FI72344C - Foerfarande foer spjaelkning av dubbelstraengat dna. - Google Patents

Foerfarande foer spjaelkning av dubbelstraengat dna. Download PDF

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FI72344C
FI72344C FI853489A FI853489A FI72344C FI 72344 C FI72344 C FI 72344C FI 853489 A FI853489 A FI 853489A FI 853489 A FI853489 A FI 853489A FI 72344 C FI72344 C FI 72344C
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stranded dna
stranded
dna
double
cleavage site
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Dennis G Kleid
Daniel G Yansura
Herbert L Heyneker
Giuseppe F Miozzari
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Genentech Inc
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C07K14/61—Growth hormone [GH], i.e. somatotropin
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/71—Expression systems using regulatory sequences derived from the trp-operon
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
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    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

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Description

1 72344
Menetelmä kaksijuosteisen DNA:n pilkkomiseksi
Jakamalla erotettu patenttihakemuksesta 810 876 5 Yhdistelmä-DNA-tekniikan menetelmissä olennainen menetelmävaihe on kaksijuosteisen DNA:n pilkkominen. Tällöin tarvitaan usein sellaista pilkkomismenetelmää, jossa DNA saadaan katkaistuksi kontrolloidulla tavalla tietystä kohdasta. Restriktioentsyymit, joita DNA:n pilkkomiseen 10 yleisesti käytetään, pilkkovat DNA:n ainoastaan tietyistä katkaisukohdista (ts. tiettyjen nukleotidisekvenssien kohdalta) , jotka ovat kullekin entsyymille spesifisiä.
Esillä olevan keksinnön mukaisesti on kehitetty menetelmä, jolla kaksijuosteinen DNA voidaan pilkkoa mistä 15 kohdasta tahansa, myöskin siinä tapauksessa, että siinä ei ole minkään restriktioentsyymin katkaisukohtaa, mikä menetelmä on käyttökelpoinen mm. muodostettaessa trp-ope-roneja, joista on poistettu muita attenuaattorialueita kuin tähänastisella mutanttiselektiolla poistetut.
20 Keksinnön mukaista kaksijuosteisen DNA:n pilkkomis menetelmää voidaan soveltaa esim. polypeptidien valmistusmenetelmiin, joissa ekspressoidaan mainittua polypeptidiä koodaava rakennegeeni bakteerissa käyttäen bakteriaalista trp-promoottori-operaattori-järjestelmää (katso FI-patent-25 tihakemus 810876) sekä menetelmään ekspressioplasmin valmistamiseksi, jonka avulla voidaan ekspressoida hetorolo-ginen geeni (katso FI-patenttihakemus 85 3488) .
Keksinnön mukaiselle menetelmälle on tunnusomaista, että menetelmään kuuluu seuraavat vaiheet: 30 a) muutetaan kaksijuosteinen DNA yksijuosteiseksi katkaisukohdan molemmilta puolita; b) hybridisoidaan vaiheessa (a) muodostuneeseen yksijuosteiseen alueeseen yksijuosteisen DNA:n komplementaarinen primeeri, jonka 5'-pää sijaitsee vastapäätä ha-35 lutun katkaisukohdan viereistä nukleotidia; 2 72344 c) muodostetaan uudelleen se osa toisesta juos-teesta, joka on poistettu vaiheessa (a) ja joka sijaitsee 3'-suunnassa mainittuun primeeriin nähden, antamalla vaiheesta (b) saadun tuotteen reagoida DNA-polymeraasin kanssa 5 adeniinia, tymiiniä, guaniinia ja sytosiinia sisältävien deoksinukleotiditrifosfaattien läsnäollessa; ja d) lohkaistaan irti jäljelle jäänyt yksijuosteinen DNA-fragmentti, joka työntyy esiin halutusta katkaisukoh-dasta.
10 Keksinnön mukaisessa menetelmässä kaksijuosteinen DNA muutetaan yksijuosteiseksi halutun katkaisukohdan molemmilta puolilta, esim. antamalla kaksijuosteisen DNA:n reagoida lambda-eksonukleaasin kanssa. Synteettinen tai muu yksijuosteinen DNA-primeeri hybridisoidaan sitten muo-15 dostuneen yksijuosteisen fragmentin kanssa Watson-Crick- emäspariutumisen kautta, jolloin primeerisekvenssin täytyy olla komplementaarinen sen yksijuosteisen DNA:n sukleoti-din kanssa,joka sijaitsee sekvenssissä juuri ennen haluttua katkaisukohtaa. Primeerin 3'-päähän liitetään seuraavaksi 20 se osa alkuperäisestä kaksijuosteisesta DNA:sta, joka sijaitsi halutun katkaisukohdan edellä ja joka poistettiin ensimmäisessä vaiheessa, antamalla primeerin reagoida DNA-polymeraasin kanssa. Samanaikaisesti tai myöhemmin se osa ensimmäisestä juosteesta, joka sijaitsee halutun 25 katkaisukohdan ulkopuolella, poistetaan. Menetelmän periaate ilmenee seuraavasta kaaviosta, jossa "v" merkitsee haluttua katkaisukohtaa: a) ....... v_ haluttu katkaisukohta "v" 30 b) ...... y_ DNA tehdään yksi juosteiseksi "v":n molemmilta puolilta 25 c)...... v_ primeerin hybridisointi ***** * li 3 72344 d) .......v_ ketjua pidennetään primee- ristä alkaen e) .......v__ yksijuosteinen osa poistetaan
Edullisimmassa suoritusmudossa vaiheet (d) ja (e) suoritetaan samanaikaisesti käyttäen polymeraasia, 10 joka samanaikaisesti poistaa esiintyöntyvän yksijuostei- sen pään 3'—^5' -suunnassa ja pidentää primeeriä (dATPrn, dGTP:n, dTTPrn ja dCTP:n läsnäollessa) 5'—>3' -suunnassa. Tähän käytetään edullisesti Klenow-polymeraasia I (fragmentti, joka on valmistettu hajoittamalla proteolyyttises-15 ti DNA-polymeraasi I, joka sisältää alkuperäisen entsyymin polymerisoivan aktiivisuuden 5'—^3' -suunnassa sekä sen eksonukleolyyttisen aktiivisuuden 3'—>5' -suunnassa, mutta josta puuttuu alkuperäisen entsyymin eksonukleolyyt-tinen aktiivisuus 5'—»3' -suunnassa. Katso A. Kornberg, 20 DNA Synthesis 98, W.H. Freeman and Co., SFO (1974).
Edellä kuvatussa menetelmässä attenuaattoridelee-tiot voidaan tehdä millä tavalla tahansa trp-operonin sisältävässä plasmidissa, joka on ensin linearisoitu esimerkiksi katkaisemalla molekyyli restriktiokohdasta, joka 25 sijaitsee alavirtaan pisteestä, josta molekyyli on tarkoitus tehdä tylppäpäiseksi ("v" edellä mainitussa tapauksessa) . Kun heikentäjäalue on poistettu, molekyyli voidaan syklisoida uudelleen esimerkiksi liittämällä tylpät päät yhteen tai muilla asiantuntijalle itsestään selvillä ta-30 voilla.
Seuraava esimerkki havainnollistaa keksinnön mukaisen menetelmän suoritusta käytännössä.
Esimerkki:
Liitteenä olevaan kuvioon viitaten plasmidi pSom7 Δ 2 35 (katso FI-patenttihakemukset 810876 ja 853488) käsiteltiin Hind ΙΙΙ-restriktioentsyymillä ja sen jälkeen lambda-ekso-nukleaasilla (5’-3’-eksonukleaasi) sellaisissa olosuhteissa, 4 72344 joissa plasmidi saatiin hajoitetuksi Bgl II-restriktiokoh-dan yli, joka sijaitsi LE'-sekvenssiä koodaavalla alueella. 20 ng Hind 111:11a hajoitettua pSom7 Δ 2 -plasmidia ‘ -3
liuotettiin puskuriin (20 x 10 -m glysiinipuskuri, pH
-3 -3 5 9,6, joka sisälsi 1 x 10 -m MgC^ ja 1 x 10 -m yS-merkap- toetanolia). Muodostunut seos käsiteltiin 5 yksiköllä lambda-eksonukleaasia 60 minuutin ajan huoneen lämpötilassa. Näin saatu reaktioseos uutettiin fenolilla ja kloroformilla, ja DNA saostettiin etanolilla.
10 Jotta EcoRI-jäännös saataisiin muodostetuksi LE'- geenifragmentin distaaliseen päähän, syntetisoitiin 32 pCCTGTGCATGAT-primeeri parannetulla fosfotriesterime-netelmällä (R. Crea et ai., Proc. Nat'l Acad. Sei. USA 75 (1978) 576) ja se hybridisoitiin LE'-geenifragmentin 15 yksijuosteiseen päähän, joka oli muodostunut käsiteltäes sä plasmidia lambda-eksonukleaasilla. Hybridisointi suoritettiin seuraavaksi kuvatulla tavalla.
20 yug Hind ΙΙΙ-restriktioentsyymillä ja labda-eksonukleaasilla käsiteltyä pSom7 A 2 -plasmidituotetta 20 liuotettiin 20 yal:aan vettä, ja näin saatu liuos yhdistettiin 6 jul:aan liuosta, joka sisälsi noin 80 pikomoolia edellä kuvattua 5'-fosforyloitua oligonukleotidia. Synteettinen fragmentti hybridisoitiin LE'-koodaussekvenssin 3'-päähän, ja LE'-fragmentin jäljelle jäänyt yksijuosteinen 25 osa täytettiin käyttäen Klenow-polymeraasia I, dATP:n, dTTP:n, dGTP:n ja dCTP:n läsnäollessa.
Reaktioseos kuumennettiin 50°C:seen, minkä jälkeen sen annettiin jäähtyä hitaasti 10°C:seen, ja sen jälkeen lisättiin 4 jul Klenow-entsyymiä. Seosta inkuboitiin 15 mi-30 nuuttia huoneen lämpötilassa ja sen jälkeen 30 minuuttia 37°C:ssa, minkä jälkeen reaktio pysäytettiin lisäämällä 5 μΐ 0,25-m EDTA. Reaktioseos uutettiin fenolilla ja kloroformilla, ja DNA saostettiin etanolilla. Sen jälkeen DNA käsiteltiin resktriktioentsyymillä Bgl II. Muodostu-35 neet fragmentit fraktioitiin PAGE:11a. Geelin autoradio- grammi osoitti, että oli muodostunut halutun pituinen noin 32 5 72344 470 emäsparia sisältävä P-merkitty fragmentti, joka erotettiin elektroeluoimalla. Odotetusti tämän fragmentin (LE'(d)) Bgl II-pää ja tylppä pää osuivat primeerin alkukohtaan.
5 Keksinnön mukaista menetelmää voidaan käyttää esim.
FI-patenttihakemuksissa 810876 ja 853488 kuvatulla tavalla ekspressiovektorin muodostamiseen, joka sisältää Le'(d)-fragmentin (29) (katso kuvio) promoottorin (trp-promootto-ri-operaattorin) säätelyn alaisena, johon vektoriin voidaan 10 funktionaalisesti insertoida heterologista polypeptidiä, kuten tymosiini-alfa-1:tä, ihmisen insuliinin esiastetta tai ihmisen insuliinin A- tai B-ketjua koodaava sekvenssi. Muodostunut ekspressiovektori voidaan sitten transformoida sopivaan isäntäorganismiin (E. coli 294) heterolo-15 gisen polypeptidin ekspressoimiseksi.

Claims (3)

1. Menetelmä kaksijuosteisen DNA:n pilkkomiseksi mistä tahansa halutusta kohdasta, tunnettu siitä, 5 että menetelmään kuuluu seuraavat vaiheet: a) muutetaan kaksijuosteinen DNA yksijuosteiseksi katkaisukohdan molemmilta puolilta; b) hybridisoidaan vaiheessa (a) muodostuneeseen yksijuosteiseen alueeseen yksijuosteisen DNA:n komplemen- 10 taarinen primeeri, jonka 5'-pää sijaitsee vastapäätä halutun katkaisukohdan viereistä nukleotidia; c) muodostetaan uudelleen se osa toisesta juos-teesta, joka on poistettu vaiheessa (a) ja joka sijaitsee 3'-suunnassa mainittuun primeeriin nähden, antamalla vai- 15 heesta (b) saadun tuotteen reagoida DNA-polymeraasin kanssa adeniinia, tymiiniä, guaniinia ja sytosiinia sisältävien deoksinukleotiditrifosfaattien läsnäollessa; ja d) lohkaistaan irti jäljelle jäänyt yksijuosteinen DNA-fragmentti, joka työntyy esiin halutusta katkaisukoh- 20 dasta.
2. Patenttivaatimuksen 1 mukainen menetelmä, tunnettu siitä, että vaiheet (c) ja (d) suoritetaan samanaikaisesti antamalla vaiheessa (b) muodostuneen tuotteen reagoida DNA-polymeraasin kanssa, joka polymeri- 25 soi suunnassa 5' —> 3', on eksonukleolyyttinen suunnassa 3'—> 5', mutta ei-eksonukleolyyttinen suunnassa 5'—> 3’.
3. Patenttivaatimuksen 2 mukainen menetelmä, tunnettu siitä, että polymeraasina käytetään Klenow-polymeraasia I.
FI853489A 1980-03-24 1985-09-12 Foerfarande foer spjaelkning av dubbelstraengat dna. FI72344C (fi)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US13329680A 1980-03-24 1980-03-24
US13329680 1980-03-24
FI810876 1981-03-20
FI810876A FI810876A7 (fi) 1980-03-24 1981-03-20 Polypeptidien ekspressoituminen bakteereissa tryptofaanigeenin promoottori/operaattorin avulla

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FI853489A0 FI853489A0 (fi) 1985-09-12
FI853489L FI853489L (fi) 1985-09-12
FI72344B FI72344B (fi) 1987-01-30
FI72344C true FI72344C (fi) 1987-05-11

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FI810876A FI810876A7 (fi) 1980-03-24 1981-03-20 Polypeptidien ekspressoituminen bakteereissa tryptofaanigeenin promoottori/operaattorin avulla
FI853488A FI853488A0 (fi) 1980-03-24 1981-03-20 Foerfarande foer framstaellning av en expressionsplasmid.
FI853489A FI72344C (fi) 1980-03-24 1985-09-12 Foerfarande foer spjaelkning av dubbelstraengat dna.

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FI810876A FI810876A7 (fi) 1980-03-24 1981-03-20 Polypeptidien ekspressoituminen bakteereissa tryptofaanigeenin promoottori/operaattorin avulla
FI853488A FI853488A0 (fi) 1980-03-24 1981-03-20 Foerfarande foer framstaellning av en expressionsplasmid.

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ATE34183T1 (de) 1988-05-15
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OA06774A (fr) 1982-06-30
EP0154133B1 (en) 1990-05-16
EP0154133A3 (en) 1986-05-28
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