Download Introduction 1.1 The Importance of Homeostasis in Maintaining

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?