Inligting

Hoe word die twee paringsoorte, a en α, vir S. cerevisiae uitgespreek?

Hoe word die twee paringsoorte, a en α, vir S. cerevisiae uitgespreek?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

By die lees van S. cerevisiae is daar twee paringsoorte, waarvan een die Latynse letter isaen die ander is die Griekse letterα.

Hoe word hierdie twee tipes so uitgespreek dat hulle onderskei kan word?

Met ander woorde wanneer ek die teks uitspreek wil ek nie vir albei 'a' sê nie; daar moet 'n ander woord of frase vir elkeen wees, sodat hulle bekend is dat hulle anders is.

Is dit bloot ditaword uitgespreek as 'n enαword as alfa uitgespreek?


Korrek.

Haploid paring tipes in Saccharomyces cerevisiae is MATa (spreek a uit soos in day) en MATα (d.i. MAT-alfa).

Gerieflikheidshalwe kan jy ook na die ooreenstemmende allele verwys as MATa of MATalpha (sien ook hier)


'N Konstitutief aktiewe GPCR beheer morfogene oorgange in Cryptococcus neoformans

Seks by swamme word aangedryf deur peptiedferomone wat deur sewe-transmembraan feromoonreseptore waargeneem word. In Cryptococcus neoformans, seksuele voortplanting vind plaas deur 'n outcrossing/heterothallic a- seksuele siklus of 'n inteling/homotaliese – eenslagtige paringsproses. Feromoonreseptore gekodeer deur die paringstipe lokus (MAT) bemiddel wedersydse feromoonwaarneming tydens paring van teenoorgestelde geslagte en dra by tot, maar is nie noodsaaklik vir eenseksuele paring nie. 'n Feromoonreseptoragtige geen, KPR2, is ontdek wat nie deur MAT en wie se uitdrukking veroorsaak word tydens a- paring. cpr2 mutante is vrugbaar maar het 'n samesmeltingsdefek en produseer abnormale hipale strukture, terwyl KPR2 oormatige uitdrukking veroorsaak ongeslagtelike voortplanting. Wanneer heterologies uitgedruk in Saccharomyces cerevisiae, Cpr2 aktiveer feromoonreaksies in die afwesigheid van enige ligand. Hierdie konstitutiewe aktiwiteit is die gevolg van 'n onkonvensionele residu, Leu 222, in die plek van 'n bewaarde prolien in transmembraandomein ses, 'n Cpr2 L222P -mutant is nie meer konstitutief aktief nie. Cpr2 betrek dieselfde G-proteïen-geaktiveerde seinkaskade as die Ste3a/α feromoonreseptore, en ding daarmee mee om die aktivering van die pad. Hierdie studie het 'n nuwe paradigma gevestig waarin 'n natuurlike voorkomende aktiewe G-proteïen-gekoppelde reseptor morfogenese in swamme beheer.


Sel-tipe spesifikasie in Saccharomycotina

Die lewensiklus van ontluikende giste (Figuur 1) bestaan ​​hoofsaaklik uit drie seltipes: haploïede van twee isogame paringsoorte, a en α, en a/α diploïede (Herskowitz 1988 Madhani 2007). Die twee tipes haploïde word dikwels paringsoorte genoem omdat hulle paringsgedrag beskryf: paring vind slegs tussen a selle en α -selle. Paring-tipe skakel is die proses waardeur 'n haploïede a sel kan 'n haploïede α-sel word deur sy genotipe by die paringstipe te verander (MAT) lokus van MATa aan MATα, of andersom. Alhoewel dit histories paring-tipe skakel genoem word, kan die proses ook sel-tipe skakel genoem word.

Skematiese lewensiklus van S. cerevisiae.

Al drie seltipes kan mitoties verdeel gegewe gunstige omgewingstoestande, maar in S. cerevisiae, vegetatief groeiende haploïede selle van teenoorgestelde paringstipes sal maklik paar as hulle mekaar ontmoet (Merlini et al. 2013). Haploid a selle druk die G-proteïengekoppelde reseptor Ste2 uit, wat die α-faktor-paringferomoon wat deur haploïede α-selle uitgedruk word, opspoor. Wederkerig druk haploïede α -selle die reseptor Ste3 uit, wat die bind a-faktorferomoon uitgedruk deur haploïed a selle. Interaksie tussen 'n feromoon en sy reseptor in óf die haploïede seltipe veroorsaak 'n MAP-kinase sein waterval wat lei tot G1fase -arrestasie van mitotiese verspreiding, vorming van 'n paringsprojeksie (shmoo) wat gepolariseer is na die feromoonbron, en uiteindelik paring deur sel- en kernfusie om 'n diploïede sigoot te genereer (Bardwell 2005 Jones en Bennett 2011 Merlini et al. 2013). Diploïede word veroorsaak om meiose en sporulasie te ondergaan deur voedingstofbeperkende toestande in die omgewing (spesifiek honger vir stikstof in die teenwoordigheid van 'n nie-fermenteerbare koolstofbron), wat lei tot die vorming van 'n ascus. Die ascus bevat gewoonlik vier haploïede spore (twee aEn twee α’s) wat ontkiem na herstel van gunstige toestande (Honigberg en Purnapatre 2003 Piekarska et al. 2010 Neiman 2011).

Spesies in die swamfilm Ascomycota wissel of hulle verkies om vegetatief te groei as haploïede ("haplonties") of as diploïede ("diplontiese") (Phaff et al. 1966). Terwyl natuurlike isolate van S. cerevisiae is hoofsaaklik diploïed, baie ander gissoorte is hoofsaaklik haploïed, insluitend K. lactis, S. pombe, en die metielotrofiese giste soos Ogataea (Hansenula) polimorfa (Dujon 2010). In ooreenstemming met hierdie ploidy voorkeure, S. cerevisiae trou spontaan (selfs in ryk media) en gebruik slegs 'n omgewingswysie vir sporulasie. Daarteenoor, in haplontiese giste, word paring en sporulering gesamentlik deur swak omgewings geïnduseer en kom gewoonlik agtereenvolgens voor sonder ingrypende diploïede mitotiese seldelings, byvoorbeeld in Zygosaccharomyces, Kluyveromyces, Ogataea, Clavispora, en Skizosaccharomyces (Herman en Roman 1966 Gleeson en Sudbery 1988 Booth et al. 2010 Merlini et al. 2013 Sherwood et al. 2014). Sporulasie van die sigoot onmiddellik na kernfusie lei tot 'n kenmerkende "haltervormige" askus wat die buitelyn van die twee shmooing haploïede selle behou wat dit gevorm het (Kurtzman et al. 2011).

Die MAT lokus beheer prosesse wat sel-tipe identiteit bepaal (Herskowitz 1989 Johnson 1995). Vir haploïede selle (beide a en α), seltipe-spesifieke prosesse sluit in die induksie van bevoegdheid om te paar en die onderdrukking van sporulasie, terwyl diploïede selle onderdrukking van paring vereis en die vermoë om meiose en sporulasie te begin. Ander prosesse verskil ook tussen haploïede en diploïede selle, soos die keuse van ligging vir die vorming van die volgende knop (aksiale vs. bipolêre patrone Chant en Pringle 1995), en die voorkeurmeganisme vir dubbelstring DNA (dsDNA) breekherstel (homologe rekombinasie in diploïede vs. nie-homologe einde verbind in haploïede Kegel et al. 2001 Valencia et al. 2001).

Die MATa en MATα -allele (soms idiomorfe genoem) van die MAT lokus is in volgorde heeltemal anders. In S. cerevisiae die MATα-alleel bevat twee gene, MATα1 en MATα2, en die MATa alleel bevat 'n enkele geen, MATa1 (Figuur 2). Hierdie drie gene kodeer vir transkripsiereguleerders. Hulle bepaal die seltipe van die haploïed deur die uitdrukking van a-spesifiek (asg) en α-spesifieke (αsg) gene (Johnson 1995 Galgoczy et al. 2004 Haber 2012). Daar is –5–12 asg's en αsg's, afhangende van die spesie (Sorrells et al. 2015). Benewens hierdie, word 'n gedeelde stel haploïedspesifieke gene (hsg's) (∼12–16 in getal) wat paring vergemaklik, konstitutief uitgedruk in beide a en α -selle maar nie in nie a/α diploïede (stand et al. 2010), en 'n groter groep van ~100 algemene feromoon-geaktiveerde gene word in haploïede van beide tipes geïnduseer sodra 'n feromoonsein van die teenoorgestelde tipe haploïed opgespoor word (Sorrells) et al. 2015). Byvoorbeeld, in S. cerevisiae, die feromoongene MFA1 en MFα1 is 'n asg en 'n αsg, onderskeidelik die feromoon seinroete G proteïene-subeenheid gene GPA1, STE4, en STE18 is hsg's, en die MAP kinase FUS3 is 'n algemene feromoon-geaktiveerde geen (Sorrells et al. 2015).

Gene organisasie in die MAT, HML, en HMR lokusse op S. cerevisiae chromosoom III. Skakering dui op gene waarvan die transkripsie onderdruk word.

In S. cerevisiae haploïede α-selle, die MATα1 geen kodeer vir die HMG-domein transkripsie-aktiveerder α1 (voorheen na verwys as 'n "α-domein" proteïen maar word nou erken as 'n divergente HMG domein Martin et al. 2010), en die MATα2-geen vir die homeodomein-transkripsierepressor α2. Die α1 en α2 proteïene kan beide komplekse afsonderlik vorm met die konstitutief uitgedrukte Mcm1 (MADS domein) proteïen wat stroomop bind asg's en αsg's. In α-selle word transkripsie van αsg's geaktiveer omdat die α1-Mcm1-kompleks die transkripsiefaktor Ste12 na hul promotors werf, terwyl transkripsie van asg's word onderdruk omdat die α2-Mcm1-kompleks die Tup1-Ssn6-corepressor werf (Figuur 3).

Logika van die seltipe-spesifikasiekringe in Saccharomycetaceae spesies. Vaste kleure verteenwoordig seltipes (groen, a pienk, α bruin, a/α), en buitekleure verteenwoordig gene stelle. Die genotipe (1) van 'n sel s'n MAT locus spesifiseer die regulatoriese proteïene wat in daardie sel voorkom (2 blokkies), wat by promotors (3) inwerk om gepaste transkripsie van die drie gene stelle te genereer (4 asg's, αsg's en hsg's) en bepaal die seltipe (5). Die geel blokkies beskryf die herbedrading gebeurtenis wat plaasgevind het wanneer MATa2 verlore gegaan het, wat saamval met die WGD. Die diagram som inligting van die post-WGD spesies op S. cerevisiae en die nie-WGD spesies K. lactis, L. kluyveri, en C. albicans (Tsong et al. 2003, 2006 Stand et al. 2010 Bakker et al. 2012 Sorrells et al. 2015).

In S. cerevisiae haploïed a selle, die MAT locus bevat slegs die MATa1 geen kodeer vir die homeodomein proteïen a1, maar hierdie proteïen is nie nodig vir a seltipe identiteit. Die identiteit van a selle word eerder gedefinieer deur die afwesigheid van beide α1, die aktiveerder van αsg's en α2, die onderdrukker van asg se. In plaas daarvan om 'n a-spesifieke aktiveerder, asg's word geaktiveer deur Mcm1 en Ste12, wat konstitutief in alle seltipes uitgedruk word (Figuur 3). Dus in S. cerevisiae, die a seltipe is die verstektipe, en gisselle wat a MAT lokus sal met haploïede α-selle paar.

In a/α diploïede selle van S. cerevisiae, αsg's, asg's en hsg's word almal onderdruk. Hierdie selle het MATα1 en MATα2 gene by die MAT lokus op een chromosoom, en MATa1 aan die ander kant (figuur 2), wat lei tot die vorming van die a1-α2 heterodimer van die twee homeodomein proteïene. Die a1-α2 dimeer onderdruk direk transkripsie van hsg's en onderdruk indirek αsg's deur onderdrukking van MATα1 (Figuur 3). Transkripsie van asg's in diploïede word deur α2-Mcm1 onderdruk soos in haploïede α-selle. Want S. cerevisiae gebruik die vorming van 'n heterodimeer om die heterosigositeit daarvan te bepaal MAT lokus, en omdat hierdie heterodimeer 'n onderdrukker is, is daar geen "diploïed-spesifieke" gene in S. cerevisiae (Galgoczy et al. 2004). In plaas daarvan word diploïdespesifieke prosesse soos meiose en sporulasie in haploïede onderdruk. Hierdie onderdrukking word bereik via die hsg RME 1, 'n haploïed-spesifieke aktiveerder wat transkribeer IRT1, 'n niekoderende RNA wat op sy beurt onderdruk IME1, die meester-induktor van meiose (van Werven et al. 2012). Dus die gekombineerde optrede van RME 1 en IRT1 keer die uitset van die hsg regulatoriese logika om om te beperk IME1 uitdrukking aan diploïede (Figuur 3). IME1 uitdrukking vereis ook die omgewingseine van stikstof- en glukose-uitputting wat meiose inisieer (Neiman 2011). Geen gene het konstitutiewe diploïed nie (a/α) spesifieke uitdrukking op dieselfde manier as hsg's, αsg's, en asg's het konstitutiewe seltipe-spesifieke uitdrukking in haploïede.

Die asg's en αsg's gereguleer deur die MAT lokus sluit hoofsaaklik gene vir feromone, hul reseptore en seinproteïene in wat nodig is vir die herkenning van selle met die teenoorgestelde paringstipe (Johnson 1995 Galgoczy et al. 2004). Binding van die feromone aan hul reseptore veroorsaak 'n seinkaskade wat die feromoonreaksie-weg genoem word, wat verdere gametiese differensiasie na paringsbevoegdheid in haploïede veroorsaak (Wittenberg en La Valle 2003 Merlini et al. 2013). Hierdie kaskade kulmineer in die aktivering van die transkripsiefaktor Ste12, wat nodig is vir die uitdrukking van 'n groot aantal gene wat verantwoordelik is vir paring—die algemene feromoon-geaktiveerde gene (Roberts) et al. 2000 Sorrells et al. 2015). Ste12 word in alle seltipes uitgedruk, maar as haploids 'n feromoon opspoor, word Ste12 aansienlik meer aktief by feromoon-responsiewe promotors omdat die Fus3 MAP-kinase twee proteïene, Dig1 en Dig2, wat Ste12 inhibeer, inaktiveer. et al. 2001). Ste12 word vereis vir uitdrukking van almal asg's, αsg's en hsg's. Dit bind aan hsg -promotors as 'n dimeer wat deur die a1-α2-kompleks in diploïede, en dit word na αsg-promotors gebring deur α1-Mcm1. Ste12 aktiveer ook asg -promotors in samewerking met Mcm1, alhoewel asg-regulering het onlangse dramatiese evolusionêre verandering ondergaan (Sorrells et al. 2015).


Herbedrading van die logiese kring na duplisering van die hele genoom in Saccharomycetaceae

Die seltipe-spesifikasie stroombaan van S. cerevisiae het uitgebreide herorganisasie ondergaan sedert dit afgewyk het van ander spesies in die gisfamilie Saccharomycetaceae, soos bv. Candida albicans en K. lactis. Die herorganisasie het drie afsonderlike stappe behels: die wins van α2-bindingsplekke stroomop van asg's om hulle in α haploids (Tsong et al. 2006), die wins van Ste12 -bindingsplekke stroomop asg's sodat dit standaard uitgedruk word (Sorrells et al. 2015), en die volledige verlies van die MATa2 geen. MATa2 kodes vir 'n HMG-domein transkripsie-aktiveerder 1 genoem a2, wat dien as 'n aktiveerder van asg is in C. albicans en K. lactis (Tsong et al. 2003, 2006 Baker et al. 2012 Sorrells et al. 2015). Interessant genoeg het die tweede en derde van hierdie stappe op dieselfde tak van die filogenie plaasgevind as die hele-genoom duplisering (WGD) (Sorrells et al. 2015), maar dit is nie bekend of dit die WGD voor of na die datum was nie. Onlangse filogenomiese ontleding toon aan dat die WGD in werklikheid 'n hibridisasie tussen twee spesies was: een wat verband hou met Zygosaccharomyces/Torulaspora, en een wat verband hou met Kluyveromyces/Lachancea/Eremothecium (Marcet-Houben en Gabaldon 2015). MATa2 is teenwoordig in die meeste nie-WGD-spesies, insluitend hierdie twee ouerlike afstammelinge, maar is afwesig in S. cerevisiae en alle ander afstammelinge na WGD, insluitend vroeë uiteenlopende, soos Vanderwaltozyma polyspora (Scannell et al. 2007 Wolfe et al. 2015).

Figuur 3 gee 'n opsomming van die logika van die seltipe-spesifikasie stroombaan in verskeie gis spesies, en hoe die uitset van hierdie logiese kring onveranderd gebly het deur die herbedrading gebeurtenis (Tsong et al. 2006 Sorrells et al. 2015). By spesies soos C. albicans en K. lactis wat die behou MATa2 geen, onderdrukking van asg -uitdrukking is nie nodig in α -selle nie, want asg's is standaard nie aan nie, dus is die onderdrukking van asg's deur α2 kom slegs voor in S. cerevisiae. Daarom C. albicans MAT-vee -stamme is steriel eerder as om haploïed te word a paringsgedrag (Tsong et al. 2003). Die filogenetiese verwantskap (Figuur 4) dui daarop dat die selspesifikasiebaan in C. albicans en K. lactis (Figuur 3) verteenwoordig die voorvaderlike toestand van die netwerk. Verlies van a2 in die post-WGD-afstamming was slegs moontlik omdat die promotors van asg's in hierdie geslag het direkte DNA-bindingsplekke vir Ste12 (Sorrells et al. 2015). In die voorvaderlike situasie is Ste12 gebring na asg promotors indirek deur 'n proteïen-proteïen interaksie met a2, soos voorkom in K. lactis. 'n Tussentoestand oorleef in K. wickerhamii en Lachancea kluyveri, waar sommige asg's het "hibriede" promotors wat albei deur α2 in α -selle onderdruk word en geaktiveer word deur a2 in a selle (Baker et al. 2012). Benewens die verlies van MATa2 in die post-WGD-klade van Saccharomycetaceae, ander variasies in MAT geeninhoud kan gevind word in die CUG-Ser (Candida) klade, waarin veelvuldige spesies nie die tuisdomein -gene MATa1 en/of MATα2 het nie, en dit lyk asof een spesie geen MAT gene enigsins (Butler et al. 2009 Butler 2010).

Filogenetiese boom van filum Ascomycota wat groot klades toon, MAT-fokus organisasie, en bekende of afgeleide paring-tipe skakel meganismes. Gebaseer op Riley et al. (2016), met plasing van A. rubescens soos in Shen et al. (2016). Paringstipe-wisseling vind nie plaas by spesies met slegs een nie MAT-soos lokus of in Aspergillus nidulans, wat 'n primêre homotalliese spesie is.

Filogenetiese boom van filum Ascomycota wat groot klades toon, MAT-fokus organisasie, en bekende of afgeleide paring-tipe skakel meganismes. Gebaseer op Riley et al. (2016), met die plasing van A. rubescens soos in Shen et al. (2016). Paringstipe-wisseling vind nie plaas by spesies met slegs een nie MAT-agtig locus of in Aspergillus nidulans, wat 'n primêre homotalliese spesie is.

Sel-tipe spesifikasie in giste word bereik deur kombinatoriese regulatoriese geen teenwoordigheid of afwesigheid (Figuur 3). Die vereiste vir een kopie van elk MAT alleel (MATa of MATα) om die heterodimeriese onderdrukker te vorm, dien as 'n sensor van ploïdie, wat verseker dat slegs diploïede selle bevoeg is vir meiotiese toetrede (Haag 2007), en dat diploïede selle onbevoeg is vir bykomende paringsgebeure wat aneuploïdie tot gevolg sal hê. Verder, die teenwoordigheid van slegs een aktiewe MAT allel in haploïede selle verhoed die uitdrukking en herkenning van selfferomoon wat andersins paringsprosesse sou veroorsaak in die afwesigheid van 'n paringsvennoot. Daar is dus voorgestel dat die struktuur van die MAT locus dien as 'n 'ontwikkelingsskakelaar' wat parings- en sporuleringsreaksies in die toepaslike stadiums van die lewensiklus van gis veroorsaak (Perrin 2012).


MATERIAAL EN METODES

Alle gisstamme is afkomstig van wilde tipe S.cerevisiae W303 (DDY2, DDY3 en DDY4, oorspronklik JRY4012, JRY4013 en JRY2334, verkry van Jasper Rine, Universiteit van Kalifornië by Berkeley genotipes van alle gisstamme wat in hierdie studie gegenereer word, word in tabel 1 gelys). Sedert TRT2 is 'n noodsaaklike enkelkopie tRNA geen, 'n 0,32 kb fragment van TRT2 (SGD -chromosoom XI -koördinate 46596–46919) is deur PCR gekloneer in plasmiede pRS414 en pRS415 (26) om verwyderings van die geen te dek (plasmiede pDD675 en pDD676, onderskeidelik). Om die trt2cbt1Δ ::URA3 verslaggewer stamme beskryf in Figuur 1, 'n 2.1 kb segment van die TRT2 lokus (koördinate 46162–48248) is geamplifiseer deur PCR en gekloneer in pCR2.1-TOPO (Invitrogen) om plasmied pDD689 te maak. Die resulterende plasmied is gesny Spe Ek en Xho Ek moet verwyder TRT2 en CBT1, en is vervang met die Spe I-Xho I URA3 fragment uit pDD588 (URA3 gekloon in Bluescript SK+) om plasmied pDD694 te skep, trt2cbt1Δ ::URA3. Die gemodifiseerde lokus is uit pDD694 gesny en omskep in die diploïede stam DDY2, en URA+ rekombinante is gekies en deur PCR gekeur om die korrekte integrasie te verifieer. Hierdie diploïede stam is daarna getransformeer met TRT2 plasmiede pDD675 of pDD676 om die delesie te bedek, gesporuleerde en URA+ haploïede is herwin. Die cbt1Δ ::URA3 beheer stamme is gemaak deur direkte PCR uitklophou van CBT1 met URA3, met pRS406 as sjabloon. Selle is gegroei op gis minimale medium (YMD + 2% dekstrose) sonder urasiel om te toets vir onderdrukking van die URA3 merkergeen. Gistestikstofbasis is by U.S. Biologicals gekoop, en die mengsel YMD + bevat slegs die voedingstowwe wat nodig is vir die groei van W303 -stamme (adenien, histidien, leucien, lysien, tryptofaan en urasil).

A URA3 merkergeen word onderdruk wanneer dit stroomop van die STE6 α2 operateur webwerf in S.cerevisiae chromosoom XI. (A) Die wilde tipe STE6CBT1 gebied van chromosoom XI word bo-op uitgebeeld. URA3 is ingevoeg deur homoloë rekombinasie stroomop van die STE6 α2 -operateur om die TRT2 tRNA Thr -geen (DDY890, DDY891, DDY902 en DDY903), of om die ingryping te behou TRT2 geen (DDY974 en DDY 975). (BElke stam is gestreep op YMD wat uracil ontbreek (YMD - ura) en vir 2 dae geïnkubeer. MATα -stamme ontbreek TRT2 het 'n geïnhibeerde groei getoon op 'n medium wat nie uracil het nie, terwyl alle stamme gelyk gegroei het met minimale uracil wat YMD bevat (YMD + alles).

A URA3 merkergeen word onderdruk wanneer dit stroomop van die ingevoeg word STE6 α2 operateur werf in S.cerevisiae chromosoom XI. (A) Die wilde-tipe STE6CBT1 gebied van chromosoom XI word bo-op uitgebeeld. URA3 is ingevoeg deur homoloë rekombinasie stroomop van die STE6 α2 operateur om óf die te verwyder TRT2 tRNA Thr -geen (DDY890, DDY891, DDY902 en DDY903), of om die ingryping te behou TRT2 geen (DDY974 en DDY 975). (BElke stam is gestreep op YMD wat uracil ontbreek (YMD - ura) en vir 2 dae geïnkubeer. MATα-stamme ontbreek TRT2 het 'n geïnhibeerde groei getoon op 'n medium wat nie uracil het nie, terwyl alle stamme gelyk gegroei het met minimale uracil wat YMD bevat (YMD + alles).

Genotipes van alle gisstamme wat in hierdie studie gegenereer word

. S.cerevisiae W303 stamme. Bron .
DDY2 MATα/MATaade2-1/ADE2 his3-11/his3-11 leu2-3, 112/leu2-3, 112 LYS2/lys2Δ trp1-1/trp1-1 ura3-1/ura3-1J. Rine
DDY3 MATaADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1J. Rine
DDY4 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1J. Rine
DDY889 MATα ADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ:: URA3 pTRT2: LEU2Hierdie studie
DDY890 MATaade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ::URA3 ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY891 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ::HIS3 pTRT2:TRP1Hierdie studie
DDY902 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY 903 MATα ade2-1 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY974 MATα ade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 cbt1Δ::URA3 ppr1Δ:: HIS3Hierdie studie
DDY975 MATaade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 cbt1Δ:: URA3 ppr1Δ::SY3Hierdie studie
DDY1022 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1024 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1026 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1028 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY1261 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ α2 operateurΔ ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY1262 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ α2 operateurΔ ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY1737 MATα ADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1739 MATaADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1740 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1742 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1805 MATα ade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ pTRT2: URA3 hos1 :: HIS3 hos2 :: TRP1 rpd3 :: LEU2Hierdie studie
DDY1825 MATα ADE21 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ pTRT2:URA3 hos1::HIS3 hos2::TRP1 rpd3::LEU2Hierdie studie
DDY1956 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2 hda1Δ:: KanMXHierdie studie
DDY2021 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2 hda1Δ:: KanMXHierdie studie
. S.cerevisiae W303 stamme. Bron .
DDY2 MATα/MATaade2-1/ADE2 his3-11/his3-11 leu2-3, 112/leu2-3, 112 LYS2/lys2Δ trp1-1/trp1-1 ura3-1/ura3-1J. Rine
DDY3 MATaADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1J. Rine
DDY4 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1J. Rine
DDY889 MATα ADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ:: URA3 pTRT2: LEU2Hierdie studie
DDY890 MATaade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY891 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ::HIS3 pTRT2:TRP1Hierdie studie
DDY902 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY 903 MATα ade2-1 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY974 MATα ade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 cbt1Δ::URA3 ppr1Δ:: HIS3Hierdie studie
DDY975 MATaade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 cbt1Δ::URA3 ppr1Δ:: HIS3Hierdie studie
DDY1022 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1024 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1026 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY1028 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1261 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ α2 operateurΔ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1262 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ α2 operateurΔ ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY1737 MATα ADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1739 MATaADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1740 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1742 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1805 MATα ade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ pTRT2: URA3 hos1 :: HIS3 hos2 :: TRP1 rpd3 :: LEU2Hierdie studie
DDY1825 MATα ADE21 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ pTRT2: URA3 hos1 :: HIS3 hos2 :: TRP1 rpd3 :: LEU2Hierdie studie
DDY1956 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2 hda1Δ:: KanMXHierdie studie
DDY2021 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2 hda1Δ:: KanMXHierdie studie

Genotipes van alle gisstamme wat in hierdie studie gegenereer word

. S.cerevisiae W303 stamme. Bron .
DDY2 MATα/MATaade2-1/ADE2 his3-11/his3-11 leu2-3, 112/leu2-3, 112 LYS2/lys2Δ trp1-1/trp1-1 ura3-1/ura3-1J. Rine
DDY3 MATaADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1J. Rine
DDY4 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1J. Rine
DDY889 MATα ADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ::URA3 pTRT2:LEU2Hierdie studie
DDY890 MATaade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY891 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ:: HIS3 pTRT2: TRP1Hierdie studie
DDY902 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY 903 MATα ade2-1 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2-cbt1Δ::URA3 ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY974 MATα ade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 cbt1Δ:: URA3 ppr1Δ:: HIS3Hierdie studie
DDY975 MATaade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 cbt1Δ:: URA3 ppr1Δ::SY3Hierdie studie
DDY1022 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY1024 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY1026 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1028 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY1261 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ α2 operateurΔ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1262 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ α2 operateurΔ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1737 MATα ADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1739 MATaADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1740 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1742 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1805 MATα ade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ pTRT2:URA3 hos1::HIS3 hos2::TRP1 rpd3::LEU2Hierdie studie
DDY1825 MATα ADE21 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ pTRT2: URA3 hos1 :: HIS3 hos2 :: TRP1 rpd3 :: LEU2Hierdie studie
DDY1956 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2 hda1Δ:: KanMXHierdie studie
DDY2021 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2 hda1Δ:: KanMXHierdie studie
. S.cerevisiae W303 stamme. Bron .
DDY2 MATα/MATaade2-1/ADE2 his3-11/his3-11 leu2-3, 112/leu2-3, 112 LYS2/lys2Δ trp1-1/trp1-1 ura3-1/ura3-1J. Rine
DDY3 MATaADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1J. Rine
DDY4 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1J. Rine
DDY889 MATα ADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ:: URA3 pTRT2: LEU2Hierdie studie
DDY890 MATaade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ::URA3 ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY891 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2-cbt1Δ::URA3 ppr1Δ:: HIS3 pTRT2: TRP1Hierdie studie
DDY902 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY 903 MATα ade2-1 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2-cbt1Δ:: URA3 ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY974 MATα ade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 cbt1Δ:: URA3 ppr1Δ:: HIS3Hierdie studie
DDY975 MATaade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 cbt1Δ:: URA3 ppr1Δ:: HIS3Hierdie studie
DDY1022 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY1024 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1026 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1028 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ::HIS3 pTRT2:LEU2Hierdie studie
DDY1261 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ α2 operateurΔ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1262 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ α2 operateurΔ ppr1Δ:: HIS3 pTRT2: LEU2Hierdie studie
DDY1737 MATα ADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1739 MATaADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1740 MATaADE2 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1742 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 α2 operateurΔ Hierdie studie
DDY1805 MATα ade2-1 his3-11 leu2-3,112 LYS2 trp1-1 ura3-1 trt2Δ pTRT2: URA3 hos1 :: HIS3 hos2 :: TRP1 rpd3 :: LEU2Hierdie studie
DDY1825 MATα ADE21 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ pTRT2:URA3 hos1::HIS3 hos2::TRP1 rpd3::LEU2Hierdie studie
DDY1956 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2 hda1Δ:: KanMXHierdie studie
DDY2021 MATα ADE2 his3-11 leu2-3,112 lys2Δ trp1-1 ura3-1 trt2Δ ppr1Δ:: HIS3 pTRT2: LEU2 hda1Δ:: KanMXHierdie studie

Om die gemodifiseerde chromosomale loci te maak, is pDD689 gemutageniseer met behulp van die Quik-change kit (Stratagene) om te verwyder TRT2 (oligonukleotiede DDO-96/97) van boks A aan die boks B (chromosoom XI koördinate 46747–46800). Die α2-operateur van koördinate 46472–46489 is op dieselfde manier met oligonukleotiede DDO-123/124 verwyder. Plasmiede wat skrape bevat TRT2 en/of die α2 -operateur is omskep in DDY889 (trt2cbt1Δ ::URA), gekies op 5-FOA, en behoorlike integrasie geverifieer deur PCR. Gevolglike stamme wat gemodifiseer is STE6 – CBT1 lokusse is teruggekruis na trt2cbt1Δ ::URA3 probeer om 'n broer te kry MATa en MATα weergawes.

Vir noordelike klad-analise is RNA voorberei soos beskryf in Iyer en Struhl (27). Northern blots het 10 μg totale RNA per baan bevat, en is uitgevoer met behulp van Northern Max reagense (Ambion). CBT1 noordelike vlekke is uitgevoer op 1.0% agarosegel, en die TRT2 vlek in figuur 4 is uitgevoer op 'n 1,2% agarosegel. Noordelike probes is gegenereer uit PCR-produkte van die eerste 600 bp van elke geen (behalwe vir TRT2, waar die hele geen versterk is) wat 'n T7 RNA -polimerase -promotor bevat wat aan die stroomaf -primer geheg is. Hierdie PCR-produkte is as sjablone gebruik om radio-gemerkte riboprobes te sintetiseer met behulp van die Ambion Strip-EZ-stel. Alle oligonukleotiedreekse wat gebruik word vir uitklophoue, PCR -klonings, sondesjablone en mutagenese reaksies is op aanvraag beskikbaar.

HDA1 skrapping in die trt2Δ stam is gemaak deur standaard PCR uitklop protokolle met behulp van die plasmied pUG6 as 'n sjabloon (28). Die hos1 hos2 rpd3 stamme is gemaak deur kruising trt2Δ stamme met stam DY6445 (MATα ade2 kan1 his3 leu2 trp1 ura3 hos1::HIS3 hos2::TRP1 rpd3::LEU2), 'n geskenk van David Stillman (Universiteit van Utah).

Chromatien immunopresipitasie is uitgevoer soos beskryf in Kuo en Allis (29). Teenliggaampies wat gebruik is, was anti-asetiel-histoon H3 en anti-asetiel-histoon H4 van Upstate (katalogus No. 06–599 en 06–866). 'N Aliquot van 5 ul van 'n 1: 10 -verdunning van DNA wat uit die immunopresipitate herwin is, is gebruik om PCR -reaksies te programmeer (Taq polimerase gekoop by Promega), en dieselfde volume van 'n 1: 40 -verdunning is gebruik vir die insetkontroles. PCR -toestande was 95 ° C vir 2 minute (aanvanklike denaturasie), 95 ° C × 30 s, 55 ° C × 30 s, 72 ° C × 60 s (28 siklusse).


Ingenieurswese van Poliploïed Saccharomyces cerevisiae vir die afskeiding van groot hoeveelhede swamglukoamilase

FIG. 1. Struktuur van pSAK068-GAI. pSAK068-GAI is 'n δ-integrerende plasmied wat gebou is vir GAI-afskeiding soos beskryf in materiale en metodes. P mf α, S. mf α, en T. mf α dui onderskeidelik promotor-, sein- en terminatorvolgorde van paringsfaktor α aan. FIG. 2. Lokalisering van δ-geïntegreerde GAI-gene op die chromosome in verskillende haploïede selle. Die chromosomale DNA's geskei deur gel-elektroforese met polsveld is met GAI cDNA as 'n sonde gehibrideer. GHM-4925, GHM-7244, GHM-7943 en GHM-6859 is verkry deur transformasie van stam CG379 met pSAK068-GAI en pSAKL068-GAI. GHT-127 is gegenereer uit diploïede kruisings tussen GHM-4925 en GHM-7244a. GHT-478 is gegenereer uit diploïede kruisings tussen GHM-6859 en GHM-7943a. GHP-251 is gegenereer uit diploïede kruisings tussen GHT-127 en GHT-478a. Lane 1, GHM-4925 bane 2, GHM-7244 bane 3, GHM-7943 bane 4, GHM-6859 bane 5, GHT-127 bane 6, GHT-478 bane 7, GHP-251. FIG. 3 . Glukoamilase -aktiwiteite van haploïede selle wat verskeie GAI -gene op chromosome dra. Glukoamilase (GA) aktiwiteite van verskillende haploïede selle word getoon. GHM-4925, GHM-7244, GHM-7943 en GHM-6859 bevat GAI-gene op twee chromosome. GHT-127 en GHT-478 bevat GAI-gene op drie chromosome. GHP-251 bevat GAI-gene op vyf chromosome. The error bars indicate the standard deviations of three separate experiments. FIG. 4 . Construction of the haploid yeast cells carrying GAI genes on multiple chromosomes.

Laai hierdie artikel af en druk dit uit vir u persoonlike wetenskaplike, navorsings- en opvoedkundige gebruik.

Koop 'n enkele uitgawe van Wetenskap vir slegs $ 15 dollar.

Wetenskap

Vol 287, Issue 5454
04 February 2000

Artikel Gereedskap

Meld asseblief aan om 'n waarskuwing vir hierdie artikel by te voeg.

By Christopher J. Roberts , Bryce Nelson , Matthew J. Marton , Roland Stoughton , Michael R. Meyer , Holly A. Bennett , Yudong D. He , Hongyue Dai , Wynn L. Walker , Timothy R. Hughes , Mike Tyers , Charles Boone , † Stephen H. Friend

Wetenskap 04 Feb 2000 : 873-880


Data availability

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for choosing to send your work entitled “Mechanism for priming DNA synthesis by yeast DNA Polymerase α” for consideration at eLife. Your article has been favorably evaluated by a Senior editor and 2 reviewers. The Reviewing editor and the other reviewer discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. We anticipate that you will be able to deal with all of these comments by revising the text of the manuscript, and we look forward to receiving your revised manuscript soon.

This study investigates one of the most frequent DNA transactions in a eukaryotic cell, the initiation of Okazaki fragment synthesis during nuclear DNA replication. The manuscript describes X-ray crystal structures of the catalytic subunit of yeast DNA polymerase alpha in three states (apo, bound to RNA-primed DNA, and a complete catalytic complex). The polymerase is shown to bind to an RNA/DNA helix containing a turn of A-form base pairs in the duplex upstream of the active site. Biochemical and computational results are presented that lead the authors to suggest a mechanism for the termination of primer synthesis by polymerase alpha after one helical turn, allowing transfer of the primer-template to the DNA polymerases that perform most of nuclear DNA replication. The enzyme binds selectively to the A-form helix embodied by the RNA-DNA substrate. Primer extension with deoxynucletides is expected to change the conformation of the primer-template towards a B-form helix, which would not be optimal for the observed contacts seen with the A-form primer-template in the crystal structure. Release of Pol α enables Pol δ to proceed to complete the faithful synthesis of Okazaki fragments.

This is an excellent paper in which the structural and biochemical analyses are augmented by molecular dynamics simulations that show that the polymerase is so constructed that it requires the presence of the RNA-DNA hybrid to maintain a closed form (in contrast to a bacteriophage DNA polymerase of the same family). The crystallographic data presented are impressive and are technically sound, and the structures are novel. The observations presented here are consistent with the authors' hypothesis about DNA release from Pol α, which is logical and elegant in its simplicity. This manuscript elucidates a fundamental mechanism of eukaryotic replication and will therefore be of general interest to a large group of biologists. Publication in eLife is recommended after the manuscript is revised to address the following comments.

1) A key point of emphasis in the manuscript is that Pol α synthesizes about 10 nt. of DNA (i.e., a turn of duplex) before dissociation. This is a key point because that length of DNA suffices to release the RNA from the Pol binding site. The reviewers are confused by apparently contradictory statements about the length of the DNA segment synthesized in the literature. For example, reviews by Burgers (2009) [JBC 284:4041] and Arezi and Kuchta (2000) [TIBS 25: 572] both state that ∼20 nt of DNA are synthesized. If 20 nts of DNA are synthesized, the duplex bound by DNA polymerase α would be all DNA, and the structural features that are suggested to be important for termination would no longer be relevant. We recognize that both reviews simply state this “fact” without attribution, but a review by Balakrishnan and Bambara (2011) [JBC 286:6865] clearly states that experiments with the SV40 system shows synthesis of ∼20 nucleotides of DNA by the primase. What is the length of DNA synthesized by Pol α before dissociation? The authors should clear up this point of confusion by referring clearly to the literature concerning the length of DNA synthesized by Pol α as opposed to SV40. If this is a matter of uncertainty then the manuscript should be revised accordingly to reflect the uncertainty.

2) The authors indicate that the structure of the ternary complex depicts an actively copying polymerase. The protein was pre-incubated with RNA-DNA and dGTP. Could the observed incorporation have occurred prior to crystal formation? Why is the incoming dGTP observed in the ternary complex not incorporated? Are all atoms needed for catalysis observed and in the correct geometry? If not, and/or if the resolution is insufficient, then the claim is premature. It would be very useful, and standard in this field, to shown the geometry of the polymerase active site in detail, perhaps superimposing it with that of another polymerase (e.g., RB69 Pol), to show that the primer terminus and the 3´-O is the correct position with respect to the catalytic metals and incoming nucleotide.

3) The models presented in this paper are plausible, rather than completely convincing. The model in Figure 6 might be more convincing (or not) depending on the outcome of more precise mapping experiments using a natural DNA sequence, ideally the sequence used for the crystallography, rather than the artificial template used in Figure 4. It would be straightforward to quantify termination of processive synthesis at each nucleotide position as synthesis proceeds, and map this pattern against the structural features that are hypothesized to be important for switching. This experiment could be applied to derivatives containing amino acid substitutions for residues thought to be important for switching. Even one positive result of this type would lend confidence to the model that is the main take home message of the manuscript.

It would also be great to have genetic evidence that the proposed mechanism limits genome instability, which is stated in the last sentence of the Abstract more like a fact than a possibility worth investigating. In the absence of such experiments, the authors should adjust the language to reflect the fact that they are proposing plausible but not proven models.


Bespreking

The major outcome of our study is identification of a new functional redox motif, CxxS, that is used by structurally distinct families for redox function. The specificity and selectivity of our searches were such that this simple redox motif, when conserved and present in the context of a simple secondary structure pattern, could be used as a predictor of both redox function and location of redox sites in proteins. Some of our predictions are illustrated in Figures 2–5, , , , and several other predictions are discussed above.

Identification of redox function in proteins is difficult because of the multiplicity of families of redox enzymes and the extreme divergence of their members. Even within the most studied family of thiol-dependent enzymes, thioredoxin-fold proteins, intergroup homology analyses can rarely identify functional relationships (Martin 1995 ).

To assist in homology-based analyses, various function prediction approaches have been used for identification of redox function, but these are restricted to specific subsets of redox proteins. For example, a sequence—to structure—to function paradigm has been used to identify thiol-disulfide oxidoreductases by threading sequences to a fuzzy functional form descriptor (Fetrow et al. 1998 , 2001 ). However, this approach was limited to thioredoxin-fold proteins containing a conserved proline residue located in close proximity to the active-site cysteine(s).

In contrast to homology- and threading-based algorithms, our approach is not limited to a specific structural fold rather, it identifies redox function in structurally unrelated proteins. Interestingly, the CxxS groups were typically located in these proteins downstream of an α-helix, implying that this structural element assists in redox function (Fig. 6). The molecular basis behind the flanking occurrence of an α-helix is not known, but may involve generation of a dipole and stabilization of a cysteine thiolate, or simply be indicative of preferential presence of helices downstream of enzyme active sites.

In any event, the finding that α-helices are frequently located next to cysteine-based redox motifs may be very useful for identification of thiol/disulfide proteins, including those that do not contain the CxxS motif. For example, searches for CxxC-containing thiol/disulfide oxidoreductases are difficult because of the presence of a large number of zinc-containing proteins. However, most of these do not have α-helices downstream of the CxxC, which may allow efficient filtering of these proteins during the searches.

The finding of CxxS motifs in thioredoxin-fold proteins that were located in place of CxxC motifs resulted in considerable debate within the field in regard to the ability of CxxS proteins to catalyze redox reactions that require two cysteines. Recent studies illustrate that at least glutaredoxins and protein disulfide isomerases of this family are active even when only one cysteine is present (Anelli et al. 2002 ).

We showed that a search for the conserved CxxS motif allows prediction of redox function on a genome-wide basis. It will be of interest to determine how many such proteins are present in the human genome. Considering our analyses of bacterial, archaeal, and eukaryotic genomes, a large number of human CxxS-containing redox proteins may be expected. Further studies are needed to identify such proteins and characterize their functions.

Although our method can be used to identify both redox proteins and redox sites in proteins, it would be incorrect to view these predictions as a proof of identification of thiol-dependent processes. Because the CxxS motif involves only two residues, these can be conserved in some proteins for various reasons. The presence of conserved CxxS sequences upstream of an α-helix is a strong indication of redox function, but further studies may be needed to confirm such predictions.

Finally, our study illustrated that a large number of reactions use CxxS motifs, either with or without the help of other redox cysteines. CxxS motifs can form intermolecular disulfide bonds that may help to retain target proteins in a specific compartment or to serve as intermediates in disulfide-bond formation, isomerization, or reduction (Cotgreave and Gerdes 1998 Carmel-Harel and Storz 2000 Holmgren 2000 Rhee et al. 2000 Tanaka et al. 2000 Finkel 2001 Ritz and Beckwith 2001 ). Protein disulfide isomerases are examples of such proteins. The CxxS-containing proteins may also provide antioxidant defense using small redox molecules for reduction of various oxidized compounds or proteins. Glutaredoxins that use glutathione as an electron donor are examples of this function (Cotgreave and Gerdes 1998 Carmel-Harel and Storz 2000 Holmgren 2000 Ritz and Beckwith 2001 ). CxxS may also directly attack certain other forms of oxidized sulfur, such as sulfoxides, with formation of sulfenic acid intermediates, and methionine sulfoxide reductases are an example of such function (Kryukov et al. 2002 ). Furthermore, in certain proteins, CxxS may be used for both redox function and to coordinate metals, as evident from the analysis of HSP33 proteins containing CxxS and CxxC motifs (Jakob et. al. 2000 ). Further experimental analyses will undoubtedly provide additional important roles for CxxS, a redox motif present in structurally distinct proteins that was defined in our study.


BESPREKING

The realization that the βγ as well as the α subunits of heterotrimeric G proteins are active signaling elements in numerous systems (C lapham and N eer 1993 I niguez -L luhi et al. 1993) raises the question as to how the independent branches of G protein–mediated signaling pathways are functionally related, and how they are regulated. A priori, one can imagine systems in which Gα en G.βγ regulate different effectors and systems in which they act on the same effector. In principle, the effects of Gα en G.βγ stimulation could be additive, synergistic, or antagonistic. In fact, examples of each type are known (C lapham and N eer 1993).

The yeast mating response, which is driven by the βγ subunit of a G protein, provides an example of antagonistic regulation. We have shown that the pheromone-inducible Gα protein Gpa1p stimulates an adaptive signal that downregulates the Gβγ-induced mating signal independently of Gβγ sequestration (S tratton et al. 1996). To identify elements downstream of Gpa1p in this pathway, we conducted a genetic screen for adaptive defects in a strain expressing a hyperadaptive allele of GPA1. Both recessive and dominant Adp − mutations were recovered. Eight out of the 10 dominant mutations tested showed tight linkage to STE4. Sequence analysis of the STE4 locus in these mutant strains revealed seven novel STE4 alleles—F115S, L138F, A405V, G409D, S410L, W411L, and W411S—each of which were shown to disrupt proper regulation of the pheromone response. The genetic characterization of these mutant forms of Gβ led to two conclusions: (1) Gβγ is a target of Gα-mediated adaptation and (2) the adaptive mechanism or mechanisms impaired by the lesions in STE4 do not involve Gβ phosphorylation.

Gβγ is a target of Gpa1p-mediated adaptation: The mutant alleles of STE4 isolated in this work confer a defect in regulation of the mating signal, especially in recovery from exposure to pheromone. It has been suggested that Ste4p negatively regulates the pheromone response in addition to stimulating it (G rishin et al. 1994). If Ste4p works in concert with Gpa1p to stimulate adaptation, mutations resulting in the loss of this function should be recessive. The discovery of mutations in STE4 that disrupt Gpa1p-mediated adaptation and that are dominant suggests that Ste4p is a target of, rather than a cofactor in, the Gα-induced desensitization mechanism. The lesions in question presumably render Gβ refractory to this adaptive mechanism.

Could the Adp − mutations in STE4 simply disrupt α–βγ binding? Two pieces of evidence argue against this possibility. First, the mutant forms of Ste4p have relatively large effects on the induced signal in comparison to their effects on the basal signal. With the exception of the F115S mutation, the alterations in STE4 had virtually no significant impact on the growth and morphology of cells cultured in the absence of pheromone, and only small effects on the steady-state levels of mating-specific transcription and Ste4p phosphorylation (Table 1 and Figures 3, 5, and 6). Een STE4 Adp − strain, STE4-A405V, was almost indistinguishable from wild type when grown in the absence of pheromone. After stimulation with pheromone, however, the effects of the STE4 Adp − alleles were dramatic: cells coexpressing a STE4 Adp − allele with wild-type Gpa1p formed larger than normal halos (Table 1 and Figure 4, top row) those expressing a STE4 Adp − allele with the hyperadaptive allele of GPA1, E364K, showed significantly less colony formation within the halos than cells expressing GPA1-E364K and wild-type STE4 (Figure 4, middle row). Thus, it appears that inactivation of the aberrant Gβγ subunits is normal or nearly normal in the absence of pheromone, but is defective after pheromone treatment.

The results of the experiments designed to measure the effects of the STE4 Adp − alleles on mating-specific transcription, which is more sensitive to pheromone stimulation than are the cellular responses, also suggest that the mutations primarily affect downregulation of the induced mating signal rather than basal pathway activity. In examining the transcriptional data, it is helpful to rank the mutants according to their relative β-galactosidase levels. The effects of the various STE4 mutations on basal FUS1-lacZ transcription are widely variable: the STE4-F115S allele induces the highest constitutive activity, STE4-A405V has the least effect on transcription, and the other alleles are intermediate in this regard (Figure 5A). After treatment with a low dose of pheromone, the mutant strains expressing wild-type Gpa1p exhibited between 2- and 10-fold supersensitivity (Figure 5B) those expressing GPA1-E364K all hyperinduced FUS1-lacZ to a similar level, 11–16-fold higher than the control (Figure 5C). Moreover, the rank order of the induced and basal FUS1-lacZ activities did not correlate in cells expressing either wild-type Gpa1p or Gpa1-E364Kp. In the wild-type GPA1 background, for example, the allele that showed the greatest supersensitivity, STE4-S410L, exhibited one of the lowest basal activities. Similarly, the mutants that exhibited the highest basal activities were most effectively downregulated by Gpa1-E364Kp, whereas FUS1-lacZ was not downregulated as well by Gpa1-E364Kp in those mutants with the lowest basal activities. The lack of correspondence between the effect of a given STE4 allele on basal and induced mating-specific transcription is significant. If the regulatory defect were simply caused by poor α–βγ binding, then the mutations that most dramatically affect Gβγ sequestration in the absence of pheromone might be expected to have the most adverse effect on recovery.

The second argument against the idea that the STE4 Adp − mutations merely cause dissociation of the G protein subunits relies on experiments with G322E, a mutant form of Gpa1p that confers insensitivity to pheromone by sequestering Gβγ (S tratton et al. 1996 M. C ismowski and D. S tone , unpublished results). As discussed above, the hyperadaptive form of Gpa1p, E364K, cannot fully stimulate recovery from pheromone treatment in cells expressing any of the STE4 Adp − alleles (Figures 4, middle row, and 5). Die STE4 Adp − mutations are epistatic to GPA1-E364K. In contrast, the ability of Gpa1-G322Ep to sequester Gβγ and block the mating response, as assayed in halo tests, is unaffected in all but one of the mutant strains (Figure 4, bottom row). Die STE4-F115S mutant cells were the exception, forming detectable halos in spite of Gpa1-G322Ep expression. The difference in the effects of the STE4 Adp − alleles on cells expressing these two functionally dissimilar forms of Gpa1p is consistent with the idea that the Adp − mutations affect the mating signal primarily through a defect in Gpa1p-mediated adaptation rather than by disrupting the sequestration of Gβγ by Gα. The transcriptional and Ste4p phosphorylation assays, however, demonstrate that the mutant forms of Gβ do have some impact on basal signaling (Figures 5 and 6). Die vlakke van FUS1-lacZ activity and Ste4p phosphorylation are elevated to a variable degree, depending on which allele is tested. Presumably, the small increases in constitutive FUS1-lacZ activity result from the release of Gβγ van Gα. Consistent with this inference, Gpa1-G322Ep suppressed the various forms of Gβ in an allele-specific manner. When the effect of coexpressing GPA1-G322E en die STE4 Adp − alleles in cells treated with pheromone was examined at the transcriptional level, a striking similarity to the basal signaling activities of the STE4 Adp − mutants expressing wild-type Gpa1p was seen (Figure 5, A and C). Die STE4 Adp − mutations that manifested the highest constitutive FUS1-lacZ activity, presumably those that are most disruptive to α–βγ binding, were also least well suppressed by Gpa1-G322Ep in cells exposed to pheromone. In fact, the bar graphs representing these two data sets are almost superimposable. This correlation between basal FUS1 activity in wild-type cells and induced FUS1 activity in cells expressing Gpa1-G322Ep supports the idea that these measurements reflect α–βγ affinity. Thus, the A405V form of Ste4p is the least disruptive, and the F115S form of Ste4p is most disruptive to α–βγ binding. Note that although STE4-A405V has almost no impact on basal signaling, it confers as severe a defect in recovery from pheromone treatment as do any of the other alleles.

Taken together, the data discussed in this section indicate that one target of Gpa1p-mediated adaptation is Gβγ. The observed defect in downregulation conferred by the STE4 Adp − alleles can be explained in two ways. Gpa1p might stimulate the binding of an unknown regulator to Gβγ, and the Adp − mutations might disrupt this interaction. Candidates for this regulator include Akr1 (K ao et al. 1996 P ryciak and H artwell 1996), Syg1 (S pain et al. 1995), Cdc24 (S imon et al. 1995 Z hao et al. 1995), and Ste5 (W hiteway et al. 1995)—proteins that are all thought to interact with Ste4p in vivo. Alternatively, the Adp − forms of Gβγ may simply be refractory to sequestration by Gpa1p after pheromone treatment, but more easily bound by Gpa1p during vegetative growth. Although our data are inconsistent with the idea that the STE4 Adp − mutations merely cause the release of Gβγ from Gpa1p, we cannot eliminate the possibility that they differentially affect the ability of Gpa1p to bind Gβγ in vegetative and pheromone-treated cells. Slight defects in α–βγ affinity could also be overcome by the induction of downstream adaptive mechanisms in dividing cells. In other words, our failure to observe projection formation (shmooing) and poor growth (Table 1 and Figure 3) in cells newly transformed with the various STE4 Adp − alleles could conceivably be caused by a rapid change in cellular physiology.

Ste4p phosphorylation and Gpa1p-mediated adaptation: Like metazoan Gβ subunits, Ste4p is made up of a repeating amino acid sequence motif of

40 residues. In addition to the seven repeat motifs found in metazoan Gβ subunits, however, Ste4p contains two unique insertions, one at the amino terminus and another between repeat motifs five and six. When cells are exposed to pheromone, Ste4p is rapidly phosphorylated within the internal domain, residues 310–346 (C ole and R eed 1991 and E L i and D. S tone , unpublished results). Because this domain has been implicated in adaptation, we asked whether the STE4 Adp − mutations affect the phosphorylation state of Ste4p. A priori, the Adp − phenotypes could be caused by an inability to phosphorylate Ste4p. As shown in Figure 6, this is clearly not the case. After treatment with a submaximal dose of pheromone, the mutant forms of Ste4p were all fully phosphorylated. Our data are also inconsistent with the idea that Gpa1p stimulates Ste4p phosphorylation. Rather than augmenting phosphorylation of Gβ, a hyperadaptive form of Gpa1p actually inhibits it (Figure 7). Thus, although these data are strictly correlative, our failure to observe a correspondence between adaptation and Ste4p phosphorylation indicates a need to reexamine the role of Gβ modification in pheromone signaling.

Location of the Adpmutations in the three-dimensional structure of Gβ: The crystal structures of the Gβγ dimer from mammalian retinal cells (transducin βγ) in its free form (S ondek et al. 1996), complexed with a chimeric form of α (L ambright et al. 1996), and complexed with retinal phosducin (G audet et al. 1996) have recently been solved. Because most of the contacts between α and β involve residues that are conserved in both proteins, the interactions observed in the α–βγ crystal are likely to occur in other members of the heterotrimeric G protein family, including Gpa1p and Ste4p (L ambright et al. 1996). Structural information about the Ste4p residues implicated in adaptation is summarized in Table 2, and the locations of the corresponding residues in the transducin βγ crystal are shown in Figure 8.

The transducin βγ dimer is primarily a seven-bladed β-propeller. Each of the blades is a β sheet made of four antiparallel strands. The N-terminus of Gβ forms an α helix that interacts with the first helix of the Gγ subunit, and the second helix and extended portions of Gγ partially encircle the β propeller. Interaction between Gα en G.βγ occurs at two distinct interfaces. Residues in the loops and turns at the top of the β propeller domain of Gβ (blades 2–5, and 7) interact with residues in or adjacent to the switch I and switch II region of Gα. This is called the switch interface. The second α–βγ interface is formed by interaction between the N-terminal helix of Gα and the side of the first blade of the β propeller. This is called the N-terminal interface. The surface area of the N-terminal interface is about half that of the switch interface.

Because the structures of the free and α-bound forms of transducin βγ are not significantly different, it is unlikely that the signaling activity of Gβγ depends on a change in its conformation. Rather, key contact sites for Gβγ effectors and regulators may be unmasked when the G protein subunits dissociate. By analogy to the mammalian α–βγ crystal structure, three of the Ste4p residues identified in this study are located in the regions of α–βγ interaction and are predicted to lie on the exposed surface of the free Ste4p-Ste18p dimer. Residues L138 (in blade 2) and W411 (in blade 7) are predicted to contact the switch region of Gpa1p residue F115 (in blade 1) is predicted to contact the N-terminal helix of Gpa1p. In addition to interacting with Gpa1p, all three of these residues are readily accessible to effector and regulatory molecules. This raises the possibility that the Adp − forms of Ste4p prevent proper downregulation of the mating signal by hyperstimulating the mating effector. It is not necessary, however, to invoke gain-of-function mutations to explain the data. It is more plausible that F115S, L138F, W411L, and W411S are loss-of-function mutations that prevent downregulation of Gβγ by Gpa1p or by an unknown negative regulator. A precedent for such a molecule is phosducin, which downregulates Gβγ moieties in a variety of metazoan systems. The recently solved crystal structure of the transducin βγ-retinal phosducin complex (G audet et al. 1996) reveals direct contacts between the N-terminal domain of phosducin (helices 1 and 3) and the residues of transducin Gβ that are analogous to residues L138 and W411 of Ste4p. The structure of the transducin βγ–phosducin complex also suggests that phosducin induces a conformational shift in Gβγ. The positions of three loops at the top of blades 6 and 7 of the β propeller are changed upon Gβγ–phosducin binding. Interestingly, the most C-terminal of these loops corresponds to residues 408–417 of Ste4p. This suggests a mechanism by which the A405V, G409D, and S410L substitutions in Ste4p might prevent proper downregulation of the mating signal. Although the analogous residues of transducin Gβ contact neither Gα or phosducin directly, substitutions at these positions might affect a local change in the conformation of the β propeller such that the binding of a negative regulator is inhibited.

Ribbon diagram of the transducin βγ crystal structure. The N-terminal helix of transducin β and the blades of the propeller are distinguished by color. Transducin gamma is shown in purple. The transducin β residues that correspond to the substituted residues in the Adp − forms of Ste4p are shown in red and are labeled. The analogous residues of Ste4p are indicated in parentheses. The label 330-332 refers to G330, S331, and W332 of transducin β 409-411 refers to G409, S410, and W411 of Ste4p.



Kommentaar:

  1. Doudal

    What necessary words ... Great, an excellent phrase

  2. Faukree

    U laat die fout toe. Voer ons bespreek. Skryf vir my in PM.

  3. Montrelle

    Dit is jammer dat ek myself nie nou kan uitdruk nie – daar is geen ontspanning nie. Ek sal terugkom - ek sal absoluut die mening oor hierdie kwessie uitspreek.

  4. Thamyris

    Kulny beeldjies))))))

  5. Ohcumgache

    Na my mening is dit werklik, ek sal aan bespreking deelneem. Ek weet dat ons saam tot 'n regte antwoord kan kom.

  6. Felamaere

  7. Akinogrel

    Nie 'n slegte vraag nie

  8. Jamel

    Jy het die kol getref. Daar is iets hierin en ek hou van jou idee. Ek stel voor om dit vir algemene bespreking te bring.



Skryf 'n boodskap