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Introduction 1.1TheImportanceofHomeostasisinMaintainingCellularFunction. Organismsneedtobeabletomaintainnearlyconstantinternalenvironmentsinordertosurvive, growandfunctioneffectively(Guyton&Hall,2006).Homeostaticmechanismsresistchangesto theorganism'sinternalenvironment.Thesecomplexmechanismsarespecifictoeachindividual factor,andactviaoneoftwodistinctpathways:positiveandnegativefeedback.Intheyeast species,InternalpH,waterpotentialandtemperature(Walker,1998)areamongthemanyfactors wherebyhomeostaticmaintenanceisvitalfornormalcellfunction. Likeotherorganisms,yeastshavevariousphysiologicalrequirementstopermitnormalgrowth andsurvival.Yeastrequiressubstratesandenzymesforcellfunction,andthesearefoundin aqueoussolution(Walker,1998).Withouthighwaterconcentrations,enzymaticactivitywould ceaseandthereforeimpairnormalcellfunction.Differentyeaststrainsexhibitdifferenttolerance levelstochangesinwaterpotentialoftheyeastgrowthmedia.Forexample,Saccharomyces cerevisiae(S.cerevisiae)requiresawaterpotentialbetween-5to-20(Mpa)fornormalcell growth(Jennings,1995). 6 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. Yeastgrowthmediawithhighlevelsofexternalundissociatedorganicacidshaveaninhibitory effectonyeastgrowthasaresultoftranslocationacrossthecellmembrane,loweringthe intracellularpH(Walker,1998).YeastcellsinstationaryphaseareabletomaintainaconstantpH whenextracellularpHisdecreasing(Vallietal,2004). Oneofthemostimportantphysiologicalfactorsinfluencingyeastgrowthistemperature(Walker, 1998).Yeastspeciespossessoptimumgrowthranges,withbothminimumandmaximumgrowth temperatures.Deviationfromthesephysiologicalparametersleadstotheonsetofmechanisms thatmaintainhomeostasis,thusprotectingtheorganismfromirreversibledamage. 1.2TheUniversalEukaryoticHeatShockResponse Itiswellknownthatlivingeukaryoticcellspossessmechanismstoprotectthemselvesagainst changesinexternalenvironment(Mageretal,1993).Theseeukaryoteselicitacomplexresponse tothermalstressinordertoprotectthecellproteomefromdegradation.Firstobservedin DrosophilaMelanogasterbyRitosain1962,thisresponseisknownastheheatshockresponse (HSR).Wheneukaryoticorganismsaresubjectedtothermalstressi.e.temperaturesabove optimumgrowthandsurvival,cellularproteinsandnucleicacidsbecomedamagedashydrogen bondingandhydrophilicinteractionsaredisrupted(Walker,1998). Cellssubjectedtosub-lethaltemperaturesrespondbyincreasingthesynthesisofheatshock proteins(Craig,1985)duetoincreasedtranscriptionofheatshock(HS)genesTheseHSgenesare presentinalllivingorganisms(Khalawanetal,1997).HSgenesareinducedbytheactivationof heatshocktranscriptionfactors(HSF)(Sorger&Pelham1988)thatbindtoheatshockelements (Pelham&Beinz1982)atthepromoterregionsoftheHSgenes(Pelham,1982).Anumberof 7 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. otherstressingagentsarefoundtoinducemanyofthesameHSgenes,includingexposureto increasedethanolconcentrations,exposuretoheavymetal-ionconcentrations,(Parsell& Lindquist,1993)oxidativeandosmoticstress(Mager,Ferreira,1993). TheseHSgenesencodehighlyconserved,ubiquitousheatshockproteins(Hsps)thatactas molecular'chaperones'.TheseHspsbindtocellularproteinstocopewithdenaturationinduced bythermalstress(Federetal,1999).Theyactby"folding,trafficking,maturing,anddegrading" cellularproteins(Hashikawa,etal2004).Heatshockproteinsdisplayavarietyoffunctions.Hsps functiongenericallyby"assistingpolypeptidestoself-assemblebyinhibitingalternativeassembly pathwaysthatproducenonfunctionalstructures"(Ellisetal,1991).Hspsacttomaintainnormal cellularfunctions,andareinducedatdifferenttemperaturesindifferentorganisms(Lindquist, 1986). 1.3TheMolecularResponseInducedByHeatStress Theheatshockfactors(HSF)andheatshockelements(HSE)involvedintheHSRarehighly conservedtranscriptionalsequencespresentinnature(Hahnetal,2004).S.cerevisiaecontains manyofthesameHSgenesasotherorganisms.Thehsp70geneappearstohavebeenconserved throughoutevolutionandispresentamongstvariousorganisms,includingbothDrosophilaandS. cerevisiae(Lindquist,1984).S.cerevisiaehasthereforebeenutilisedasamodeleukaryotic organismtoattempttounderstandthemolecularmechanismsinvolvedinactivation,durationof responseandthefunctionsofheatshockproteins. Theheatshockresponseiscontrolledatthetranscriptionallevel.InS.cerevisiae,themolecular responsehasbeenhighlycharacterised.Investigationsonamolecularlevelhavehighlighteda 8 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. numberofcomplexprocessesandeventsdetailingthewholeresponse.Thephosphorylationstate oftheheatshocktranscriptionfactorinS.cerevisiae(ScHsf1)isfoundinthenucleusboundtoHSE undernormalconditions(Pelham&Jakobsen,1988).TwotranscriptionaldomainsnamelyAR1 andAR2locatedontheCandN-terminalofScHsf1haveactivationdomainsthatarerepressed undernormalconditions.Ithasbeenfoundthathyper-phosphorylationoftheC-terminalofthese activationdomainsresultsinactivationofScHsf1andsoinductionofHSgenes(Hashikawaetal, 2004).ScHsf1belongstoafamilyofwingeddomainproteins(Littlefieldetal,1999).These 'wingeddomains'areknowntointeractwithDNA.Morespecifically,'wingeddomains'interact upstreamofthetranscriptionstartsitesofgenesencodingHsps;thesepromotersarecalledHSE. ThesewingeddomainsareimportantforoptimalbindingtoHSEs(Ciceroetal,2001).Anincrease intheexpressionofHspgenes,resultinaconcomitantincreaseinHspmRNAs,whichmaythen subsequentlybetranslatedintoHsps.ThesynthesisofHspmRNAandHspspeak10-20minutes aftercellsaresubjectedtoheatshock(Broachetal,1992). Hspspossessavarietyoffunctionsinprotectingthecellfromproteindegradation.Thenamesand functionsofthemajorHspsinS.cerevisiaehavebeensummarizedinthetablebelow. FunctionsoftheMajorHeatShockProteinsinS.cerevisiae. HeatShockProtein Hsp104 Hsp83 Hsp70Family Hsp60 ProposedPhysiologicalFunction Acquisitionofstresstolerance.Constitutivelyexpressed inrespiring,notfermentingcellsandonentryinto stationaryphase. Chaperone(s)function. Interactwithdenatured,aggregatedproteinsandassists insolubilisingthemwithsimultaneousrefolding(i.e. chaperones(s)function).Alsoinvolvedinposttranslationalimportpathways. SimilartoHsp70,Thischaperoninfamilyfacilitateposttranslationalassemblyofproteins.Hsp60facilitatesthe 9 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. SmallHsps Hsp30 Hsp26 Hsp12 foldingandassemblyofunfoldedproteinsinanATPdependentmannerbydirectlybindingtotheunfolded protein(Craig,1994). Cellularrolestillelusive,butmaybeinvolvedinentry intostationaryphaseandtheinductionofsporulation. Hsp30mayregulateplasmamembraneATPase. Table1.FunctionsofmajorheatshockproteinsinS.cerevisiae.Adaptedfrom:Walker,G (1998).YEASTPhysiologyandBiotechnology.WestSussex,England:Wiley&SonsLtd.151 DespitedetailedmolecularcharacterisationoftheresponseinS.cerevisiaethetriggerfor activationofthetranscriptionfactorhasnotyetbeendiscovered.Asaresult,theregulatorofthe molecularHSRisunknown. 1.4TemperatureastheDirectInduceroftheHeatShockResponse. TheHSRisaresponsetosub-lethaltemperatureasameansofprotectionfromthermaldamage (Sorger&Pelham1988).S.cerevisiaecharacterisesanormaltemperaturerangebetween35-43°C (Walker,1998)containingminimum,maximumandoptimumtemperatures(T min,Tmax,andTopt). Itisthereforereasonabletoinferthatthermalstressingagentsareresponsibleforinductionof theHSR.Exposuretosub-lethaltemperatures,resultinproteindenaturationanddamage.Cells respondtothepresenceofthermallydenaturedproteinsandinducearesponsetosynthesise Hsps.Untilthelateeightiesthe'classical'viewwasinfactthattemperatureitselfwasthoughtobe thedirectinduceroftheHSR(Lindquist,1986).Severalstudiesprovidedevidenceinfavourof this.IfthereweresecondarymessengersinvolvedinHsptranscriptionotherthantemperature, thentemperaturewoulddamagethesesecondarymessengersandhenceaffecttranscription. VariousstudiesdemonstratedthelackofsecondarymessengersininductionoftheHSR.When 10 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. heatshockedcellsaresubjectedtonormaltemperatures,theydonotimmediatelyceaseto synthesiseHsps(Lindquist,1981).Thelackofasecondarymessengerintranscriptional regulationcontributedtotheideaoftemperatureastheprimarytranscriptionaltrigger. 1.5TheTransientNatureoftheHeatShockResponse,andtheAbilitytobeinducedby OtherStressAgents. Despitethereasonableargumentthattemperatureactsasthetrigger/induceroftheheatshock response,thediscoveryofakeyphenomenonintheHSRcontradictedthis'classicallyheld'view (Lindquist,1986).TheHSRwasdiscoveredtobehaveintransientmanner(Miller,etal,1990). Whensubjectedtosub-lethaltemperatures,theHSRwasfoundtodiminishwithinonehourof inductionandreturntonormalproteinsynthesis(Milleretal,1990).MorespecificallyintheS. cerevisiaetranscriptionfactor(ScHsf1)theN-terminalregionoftheAR2domainofScHsf1was foundtohaveseparablepropertiestotheC-terminalregion,inthatitwasabletoinducea transientresponse(Sorger,1990).Thediscoveryofthisphenomenonchallengedtheideaof temperatureasadirecttriggeroftheHSR.TheveryfactthatthenatureoftheHSRistransient, suggeststhatthetriggerfortheHSRis'short-lived'. Temperature,bydefinitioncannotactasthetriggerfortranscriptionalactivationoftheHSR. TemperatureisaconstantfactorintheHSR.Ifindeedtemperaturedoesactasadirect transcriptionaltrigger,itshouldlogicallyresultinaconstantheatshockresponse,andtherefore nodiminutionoftheresponseshouldbeobserved.However,thisisnottheobservedresponse (Miller,etal1979).Asaresult,itislogicaltosuggesttemperaturepossiblyplaysanindirectrole inHSRinductionbutnotadirectrole.Thisdiscoverychallengedexistingideasandfurthermore 11 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. developedresearchtargetswiththeaimofdiscoveringthe'directtrigger'ofthisuniversalstressresponse. (Milleretal,1979)presentedevidencetocharacterizeatransientHSR.Itcanthereforebeargued thatthemechanismthatsensestemperaturebecomesdesensitizedovertime.Another phenomenondescribed,indicatesthatorganismsexposedtomildheatshockexhibitinduced thermotolerance(Parsell&Lindquist,1993).S.cerevisiaecellssubjectedtobriefheatshockat mildtemperatures,exhibitresistancetothermaldenaturationatotherwiselethaltemperatures. CellssubjectedtoHSat37°CdisplayatransientHSR.Whenbrieflyexposedtolethal temperatures,thesecellsexhibitresistancetothermaldamageasaresultofinduced thermotolerance(McAlister&Finkelstein,1980) Furthermoreinthepresenceofethanol,theHSRincreasesinsensitivityasthetemperature requiredformaximalHSinductionisdecreased(Curran&Khalawan,1994).Plasmamembrane ATPaseactivityinfluencestheHSR(Panaretou&Piper,1990)aswellasosmoticstress(Varelaet al,1992).MorespecificallyHsp-104hasshowntoplayanimportantroleinthermotolerance, includingcellswithmutatedHsf1.(Lindquistetal,1996).Neitherthetransientresponseor ethanolsensitivephenomenonsupportstheclassicalideathatorganismsresponddirectlyto thermallydenaturedproteins,toinducetheHSR.Instead,theseconceptssuggestthatthe'primary sensor'thatdetectscellularproteindenaturationisanactive,adaptabletrigger(Chatterjeeetal, 1997)thatisabletochangetovarysensitivityoftheHSRtothesametemperature (thermotolerance). 12 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. 1.6Evidencetosuggesttheheatshockresponseislipidmediated(S.cerevisiae). InductionoftheHSRinvolvesphosphorylationandthereforeactivationofSchsf1.HSisalso inducedattemperaturesspecifictoeachindividualcell(Carratuetal,1996).Howeverthe phosphorylatingagentisunknown.ThemolecularstructureofthecellmembraneinS. cerevisiaewasinvestigatedasapotentialregulatorofHSRinduction.Commonphysiological responsestoachangeinexternalenvironmentalconditionsconsistoflipidmembrane reorganizationandmodification(Rogers&Glasser,1993).Responsestotemperaturechanges areknowntoinvolvefattyaciddesaturation.(Lee&Cossins,1990).S.cerevisiaewasuseda modeltodeterminewhethertheHSRislipidmediated.Exposureofatemperaturesensitive strainofS.cerevisiaetosaturatedfattyacids(SFA)resultedinanincreasedtranscriptionofHS genetranscriptionat37°C,andadditionofunsaturatedfattyacids(UFA)downregulatedHS genetranscription(Carratuetal,1996). Furtherinvestigationsdevelopedthesefindingstolinkcellularlipidconformationsas responsiblefordesensitisationandthereforethetransientnatureoftheHSR.Unsaturated fattyacid(UFA)levelsweremonitoredduringsub-lethalheatshockatvaryingtemperatures (Chatterjeeetal,1997).Followingashiftintemperatureofyeastcellsfromoptimumtosublethaltemperatures,anincreaseincellularfattyacidunsaturationisassociatedwithan increasedtemperatureatwhichmaximalHSoccurs.Unsaturatedfattyacidlevelsandthe maximalHSRbothdeclinewhenreacclimatizedfromsub-lethaltooptimumtemperatures (Khalawanetal,1996).ThesefindingsdemonstratedthatdensitisationoftheHSRwaslinked toUFAcellularlevels.FurthermorefindingssuggestedthekineticsofUFAlevelswere consistentwiththekineticsfordownregulationofHSgeneinduction,thetransientresponse (Khalawanetal,1996). 13 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. WithevidenceproposinglinksbetweenUFAlevelsandHSinduction,itcanbereasonedthat UFAlevelsplayaregulatoryroleintheHSR.InS.cerevisiaetheOLE1geneencodesthedelta-9 desaturaseenzymeresponsibleforfattyaciddesaturation(Stukey,etal,1990).IfUFA desaturationactsasthe'primarysensor'inHSgenetranscription,knockoutoftheOLE1gene wouldresultinaninabilityoffattyacidstobecomeunsaturated.Ifthecellularlipidsremain saturatedandifdesaturationisthetriggerforHSdownregulation,aconstantlysaturatedFA cellularprofilewouldresultintheinabilityoftheHSRtobedownregulated.TheHSRwould remainconstant,thereforehighlightingUFAlevelsasresponsibleformediatingthetransient natureoftheHSR. 1.7YeastCharacteristicsandCellMembranePhysiology "Yeastsareascomycetousorbaidomycetousfungithatreproducevegetativelybybuddingor fission,andthatformsexualstateswhicharenotenclosedinafruitingbody."(Boekhoutand Kurtzman,1996).Yeastcellmembranesactsasimpermeablebarriersagainsthydrophilic moleculestopreventthemixingofthecytoplasmandexternalenvironment.Around7.5nthick, thecellmembraneiscomposedofalipidbilayer(Walker,1998).Aswithalleukaryotic membranes,thelipidbilayercontainsglobularproteinsdispersedthroughoutalipidmembrane, toformafluidmosaicstructure(Nicholson&Singer,1972).Thecellmembraneconsistsof discontinuousamphipathiclipidbilayers,withthepolarhydrophobictailsfacinginternally,and theirnon-polarhydrophilicheadsfacingoutwards. Bothintegralandmembranespanningproteinsarefounddispersedthroughoutthemembrane. Proteinsplayaroleintransportthroughactingascarrierorchannelproteins(Guyton&Hall, 14 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. 2006).Forexample,theprotonpumpingplasmamembraneATPase,utilizesATPtoexpelcellular proteinstocreateanelectrochemicalgradientfornutrientuptake(Cooteetal,1994).Thelipid bilayerisprimarilycomposedofphospholipids,mainlyphosphatidylcholineandsterols(Walker, 1998).Thephosphatidylcontentincreasedbyapproximately10-foldinS.cerevisiae(Walker, 1998).Sterolsstabilizethelipidbilayerwhereasphospholipidsaddfluidity(Walker,1998).The selectivepermeability,mediatedbycertainproteinsandlipidsexhibitinghydrophobicand hydrophilicinteractions(Nicholson&Singer,1972)ofthesemembranesservetocontrolwhat canenterandleavethecell. LipidComponentsOfThePlasmaMembrane Figure1.Lipidcomponentsoftheplasmamembrane.Theouterleafletconsistspredominantlyof phosphatidylcholine,sphingomyelin,andglycolipids,whereastheinnerleafletcontains phosphatidylethanolamine,phosphatidylserine,andphosphatidylinositol. Cholesterolisdistributedinbothleaflets.Thenetnegativechargeoftheheadgroupsof phosphatidylserineandphosphatidylinositolisindicated. Adaptedfrom:Cooper,GM(2000).StructureofthePlasmaMembrane,TheCell:AMolecular Approach.2nded.SunderlandMA:SinauerAssociates.1. 15 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. Thecellmembranehasavastrangeoffunctions,ofwhichthemainonesarementionedbelow withrelevantexamples. MainFunctionsoftheCellMembraneinS.cerevisiae. RelevantExamplesWhereThisIs FunctionsOfTheYeastCellMembrane Shown AphysicalbarriertopreventthemixingofPlasmamembraneprotonpump(ATPase) aqueousandcytoplasmicmolecules,andtoisvitalinexpellingcellularproteinsin controlwhatentersandleavesthecell.ordertocreatetheelectrochemical Membraneproteinsmediatethisresponse.gradientneededforuptakeofessential solutes(e.gH+K+Ca2+ CellSignalling.InS.cerevisiaePhosphatidylinositol4,5-bisphosphateis phosphoinositidephopsphorylationformsresponsibleforrolesincellular moleculesthatarefurtherbrokendowntoproliferation. formmoleculesthatactassecondary messengersinmajorsignalingpathways. Exocytosis Endocytosis Secretoryvesiclesaresecretedbythe GolgiapparatusandtheEndoplasmic Reticulum.Thesevesiclesfusewiththe plasmamembranetoexpeltheunwanted molecule. Endosomesinternalizestructures requiredbythecell.Invaginationsfrom thecellmembraneare'pinched'awayto formvesiclesthatarethentransported throughthecytoplasm.InS.cerevisiae thisprocessisimportantininternalizing matingpheromes. Table2:MainFunctionsoftheCellMembraneinS.cerevisiae..Source:Walker,G(1998).YEAST PhysiologyandBiotechnology.WestSussex,England:Wiley&SonsLtd.19-21. 1.8PrinciplesofOsmosis 16 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. Osmosisiscommonlyknownasthemovementofparticlesthroughasemi-permeablemembrane, fromareasofhighwaterpotential(lowsoluteconcentration)toareasoflowwaterpotential(high soluteconcentration).Thefactthatthecellmembraneissemi-permeableallowstheexistenceof thisphenomenon.(Roseetal). Duetothesmallsizeofwatermolecules,theyexistathighconcentrationsinsolution.Purewater existsataconcentrationof55.4Mat20°C)(Roseetal).Thishighconcentrationdoesn'tappearto bedramaticallyalteredinsolutionsmixedwithothersolutes.Itistheassociationofwater moleculeswiththesesolutesthatchangethestateofwater.(Roseetal).Achangeinthestateof wateraffectstheamountofthermodynamicallyavailablewater.Thewaterpotentialisdefinedas "thefunctionoftheconcentrationofsoluteparticles."(Roseetal). OsmoticpotentialsinS.cerevisiaewerededucedbydegradingthecellwalltoformprotoplasts. Investigationshighlightedthataprotoplastconcentrationof0.5M,equivalenttoawaterpotential of-1.5mpawassufficienttomaintainnormalturgorpressure,andnormalcellularstructure.(Rose etal).Externalwaterpotentialandcellularosmoticpotentialformthebasisofturgorpressure: pressureofthecellconstituentsagainstthecellwalloftheorganism. 1.9UsingOsmosistoInduceStructuralChangestoYeastCellMembranes Evidence(seesection1.7)clearlyillustratesthatthecellmembraneofyeasts,andalleukaryotes arevitalinperformingessentialcellularfunctions.Lipidsformanintegralpartofthecell membrane,structureandfunction.Evidence(seesection1.6)suggeststhetransientHSRislipid mediated,(Chatterjeeetal,1997).Changingthestructureoftheyeastcellmembranewould disruptlipidstructureandmetabolism.IftheHSRislipidmediated,thenchangestocellularlipid 17 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. content,wouldhaveaneffectontheHSR.Toexperimentallyinducethesestructuralchanges,cells couldbesubjectedtoosmoticshock,followedbyHStomonitorchangesintheHSR. 1.10UtilisingGeneDeletionTechniquesToMeasureHeatShockRegulation Homologousrecombinationistheabilityofanorganismtoexchangenucleotidesequences betweensimilarsectionsofDNA.Thischaracteristichasbeenexploitedtoformthebasisofgene deletiontechnology.Homologousrecombinationcanbeutilisedtopinpointregulationofcellular responsesonamolecularlevel.MethodsweredevelopedtoenablefragmentsofDNAtobe integratedintothegenomeofcells(Tropp,2004)toknockoutaparticulargene:eitherviagene replacementorgeneinsertion(Tropp,2004).Genereplacementinvolvesthereplacementofthe wholecodingsequenceofatargetgenewithaselectablemarker.Thisresultsingeneknockout anddisruptionofgenefunction.Knockoutgenesarereplacedwithacodingsequencethatcanbe detected.SequencehomologybetweentherecipientgeneandtheDNAfragmentenabletheDNA constructtointegrateatthegeneyouwishtoreplace. ConstructionoftheDNAfragmentcanbepreparedviaPCRmethods.Primerscontain approximately'50bpofhomologytothegeneofinterestand20bofhomologytotheselectable marker'(Tropp,2004)resultinginaPCRproductwith50bpofsequencehomologytothegene targetedforknockout. TheOLE1genecanbesubjectedtogenedeletionviathesamemethodology.Usingtheamplified HIS4genethathasbeendesignedtosharesequencehomologywiththeOLE1gene,theOLE1gene canbedeleted.InsertionoftheHIS4DNAfragmentintotheS.cerevisiaeDBY747lacZstrainresults 18 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. inhomologousrecombinationtoreplacetheOLE1gene.TheHIS4geneencodestheaminoacid histidine.TheoriginalDBY747lacZstraincannotgrowonmedialackinghistidine,leucine,or tryptophanastheplasmiddoesnotcontainthegenesabletosynthesisetheaminoacids. However,replacementoftheOLE1genewithHIS4resultsintheabilityoftherecombinant plasmidtosynthesishistidine.Thisresultsinthefollowingphenotypes: 1.Wild-typeDBY747lacZphenotype:His -Leu-Tryp2.Mutant-DBY747lacZphenotype:His +Leu-TrypHIS4isthenusedasadetectablemarkerasitisknownthatthemutant-DBY747lacZstraincan synthesiseitsownhistidine,andcanthereforegrowonmedialackinghistidine,onlyrequiring leucineandtryptophan. HavingsuccessfullyknockedouttheOLE1gene,themutantstaincanbetestedforexpected behaviourstoindicatewhetherofnotthetransientnatureoftheHSRislipidmediated(see section1.6). 1.11AimsandHypotheses Theaimsofthisprojectaretwofold.Evidencesuggeststhatthetransientnatureoftheheatshock responseappearstobelipidmediated.'Knockingout'theOLE1generesponsibleforthistransient natureandmonitoringforanexpectedchangeinheatshockactivity,couldestablishwhetherthis isthecase,onamolecularlevel.Secondly,cellsunderosmoticstressundergophysiological changesacrossthecellmembraneandinothercellcomponents.Iftheheatshockresponseislipid mediated,structuralchangestotheselipidsasaresultofosmoticstressshouldinduceachangein thedynamicsoftheHSR. 19 InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae. Theprojectaimsleadstothefollowingtwohypotheses: 1.DoestheknockoutoftheOLE1geneaffecttheheatshockresponse?Ifso,istheOLE1gene responsibleforthetransientnatureoftheHSR? 2.IstheHSRaffectedwhenlipidstructureandmetabolismaredisruptedinS.cerevisiaevia osmoticpressurechanges?