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Wat is 'n goeie eenvoudige toets vir cDNA-biblioteekkwaliteit?

Wat is 'n goeie eenvoudige toets vir cDNA-biblioteekkwaliteit?



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Hoe beoordeel ek die kwaliteit van 'n cDNA -biblioteek?

Ek wil CDS-kopieë van gene uit 'n biblioteek kloon, maar ek weet nie wat 'n tipiese verwagting is om 'n volle lengte kloon te kry selfs vir 'n korter geen (~1000 bp). Ek weet dat dit nie vir die volgorde van cDNA uit 'n volledige lengte bestaan ​​nie, maar ek dink dat die kwaliteit van die beginmateriaal ook belangrik is.

Is daar 'n paar algemene transkripsies wat ek van PCR kan probeer verkry wat my sal vertel hoe gefragmenteerd die rye daarin is?

Wat is 'n tipiese resultaat van 'n kommersiële cDNA-biblioteek - is daar enige QC-toetse wat vinnig in die laboratorium uitgevoer kan word?


Aan die gang met RNA-volgorde (RNA-sekwensie)

Standaardmetodes vir die voorbereiding van RNA -biblioteke behou nie inligting oor die DNA -string waaruit die RNA -string getranskribeer is nie. Die vermoë om inligting oor die oorsprongstreng te bekom, is om baie redes nuttig, insluitend die identifisering van antisense-transkripsies, bepaling van die getranskribeerde string van nie-kodende RNA's en bepaling van die uitdrukkingsvlakke van koderende of nie-koderende oorvleuelende transkripsies. In die algemeen kan die vermoë om die oorsprongstreng te bepaal, die waarde van 'n RNA-seq-eksperiment aansienlik verhoog.

Selfs buite die besluit om rigting of nie-rigtinggewende biblioteekvoorbereiding uit te voer, kan verskeie metodes gebruik word om 'n RNA-seq-biblioteek te genereer, en die besonderhede van hierdie metodes is afhanklik van die platform wat gebruik word vir die volgorde van hoë deurset. Daar is egter algemene stappe.

  • Oorvloedige verwydering van transkripsie: Die meerderheid RNA -molekules wat in 'n sel voorkom, is ribosomaal RNA (rRNA), en aangesien dit oor die algemeen nie van belang is nie, moet dit verwyder word voordat 'n biblioteek gemaak word uit die RNA van belang. Net so word globien -RNA gewoonlik uit bloedmonsters verwyder en chloroplast -RNA word dikwels uit plantblaarmonsters verwyder. Twee gewilde opsies vir hierdie stap is:
  • Fragmentasie: Fragmente van 'n gepaste grootte vir volgordebepaling word gegenereer deur fragmentasie van RNA voor omgekeerde transkripsie en cDNA -sintese, eerder as deur fragmentasie van cDNA.
  • Omgekeerde transkripsie en tweede-string cDNA sintese: Aanvullende DNA (cDNA) word gegenereer uit die RNA -sjabloon deur 'n omgekeerde transkriptase. Hierdie eerste string cDNA word dan dubbelstrengs gemaak deur 'n DNA-polimerase te gebruik.
  • Eindherstel, dA-Tailing en Adapter Ligation: Die einde van die herstel van die ds cDNA-biblioteek en die opsionele dA-tailing (afhangende van die volgordeplatform wat gebruik gaan word) word gevolg deur ligering aan adapters. Die biblioteek is dan gereed vir versterking en volgordebepaling.


Belangrike faktore om in ag te neem by die uitvoering van RNA-seq
Biblioteekvoorbereiding is 'n belangrike deel van die RNA-seq-werkvloei en metodes is tans beskikbaar vir biblioteekvoorbereiding vir RNA-seq wat vereenvoudigde protokolle en verbeterde opbrengste bied. Die kwaliteit en akkurate kwantifisering van inset -RNA bly egter steeds van kritieke belang om suksesvolle cDNA -sintese en biblioteke te verseker. Die volgende is 'n paar belangrike faktore om te oorweeg:


INLEIDING

Identifikasie en isolasie van cDNA -klone is belangrik vir die interpretasie en funksionele ondersoek van genome in volgorde. cDNA -volgorde -inligting vergemaklik die identifisering van eksone en dus die definisie van die koderingspotensiaal van gene. cDNA-klone verskaf probes vir 'n verskeidenheid studies van genomiese uitdrukking, en klone wat volle oop leesrame (ORF's) bevat, laat funksionele ondersoek van hul proteïenprodukte toe. Die Mammalian Gene Collection-projek (MGC) is opgestel as 'n publieke domein-program met die doel om vollengte cDNA-volgorde-inligting en fisiese klone vir alle gene te verskaf, begin met die mens- en muisgenome (1). Die Riken Instituut (2) en 'n aantal private sektor groepe het ook groot programme wat daarop gemik is om vollengte klone te versamel. Aanvanklik is cDNA -biblioteke in die MGC uit verskillende sellyne en weefsels voorberei. Klone wat ewekansig uit hierdie biblioteke gekies is, was eindvolgorde en unieke klone wat vermoedelik volledige ORF's bevat, is volledig op volgorde geplaas en beskikbaar gestel vir publieke verspreiding. Die MGC was baie suksesvol. Vanaf Oktober 2004 is gerapporteer dat gt12 000 menslike en 10 000 muisunieke, volledige lengte-klone in volgorde geplaas en versprei word, en 'n bykomende 3000 klone van elke spesie wat geëvalueer word as waarskynlik volledige ORF's bevat, is in die volgordepyplyn (3) (http://mgc.nci.nih.gov/). Soos aanvanklik voorspel is, het die opbrengs van nuwe klone egter afgeneem namate die projek vorentoe beweeg het. Gene wat nie deur geselekteerde cDNA -klone voorgestel word nie, kan lae uitdrukkingsvlakke hê en kan dus seldsame transkripsies wees. Verskeie benaderings tot isolasie van skaars klone word gebruik. Die een is generasie en willekeurige volgorde van afgetrokke cDNA -biblioteke. Nog 'n ander is voorspelling van mRNA-volgordes gebaseer op genomiese volgorde, en amplifikasie van hierdie spesifieke volgordes vanaf cDNA met behulp van die PCR. 'N Derde benadering is gebaseer op die idee dat transkripsies wat skaars is in cDNA -biblioteke wat uit beskikbare sellyne en maklik verkrygbare weefsels bestaan, teenwoordig kan wees in 'n veel groter relatiewe oorvloed in spesifieke weefsels wat slegs in klein hoeveelhede verkry kan word. In die brein is daar byvoorbeeld bekende gevalle van gene wat sterk uitgedruk word in 'n relatief klein aantal neurone in hoogs diskrete streke. Daar is byvoorbeeld ongeveer 10 000 vasopressienbevattende selle in die rot paraventrikulêre kern (verdeel tussen twee hoofseltipes) en 7000 in suprachiasmatiese kern (4, 5). 'n Mikrodisseksie wat uiteindelik 'n baie klein hoeveelheid RNA sou oplewer, is nodig om 'n monster te verkry wat in hierdie selle verryk is. Metodes van cDNA-biblioteekproduksie wat effektief is met baie klein hoeveelhede beginmateriaal is dus belangrik om biblioteke te verkry met beduidende voorstelling van hierdie tipe skaars transkripsies.

Die meeste prosedures vir die opwekking van cDNA-biblioteke gebruik 'n oligonukleotied wat 'n kort poli (dT) ry bevat as die onderlaag in 'n omgekeerde transkripsiereaksie wat gesuiwerde poli-gedenileerde RNA as sjabloon gebruik. Na 'n tweede-streng vervangingsreaksie, word sintetiese oligonukleotied-skakelers of -adapters aan die dubbelstrengs cDNA's geligeer. Hierdie produkte, in baie gevalle na restriksie-ensiemvertering, word aan 'n toepaslik voorbereide vektor geligeer. Die adaptor/linker ligasie is 'n relatief ondoeltreffende bi-molekulêre stomp-end ligasie. Dit word uitgevoer met 'n oormaat adapter/skakelaar, wat uit die cDNA-produk verwyder moet word voordat dit in die vektor ingebring kan word. Hierdie suiweringstap lei tot materiaalverlies. Die daaropvolgende ligering van die cDNA in vektor, selfs in die geval wanneer 'taai punte' geskep word, is 'n bimolekulêre reaksie met beperkte doeltreffendheid. Deur die oligo-dT-primingsekwensie in die vektor op te neem om 'n 'vectorprimer' te produseer, word een of albei die ligeringsreaksies en die suiweringstap uitgeskakel. Okayama (6) het 'n hoogs doeltreffende vektor-primer-gebaseerde cDNA-biblioteekkonstruksieprosedure beskryf in 1982. Hierdie metode is nie algemeen aangeneem nie, deels omdat dit tegnies veeleisend is. Eenvoudiger vektor-primer metodes is voorheen beskryf (7, 8), maar dit word ook nie algemeen gebruik nie. Miskien is dit te wyte aan die feit dat sommige tegniese beperkings nie voldoende aangespreek is nie, of dat kommersiële kits wat oligonukleotied -aanvangsmetodes gebruik, wyd beskikbaar en maklik om te gebruik geword het. Ons het teruggekeer na 'n vektor-primer metode om cDNA biblioteke uit baie klein hoeveelhede weefsel te genereer. Ons bied nou die evaluering van verskeie cDNA-biblioteke aan wat voorberei is uit mikrogram hoeveelhede totale RNA.


Wenke vir twee-hibriede sifting

Minimaliseer vals positiewe:

  • Begin herhalings van die eksperiment om die waarskynlikheid van onoordeelkundige aktivering van die verslaggewer se gene te verminder. Deur 'n uitsluitlike prooi-beheer in te sluit, bied dit ook 'n basiese vlak van verslaggeweraktivering.
  • Varieer die uitdrukkingsvlakke van aas- en prooi -proteïene - ooruitdrukking kan tot vals interaksies lei. Verlaagde uitdrukkingsvlakke verhoog die stringensie van u binding.
  • En die belangrikste: Bekragtig geïdentifiseerde bindingsvennote onafhanklik met ander tegnieke!

Minimaliseer vals negatiewe:

Interaksies wat lei tot waarneembare verslaggewerseine hang af van 'n aantal faktore, insluitend proteïenuitdrukking en korrekte vou, post-translasiemodifikasie, proteïenafbreking, toegang tot die kern in eukariotiese skerms en samesmeltingskonfigurasie. Die moontlikheid van probleme met een of meer van hierdie parameters kan lei tot 'n groot aantal vals negatiewe in Y2H -skerms.

  • Om jou verslaggewerstelsel te toets met 'n paar proteïene wat bekend is om te bind, dien as 'n goeie positiewe beheer om te verseker dat jou opstelling werk. Die laaste ding wat jy wil hê, is om jou pragtige biblioteekskerm te laat loop net om te sien dat die stelsel self nie werk nie omdat jou verslaggewerproteïen nie in gis funksioneer nie!
  • Baie vals negatiewe spruit voort uit of word vererger deur uitdrukking van teikenproteïene in heteroloë sisteme (bv. soogdierproteïene in S. cerevisiae).
    • Alhoewel dit moeilik kan wees om op te los vir 'n wye reeks prooiproteïene, is daar nou 'n aantal twee-hibriede stelsels beskikbaar in model organismes, insluitend bakterieë, alternatiewe swamme ( C. albicans , pC2HB ) (7) en soogdierselle.
    • As die probleem is dat u proteïene nie na-translasioneel aangepas is nie, kan mede-uitdrukking van die ensiem wat verantwoordelik is vir die verandering in die assay-gasheer stam help.
    • Om die kans te verminder dat die opset van u samesmeltingsproteïene die bindingsplekke vir die proteïenvennoot of die UAS/verslaggewer-gene fisies blokkeer, kan dieselfde aas- en prooi-biblioteke gekeur word met beide N- en C-terminale samesmeltings van hierdie proteïene . Op hierdie manier word albei 'punte' van jou proteïen gekeur vir binding.
    • Deur 'n verskeidenheid uitdrukkingsvlakke en -stelsels te gebruik, kan vals negatiewe verlig word as gevolg van 'n lae of foutiewe uitdrukking van u teikenproteïene. 'N Goeie algemene strategie om vals negatiewe te verminder en 'n meer kragtige skerm te produseer, is om verskillende aas- en prooivektore te gebruik. Dit is bewys dat dit net so effektief is as die gebruik van vyf onafhanklike metodes om proteïeninteraksie op te spoor (6).
    • Om DNA te bind en 'n verslaggewergeen in eukariotiese selle te aktiveer, moet fusieproteïene toegang tot die kern verkry. Om hierdie vereiste vir transmembraanproteïene te omseil, is 'n gesplete ubiquitin-stelsel bedink (8).

    1. Casadaban M.J., Martinez-Arias A., Shapira S.K., Chou J. Beta-galaktosidase-genfusies vir die ontleding van geenuitdrukking in Escherichia coli en gis. Metodes Ensiemol. 1983100: 293-308. PubMed PMID: 6312261.

    2. Keegan, L., Gill, G., en Ptashne, M. Skeiding van DNA-binding van die transkripsie-aktiverende funksie van 'n eukariotiese regulatoriese proteïen. Wetenskap. 1986 231:699-704. PubMed PMID: 3080805.

    3. Brent, R., en Ptashne, M. 'n Eukariotiese transkripsie-aktiveerder wat die DNA-spesifisiteit van 'n prokariotiese onderdrukker dra. Sel. 1985 43: 729-736. PubMed PMID: 3907859.

    4. Fields, S., Song, O. 'n Nuwe genetiese stelsel om proteïen-proteïeninteraksies op te spoor. Natuur. 1989 340: 245-246. PubMed PMID: 2547163.

    5. Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., Umezawa, Y.l. Gesplete luciferase as 'n optiese sonde vir die opsporing van proteïen-proteïen-interaksies in soogdierselle gebaseer op proteïensplywing. Anale Chem. 2001 73: 2516-2521. Gepubliseerde PMID: 11403293.

    6. Chen, Y.C., Rajagopala S.V., Stellberger T., Uetz P. Uitputtende maatstaf van die gis-tweebasterstelsel. Natuurmetodes. 2010 7: 667-668. Gepubliseer PMID: 20805792.

    7. Stynen B., Van Dijck P., Tournu H. 'n CUG-kodon aangepaste twee-baster sisteem vir die patogene swam Candida albicans. Nukleïensure Res. 201038(19):e184. Pubmed PMID: 20719741. Pubmed Central PMCID: PMC2965261.


    Wat is 'n goeie eenvoudige toets vir cDNA-biblioteekkwaliteit? - Biologie

    Illumina bied drie benaderings aan om navorsers te help met die volgorde van SARS-CoV-2. Volgende generasie volgordebepaling (NGS) bied 'n effektiewe manier om monsters te toets en virusse te karakteriseer sonder vooraf kennis van die aansteeklike middel. Die Illumina SARS-CoV-2-werkstrome wat hieronder beskryf word, is nie bedoel vir diagnostiese doeleindes nie (hierdie bulletin handel nie oor die COVIDSeq-werkstroom nie). Aansoeknotas wat gedetailleerde inligting en voorbeeldresultate bevat, is ingesluit vir elke werkstroom hieronder.

    Totale RNA-volgordebepaling met die TruSeq™ Stranded Total RNA Gold-biblioteekvoorbereidingswerkvloei

    • Omvattende werkstroom vir die opsporing van koronavirus met Illumina tafelbladstelsels se aansoeknota.
    • Voorbeeldvoorbereidingswerkstroom:
      • Virale RNA word onttrek met die QIAGEN QIAmp Viral Mini Kit (Katalogus No. 52904).
      • Biblioteke word voorberei met TruSeq Stranded Total RNA Library Prep Gold-kit (Illumina, kit met 48 monsters, katalogusnr. 20020598 96-monsterstel, katalogusnr. 20020599).
      • Monsters wat direk uit deppers of soortgelyke monsterbronne gemaak is, is die beste geskik vir die NextSeq ™ -reeks stelsels weens die aanbevole 10 miljoen lesings per monster.
      • Biblioteke wat uit virale kultuur voorberei is deur dieselfde biblioteekvoorbereidingswerkvloei te gebruik, is goed geskik vir ander werkbankinstrumente, insluitend die iSeq™ 100-, MiniSeq™- en MiSeq™-stelsels as gevolg van die laer aanbevole leestelling van 500 000 leeswerk per monster.
      • Plaaslike data-analise word uitgevoer met die Illumina Local Run Manager (LRM) Resequencing Module, met die SARS-CoV-2 verwysingsgenoom.
      • Stroomopwaartse ontledings kan uitgevoer word met die IDbyDNA Explify-platform (www.idbydna.com/explify-platform).

      Verrykingsgebaseerde volgordebepaling met die Illumina DNA Prep with Enrichment (voorheen bekend as Nextera™ Flex for Enrichment) biblioteekvoorbereidingswerkvloei

      • Verrykingswerkvloei vir die opsporing van koronavirus met behulp van Illumina NGS-stelseltoepassingsnota
      • Voorbeeldvoorbereidingswerkstroom:
        • Virale RNA word onttrek met die QIAGEN QIAmp Viral Mini Kit (Katalogus No. 52904).
        • Virale RNA word omgekeerd getranskribeer deur gebruik te maak van Thermo Fisher se Scientific Maxima H Minus Dubbelstring cDNA Sintese Kit (Thermo Scientific, Katalogus No. K2561). Ander werkstrome vir omgekeerde transkripsie kan verenigbaar wees, insluitend die sintetiemodule van die eerste streng en die tweede streng (nie -gestandaardiseerde) sintesemodule van NEB.
        • Virale cDNA word gebruik as invoer vir die Illumina DNA Prep with Enrichment -biblioteekvoorbereidingskit (voorheen bekend as Nextera Flex for Enrichment) en verryk met die respiratoriese virus -oligopaneel (Illumina, katalogus nr. 20042472).
        • Volgordebepaling word uitgevoer op die benchtop iSeq 100-, MiniSeq- of MiSeq-stelsels wat goed geskik is vir die lae leesvereistes vir hierdie monsters
        • Plaaslike data-analise word uitgevoer met die Illumina Local Run Manager (LRM) Hervolgordemodule, met die SARS-CoV-2 verwysingsgenoom.
        • Stroomopwaartse ontledings kan uitgevoer word met die IDbyDNA Explify-platform (www.idbydna.com/explify-platform).

        Amplicon-volgordebepaling met die AmpliSeq™ vir Illumina SARS-CoV-2 Navorsingspaneel

        • AmpliSeq vir die produkbladsy van Illumina SARS-CoV-2 Research Panel
        • Paneeloorsig:
          • Hierdie AmpliSeq-gemeenskapspaneel (op bestelling) bevat 247 amplikone in twee poele wat gerig is op die SARS-CoV-2-genoom. Die paneel is ontwerp vir & dekking van 99% van die SARS-CoV-2 genoom (

          • Virale RNA word onttrek met die QIAGEN QIAmp Viral Mini Kit (katalogus nr. 52904).
          • Raadpleeg die AmpliSeq for Illumina Community Panel Reference Guide, Hoofstuk 3, vir die Protokol vir RNA -panele.
          • Na voorbereiding van die biblioteek word opeenvolging gewoonlik uitgevoer op die volgorde van die tafel, iSeq 100, MiSeq, MiniSeq of NextSeq 500/550 as gevolg van die lae leesvereistes vir hierdie monsters
          • Die DNA Amplicon -app in BaseSpace ™ of die DRAGEN ™ Metagenomics -app kan vir data -analise gebruik word.

          Hierdie paneel is bedoel vir slegs navorsingsgebruik (RUO) toepassings, en nie vir gebruik in diagnostiese prosedures nie.


          Wat is 'n goeie eenvoudige toets vir die kwaliteit van cDNA -biblioteek? - Biologie

          Sentrum vir Molekulêre Geneeskunde en Genetika
          Wayne State University School of Medicine
          540 E. Canfield
          Detroit, MI 48201

          ONDERSOEK VAN DIE FUNKSIE VAN PROTEÏEN EN PROTEÏENNETWERKE MET DIE GIS TWEE-HIBRIEDE STELSEL

          Die gis-twee-hibriede stelsel bied 'n relatief eenvoudige benadering om proteïenfunksie te verstaan. Afdeling II gee 'n uiteensetting van die basiese komponente van die interaksieval, 'n gis-tweebasterstelsel wat in die Brent-laboratorium ontwikkel is (Gyuris et al., 1993). Meer gedetailleerde agtergrondinligting kan verkry word in 'n aantal onlangse resensies (Ausubel et al., 1987-1996 Finley en Brent, 1995 Mendelsohn en Brent, 1994). Afdeling III bevat 'n interaktorjagprotokol, wat 'n verkorte en bygewerkte weergawe is van die oorspronklike protokol wat ons die eerste keer in 1992 op die internet geplaas het en daarna opgedateer het (Finley en Brent, 1995 Finley et al., 1997). Die weergawe wat hier aangebied word, is die weergawe wat ons tans in ons laboratorium gebruik en verteenwoordig ons pogings om hierdie tegnieke te vaartbelyn en op te skaal om die karakterisering van groot netwerke van interaksie -proteïene te vergemaklik. Dit is ook nuttig vir individuele jagte. Afdeling IV bespreek alternatiewe benaderings wat spesifiek ontwerp is om na groot proteïennetwerke te kyk. Die uiteindelike doel van die ontwikkeling van hierdie en verwante benaderings is om uiteindelik al die interaksies wat deur 'n genoom gekodeer word, in kaart te bring. Afdeling V bespreek kortliks twee-hibriede benaderings om die funksies van individuele proteïeninteraksies te verstaan.

          Verskeie verskillende twee-hibriede sisteme is ontwikkel om proteïenfunksie te bestudeer. Die tuinsoortoepassing is om te leer oor die funksie van 'n gegewe proteïen deur proteïene wat daarmee interaksie het, te isoleer, gewoonlik deur 'n cDNA-biblioteek te ondersoek. Om so 'n interaktorjag uit te voer, word 'n proteïen in gis uitgedruk as 'n samesmelting van die DNA-bindende domein van 'n transkripsiefaktor sonder 'n transkripsie-aktiveringsdomein. Die DNA-bindende samesmeltingsproteïen word algemeen die aas genoem. Die gisstam bevat ook een of meer verslaggewergene met bindingsplekke vir die DNA-bindende domein. Om proteïene te identifiseer wat met die aas in wisselwerking is, word 'n plasmiedbiblioteek wat cDNA-gekodeerde proteïene tot 'n transkripsie-aktiveringsdomein uitdruk, in die stam ingebring. Interaksie van 'n cDNA-gekodeerde proteïen met die aas lei tot die aktivering van die verslaggewergene, sodat selle wat die interaktore bevat, geïdentifiseer kan word.

          Die tweebasterstelsel wat in die Brent-laboratorium ontwikkel is (die interaksie-val) gebruik die E.coli-proteïen LexA as die DNA-bindende domein en 'n proteïen wat deur ewekansige E. coli-rye, die B42 "acid blob", gekodeer word as die transkripsie-aktivering domein. Beide proteïene word uitgedruk vanaf multikopie (2µ) plasmiede die LexA-fusie, of lokaas, word uitgedruk vanaf 'n plasmied wat die HIS3-merker bevat, en die aktiveringsdomein-gefuseerde proteïen, soms die prooi genoem, word uitgedruk vanaf 'n plasmied wat die TRP1-merker bevat. . In die mees gebruikte lokaasplasmied, pEG202, word die lokaas uitgedruk uit die konstitutiewe gis ADH1 -promotor. Verwante aasplasmiede is beskikbaar wat die aas uitdruk wat saamgesmelt is tot 'n kernlokalisasie sein. Die mees gebruikte prooi-plasmied, pJG4-5, druk proteïene uit wat saamgesmelt is met die B42-aktiveringsdomein, die SV40-kernlokalisasie-sein en 'n epitoop-tag afgelei van hemagglutinien, alles aangedryf deur die GAL1-promotor van gis wat slegs aktief is in gis wat op galaktose verbou word. . Deur die GAL1 -promotor te gebruik om die prooi uit te druk, kan toksiese proteïene kortstondig uitgedruk word en help dit om baie vals positiewe aspekte in interaktiewe jagte uit te skakel. Die interaksielokval gebruik twee verslaggewergene wat stroomop LexA-bindingsplekke of operateurs dra: LEU2 en lacZ. Die LEU2 -verslaggewers is geïntegreer in die gisgenoom, die lacZ -verslaggewers woon gewoonlik op 2 µ plasmiede wat die URA3 -merker dra, hoewel geïntegreerde weergawes ook beskikbaar is. Verskeie weergawes van die LEU2- en lacZ-verslaggewers bestaan ​​wat 'n reeks sensitiwiteite het gebaseer op die aantal stroomop LexA-operateurs. Oor die algemeen is die LEU2-verslaggewers meer sensitief vir 'n gegewe interaksie-paar proteïene as die lacZ-verslaggewers (Estojak et al., 1995), maar hoogs sensitiewe lacZ-verslaggewers is gebruik wat verskeie LexA-operateurs en transkripsieterminatorreekse stroomaf van die lacZ-geen bevat (S. Hanes, persoonlike mededeling).

          Meer besonderhede oor die verskillende stamme en plasmiede wat beskikbaar is vir die interaksieval kan elders gevind word (Ausubel et al., 1987-1996 Brent et al., 1997 Estojak et al., 1995 Finley en Brent, 1994 Finley en Brent, 1995 Finley et al., 1997 Gyuris et al., 1993)

          III. INTERAKTOR JAG PROTOKOL

          Hieronder verwys ek na tipiese stamme en verslaggewers wat nodig is vir 'n interakteurjag. Dit sluit in die sensitiewe LEU2-verslaggewerstam EGY48, die sensitiewe lacZ-verslaggewerplasmied pSH18-34, 'n plasmied om LexA-fusies soos pEG202 uit te druk, en die biblioteekplasmied pJG4-5. Die volgende is 'n verkorte weergawe van 'n voorheen gepubliseerde protokol (Finley en Brent, 1995). Dit is bedoel om 'n paar belangrike punte in die oorspronklike protokol te verduidelik en uit te brei. Meer besonderhede kan by die webwerwe gevind word (Brent et al., 1997 Finley et al., 1997)

          A. Toets van lokaas Deel 1: Aktiveer die aas transkripsie?

          Voordat 'n interaktorjag uitgevoer word, is dit baie belangrik om die vlak van agtergrondaktivering deur die aasproteïen self te ken. Byna elke LexA -samesmelting sal die LEU2 -verslaggewer in EGY48 tot 'n mate op sigself aktiveer. Die hoeveelheid aktivering deur 'n aas bepaal hoe, en of 'n interakteurjag gedoen word. Die nuttigste manier om die vlak van aktivering te meet, is om die fraksie van lewende selle te bepaal wat in staat is om te groei in die afwesigheid van leucine (op leu- plate). Alhoewel dit nie onmiddellik duidelik is waarom 'n sterker aktiveerende aas 'n groter fraksie EGY48 -selle laat groei in die afwesigheid van leucine nie, is die bepaling van hierdie breuk noodsaaklik vir 'n interaktiewe jag. Die breuk kan voorgestel word as die aantal kolonies wat op 'n leuplaat (Leu+ kolonies) groei per lewende gissel. Die aantal lewende selle, of kolonievormende eenhede (CFU), in 'n hoeveelheid selle word bepaal deur plateringsverdunnings te plaas op plate wat leucien bevat. Die frekwensie van Leu+ kolonies (of Leu+/CFU) is dus 'n verhouding tussen die aantal kolonies wat op leuplate vorm, bo die getal wat vorm op plate wat leucine bevat. Die toets word uitgevoer met die seleksiestam (die stam wat reeds die lacZ-verslaggewer en lokaasplasmiede bevat) wat met die leë biblioteekplasmied getransformeer word, dit pJG4-5 naboots die omstandighede waaronder die seleksie vir interaktore uiteindelik uitgevoer sal word. Vir 'n lokaas wat feitlik nie in staat is om die LEU2-geen self te aktiveer nie, sal die frekwensie van Leu+ kolonies in die toets minder as 10 -6 wees (dws minder as 1 Leu+ kolonie sal vorm wanneer 10 6 CFU op die leu- plate). Aas wat 'n matige transkripsievlak aktiveer, sal lei tot kolonies van Leu+ met frekwensies van 10 -4 tot 10 -5.

          Dit is belangrik om ten minste 10 6 CFU op die leuplate te plaas wanneer 'n lokaas getoets word vir aktivering van LEU2. Om 'n tipiese biblioteek van 106 individuele cDNA's te vertoon, sal dit nodig wees om meer as 106 CFU van die seleksiestam wat met die biblioteek getransformeer is, op die leuplate te plaas om vir interaksies te selekteer. As die agtergrondaktivering deur 'n aas getoets word deur slegs 10 3 of 10 4 CFU op leuplate te plaas, en slegs een of 'n paar Leu+ kolonies vorm, sou dit aanloklik wees om tot die gevolgtrekking te kom dat die aas LEU2 op 'n voldoende lae vlak aktiveer gebruik word vir 'n interaktiewe jag. As 'n mens egter dan sou probeer om 'n biblioteek van 3 x 10 6 individuele cDNA's deeglik te skerm deur 3 x 10 6 CFU op die leu-seleksieplate te plaat, sal ten minste 3000 kolonies vorm, dit sal na verwagting almal vals positief wees (dws gevorm as gevolg van aktivering deur die lokaas en nie as gevolg van interaksie van die lokaas met cDNA-gekodeerde proteïene nie). Soos hieronder bespreek, sal kennis van die frekwensie van Leu+ -kolonies wat voortspruit uit aktivering deur die aas self ook belangrik wees by die bepaling van die aantal Leu+ -kolonies wat hulle moet kies vir verdere analise tydens 'n interaktorjag.

          'N Tweede belangrike toets van die aktiveringspotensiaal van 'n aas is die vermoë om die lacZ -verslaggewer te aktiveer. Oor die algemeen is die sensitiefste lacZ-verslaggewers (bv. Plasmied pSH18-34) nie so sensitief as die LEU2-verslaggewers nie. In die meeste gevalle sal 'n aas wat Leu+ -kolonies produseer met 'n frekwensie van minder as 10 -4 nie die lacZ -geen aktiveer nie, gemeet aan die vermoë van 'n kolonie om blou te word op 'n X -Gal -plaat. In seldsame gevalle en om onbekende redes, sal 'n aas wat 'n baie lae vlak van die LEU2 -verslaggewer aktiveer, die lacZ -verslaggewer tot 'n beduidende vlak aktiveer. Dit is dus noodsaaklik om te toets vir die aktivering van die lacZ -verslaggewer by die karakterisering van die aas.

          Protokol 1 Toets of 'n aas transkripsie aktiveer

          • Mediaresepte kan op die webwerf gevind word (Finley et al., 1997) en elders (Ausubel et al., 1987-1996 Finley en Brent, 1995 Guthrie en Fink, 1991).
          • Vloeibare YPD media
          • Vloeibare uitvalmedia (Glu ura-, Glu ura-his-)
          • Uitvalplate (Glu ura-, Glu ura-his-, Glu ura-his- trp-, Gal/Raf ura-his- trp-, Gal/Raf ura-his- trp-leu-)
          • X-Gal plate (Gal/Raf ura-his- trp- X-Gal)
          • Gisstam EGY48 (MAT µ ura3 his3 trp1 3LexAop- LEU2 :: leu2 ) of een van die minder sensitiewe LEU2 verslaggewerstamme EGY191 of EGY189 (MAT µ ura3 his3 trp1 1LexAop- LEU2) (E::leusto2k- LEU2). , 1995)
          • Die URA3 2 & # 181 lacZ verslaggewer plasmied pSH18-34, of 'n minder sensitiewe lacZ verslaggewer (Finley en Brent, 1995)
          • HIS3 2 µ aasplasmied (bv. 'n afgeleide van pEG202) wat jou aasproteïen uitdruk wat aan LexA saamgesmelt is
          • Twee beheeraasaasplasmiede: een wat vir LexA kodeer wat saamgesmelt is aan 'n aktivator soos Gal4 soos in die plasmied pSH17-4, en een wat kodeer vir 'n transkripsioneel inerte aas soos LexA-Max (Zervos et al., 1993)
          • Die TRP1 2 µ biblioteekplasmied, pJG4-5, sonder cDNA
          • Sien aangehegte transformasieprotokol vir bykomende reagense

          1. Bou die seleksiestam óf deur seriële transformasie van EGY48 met pSH18-34 gevolg deur u lokaasplasmied, óf deur ko-transformasie van EGY48 met u lokaasplasmied en pSH18-34. Die seleksiestam (EGY48/pSH18-34/lokaasplasmied) moet op ura-sy-medium gekweek word in alle daaropvolgende stappe om seleksie vir die aas- en lacZ-verslaggewerplasmiede te behou. Kies drie individuele transformante kolonies en trek na 'n ander Glu ura-sy-bord vir berging en later herstel. Al drie moet identies optree in die onderstaande toetse, in welke geval enige een sal dien as die seleksiestam waarin die biblioteek ingevoer sal word.

          2. Transformeer die seleksiestam met pJG4-5 en selekteer transformante op Glu ura-his-trp-plate. Maak van hierdie geleentheid gebruik om die seleksiestam met 'n hoë doeltreffendheid te transformeer, dit sal nodig wees vir transformasie met die biblioteek -DNA (protokol 3).

          3. Kies twee of drie transformantkolonies en ent 10 ml vloeibare Glu ura-his-trp- medium (weereens moet alle kolonies dieselfde optree, maar om die toets op meer as een uit te voer kan help om te verseker dat die resultate nie te wyte is aan sommige skelm mutante gis of kontaminant). Kweek die vloeibare kulture by 30oC met skud tot OD600=1.0 (wat ooreenstem met ongeveer 107 selle/ml). Dit is middel-log fase, mits die kultuur begin by OD600<0.2. As oornagkulture tot 'n digtheid groter as OD600 = 1.0 groei, verdun tot minder as OD600 = 0.2 en groei dan tot OD600 = 1.0 sodat die selle in die middel-logfase is wanneer hulle geoes word.

          4. Maak reeksverdunnings van 10-1 tot 10-6 van elke kultuur in steriele water.

          5. Plaas 100 ml van die kultuur en 100 ml van elke verdunning op twee plate:

          6. Monitor die opkoms van kolonies gedurende die volgende paar dae. Bereken die aantal CFU wat op elke Gal/Raf ura-his-trp-leu-plaat uitgeplaat is deur die aantal kolonies te tel wat op die Gal/Raf ura-his-trp- plate vorm. Bereken die aantal Leu+ kolonies/CFU. Dit is ook die moeite werd om kennis te neem van die grootte van kolonies na 2, 3 en 4 dae (sien hieronder).

          7. Toets vir lacZ uitdrukking. Een manier om dit te doen, is eenvoudig om individuele transformante van stap 2 na Gal/Raf ura-his- trp- X-Gal plate (ongeveer 1 cm x 1 cm kolle) te plak en by 30oC te inkubeer. Gis met 'n beheerde LexA-aktivator-samesmelting moet oornag blou word, terwyl diegene wat LexA ontbreek of 'n transkripsie-inerte aas bevat, onbepaald wit sal bly. Alternatiewelik, as die frekwensie van Leu+/CFU hoër as 10-4 is, kan dit nuttig wees om plaat van een van die Gal/Raf ura-his-trp-leu-plate (een met 200-500 kolonies) na Gal/ te replikeer. Raf ura-his- trp- X-Gal. Dit sal die frekwensie van blou kolonies onder die Leu+ -kolonies openbaar, 'n getal wat nuttig kan wees om jagstrategieë te bepaal (sien hieronder).

          'n Galaktose word in die medium gebruik omdat die werklike seleksie uiteindelik op galaktose-plate gedoen sal word om die uitdrukking van die aktiveringsgemerkte cDNA-proteïen te veroorsaak. Raffinose word bygevoeg om gisgroei aan te help dit verskaf 'n beter koolstofbron as galaktose alleen maar blokkeer nie die vermoë van galaktose om die GAL1 promotor te induseer nie.

          B. Toets van lokaas Deel 2: Kom die aasproteïen die kern binne en bind dit aan LexA-operateurs in die verslaggewers, en word die fusieproteïen in die volle lengte gemaak?

          Daar is skaars berigte van lokaas wat uitgesluit is van die giskern, dit is gewoonlik moontlik om dit in die kern in te dwing deur 'n kernlokaliseringsdomein N-terminaal by LexA in te sluit. Enige klein transkripsie -aktivering deur 'n aas kan beskou word as 'n aanduiding dat die lokaasproteïen die giskern binnedring. Die ideale lokaas aktiveer egter nie transkripsie nie, dus is nog 'n toets nodig om te wys dat dit operateurs in die giskern kan beset. Een eenvoudige toets is die onderdrukkingstoets. Hierdie toets is gebaseer op die vermoë van die meeste transkripsioneel inerte LexA -samesmeltings om transkripsie te belemmer wanneer dit gebind is aan LexA -operateurs wat tussen die TATA -boks en die stroomop -aktiverende volgorde (UAS) van 'n verslaggewer geleë is. Die verslaggewer wat vir hierdie toets gebruik is, is die lacZ -verslaggewer in plasmied pJK101. Hierdie URA3 2 µ plasmied verskil van pSH18-34 deurdat die GAL1 UAS stroomop van die LexA-operateurs geleë is. Die GAL1 UAS aktiveer die lacZ-verslaggewer op 'n hoë vlak in die teenwoordigheid van galaktose, en vir hierdie spesifieke afgeleide aktiveer dit ook op 'n lae vlak in gis wat op glukose gekweek word. Elke mate van onderdrukking van die GAL UAS deur 'n aas, hetsy in galaktose of glukose, dui aan dat die aas die kern binnedring en LexA -operateurs inneem.

          Protokol 2 Die onderdrukkingstoets

          • Vloeibare YPD media
          • Vloeibare uitvalmedia (Glu ura-, Glu ura-his-)
          • Uitvalplate (Glu ura-, Glu ura-his-)
          • X-Gal plate (Glu ura-his- X-Gal, Gal/Raf ura-his- X-Gal)
          • Gisras EGY48 of 'n verwante ras
          • Die URA3 2 µ lacZ repressie toets verslaggewer plasmied pJK101
          • HIS3 2 µ aasplasmied wat u lokaasproteïen uitdruk, versmelt met LexA
          • Twee HIS3 2 µ beheer lokaasplasmiede: een wat LexA kodeer wat saamgesmelt is aan 'n transkripsie-inerte proteïen, soos Bicoid in pRFHM1, of LexA-Max (Zervos et al., 1993), en een wat geen LexA kodeer nie, byvoorbeeld pRFHM0.

          1. Transformeer EGY48 met pJK101 en kies transformante op Glu ura- plate.

          2. Combine three colonies from these plates and transform them with the HIS3 bait plasmid (and the HIS3 control plasmids). Select transformants on Glu ura-his- plates.

          3. Pick four individual colonies from each transformation and streak a patch of them onto Glu ura-his- and Gal/Raf ura-his- plates containing X-Gal. Incubate at 30oC.

          4. Examine the X-Gal plates after 1, 2, and 3 days. Yeast lacking LexA will begin to turn blue on the Gal/Raf plates after one day and will appear light blue on the glucose plates after two or more days. Yeast containing a bait that enters the nucleus and binds operators will turn blue more slowly than the yeast lacking LexA.

          5. Baits that repress transcription of lacZ in pJK101 by 2-fold or less may not cause a visible reduction in blue on X-Gal plates. If no repression is observed on the X-Gal plates, perform the more sensitive liquid ß-galactosidase assays with transformants from step 2. Grow the transformants in 5 ml Glu ura-his- and Gal/Raf ura-his- liquid media, or on Glu ura-his- and Gal/Raf ura-his- plates for 2 days, before doing ß-galactosidase assays (Miller, 1972).

          An ideal bait protein for an interactor hunt is one that does not itself activate transcription but does repress in the repression assay. It is also useful to verify that the full-length fusion protein is made. In some instances, proteases in yeast will cleave specific portions of a bait, leaving a truncated LexA fusion that still binds to operators. To demonstrate that the full-length bait protein is made one can perform a Western blot on extracts from yeast cells that harbor the bait plasmid, immunoblotting with either an antibody to LexA or one specific to the protein fused to LexA. The simplest way to do this is to prepare yeast cell extracts by growing yeast in liquid culture (lacking histidine to maintain selection for the bait plasmid) to OD 600 = 0.5, spinning 1 ml of the

          culture to pellet the cells, and resuspending the cells in 50 m l of 2X Laemmli sample buffer (Laemmli, 1970). The cells can then be broken by freezing on dry ice followed by boiling for 5 min prior to loading on an SDS polyacrylamide gel (about 15 m l/lane). The proteins can then be transferred to a filter and blotted with standard immunoblotting (Western) methods (Ausubel et al., 1987-1996 Harlow and Lane, 1988).

          C. Screening a library for interactors

          Most cDNA libraries available for the Brent lab version of the yeast two-hybrid system contain over 10 6 individual cDNAs (in plasmid pJG4-5). In theory, a library with 10 6 individual cDNAs includes cDNAs for messages that were more frequent than 1 in 10 6 mRNA molecules in the mRNA population used to make the library. To have a chance at isolating the rarest cDNAs in a library, it is important to collect more yeast transformants than there are individual cDNAs in the library. Thus, for a library with 10 6 individual cDNAs, one might try to obtain 2-3 x 10 6 yeast transformants. With the most common yeast two-hybrid strains one can obtain up to 10 5 transformants per µg of library plasmid DNA using the attached transformation protocol.

          A pilot transformation should be performed with the selection strain to determine the transformation efficiency that can be obtained. This allows one to calculate how many individual transformations to set up to obtain the desired number of total transformants. The transformation mixes are plated onto 22cm x 22cm Glu ura-his-trp- plates, attempting to get 1-2 x 10 5 transformants/plate. Again, the number of individual transformation mixes to put on each plate is calculated from the expected transformation efficiency derived from pilot experiments. The transformants are collected and stored frozen. Aliquots are then plated to ura-his-trp-leu- Gal/Raf plates to select interactors.

          Protocol 3 Transforming the selection strain and selecting potential interactors

          • Liquid dropout media (Glu ura-his-, Gal/Raf ura-his-trp-)
          • Dropout plates (Glu ura-his-trp-, Gal/Raf ura-his-trp-leu-, Glu ura-his-trp-leu-)
          • X-Gal plates (Glu ura-his-trp- X-Gal, Gal/Raf ura-his-trp- X-Gal)
          • Sterile water
          • Sterile glycerol solution (65% (v/v) glycerol, 0.1 M MgSO4, 25 mM Tris-HCl 7.4).
          • Glass beads (4 mm diameter Fisher Scientific), sterilized by autoclaving.
          • Sterile 50 ml Falcon tubes
          • Sterile 50 ml round-bottom polypropylene centrifuge tubes

          1. Using the selection strain prepared in Protocol 1, perform pilot transformations (as suggested in Protocol 1 step 2) to determine transformation efficiency.

          2. Based on your transformation efficiency, calculate the number of transformations to obtain the desired number of total transformants (i.e., each transformation = 1 µg library DNA/50 µl of cells as described in the transformation protocol). Also, calculate the number of transformations to be plated on each 22cm x 22cm Glu ura-his-trp- plate to get 1-2 x 10 5 transformants/plate (e.g., if your efficiency in pilot experiments is 5 x 10 4 transformants/µg you should set up 2 transformations for each 22cm x 22cm plate).

          3. Based on the above calculations, grow the appropriate amount of the selection strain in liquid Glu ura-his- medium and set up the necessary number of transformations (see attached transformation protocol).

          4. After the heat shock, invert the tubes several times to mix - gently. Remove 10 µl from several of the transformation mixes and make three dilutions (10 -1 , 10 -2 and 10 -3 ) each in sterile water. Plate 100 m l of each dilution onto 100 mm diameter Glu ura-his-trp- plates and incubate at 30oC. This will allow the total number of transformants to be calculated.

          5. Plate the remainder of the transformation mixes (less then 2 ml total/plate) onto 24cm X 24cm Glu ura-his-trp- plate. There is no need to spin the cells or remove the PEG. The medium in these plates should be at least 0.6 cm thick, level, and free of bubbles. To achieve an even distribution of cells, pour about 100 sterile glass beads (4 mm diameter) onto the plate with the cells. Gently roll the beads around the plate to distribute the transformation mix, then pour the beads off, or onto the next plate. This technique works best when the surface of the plates is not too wet so that the medium absorbs the transformation mix. To achieve this moisture content, put newly solidified plates into a laminar flow hood with the lids ajar for about 1 h before plating.

          6. Incubate the plates at 30oC. Colonies should appear after about 24 h. Continue incubation until colonies are 1 - 2 mm in diameter, which should take a total of approximately 2 days.

          7. Place the plates at 4oC for 2 - 4 hours to harden the agar. Using the long edge of a sterile 75mm x 50mm glass microscope slide (and sterile technique!), scrape the yeast from the plate. Try not to scrape any agar as this will interfere with pipetting. Collect the yeast from the glass slide by wiping it on the lip of a sterile 50 ml Falcon tube.

          8. Wash the cells twice with 2 or 3 volumes of sterile TE. It may be necessary to split into two or more tubes to effectively pellet. It is best to pellet the cells each time in a sterile round bottom polypropylene tube at 2000 g for 4 min so they may be easily resuspended. The pellet volume for 500,000 transformants will be about 8 ml.

          9. Resuspend the cells thoroughly by swirling in 1 pellet volume of sterile glycerol solution. Mix well by vortexing on low speed. Freeze 1 ml aliquots at -70oC.

          10. Determine the plating efficiency by thawing an aliquot of library transformants and making serial dilutions in sterile water. Plate 100 m l of each dilution onto 100 mm diameter Gal/Raf ura-his-trp- plates. Count the colonies that grow after 2 - 3 days at 30oC. Represent the plating efficiency in c olony f orming u nits (CFU) per unit volume of frozen cells. Note: to save time one can estimate the plating efficiency as

          108 CFU/100 m l, and immediately proceed to steps 11 and 12. Once the actual plating efficiency is known, calculate the number of CFU that were actually plated in steps 11 and 12.

          11. Thaw a 1 ml aliquot of transformed yeast and dilute 10-fold into 9 ml Gal/Raf ura-his-trp- liquid medium. Incubate at 30oC with shaking for 6 to 8 h to induce the GAL1 promoter and expression of the library encoded proteins. Pellet the cells by centrifugation at 2000 g for 4 min at 20 - 25oC and resuspend in 10 ml sterile water.

          13. Plate less than 106 CFU (determined from the plating efficiency test in step 10) onto each 100 mm diameter Gal/Raf ura-his-trp-leu- plates. To avoid overcrowding of Leu+ colonies, do not plate more CFU than are expected to produce

          20 background Leu+/plate (as determined in Protocol 1). Incubate the selection plates at 30oC. Colonies should appear in 2 - 5 days. To keep the plates from drying out after two days, it may be helpful to put them in plastic bags or containers, or put parafilm around each plate.

          14. Pick colonies (see discussion below for number to pick) with sterile toothpicks or applicator sticks and patch, or streak for single colonies, onto another Gal/Raf ura-his-trp-leu- plate. If the Leu+ colonies are closely spaced it will be necessary to streak purify to single colonies to separate the different Leu+ clones. Ideally the Leu+ yeast should be streaked for single colonies to isolate them from contaminating Leu- yeast. However, when there are large numbers of Leu+ colonies to pick, it may be inconvenient to streak purify every one in this case, growing patches on a second selection plate will at least enrich for the Leu+ cells.

          15. To show that the Leu+ phenotype is galactose-dependent, patch (or replica plate) the Leu+ yeast onto Glu ura-his-trp- master plates to turn off the GAL1 promoter and stop expression of the activation-tagged cDNA protein. Grow at 30oC for about 24 h.

          16. Replica the master plates to the following five plates, in order: 1. Gal/Raf ura-his-trp- X-Gal 2. Glu ura-his-trp- X-Gal 3. Glu ura-his-trp-leu- 4. Gal/Raf ura-his-trp-leu- 5. Glu ura-his-trp-. Incubate at 30oC and examine the results after 1, 2, and 3 days.

          17. Pick only those yeast that are Leu+ on galactose but not glucose. Keep in mind that if Leu+ clones were not purified in step 14, some patches may be contaminated with background Leu+ yeast, which will not be galactose-dependent. The galactose-dependent Leu+ phenotype indicates that reporter activation depends on expression of the library protein. Further characterize these by isolating the library plasmid and determining the interaction specificity.

          Alternate protocol - liquid selection and amplification of Trp+ library transformants. We have had some success at selecting and amplifying library transformants in liquid culture (M. Kolonin and R. Finley, unpublished). To do this, we dilute individual transformation mixes after heat shock (from Protocol 3 step 4) 50-fold into liquid Glu ura-his-trp- medium and grow shaking at 30 o C until the OD 600 is

          2.0. The OD 600 of this culture begins at less than 0.2 and usually takes 30-48 hours to reach 2.0. We then harvest the cells and proceed as in Protocol 3 step 8. By removing aliquots immediately after dilution and before harvesting and plating on Gal/Raf-ura-his-trp- we have estimated that transformants are amplified approximately 100-fold in this procedure. This approach eliminates the cost and inconvenience of selecting transformants on plates. The disadvantage is that there is no reliable way to verify that library transformants are evenly amplified.

          How many Leu+ colonies should be picked? When considering how many Leu+ colonies to pick at step 14 of Protocol 3, it is important to take into account the background frequency of Leu+ colonies that the bait itself produces (represented as Leu+ colonies/CFU), as determined in Protocol 1, and the total number of library transformants obtained. To completely screen all of the library transformants, the minimum number of Leu+ colonies one would need to pick and characterize can be estimated by:

          # to pick > (# Leu+ colonies/CFU) X (total # of library transformants)

          If, for example, the background for a given bait were 10 -5 Leu+ colonies/CFU, one would need to pick and characterize at least 10 colonies to screen through 10 6 library transformants. More to the point, the first 10 colonies picked would be expected to be background, so to get an interactor that is rare in the library one might need to pick and characterize 20 or 30 Leu+ colonies.

          Should galactose-dependent Leu+ colonies that do not turn blue on the X-Gal plates be further characterized? Ja. Of the galactose-dependent positives, several different classes of Leu and lacZ phenotype are possible. Byvoorbeeld:

          Class I. galactose-dependent Leu+ galactose-dependent dark blue on X-Gal

          Klas II. galactose-dependent Leu+ galactose-dependent light blue on X-Gal

          Class III. galactose-dependent Leu+ white on X-Gal

          Many hunts will yield Leu+ colonies from each class. Often this indicates that at least three different interactors are represented among the positives. A common mistake is to concentrate on only the "strongest" class (Class I above) and ignore the "weaker" class (Class III) which can include biologically significant interactors (Finley et al., 1996).

          The next step for the galactose-dependent positives is to isolate the library plasmid from each and re-introduce it into the selection strain to show that the putative interaction phenotype depends on the library plasmid and not on mutations in the yeast or reporter genes. This test can often be performed at the same time as the specificity test described below. If the library has been properly screened to exhaustion, each interactor cDNA should be represented more than once in the putative positives. cDNAs corresponding to abundant messages may have been isolated many times. To reduce the amount of work in subsequent steps it is useful to determine which yeast contain identical cDNAs. This can be easily done by performing PCR with primers flanking the cDNA insertion site using DNA template from a quick yeast miniprep (Finley and Brent, 1995). PCR products can be digested with HaeIII and AluI and run on an agarose gel to reveal unique restriction fragment patterns for each cDNA (Finley and Brent, 1995). One or two of each unique library plasmid can then be rescued in E.coli and used in the specificity test.

          D. Determining the specificity of interactors

          Many of the proteins identified in interactor hunts are non-specific interactors: they appear to interact with a number of different unrelated LexA fusions. Non-specific interactors are frequently isolated in hunts using unrelated baits. They can be identified and discarded by testing the ability of the cDNA-encoded proteins to interact with a handful of bait proteins unrelated to the original bait. cDNA-encoded proteins that interact only with the original bait and not with unrelated baits are considered true specific interactors. The specificity test can be performed by introducing rescued library plasmids into different selection strains that each harbor a different bait plasmid. Transformants are picked and patched onto a Glu ura-his-trp- plate and then replica plated to indicator plates as in Protocol 2 steps 15 and 16. This method of testing specificity can be somewhat cumbersome if a large number of different library plasmids were isolated, and if these are to be tested for interaction with several different baits. For this reason we use the interaction mating assay (Finley and Brent, 1994) to perform the specificity test, as described in Protocol 3.

          Interestingly, the commonly isolated non-specific interactors, which include heat shock proteins, ribosomal proteins, proteasome subunits, and other proteins, are not isolated in every interactor hunt, and in fact do not appear to interact with every bait. This highlights the importance of using several different bait proteins to test the specificity of an interactor. For example, frequently a non-specific interactor will interact with just 30% of the bait proteins tested. If only one or a few bait proteins are tested, a non-specific interactor could appear to be specific.

          Protocol 4 The interaction mating assay

          • Rescued library plasmid DNA
          • Liquid YPD medium
          • Liquid dropout media (Glu ura-)
          • YPD plates
          • Dropout plates (Glu trp-, Glu ura-his-, Glu ura-his-trp-, Gal/Raf ura-his-trp-leu-, Glu ura-his-trp-leu-)
          • X-Gal plates (Glu ura-his-trp- X-Gal, Gal/Raf ura-his-trp- X-Gal)
          • Applicator sticks (e.g. FisherBrand 01-340), or toothpicks, sterilized by autoclaving.
          • Replica plating apparatus and sterile velvets or filters.
          • Yeast strain RFY231 (MAT a ura3his3 leu2 ::3LexAop- LEU2 trp1::hisG LYS2) or EGY48. Note: RFY231 is EGY48 with the trp1-1 allele deleted (R. Finley, unpublished).
          • Bait strains: S. cerevisiae strain RFY206 (MATa ura3-52 his3Æ200 leu2-3 lys2Æ201 trp1::hisG ) transformed with a URA3 plasmid containing a lacZ reporter, such as pSH18-34, and various HIS3 bait plasmids, such as derivatives of pEG202 that produce different LexA fusions. Each bait strain will contain a different bait plasmid. One strain should contain the original bait used in the interactor hunt.

          1. Transform yeast strain RFY231 with the rescued TRP1 library plasmids and select transformants on Glu trp- plates (if EGY48 is substituted for RFY231, more than one Trp+ transformant should be analyzed to ensure than a trp1-1 revertant has not been selected). As a control, transform RFY231 with a library plasmid pJG4-5 that has no cDNA insert.

          2. Use sterile applicator sticks or toothpicks to streak individual RFY231 transformants onto standard 100 mm Glu trp- plates in parallel lines (see Figure 1). Streaks should be at least 3 mm wide and at least 5 mm apart, with the first streak starting about 15 mm from the edge of the plate. A 100 mm plate will hold up to 8 different bait strains. Include at least one streak of the transformants with the control plasmid (no cDNA). Create a duplicate plate of streaked RFY231 transformants for each plate of bait strains to be used.

          3. Likewise, streak different bait strains in vertical parallel stripes on a Glu ura-his- plate. Create a duplicate plate of bait strains for each different plate of prey strains to be used. Incubate both sets of plates at 30oC until growth is heavy. When taken from reasonably fresh cultures (for example, plates that have been stored at 4oC for less than a month) streaked RFY206-derived bait strains take about 48 hours to grow and RFY231-derived strains take about 24 hours.

          4. Print the RFY231 derivatives and the RFY206 derivatives onto the same replica filter or velvet so that the streaks from the two plates are perpendicular to each other (see Figure 1).

          5. Lift the print of the two strains from the filter or velvet with a YPD plate. Incubate the YPD plate at 30oC overnight. Diploids will form where the two strains intersect. One strain may grow more rapidly than the other during this time but this does not hinder formation of diploids in the intersections.

          6. Replica from the YPD plate to the following indicator plates, in order: 1. Gal/Raf ura-his-trp- X-Gal 2. Glu ura-his-trp- X-Gal 3. Glu ura-his-trp-leu- 4. Gal/Raf ura-his-trp-leu- 5. Glu ura-his-trp-. Incubate at 30oC and examine the results after 1, 2, and 3 days. Only diploids will grow on the X-Gal plates and only interactors will grow on galactose plates lacking leucine (Figure 1).

          What next? Although the methods described above allow several types of false positive to be eliminated, they do not address the biological significance of the interactions observed. In some instances the sequence of a specific interactor will suggest that its interaction with the bait may have a real in vivo function. However, two-hybrid interactions can occur between proteins that normally do not interact (for example, because they are never expressed at the same time or in the same tissue or subcellular compartment). A good first step to show biological significance is to verify the interaction by a different, biochemical technique, preferably co-precipitation from a cell in which both proteins are expressed. Ideally, the next step would involve a functional assay for the new protein, to show, for example, that the new protein is involved in the same biological process as the bait protein. The following two sections include a few additional ways to address function.

          IV. TWO-HYBRID METHODS TO STUDY LARGE SETS OF PROTEINS AND PROTEIN NETWORKS

          Finding interacting partners can reveal much about the function of a protein. Most regulatory proteins, for example, appear to function by contacting other proteins. This is true for proteins that regulate many different cellular processes, including transcription, translation, DNA replication, signal transduction, cell cycling, differentiation, and programmed cell death. All of the proteins involved in a given process together can be thought of as a network of interacting proteins. The members of each interacting network are linked through protein-protein contacts. A complete understanding of any given process can only be achieved when all of the components of the protein network regulating it are identified. Yeast two-hybrid systems offer approaches to characterizing individual interactions and whole networks of proteins.

          Isolating a new interacting protein can reveal information about function if the sequence of the new interactor indicates similarity or identity with a protein whose function has been at least partially characterized. However, it is still often the case that the sequence of a interacting protein reveals little about its function. Another approach is to assume that the new protein functions in the same network as the original bait protein and to use the new protein as a bait to identify other members of the network. Repeating this process increases the chances of isolating a previously characterized protein, or one whose sequence provides clues to function. In principle, this approach could be used repeatedly to isolate all of the components of a regulatory network. Because some regulatory proteins may be shared by different cellular processes (e.g. regulation of cell cycle and DNA replication by p21 CIP1 (Li et al., 1994)), and networks for many different processes may be connected (e.g. a signal transduction pathway and the activation of gene transcription), this approach could identify many expressed genes from a small number of starting points.

          An approach complementary to performing sequential hunts is to use the interaction mating assay to look for interactions between increasingly large sets of proteins (Bartel et al., 1996 Finley and Brent, 1996). In one variation of this approach, large panels of baits are collected in baits strains placed on plates in grids (e.g., in the standard 96-well format). The grids can then be screened simultaneously for interactions with individual prey proteins. Bait strains can be created as described in Protocol 4 using bait plasmids that express various proteins of known and unknown function. Large panels of bait strains can be collected and stored frozen indefinitely and then screened against any number of prey strains.

          One such collection contains over 700 different bait proteins from our own work and from numerous other labs that use the interaction trap. Screening a protein against such a panel enables one to quickly test its ability to interact with a large number of known proteins, most of which have been characterized to some extent, and have been chosen for study because of their known or suspected involvement in some biological process. Thus, finding an interaction between a tested protein and a member of the panel often gives an immediate clue about the biological function of both proteins. While the number of proteins in any such panel is far less than the number of proteins in a good library, this approach does offer the advantage of screening the test protein against a set of proteins enriched for those of current interest to the biological community. More restricted panels of bait proteins, for example those known or suspected to function in a particular pathway, or those isolated in sequential interactor hunts, can provide a useful resource for characterizing new proteins. Such a panel may also be useful to characterize differences in the patterns of interactions made by wild-type and mutant variants of proteins such as those created in vitro or associated with particular diseases or other phenotypes.

          For some proteins, this approach offers additional advantages over screening a library using a traditional two-hybrid scheme. Proteins that activate transcription when fused to LexA or another DNA-binding domain can be difficult to use in conventional interactor hunts. Though methods are available to reduce the sensitivity of the reporter genes (Durfee et al., 1993 Estojak et al., 1995) it is not always possible to reduce the reporter sensitivity below the threshold of activation for some baits. Moreover, reduction in reporter sensitivity carries with it the risk that the reporters will not detect weakly interacting proteins. Thus, an alternative for proteins that activate transcription as baits, is to use them as preys to screen existing panels of baits, or even libraries of baits. Interaction mating approaches also have clear advantages for proteins that are somewhat toxic to yeast the prey vector allows conditional expression of toxic proteins in the presence of a bait, and often the interaction can be observed because the reporters are activated even if the cells subsequently become inviable.

          V. TESTING THE FUNCTION OF INDIVIDUAL INTERACTIONS

          Finding the position of a protein within a network of interacting proteins can provide information about the function of the protein and the network. However, ultimately, the nature of each individual protein-protein contact must be understood. Several two-hybrid methods allow the significance of individual protein interactions to be analyzed.

          A. Mapping interaction domains

          Determining the domains within a protein that are responsible for its interaction with other proteins can provide a valuable insight into the way a protein functions. Several approaches are available to map interaction domains with yeast two-hybrid methods. All start with a bait protein and prey protein that interact and activate the reporter genes. Derivatives of one of these proteins are constructed and tested for interaction with the other. We usually make derivatives of the prey protein because derivatives of the bait protein may differ in their ability to activate the reporters by themselves, which complicates interpretation of the results. Derivatives of the prey protein can be made and tested for interaction with the bait in several ways. In any approach it is important to keep in mind that the prey is a fusion to an N-terminal activation domain and must be maintained in the correct reading frame. One approach is to subclone restriction fragments encoding parts of the prey fusion protein into the prey vector (i.e., pJG4-5 or derivative) and introduce the resulting vectors individually into selection strains harboring the bait vector or control vectors. Alternatively, derivatives can be tested for interaction using the mating assay as described in Protocol 4. A second approach is to make N-terminal or C-terminal deletion derivatives of the prey fusion protein and test them for interaction with the bait, again by individual transformation into selection strains or by the mating assay. Deletion derivatives can be constructed in a cloning vector (Ausubel et al., 1987-1996), and then subcloned into the prey vector, pJG4-5. Alternatively, the deletion derivatives can be constructed directly in a derivative of the prey vector. For example, pZP4-5o and pJF3 are derivatives of pJG4-5 that have unique, rare restriction sites downstream of the cDNA cloning sites which allow C-terminal deletions to be constructed by unidirectional exonuclease III digestion from the 3' end of the insert (R. Finley, Z. Paroush, and J. Fonfara, unpublished). Similarly, pJF2 contains unique 5' restriction sites that allow N-terminal deletions to be constructed. A third approach is to make random DNA fragments encoding parts of the prey protein, for example by sonication (e.g., ref. (Stagljar et al., 1996), and insert these into the prey vector. Finally, a variety of techniques are available to make single and multiple point mutations of one interactor, which can then be inserted into the prey vector to test for interaction with a bait.

          B. Construction of dominant negative mutants

          A powerful approach to understanding protein function is to create and express dominant negative forms of the protein that inactivate the function of the wild-type version (Herskowitz, 1987). The yeast two-hybrid system provides a method to design and assay potential dominant negatives. One type of dominant negative is a protein mutated so that it still interacts with one of its protein partners but lacks other functional domains. In this case the "partner" could be another protein or the same protein if it forms homodimers. Expression of the mutant form of the protein might be expected to bind to the partner protein and make it inaccessible to the wild-type version. One way to create such a mutant is to isolate the minimal domain of a protein that will interact with another protein partner as described in the previous section. If the interacting domain is just a fraction of the protein it would be expected to lack other functional domains, and would therefore be a candidate dominant negative. A related but more precise approach could be used for proteins that have at least two different known partners. For example, if protein A interacts with both proteins B and C, mutant varieties of protein A could be constructed and tested in the two-hybrid assay for their ability to interact with just protein B but not protein C. In this case, we would have precise knowledge of the function missing in the dominant negative (interaction with protein C).

          It is worth noting that, while the dominant negative effect is frequently open to multiple interpretations (Herskowitz, 1987), functional inferences from the type of dominant negatives referred to here may be less uncertain. This is because we know that the dominant negative interferes with a specific protein interaction we have designed it that way and tested it in the two-hybrid system.

          C. Disrupting protein interactions

          The yeast two-hybrid system provides an assay to develop reagents that disrupt protein interactions. Such reagents can be used in vivo to probe the function of individual protein interactions. Frequently a protein makes functional contacts with several other proteins. For example, the catalytic subunit of a protein kinase may interact with one or more regulatory subunits and with substrates. Deletion of the gene encoding the kinase could provide information about the function of the protein as a whole, but would not provide information about the individual interactions that it makes with other proteins. As mentioned in the previous section, certain types of dominant negative mutants may be created that interfere with specific interactions made by a wild-type protein. In the kinase example, a dominant negative kinase might be created that interacts with its regulatory subunit but not its substrate such a mutant would be expected to compete with the wild-type kinase for regulatory subunits.

          Another type of potential disrupter of protein interactions that can be identified with the two-hybrid system is a peptide that interacts tightly and specifically with one of a pair of interacting proteins. Such peptides have been isolated from a random peptide library using the interaction trap yeast two-hybrid system as described by Colas et al. (Colas et al., 1996). These authors created a peptide library using a plasmid related to pJG4-5 that expressed random peptides fused to an activation domain and an inert platform molecule, E.coli thioredoxin. To find peptides that interact specifically with a bait protein an interactor hunt is performed as described in Protocol 2. Some of the specific peptides, called aptamers , would be expected to interact with surfaces of the bait that are required for interactions with other proteins. These are potential disrupters of specific protein interactions.

          A two-hybrid assay can also be used to show that a potential disrupter can interfere with a protein-protein interaction. The two proteins can be expressed, one as a bait and one as a prey, and then the potential disrupter can be expressed to see if it reduces the ability of the bait and prey to interact and activate a reporter. We developed a method to test whether an interacting domain or a peptide aptamer can disrupt specific interactions (M. Kolonin and R. Finley, unpublished). A potential disrupter is first isolated as an interactor. The library plasmid expressing the potential disrupter is isolated and used to transform RFY231, and these transformants are mated with a special bait-prey interaction strain as described in Protocol 4. In this case, however, the bait strain expresses the original bait as a prey (activation domain fusion) from plasmid pMK1, and a protein that interacts with it as a bait. Disruption of the interaction results in loss of LEU2 transcription and inability to grow on leu- plates.

          The methods outlined here present an integrated approach to understanding the function of proteins, protein interactions, and networks of proteins. First, all of the potential partners of a protein thought to be involved in a particular biological process can be identified. Second, many additional members of the same regulatory network can be identified in successive interactor hunts. Third, interaction domains can be mapped. Fourth, mutants incapable of specific interactions can be identified, and in many cases these mutants can be expressed in vivo to provide functional information. Finally, reagents can readily be developed that disrupt specific protein interactions, and then can be used to probe the function of these interactions in vivo .

          I thank Mikhail Kolonin, Jennifer Fonfara, and Catherine Nelson, for providing comments, and Mikhail Kolonin and members of the Finley lab for contributions to the protocols. I also thank the members of the Brent lab for their many contributions to the protocols. I especially thank Roger Brent who co-wrote previous versions of the interactor hunt protocols.

          Ausubel, F. M., Brent, R., Kingston, R. E., Morre, D., Seidman, J. G., and Struhl, K. (1987-1996). Current protocols in molecular biology (New York: Greene and Wiley-interscience).

          Bartel, P. L., Roecklein, J. A., SenGupta, D., and Fields, S. (1996). A protein linkage map of Escherichia coli bacteriophage T7. Nature Genetics 12 , 72-77.

          Brent, R., and al., e. (1997). http://xanadu.mgh.harvard.edu/brentlabhomepage4.html. web site.

          Colas, P., Cohen, B., Jessen, T., Grishina, I., McCoy, J., and Brent, R. (1996). Genetic selection of peptide aptamers that recognize and inhibit cyclin- dependent kinase 2. Nature 380 , 548-550.

          Durfee, T., Becherer, K., Chen, P.-L., Yeh, S.-H., Yang, Y., Kilburn, A. E., Lee, W.-H., and Elledge, S. J. (1993). The retinoblastoma protein associates with the protein phophatase type 1 catalytic subunit. Genes and Dev. 7 , 555-569.

          Estojak, J., Brent, R., and Golemis, E. A. (1995). Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol 15 , 5820-5829.

          Finley, R. L., Jr., and Brent, R. (1994). Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators. Proc Natl Acad Sci U S A 91 , 12980-12984.

          Finley, R. L., Jr., and Brent, R. (1995). Interaction trap cloning with yeast. In DNA Cloning, Expression Systems: A Practical Approach, B. D. Hames and D. M. Glover, eds. (Oxford: Oxford University Press), pp. 169-203.

          Finley, R. L., Jr., and Brent, R. (1996). Two-hybrid analysis of genetic regulatory networks. In The yeast two-hybrid system, P. L. Bartel and S. Fields, eds. (Oxford: Oxford University Press).

          Finley, R. L., Jr., Thomas, B. J., Zipursky, S. L., and Brent, R. (1996). Isolation of Drosophila cyclin D, a protein expressed in the morphogenetic furrow before entry into S phase. Proc. Natl. Acad. Wetenskaplike. USA 93 , 3011-3015.

          Finley, R. L. J., and al., e. (1997). http://cmmg.biosci.wayne.edu/rfinley/finlab/finlab-home.html. web site.

          Guthrie, C., and Fink, G. R. (1991). Guide to yeast genetics and molecular biology. In Methods in enzymology (Boston: Academic Press, Inc.).

          Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. (1993). Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75 , 791-803.

          Harlow, E., and Lane, D. (1988). Immunoblotting. In Antibodies: A laboratory manual (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory), pp. 471-510.

          Herskowitz, I. (1987). Functional inactivation of genes by dominant negative mutations. Nature 329 , 219-22.

          Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 , 680-685.

          Li, R., Waga, S., Hannon, G. J., Beach, D., and Stillman, B. (1994). Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication. Nature 371 , 534-537.

          Mendelsohn, A. R., and Brent, R. (1994). Applications of interaction traps/two-hybrid systems to biotechnology research. Curr. Op. Biotegnologie. 5 , 482-486.

          Miller, J. (1972). Experiments in molecular genetics (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory).

          Stagljar, I., Bourquin, J. P., and Schaffner, W. (1996). Use of the two-hybrid system and random sonicated DNA to identify the interaction domain of a protein. Biotechniques 21 , 430-432.

          Zervos, A. S., Gyuris, J., and Brent, R. (1993). Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites. Cell 72 , 223-32.


          Produksie van entstowwe, antibiotika en hormone

          Traditional vaccination strategies use weakened or inactive forms of microorganisms to mount the initial immune response. Modern techniques use the genes of microorganisms cloned into vectors to mass produce the desired antigen. Doctors then introduce the antigen into the body to stimulate the primary immune response and trigger immune memory. The medical field has used genes cloned from the influenza virus to combat the constantly changing strains of this virus.

          Antibiotics are a biotechnological product. Microorganisms, such as fungi, naturally produce them to attain an advantage over bacterial populations. Cultivating and manipulating fungal cells produces antibodies.

          Scientists used recombinant DNA technology to produce large-scale quantities of human insulin in E coli as early as 1978. Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in humans because of differences in the gene product. In addition, doctors use human growth hormone (HGH) to treat growth disorders in children. Researchers cloned the HGH gene from a cDNA library and inserted it into E coli selle deur dit in 'n bakteriese vektor te kloneer.

          In Summary: Medicinal Biotechnology

          Transgenic organisms possess DNA from a different species, usually generated by molecular cloning techniques. Vaccines, antibiotics, and hormones are examples of products obtained by recombinant DNA technology. Transgenic plants are usually created to improve characteristics of crop plants.


          Landbou -biotegnologie

          Biotechnology in agriculture can enhance resistance to disease, pest, and environmental stress, and improve both crop yield and quality.

          Transgenic Animals

          Although several recombinant proteins used in medicine are successfully produced in bacteria, some proteins require a eukaryotic animal host for proper processing. For this reason, the desired genes are cloned and expressed in animals, such as sheep, goats, chickens, and mice. Animals that have been modified to express recombinant DNA are called transgenic animals. Several human proteins are expressed in the milk of transgenic sheep and goats, and some are expressed in the eggs of chickens. Mice have been used extensively for expressing and studying the effects of recombinant genes and mutations.

          Transgenic Plants

          Figure 1. Corn, a major agricultural crop used to create products for a variety of industries, is often modified through plant biotechnology. (credit: Keith Weller, USDA)

          Manipulating the DNA of plants (i.e., creating GMOs) has helped to create desirable traits, such as disease resistance, herbicide and pesticide resistance, better nutritional value, and better shelf-life (Figure 1). Plants are the most important source of food for the human population. Farmers developed ways to select for plant varieties with desirable traits long before modern-day biotechnology practices were established.

          Plants that have received recombinant DNA from other species are called transgenic plants. Because they are not natural, transgenic plants and other GMOs are closely monitored by government agencies to ensure that they are fit for human consumption and do not endanger other plant and animal life. Because foreign genes can spread to other species in the environment, extensive testing is required to ensure ecological stability. Staples like corn, potatoes, and tomatoes were the first crop plants to be genetically engineered.

          Transformation of Plants Using Agrobacterium tumefaciens

          Gene transfer occurs naturally between species in microbial populations. Many viruses that cause human diseases, such as cancer, act by incorporating their DNA into the human genome. In plants, tumors caused by the bacterium Agrobacterium tumefaciens occur by transfer of DNA from the bacterium to the plant. Although the tumors do not kill the plants, they make the plants stunted and more susceptible to harsh environmental conditions. Many plants, such as walnuts, grapes, nut trees, and beets, are affected by A. tumefaciens. The artificial introduction of DNA into plant cells is more challenging than in animal cells because of the thick plant cell wall.

          Researchers used the natural transfer of DNA from Agrobacterium to a plant host to introduce DNA fragments of their choice into plant hosts. In nature, the disease-causing A. tumefaciens have a set of plasmids, called the Ti plasmids (tumor-inducing plasmids), that contain genes for the production of tumors in plants. DNA from the Ti plasmid integrates into the infected plant cell&rsquos genome. Researchers manipulate the Ti plasmids to remove the tumor-causing genes and insert the desired DNA fragment for transfer into the plant genome. The Ti plasmids carry antibiotic resistance genes to aid selection and can be propagated in E coli cells as well.

          Flavr Savr Tomato

          The first GM crop to be introduced into the market was the Flavr Savr Tomato produced in 1994. Antisense RNA technology was used to slow down the process of softening and rotting caused by fungal infections, which led to increased shelf life of the GM tomatoes. Additional genetic modification improved the flavor of this tomato. The Flavr Savr tomato did not successfully stay in the market because of problems maintaining and shipping the crop. However, since that time numerous crop plants have been developed and approved for sale and consumption. Corn, soybeans, and cotton in particular have been widely adopted by U.S. farmers.


          Simple, rapid and reliable methods to obtain high quality RNA and genomic DNA from Quercus ilex L. leaves suitable for molecular biology studies

          Isolation of high-quality RNA and genomic DNA (gDNA) from many samples is a necessary step before accomplishing molecular biology studies. The particular composition of Quercus ilex leaves, specially hard and rich in cell wall material, polyphenolics and secondary metabolites, usually results in preparations contaminated with non-nucleic acid compounds. Although many methods have been developed, each case of study demands a protocol adapted to the specific plant sample and the pursued research objectives. We have evaluated several protocols to establish the methodology that best suited to our current genetic and molecular studies on Q. ilex. Our priority was to select the simplest methods reducing the plant starting material and the time employed, without compromising yield, quality and integrity of the isolated nucleic acids. Our results point to two protocols based on silica-membrane purification, as the most convenient for Q. ilex leaf tissue, and both procedures are greatly improved by adding insoluble polyvinyl polypyrrolidone during the isolation process. The protocols optimized here can be completed at the microfuge scale and allow a researcher to process 48 samples in 1 h, producing high quality preparations suitable for the routinely molecular biology applications with higher efficiency than other more labour and time-consuming protocols.

          Dit is 'n voorskou van intekeninginhoud, toegang via jou instelling.


          Kyk die video: cDNA. Complementary DNA (Augustus 2022).