JPH0227987A - Cdna to code n-acetylglucosamine (beta1-4) galadctosyltransferase - Google Patents
Cdna to code n-acetylglucosamine (beta1-4) galadctosyltransferaseInfo
- Publication number
- JPH0227987A JPH0227987A JP17634688A JP17634688A JPH0227987A JP H0227987 A JPH0227987 A JP H0227987A JP 17634688 A JP17634688 A JP 17634688A JP 17634688 A JP17634688 A JP 17634688A JP H0227987 A JPH0227987 A JP H0227987A
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- Prior art keywords
- cdna
- beta1
- human
- code
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- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 title claims abstract description 6
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 title claims abstract description 6
- 229950006780 n-acetylglucosamine Drugs 0.000 title claims abstract description 6
- 239000002299 complementary DNA Substances 0.000 claims abstract description 74
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- 108060003306 Galactosyltransferase Proteins 0.000 claims abstract description 4
- 102000004357 Transferases Human genes 0.000 claims 1
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Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明はN−アセチルグルコサミン(β1−4)ガラク
トシルトランスフェラーゼをコードするc DNAに関
する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to cDNA encoding N-acetylglucosamine (β1-4) galactosyltransferase.
はとんどの腫瘍マーカーは正常ではみられない特異構造
の糖鎖をもつか、正常では微量であるが腫瘍になると大
壷に産生される糖鎖構造である。Most tumor markers have specific structures of sugar chains that are not seen in normal cells, or sugar chain structures that are produced in large quantities in tumors, although the amount is small in normal cases.
その構造を合成していくものは糖転移酵素であり、癌化
による腫瘍マーカーの出現は即ち、糖転移酵素の異常発
現によると考えられる。この蛋白は微量であるが故に精
製が困難であり、物質本体の解析が遅れている。これら
の問題の解決には酵素遺伝子を単離し、癌化との関係を
分子生物学的にアプローチすることが急務である。さら
に単離した遺伝子を発現系ベクターに組み換え、酵素蛋
白の活性標品が大量に得られれば、インビ)口(inv
itro)での糖鎖合成技術の開発の糸口になる。The substance that synthesizes this structure is glycosyltransferase, and the appearance of tumor markers due to canceration is thought to be due to abnormal expression of glycosyltransferase. This protein is difficult to purify because it is in such a small amount that analysis of the substance itself has been delayed. To solve these problems, there is an urgent need to isolate enzyme genes and to approach their relationship with canceration from a molecular biological perspective. Furthermore, if the isolated gene is recombined into an expression vector and a large amount of active specimens of the enzyme protein can be obtained, it will be possible to in vitro
This will be the starting point for the development of sugar chain synthesis technology at itro.
本発明の目的は、糖転移酵素の1つであるN−アセチル
グルコサミン(β1−4)ガラクトシルトランスフェラ
ーゼ(以下β1−4GTと略記する)をコードするcD
NAを提供することにある。The object of the present invention is to obtain a cDNA that encodes N-acetylglucosamine (β1-4) galactosyltransferase (hereinafter abbreviated as β1-4GT), which is one of the glycosyltransferases.
The goal is to provide NA.
本発明は第1図に示す塩基配列のうち、376番目のT
から始まり、1197番目のAで終わる塩基配列と86
%以上の相同性を有するり、N Aを含むβ1−4GT
をコードするcDNAに関する。The present invention relates to the 376th T of the base sequence shown in FIG.
The base sequence starting from A and ending at the 1197th A and 86
β1-4GT having homology of % or more or containing NA
Concerning cDNA encoding.
本発明のcDNAの具体例としては以下のものがあるが
、これらに限定されない。Specific examples of the cDNA of the present invention include, but are not limited to, the following.
(i)第1図に示す塩基配列のうち、376番目のTか
ら始まり、1197番目の八で終わる塩基配列を有する
マウスβl−4GTをコードするcDNA。(i) A cDNA encoding mouse βl-4GT having a nucleotide sequence starting from T at position 376 and ending at 8 at position 1197 among the nucleotide sequences shown in FIG.
(ii)第1図に示す塩基配列のうち、1番目のAから
始まり、1197番目のAで終わる塩基配列を有するマ
ウスβ1−4GTをコードするcDNA。(ii) A cDNA encoding mouse β1-4GT having a base sequence starting from the 1st A and ending at the 1197th A among the base sequences shown in FIG.
(iii )第2図に示す塩基配列のうち、373番目
のCから始まり、1194番目のCで終わる塩基配列を
有するヒトβ1−4GTをコードするcDNA。(iii) A cDNA encoding human β1-4GT having a base sequence starting from C at position 373 and ending at C at position 1194 among the base sequences shown in FIG.
(iv)第2図に示す塩基配列のうち、1番目のAから
始まり、1194番目のCで終わる塩基配列を有するヒ
トβl−4GTをコードするc D N A 。(iv) A cDNA encoding human βl-4GT having a base sequence starting from A at position 1 and ending at C at position 1194 among the base sequences shown in FIG.
以下に本発明のcDNAの製造法について説明する。The method for producing cDNA of the present invention will be explained below.
(1)マウスβ1−4GTをコードするc DNAマウ
スのβl−4GTをコードするc DNAは以下のよう
にして単離することができる。(1) cDNA encoding mouse β1-4GT cDNA encoding mouse β1-4GT can be isolated as follows.
マウステラトカルチノーマF9細胞は、イ、ンビトロ(
in vitro)で分化誘導可能な腫瘍細胞株であり
、分化段階に応じてβ1−4GTの酵素活性が変化する
ことが知られている。まず第一段階としてβ9細胞株と
cDNAバンクを、ウシβ1−4GT cDNAをプ
ローブとして用いてスクリーニングする。β9 cD
NAバンクはβ9細胞をレチノイン酸にて分化を誘導し
3kb以上のmRNAを抽出精製し、λgtllファー
ジに組み換えて作成できる。ウシβ1−4GTのORF
部分中の5stI−KpnIフラグメントをプローブと
して、50万個のλgtllファージ組み換え体をスク
リーニングすることにより陽性c DNAクローンを得
ることができる。得られたcDNAクローンが全長cD
NAでない場合には、上記で得られた一部に欠失部分の
有するcDNAクローンの5′末端からSmalのフラ
グメントをプローブとして作成し、同じF 9 c D
NAバンクを再スクリーニングすることにより全長cD
NAを単離することができる。全長cDNAの全塩基配
列は常法(例えば53%を用いたM13法)により決定
できる。Mouse teratocarcinoma F9 cells were grown in vitro (
It is a tumor cell line that can be induced to differentiate in vitro), and it is known that the enzymatic activity of β1-4GT changes depending on the differentiation stage. First, as a first step, β9 cell line and cDNA bank are screened using bovine β1-4GT cDNA as a probe. β9 cD
An NA bank can be created by inducing differentiation of β9 cells with retinoic acid, extracting and purifying mRNA of 3 kb or more, and recombining it with λgtll phage. ORF of bovine β1-4GT
Positive cDNA clones can be obtained by screening 500,000 λgtll phage recombinants using the 5stI-KpnI fragment in the fragment as a probe. The obtained cDNA clone is a full-length cD
If it is not NA, create a Smal fragment as a probe from the 5' end of the cDNA clone with the partially deleted part obtained above, and use the same F 9 c D as a probe.
Full-length cD by rescreening NA banks
NA can be isolated. The entire base sequence of full-length cDNA can be determined by a conventional method (for example, the M13 method using 53%).
(2)ヒトβ1−4GTをコードするc DNAウシβ
1−4GT cDNAをプローブとして、ヒト胎盤由
来cDNAバンクをスクリーニングし、得られる25万
個のλgtl1組み換えファージからヒトβ1−4GT
の全長をコードするcDNAを得ることができる。(2) cDNA bovine β encoding human β1-4GT
A human placenta-derived cDNA bank was screened using 1-4GT cDNA as a probe, and human β1-4GT was selected from the 250,000 λgtl1 recombinant phages obtained.
cDNA encoding the full length of can be obtained.
尚、第1図及び第2図にマウスβl−4GTをコードす
るc DNA及びヒトβ1−4GTをコードするcDN
Aをそれぞれ示す。これらc DNA(マウスβ1−4
GTをコードするc DNAは1〜1197の全長cD
NA、DNAバンクGTをコードするcDNAは1〜1
194の全長cDNA)は、公知の発現系ベクターに組
み換えすることにより、β1−4GTの活性標品を大量
に合成することが可能である。ただし、組み換えによる
β1−4GTの活性標品の合成には上記全長c、 D
N Aの断片cDNAを用いることもできる。例えば、
マウスβ1−4GTをコードするcDNAの場合には、
376番目のTから1197番目のAまでのcDNAを
用いることによってもβ1−4GTの活性標品を得るこ
とができる。同様にしてヒトβ1−4GTをコードする
c DNAの場合には、373番目のCから1194番
目のCまでのcDNAを用いることによりβ1−4GT
の活性標品を得ることができる。Furthermore, Figures 1 and 2 show cDNA encoding mouse β1-4GT and cDNA encoding human β1-4GT.
A is shown respectively. These cDNAs (mouse β1-4
The cDNA encoding GT is a full-length cD from 1 to 1197.
cDNA encoding NA, DNA bank GT is 1-1
By recombining the full-length cDNA of 194) into a known expression system vector, it is possible to synthesize an active preparation of β1-4GT in large quantities. However, for the synthesis of active preparations of β1-4GT by recombination, the above-mentioned full-length c, D
Fragment cDNA of NA can also be used. for example,
In the case of cDNA encoding mouse β1-4GT,
An active preparation of β1-4GT can also be obtained by using cDNA from position 376 T to position 1197 A. Similarly, in the case of cDNA encoding human β1-4GT, by using cDNA from position C 373 to position C 1194, β1-4GT
It is possible to obtain an active preparation of .
尚、断片cDNAはプライマー伸長法、Ba131 (
制限酵素)を用いる方法により得ることができる。In addition, the fragment cDNA was prepared using the primer extension method, Ba131 (
It can be obtained by a method using restriction enzymes).
又、実施例に記載したように、マウスの全長cDNAと
ヒトの全長cDNAは、塩基配列で75%の相同性を有
することから、第1図に示すマウスの1〜1197のc
DNAを塩基配列で75%以上の相同性を有するcDN
Aを前記のような発現系ベクターに組み換えることによ
って、β1−4GTの活性標品を得ることができる。さ
らに、第1図に示すマウスの376〜11g7のcDN
Aと第2図に示すヒトの373〜1194のcDNAと
は、86%の相同性があり、第1図に示すマウスの37
6〜1197のcDNAと80%以上の相同性を有する
cDNAを発現系ベクターに組み換えても、β1−4G
Tの活性標品を得ることができる。In addition, as described in the Examples, mouse full-length cDNA and human full-length cDNA have 75% homology in base sequence.
cDNA that has 75% or more homology to DNA in base sequence
An active preparation of β1-4GT can be obtained by recombining A into an expression system vector as described above. Furthermore, the cDNA of mouse 376-11g7 shown in FIG.
There is 86% homology between A and the human cDNA 373-1194 shown in Figure 2, and the mouse cDNA 373-1194 shown in Figure 1 has 86% homology.
Even if a cDNA with more than 80% homology to cDNA 6-1197 is recombined into an expression system vector, β1-4G
An active preparation of T.
本発明のβ1−4GTをコードするcDNAはβ1−4
GTの活性標品を提供する上で極めて有用であり、β1
−4GTの活性標品を提供することにより糖鎖をインビ
トロで合成することが可能となる。The cDNA encoding β1-4GT of the present invention is β1-4
It is extremely useful in providing an active preparation of GT, and β1
By providing an active preparation of -4GT, it becomes possible to synthesize sugar chains in vitro.
以下本発明を実施例によりさらに説明する。The present invention will be further explained below with reference to Examples.
実施例1
ヒトβ1−40Tをコードする全長cDNAの単離と全
塩基配列及び蛋白−次構造の決定ヒト胎盤より抽出され
たpolyA ”m RN Aよりランダムプライマー
法によりc DNAを0合成し、λgtllファージ組
み込むことにより作成されたcDNAバンクを、ウシβ
1−40TをコードするcDNAをプローブとして、ス
クリーニングした。cDNA内の蛋白コーディング部位
である5stI−KpnIフラグメント(約0.9 k
b)を、”P −d CT Pにてランダムプライマー
法でラベルしプローブとした(第3図)。Example 1 Isolation of full-length cDNA encoding human β1-40T and determination of complete base sequence and protein structure. cDNA was synthesized from polyA''m RNA extracted from human placenta by the random primer method, and λgtll The cDNA bank created by phage integration was
Screening was performed using cDNA encoding 1-40T as a probe. The 5stI-KpnI fragment (approximately 0.9 k
b) was labeled with ``P-dCTP'' using the random primer method and used as a probe (Fig. 3).
c DNAバンク(λgtllファージバンク)の25
0.0OOPFUを宿主菌B、Co11 Y1088に
37℃、15分間吸着させ、軟寒天(0,7%Agar
) L B培地に懸濁した。これを10CIX14C1
1の角型シャーレのLB培地1.0枚に均等にまいた。c DNA bank (λgtll phage bank) 25
0.0 OOPFU was adsorbed onto host bacteria B, Co11 Y1088 at 37°C for 15 minutes, and then transferred to soft agar (0.7% Agar).
) Suspended in LB medium. This is 10CIX14C1
The mixture was evenly spread on 1.0 pieces of LB medium in a square petri dish.
37℃、8時間培養してファージプラークを出現させた
。After culturing at 37°C for 8 hours, phage plaques were allowed to appear.
1MNaC1液にあらかじめ浸しさらに風乾しておいた
ニトロセルロースフィルター(以下NcF)9X13C
1lを、ファージプラーク平板にかぶせ30秒放置する
ことによりファージをNCFにうつしとった。さらに同
じ平板に2枚目のNCFを1分間放置しファージをうつ
しとった。このようにスクリーニングはすべてデュプリ
ケートして行われた。Nitrocellulose filter (hereinafter referred to as NcF) 9X13C soaked in 1M NaCl solution and air-dried
The phage was transferred to NCF by covering 1 liter of the plate on the phage plaque plate and leaving it for 30 seconds. Furthermore, a second sheet of NCF was left on the same plate for 1 minute to transfer the phage. All screenings were performed in duplicate.
NCFをさらに0.5 M NaH,1,5M Na
C1液にて5分間アルカリ処理を施こし、0.5M T
ris−HC1pfl?、5、■、5M NaC1液
にて中和した。風乾後、80℃にてベーキングし、プレ
ハイブリダイゼーションをバッファー組成、ホルムアミ
ド50%、SD31%、デンハルト(口enhardt
)液0.1%、デキストランサルフエイト5%、ポリA
0.05%を使用して、42℃で一昼夜行った。NCF was further diluted with 0.5 M NaH, 1,5 M Na
Perform alkaline treatment with C1 solution for 5 minutes and 0.5M T
ris-HC1pfl? , 5, ■, Neutralized with 5M NaCl solution. After air-drying, it was baked at 80°C, and prehybridization was carried out using a buffer composition of 50% formamide, 31% SD, and Denhardt.
) liquid 0.1%, dextran sulfate 5%, poly A
Using 0.05%, the test was carried out at 42°C overnight.
cDNAプローブの調整;pUc19ベクターにサブク
ローンした後、プラスミドとしてCsC1密度勾配によ
り超遠心機にて精製した。制限酵素で切断後、0.7%
低融点アガロースにて電気泳動分離し、目的のバンドを
切り出しプローブ用DNAとして精製した。約50nH
のプローブDNAをマルチランダムプライマーキット法
(アマ−ジャム社製)をもちいて32pにて標識した。Preparation of cDNA probe: After subcloning into pUc19 vector, it was purified as a plasmid using an ultracentrifuge using a CsC1 density gradient. After cutting with restriction enzyme, 0.7%
Electrophoretic separation was performed using low melting point agarose, and the target band was cut out and purified as probe DNA. Approximately 50nH
The probe DNA was labeled with 32p using the multi-random primer kit method (manufactured by Amerjam).
上記プレハイブリダイゼーション用バッファーと同じ組
成の液にて標識プローブを稀釈してハイブリダイゼーシ
ョンを42℃で一昼夜行った。最終的に55℃、0、2
X 5SPEのストリンゼンシーにてNCFを洗浄後
、オートラジオグラフィーを行い、陽性のクローンを拾
った。2次、3次、4次スクリーニングとファージ数を
順にl/10数に減らしていき、4次スクリーニングで
完全に単一クローンとした。The labeled probe was diluted with a solution having the same composition as the prehybridization buffer described above, and hybridization was performed at 42° C. overnight. Finally 55℃, 0,2
After washing the NCF with X5SPE stringency, autoradiography was performed and positive clones were picked up. The number of phages was sequentially reduced to 1/10 through secondary, tertiary, and 4th screening, and a single clone was obtained in the 4th screening.
λgtllファージのミニプレプ法:Y1088宿主菌
を前夜よりYT培地にておこしておく。菌液1−に、フ
ァージをM、0,1. 0.03〜0.2の範囲で加え
、37℃20分吸着させた後、YT培地を15−加えて
37℃で13〜14時間振とう培養した。Miniprep method for λgtll phage: Incubate Y1088 host bacteria in YT medium the night before. Phage M, 0, 1. After adding in a range of 0.03 to 0.2 and adsorbing at 37°C for 20 minutes, 15-mL of YT medium was added and cultured with shaking at 37°C for 13 to 14 hours.
遠心(3000rpm15分)後、上清をとりDE52
を等量論え30分間回転板にて混合する。遠心(300
0rpm15分)、上清を取った。5MNaCβを17
8容量、冷インプロパツールを27/40容徽加えて混
合し、−20℃で2時間放置した。4℃にて遠心(30
0Grpm15分)後、上清を揄て沈渣のファージを1
mlの70%エタノールに懸濁した後、エッペンドルフ
チューブにうつす。遠心(6000rpm 5分)後、
上清を檜で、沈渣を乾燥し400μl1TEバツフアー
に懸濁する。この後は、フェノール処理2回、フェノー
ル/クロロホルム処理を行いエタノール沈殿によりファ
ージDNAを精製した。After centrifugation (3000 rpm 15 minutes), remove the supernatant and use DE52
Mix in equal amounts on a rotating plate for 30 minutes. Centrifugation (300
0 rpm for 15 minutes), and the supernatant was taken. 5M NaCβ17
8 volumes and 27/40 volumes of cold Improper Tool were added, mixed and left at -20°C for 2 hours. Centrifuge at 4°C (30
After 15 minutes at 0 Grpm, the supernatant was scraped to remove 1 phage from the precipitate.
After suspending in 70% ethanol, transfer to an Eppendorf tube. After centrifugation (6000 rpm 5 minutes),
The supernatant is dried using a hinoki cypress, and the precipitate is dried and suspended in 400 μl of 1TE buffer. After this, phage DNA was purified by ethanol precipitation after phenol treatment twice and phenol/chloroform treatment.
ファージDNAをECoRIで切断後、電気泳動にかけ
挿入された。cDNAの結果を得た。さらにS ac
I %K pn I、SmaIなどの制限酵素を組みあ
わせてこの時点で大まかな制限酵素地図を作成した。こ
の電気泳動ゲルをナイロンフィルターにブロッティング
し、ウシcDNAを各部分とノ\イブリダイズすること
により、18個の各りC−ンの位置関係を確認した。そ
れぞれのクローンを天童培養によりDNAをm製し、E
coRiで切断した後、このEcoRI断片をpUC1
9ベクターにすべてサブクローンした。After cutting the phage DNA with ECoRI, it was subjected to electrophoresis and inserted. cDNA results were obtained. Furthermore, S ac
At this point, a rough restriction enzyme map was created by combining restriction enzymes such as I%KpnI and SmaI. This electrophoresis gel was blotted onto a nylon filter, and bovine cDNA was hybridized with each portion to confirm the positional relationship of each of the 18 C-ons. DNA of each clone was prepared by Tendo culture, and E
After cutting with coRi, this EcoRI fragment was transformed into pUC1
All were subcloned into 9 vectors.
λgillファージよりpUc19プラスミドへのサブ
クローニング;大景精製したファージDNAをEcoR
Iで切断後、LMPアガロース電気泳動にて分離し切り
出し精製した。pUc19プラスミドをEC0RIで切
断、cDNAのEcoRIフラグメントとpUcL9の
DNA量をモル数で合わせ、20μ!容量にて10単位
の74 ’DNAリガーゼの存在下に16℃−昼夜保
温した。前夜より宿主菌HBIOIをおこしておき、使
用2時間前にLB培地にて20倍稀釈し振とう培養する
。Subcloning from λgill phage into pUc19 plasmid;
After cutting with I, it was separated by LMP agarose electrophoresis, cut out and purified. The pUc19 plasmid was cut with EC0RI, the cDNA EcoRI fragment and the pUcL9 DNA amount were combined in terms of moles, and 20μ! The mixture was incubated at 16° C. day and night in the presence of 10 units of 74′ DNA ligase. The host bacteria HBIOI was incubated the night before, and 2 hours before use, it was diluted 20 times with LB medium and cultured with shaking.
その後、遠心3.00 Orpm 15分にて菌をおと
し上清を172量の50mM冷CaC1xといれかえる
。さらに遠心後、上清を1720量の50 mlJ C
aC12に懸濁する。この菌液0.3 Wd2を前述の
DNA液に加え混合後40分間氷上に放置し、42℃
2分間急速に熱を加える。この菌液をLB平板(50μ
g/mlアンピシリン、X−gal 200 D g/
ml、IPTG100μg / dを含む)上にコンラ
ージ棒にて均等にひろげ、−昼夜37℃で培養、出現し
てくるコロニーのうち白色コロニーをひろった。確かに
cDNAを挿入されたか否かを確認のために、標識cD
NAをもちいてコロニーハイブリダイゼーションをも行
った。Thereafter, the bacteria were removed by centrifugation at 3.00 Orpm for 15 minutes, and the supernatant was replaced with 172 volumes of 50 mM cold CaClx. After further centrifugation, the supernatant was diluted with 1,720 ml of 50 ml J C.
Suspend in aC12. Add 0.3 Wd2 of this bacterial solution to the DNA solution mentioned above, mix and leave on ice for 40 minutes, and incubate at 42°C.
Add heat rapidly for 2 minutes. Pour this bacterial solution onto an LB plate (50μ
g/ml Ampicillin, X-gal 200 D g/
ml containing 100 μg/d of IPTG) using a Conlage stick, and cultured at 37° C. day and night. Among the colonies that appeared, white colonies were picked. To confirm whether or not cDNA has been inserted, labeled cD
Colony hybridization was also performed using NA.
pUc19にサブクローンされたcDNAを大量に得る
ために、各プラスミドクローンをもつ菌をそれぞれ11
のLB−アンピシリン培養液にて一昼夜培養し、アルカ
リ法にてプラスミドを精製した。各種制限酵素にて切断
し制限酵素地図を作成した(第4図)。この結果よりM
13ファージに組み換えるプロトコールを検討し塩基配
列のシーフェンスを行った。In order to obtain a large amount of cDNA subcloned into pUc19, 11 strains of each plasmid clone were prepared.
The cells were cultured overnight in LB-ampicillin culture solution, and the plasmid was purified by an alkaline method. A restriction enzyme map was created by cutting with various restriction enzymes (Fig. 4). From this result, M
We investigated the protocol for recombining into phage 13 and conducted a search for the base sequence.
M13ファージジデオキシ法による全塩基配列シークエ
ンス;M13ファージのmp18及びmp19をもちい
た。各RF DNAを組み換えるべき制限酵素部位で
切断した。pUc19に組み込まれたcDNAを目的の
制限酵素部位で切断した。両者をモル比を合わせて混合
し、T4リガーゼにて16℃−昼夜、結合反応を行った
。前述の様に、宿主菌JMIOIにトランスフェクショ
ンし、ソフトアガー及び指示菌JMI 01と混合し、
LB平板(X−ガル、IPTGを含む)上に均一にひろ
げ、37℃−昼夜培養した。Complete base sequence sequencing by M13 phage dideoxy method; M13 phage mp18 and mp19 were used. Each RF DNA was cut at the restriction enzyme site to be recombined. The cDNA incorporated into pUc19 was cut at the desired restriction enzyme site. Both were mixed at the same molar ratio, and a binding reaction was performed at 16° C. day and night using T4 ligase. Transfect host fungus JMIOI as described above, mix with soft agar and indicator fungus JMI 01,
It was spread uniformly on an LB plate (X-gal, containing IPTG) and cultured at 37° C. day and night.
YT培養液5r111に白プラークをひろいあげ、宿主
菌JMI 01を加え8時間振とう培養した。遠心(3
000rpm、i5分)後、上清をとり65℃、15分
の熱処理を加えた。この5μlをフィルターにとり、M
13ファージに目的のcDNAが挿入されているかどう
か確かめるために、標識cDNAにてハイブリダイズを
行った。その結果陽性に出たM13ファージの上清のみ
をシーフェンスにもちいた。White plaques were collected in YT culture solution 5r111, host strain JMI 01 was added, and cultured with shaking for 8 hours. Centrifugation (3
000 rpm, i5 minutes), the supernatant was taken and heat-treated at 65° C. for 15 minutes. Transfer 5 μl of this onto a filter and
In order to confirm whether the cDNA of interest was inserted into the No. 13 phage, hybridization was performed with labeled cDNA. Only the supernatant of the M13 phage that showed a positive result was used for Sea Fence.
上清1rn1に15%ポリエチレンゲルコール2.5M
NaCβ液を250μl加え混合後、氷上に30分放
置し、遠心(15,00Orpm、 15分)する。15% polyethylene gelcol 2.5M to supernatant 1rn1
After adding 250 μl of NaCβ solution and mixing, leave on ice for 30 minutes and centrifuge (15,000 rpm, 15 minutes).
上清を先細のパスツールピペットにて完全に吸い出し、
沈渣のファージを風乾させる。フェノール処理、フェノ
ール/クロロホルム処理、クロロホルム処理後、エタノ
ール沈澱を2回行う。乾燥後に10μlのTEバッファ
ーにとかし、ssDNAとして4℃に保存した。Aspirate the supernatant completely with a tapered Pasteur pipette,
Air-dry the phages in the sediment. After phenol treatment, phenol/chloroform treatment, and chloroform treatment, ethanol precipitation is performed twice. After drying, it was dissolved in 10 μl of TE buffer and stored at 4° C. as ssDNA.
ssD N A 4 u 1 、LO4JJ 1 、ブ
ライ7−(0,5pmol) 1 μl 、T Mバッ
フy (0,1M)リスHClpH75,0,1M
MgCl 2> 1μlを混和し、70℃、10分加
熱し、少くとも30分以上かけてゆっくりと室温にもど
す。遠心して壁面についている水滴を底におとす。2μ
m(20μCi)の[”S〕−d ATPを加えポルテ
ックス後遠心し、氷上に置く。この間に、A、G、C,
1’、それぞれの混合物を4μmずつ分注しておく。最
初のサンプルに1 unit/μlのクレノーフラッグ
メント(Klenowfragment) 1 、!j
1を加えて軽く遠心後に、2.5〜3.0μβをA、
G、C,T混合物のそれぞれに加え軽く遠心で混合して
反応を開始する。室温で20分間放置後、チエイス溶液
(chase 5olution)1μβを加え軽く遠
心し、さらに室温で20分間放置する。スピードバック
(Spaad vac)を用いて完全に乾燥させる。5
μlのストップ溶液 (Stopsolution)を
加えボーテックス (Vortex)にてよく溶かす。ssDN A 4 u 1 , LO4JJ 1 , Bly 7-(0.5 pmol) 1 μl, TM buffer y (0.1M) Lis HCl pH 75.0.1M
Mix 1 μl of MgCl2, heat at 70°C for 10 minutes, and slowly return to room temperature over at least 30 minutes. Centrifuge the water droplets on the wall to the bottom. 2μ
m (20 μCi) of [”S]-d ATP was added, centrifuged after portexing, and placed on ice. During this time, A, G, C,
1', dispense each mixture into 4 μm portions. 1 unit/μl of Klenow fragment in the first sample! j
After adding 1 and centrifuging briefly, add 2.5 to 3.0 μβ to A,
Add to each of the G, C, and T mixtures and mix gently by centrifugation to start the reaction. After being left at room temperature for 20 minutes, 1 μβ of Chase 5 solution was added, briefly centrifuged, and further left at room temperature for 20 minutes. Dry completely using a Spad vac. 5
Add μl of Stop solution and dissolve thoroughly using Vortex.
90℃で2〜3分加熱後、素早く2.5μlをgelに
アプライし電気泳動する。After heating at 90° C. for 2 to 3 minutes, 2.5 μl is quickly applied to gel for electrophoresis.
シークエンスミ気泳動;以下の様に試薬を調整した。(
1)40%アクリルアミドストック溶液;190gアク
リルアミド、10gビスアクリルアミドにdd LOを
加え500dにする。(2)高塩ゲル溶液;50g尿素
、10 gサッカロース、15−40%アクリルアミド
ストック溶液、25rnl 10xTBE、BPB粉末
を少々、以上にdd’ )+20を加え、100rnl
にした後に、フィルター(0,45um)を通し、4℃
に保存、(3)低温ゲル溶液;250g尿素、75rn
140%アクリルアミドストック溶液、25mj!10
xTBE、以上にdd 020を加え、500−にした
後に、フィルター(0,45μm)を通し4℃で保存。Sequence microphoresis: Reagents were prepared as follows. (
1) 40% acrylamide stock solution; add dd LO to 190g acrylamide, 10g bisacrylamide to make 500d. (2) High salt gel solution; 50g urea, 10g sucrose, 15-40% acrylamide stock solution, 25rnl 10xTBE, a little BPB powder, add dd')+20 to the above, 100rnl
After cooling, pass through a filter (0.45 um) and heat at 4°C.
(3) Low temperature gel solution; 250g urea, 75rn
140% acrylamide stock solution, 25mj! 10
Add dd 020 to xTBE to make it 500-, then pass through a filter (0.45 μm) and store at 4°C.
gel 5ize350 X 425 X厚さ0.4
mm (I B I社製装置)のgelを作製した。(
1) 65−の低温ゲル溶液に120μβの25%過硫
酸アンモニウムと70μITEMEDを加える。 (2
) 15 rd高温ゲル溶液に16μm25%過硫酸ア
ンモニウムと20μITEMEDを加える。まず12〜
13FR1の(1)を25dのピペットで吸い上げ、続
いてω)を同量吸い上げる。45℃に傾けたプレートの
端より静かに流し込む。さらに残りの(1]をプレート
の中央より流し込み、プレートをねかせコームを差し込
み、ゲル化するのを待つ。コームをぬき、前述のサンプ
ルをのせ電気泳動を74Wで行った。泳動後、5%メタ
ノール75%酢酸溶液に20〜30分間浸して尿素を除
く。ゲルを3MMペーパーにうつしとりラップをかけて
80℃で乾燥させた後、コダック社製XAR−5フィル
ムに感光させた。gel 5 size 350 x 425 x thickness 0.4
A gel of mm (equipment manufactured by IBI) was prepared. (
1) Add 120 μβ of 25% ammonium persulfate and 70 μ ITEMED to the 65-mL cold gel solution. (2
) Add 16 μm 25% ammonium persulfate and 20 μ ITEMED to the 15 rd hot gel solution. First 12~
Aspirate (1) of 13FR1 with a 25d pipette, and then aspirate the same amount of ω). Pour gently from the edge of a plate tilted at 45°C. Furthermore, pour the remaining (1) from the center of the plate, let the plate rest, insert a comb, and wait for it to gel.The comb was removed, the above-mentioned sample was placed, and electrophoresis was performed at 74W.After electrophoresis, 5% methanol Urea was removed by soaking in a 75% acetic acid solution for 20 to 30 minutes.The gel was transferred onto 3MM paper, wrapped in plastic wrap, dried at 80°C, and then exposed to Kodak XAR-5 film.
以上の方法を用いて、ヒトβ1−4GT cDNAの
全塩基配列を決定した(第2図)。Using the above method, the entire base sequence of human β1-4GT cDNA was determined (Figure 2).
実施例2
造の決定
ウシβl−4GT cDNAクローンの5stI−K
pn IフラグメントがF 9 c DNAバンクの
スクリーニングにプローブとして用いられた。方法につ
いては実施例1のヒトc DNAバンクのスクリーニン
グとまったく同様である。Example 2 Determination of the structure of bovine βl-4GT cDNA clone 5stI-K
The pn I fragment was used as a probe to screen the F9c DNA bank. The method is exactly the same as the screening of human cDNA bank in Example 1.
最初のスクリーニングで、5GT個のファージ数から3
個の陽性クローンを得た。前述の如く制限酵素地図を作
成した。このうち、2個は同じ地図を示し、長さもほと
んど同じであった(λmGT−10と命名)が、1個p
mG T −2は一部異なる地図を示した。前者のうち
最も長いクローンでも全長をコードしていないことが判
明したので、5′末端のEcoRI −SmaIフラグ
メントを切り出し精製し、同じF9 cDNAバンク
を再スクリーニングした。5GT個のファージのうち、
4個の陽性クローンを得た後、型通り制限酵素地図の作
成、及び全長cDNAであるヒトβ1−4 GTc D
N Aの各部分とハイブリダイズを行い、全長をコー
ドすると思われるcDNAクローン(λmGT−5)の
塩基配列決定を行った(第1図)。In the initial screening, 3 out of 5 GT phages were selected.
Positive clones were obtained. A restriction enzyme map was created as described above. Of these, two showed the same map and were almost the same length (named λmGT-10), but one
mG T-2 showed a partially different map. Since it was found that even the longest clone among the former clones did not encode the full length, the 5'-end EcoRI-SmaI fragment was excised and purified, and the same F9 cDNA bank was rescreened. Among the 5 GT phages,
After obtaining four positive clones, a restriction enzyme map was created as usual, and the full-length cDNA human β1-4 GTc D
Hybridization was performed with each part of NA, and the nucleotide sequence of a cDNA clone (λmGT-5) that was thought to encode the full length was determined (Fig. 1).
実施例2のマウス全長βl−4GT[クローン(pmG
T −5)Eの塩基配列(1〜1197)を実施例1
のヒト全長β1−4GT cDNA (1〜1194
)と比較した結果では、塩基配列で約75%、アミノ酸
配列で約83%の相同性があった。さらにC末端側20
0個のアミノ酸はきわめて相同性が高く(約90%)、
マウスβ1−4GTの376〜1197の塩基配列とヒ
トβ1−4CTの373〜1194の塩基配列とは約8
0%の相同性があった。Mouse full-length βl-4GT [clone (pmG
T-5) The base sequence (1 to 1197) of E is shown in Example 1.
human full-length β1-4GT cDNA (1-1194
), the nucleotide sequence had approximately 75% homology and the amino acid sequence had approximately 83% homology. Furthermore, C-terminal side 20
0 amino acids have extremely high homology (approximately 90%);
The 376-1197 base sequence of mouse β1-4GT and the 373-1194 base sequence of human β1-4CT are approximately 8
There was 0% homology.
チョウ−77ス−v :/ (Chow−Fasman
)のプログラムを用いて蛋白二次構造を計算したが、相
同性の高い部分はα−ヘリックス、β−シートに富み、
逆に、低い部分は、いわゆるランダムコイル構造をとる
結果を得た。マウスβl−4GTもやはりN末端に18
個の疎水性アミノ酸よりなるアンカ一部をもち、開始コ
ドン、停止コドンともヒトcDNAと同じ位置にあった
が途中、ヒトと相同性の少ない部分に1個のアミノ酸の
インサージョンがあり、全アミノ酸敗400個であり、
計算されるM、W、 は48KDaである。このこと
からも、異種間のアミノ酸配列で相同性の低い部分は、
酵素の基質特異性、活性中心部位とは関係のない部位と
思われる(第5図)。Chow-77su-v :/ (Chow-Fasman
) program was used to calculate the protein secondary structure, and the highly homologous regions were rich in α-helices and β-sheets;
On the other hand, the lower part had a so-called random coil structure. Mouse βl-4GT also has 18 at the N-terminus.
The start codon and stop codon were both at the same positions as human cDNA, but there was an insertion of one amino acid in a part with little homology to humans, and all amino acids 400 losses,
The calculated M, W, is 48 KDa. From this, it can be seen that parts of amino acid sequences with low homology between different species are
This site seems to be unrelated to the enzyme's substrate specificity and active center site (Figure 5).
第1図はマウスβ1−4GTをコードするcONAの塩
基配列及びアミノ酸配列、第2図はヒトβ1−40Tと
コードするcDNAの塩基配列及びアミノ酸配列、第3
図はウシβ1−4GTcDNAの制限酵素地図、第4図
はヒトβ1−4GTcDNAの制限酵素地図、第5図は
ヒト及びマウスβ1−4GTのアミノ酸配列の相同性を
示す。Figure 1 shows the nucleotide sequence and amino acid sequence of cONA encoding mouse β1-4GT, Figure 2 shows the nucleotide sequence and amino acid sequence of cDNA encoding human β1-40T, and Figure 3
The figure shows a restriction enzyme map of bovine β1-4GT cDNA, FIG. 4 shows a restriction enzyme map of human β1-4GT cDNA, and FIG. 5 shows the homology between the amino acid sequences of human and mouse β1-4GT.
Claims (2)
ら始まり、1197番目のAで終わる塩基配列と86%
以上の相同性を有するDNAを含むN−アセチルグルコ
サミン(β1−4)ガラクトシルトランスフェラーゼを
コードするcDNA。(1) Of the base sequences shown in Figure 1, 86% of the base sequences start from T at position 376 and end at A at position 1197.
A cDNA encoding N-acetylglucosamine (β1-4) galactosyltransferase containing DNA having the above homology.
まり、1197番目のAで終わる塩基配列と75%以上
の相同性を有するDNAを含むN−アセチルグルコサミ
ン(β1−4)ガラクトシルトランスフェラーゼをコー
ドするcDNA。(2) Among the base sequences shown in Figure 1, N-acetylglucosamine (β1-4) galactosyl contains DNA that has 75% or more homology with the base sequence starting from the 1st A and ending with the 1197th A. cDNA encoding transferase.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP17634688A JPH0227987A (en) | 1988-07-15 | 1988-07-15 | Cdna to code n-acetylglucosamine (beta1-4) galadctosyltransferase |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP17634688A JPH0227987A (en) | 1988-07-15 | 1988-07-15 | Cdna to code n-acetylglucosamine (beta1-4) galadctosyltransferase |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH0227987A true JPH0227987A (en) | 1990-01-30 |
Family
ID=16011990
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP17634688A Pending JPH0227987A (en) | 1988-07-15 | 1988-07-15 | Cdna to code n-acetylglucosamine (beta1-4) galadctosyltransferase |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0227987A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999040205A1 (en) * | 1998-02-04 | 1999-08-12 | Kyowa Hakko Kogyo Co., Ltd. | Glycosyltransferase and dna encoding the same |
-
1988
- 1988-07-15 JP JP17634688A patent/JPH0227987A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999040205A1 (en) * | 1998-02-04 | 1999-08-12 | Kyowa Hakko Kogyo Co., Ltd. | Glycosyltransferase and dna encoding the same |
US6475761B1 (en) | 1998-02-04 | 2002-11-05 | Kyowa Hakko Kogyo Co., Ltd. | Glycosyltransferase and DNA encoding the same |
US6830908B2 (en) | 1998-02-04 | 2004-12-14 | Kyowa Hakko Kogyo Co., Ltd. | Glycosyltransferase and DNA encoding the same |
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