した。
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.

結果は、第3図に示す通りであった。The results were as shown in FIG.

【図面の簡単な説明】[Brief explanation of the drawing]

第1図は、pTpls−2の制限酵素による切断地図、
第2図は1. pTP18−4の制限酵素による切断地
図、第3図は、p’rp 18−6の制限酵素による切
断地図であり、図中の符号は制限酵素を表わし、EはE
coRI、 BはBamHI、 H2はHinc Il
、 SmはSmaI、PはPst I、PvlはPvu
I、Pv2はP、vu II、AはAva I及びTは
Taq Iを示す。また数字の単位はキロ塩基対である
。 第4図は、pTP18−1のHind ]11部位下流
の塩基配列の一部と、その部位に対応するp’rp 1
8−2、p’rp 18−4及びp’rp 18−6−
の塩基配列を示す。 特許出願人 工業技術院長 川 10 裕 部第 1 
図 第 2 図
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)

【特許請求の範囲】 (1)分子量が4.9キロ塩基対であり、β−ラクタマ
ーゼ遺伝子及び転写翻訳活性を欠くジヒドロ葉酸還元酵
素遺伝子を有し、かつ、第1図に示される制限酵素開裂
地図を有し、プロモータDNAのクローニング部位とし
て制限酵素Sma I切断部位を利用することを特徴と
するプロモータ・クローニング用プラスミドp’rp 
18−2゜(2)分子量が4.9キロ塩基対であり、β
−ラクタマーゼ遺伝子及び転写翻訳活性を欠くジヒドロ
葉酸還元酵素遺伝子を有し、かつ、第2図に示される制
限酵素開裂地図を有し、プロモータ1) N Aのクロ
ーニング部位として制限酵素BamHI切断部位を利用
することを特徴とするプロモータ・クローニング用プラ
スミドp’rp 18−4゜(3)分子量が4.9キロ
塩基対であり、β−ラクタマーゼ遺伝子及び転写翻訳活
性を欠(ジヒドロ葉酸還元酵素遺伝子を有し、かつ、第
3図に示される制限酵素開裂地図を有し、プロモータD
NAのクローニング部位として制限酵素BamHI切断
部位及び制限酵素Sma I切断部位を利用することを
特徴とするプロモータ・クローニング用プラスミドp’
rp is −6゜ (4)プロモータ・クローニング用プラスミドpTP 
】8−2を含有するE、 col 1に12 C600
株。 (5) プロモータ・クローニング用プラスミドpTP
18−4を含有するE、 colt K12 C600
株。 (6)プロモータ・クローニング用プラスミドpTP]
8−6を含有するE、coli K12 C600株。
[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.
JP58234051A 1983-12-12 1983-12-12 Plasmid ptp18-2, ptp18-4 and ptp18-6 for promoter cloning Granted JPS60126087A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP58234051A JPS60126087A (en) 1983-12-12 1983-12-12 Plasmid ptp18-2, ptp18-4 and ptp18-6 for promoter cloning

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP58234051A JPS60126087A (en) 1983-12-12 1983-12-12 Plasmid ptp18-2, ptp18-4 and ptp18-6 for promoter cloning

Publications (2)

Publication Number Publication Date
JPS60126087A true JPS60126087A (en) 1985-07-05
JPS6335236B2 JPS6335236B2 (en) 1988-07-14

Family

ID=16964798

Family Applications (1)

Application Number Title Priority Date Filing Date
JP58234051A Granted JPS60126087A (en) 1983-12-12 1983-12-12 Plasmid ptp18-2, ptp18-4 and ptp18-6 for promoter cloning

Country Status (1)

Country Link
JP (1) JPS60126087A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6339586A (en) * 1986-05-02 1988-02-20 ギスト ブロカデス ナ−ムロ−ゼ フエンノ−トチヤツプ Secretion signal selection vector for extracellular protein synthesis of bacillus

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6339586A (en) * 1986-05-02 1988-02-20 ギスト ブロカデス ナ−ムロ−ゼ フエンノ−トチヤツプ Secretion signal selection vector for extracellular protein synthesis of bacillus

Also Published As

Publication number Publication date
JPS6335236B2 (en) 1988-07-14

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