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IntroductiontoYeastGeneticsForthenexttwoweekswewillbestudyingthelife-cycleandgeneticsofthebuddingyeast,Saccharomycescerevisiae.Wewillcarryoutmanydifferent,butrelatedexperiments.Inoneexperiment we will follow yeast through its entire life-cycle; from haploid to diploid tohaploidagain.Wewilldemonstratetheprocessofgenecomplementationthroughgeneticcrosses.Wewillexaminetheeffectoftheenvironmentonthephenotypeofyeast.Someoftheseexperimentswillrequireonlyafewdays,whileotherswillgoforourfulltwoweeks.To avoid confusion, it is essential that you label plates very well and come to lab wellprepared.The budding yeast, Saccharomyces cerevisiae, is aunicellular,eukaryotic fungi. Itcangrowaerobicallyoranaerobically insimplemediaandhasadoublingtimeof 1.5 hours under ideal conditions. It is used in thebakingindustrytomakebreadriseandinthebeerandwine industry to produce alcohol by fermentation.Saccharomyces cerevisiae, is a popular researchorganism because it is the simplest of all theeukaryotes. Our understanding of many essential cellprocesses, suchas cell-cycle control and the secretionpathway,hasbeengained throughstudyingSaccharomycescerevisiae.Yeastcanexist ineitheradiploidorhaploidstateandhavetheabilitytoreproduceeithersexuallyorasexually.Haploidyeastexistsasoneof2mating types;MATaorMATα. If kept separate fromeachother,thesematingtypescanbemaintainedina haploid state indefinitely, however, when these 2mating types encounter each other they fuse tobecomeadiploidcell(seefigure1).Thediploidstatecan also be maintained indefinitely as long as theculture has a good a supply of nutrients. Understarvation conditions however, the diploid cell willsporulate(undergomeiosis)toproduceanascuswithfourhaploidspores.Theascushasastrongshellthatprotects the spores from environmental conditions.Germinationoftheascusanditssporesoccurswhena nutrient supply is restored. Otherwise the ascusmayremain intact for longperiodsof time,awaitingbetterenvironmentalconditions.

Figure1.YeastLifeCycle

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Budding yeast radically change theirmorphology as they progress through their life cycle(refertofigure2below).CellsundergoingcelldivisionbymitosisformasmallbudduringSphase. The bud enlarges as the cell progresses through G2 and Mitosis. Finally the budseparates fromthemothercell.During thisprocess themothercelldoesnotchangesize.Thebuddingprocessisthesameinbothhaploidanddiploidyeastcells.

When haploid yeast cell of opposite mating types (Matα and Mata)encounter one another they produce pheromones that induce matingbehavior(seefigure3below).Initiallytheyadoptapear-likeshape.Theyarenowcalledschmoos(afteracartooncharacterfromthe1940s).Twoschmoos fuse together at their tips to become a diploid zygote (a/α).Soonafterfusion,thediploidbeginscelldivisionbybudding.Theculturewill remain in a diploid state indefinitely until harsh environmentalconditions(suchasstarvation)inducemeiosisandformationofasci.

Figure2.Celldivisionoccursbybudding.

Figure3.MatingBehaviorinBuddingYeast

Figure4.Schmoos

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ObservationofaSimpleCrossDay1Today’s experiment demonstrates the phenomenon of complementation throughmating.YouwillbegivenfourhaploidstrainsofSaccharomycescerevisiae,HA1,HAR,HB1,HBR.The“A”strainsarematingtype“a”whilethe“B”stratinsarematingtype“α”.The“1”mutantshavemutationsinADE1,whilethe“R”strainshaveamutationinADE2.Thestraincolonycolorwillbewhiteorreddependingonthepresenceorabsenceofadenineinthemedia.TheredcolonycoloroftheADEmutatntsisduetotheaccumulationofanintermediate,P-ribosylaminoimidazole(AIR), fromtheadeninebiosynthesispathway(seefigure5below).P-ribosylamino imidazole is not a redmolecule initially, but is oxidized to become a redpigmentinanaerobicenvironment.TheaccumulationofthispigmentdecreasesthegrowthrateofADEmutantcellsandthusprovidesastrongselectionforadditionalmutationsthatreducetheproductionofthepigment.Consequently,newwhitemutantsariseinotherwiseredADEmutantculturesatahighfrequency.

The growth of ADEmutant yeast strains varies depending on the type of agarmedia onwhich they are grown. The characteristicsof themediaused in theexperiment are listedbelow.

• YEDmediaisacompletemediumthatcontainsallthenutrientsyeastneedtogrow.ItcontainsasmallamountofadenineandthereforeitsupportsthegrowthofHA2andHB1strains.Althoughadenine-requiringmutantsgrowwellonthismedia,theydoaccumulatethecharacteristicredpigment.

• MVmediaisaminimalmediathatcontainstheminimumamountofpurechemicalsrequiredtosupportthegrowthofwild-typeyeast.MVlacksadenine,thereforeHA2andHB1mutantsareunabletogrowonthismedia.

• YEKACmediaisanutritionallyunbalancedstarvationmediathatinducessporulationofdiploidyeasttoformascicontaining4haploidspores.

P-ribosyl-PPP-ribosylamineP-ribosylglycinamideP-ribosylaminoP-ribosylformylP-ribosylformylimidazole(AIR)glycinamidineglycinamideP-ribosylsuccinoP-ribosylaminocarboxamideadenineimidazolecarboxylateaminoimidazole(CAIR)(SAICAIR)Figure5.AdenineBiosynthesisPathway(ADE1-8aredifferentenzymescodedforbydifferentgenes)

ADE4 ADE5 ADE8

ADE6ADE7

ADE2

ADE1

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ExperimentalTimelineforSimpleCrossThetimelineoftheexperimentislistedbelow:Day1.Matehaploidstrains(MondayJuly9th)Day2.SelectfordiploidsonMVplates(WednesdayJuly11th)Day3.TransferdiploidstoYEKACplatesforsporulation(FridayJuly13th)Day4.Observeasciandstreakforsinglecolonies.(MondayJuly16th)Day5.Observerestorationofredphenotype(WednesdayJuly18st)ObservationofaSimpleCrossDay1Theadeninebiosynthesispathwayrequiresseveralenzyme-catalyzedsteps(refertofigure5onpage3above).Mutationsinanyofthegenesencodingtheseenzymescouldpotentiallyinhibit thesynthesisofadenineand result in theconversionofcream-coloredcells to redcells.Assumeascientisthasintroducedrandommutationsintoawild-type,cream-coloredpopulationofyeastcells,thenscreenedthemutantpopulationforredcolonies,andfoundseveralredmutantcolonies.Willallofthoseredcolonieshavemutationsinthesamegeneormighttheyhavemutations indifferentgeneswithintheadeninebiosyntheticpathway.Thisquestioncanbeansweredwiththefollowingcomplementationtest.Inthisexperimentyouwillcrossfourhaploidredstrains.HA1andHARarebothmatingtypea,whileHB1andHBR are bothmating typeα. Youwill perform the following crosses:HA1withHB1,HARwithHBR,HARwithHB1,andHA1withHBR.

1. ObtainaYEDplatethathasall4haploid,redmutants.Labelitas“matingmixtures”andwithyourinitials.

2. Using sterile toothpicks transfer a small amount of eachmutant to the positionsshowninfigure6.

3. Using new sterile toothpicks,mix each pair of strains together so they canmate.Yourplateshouldnowlooklikefigure7.

4. Drawasketchof theyeastcultures inyour labnotebooks.Label theyeast strains,thestreakcolor,thekindofmedium,andthedateoftheprocedure.

5. Incubateyourcultureat30°CelsiusuntilournextclassmeetingObservationofaSimpleCrossDay2

1. Yourmatingmixtureshavenowgrownintolargecolonies.Recordtheappearanceofyourplatebymakingadrawingof it inyour labnotebooks.Label theparentsand

Figure6.

Figure7.

HA1HAR HA1HAR

HB1HBR

HB1HBR

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themixturesanddescribetheircolors.Besuretowritedownwhatkindofmedium(YED orMV) cells are growing in, the color and appearance of the yeast, and thedate.Answerthefollowingquestionsinyourlabnotebook:

a. What is the color of thematingmixture colony? Are thematingmixturesconsistentintheirappearance?Providearationaleforthiscolorphenotype.

b. On the basis of this observation, is the red phenotype dominant orrecessive?Explain.

Yourmatingmixture is likely a combination of haploids and diploids. Both of thehaploid strains can grow on the rich YED medium, but neither can grow on thenutritionally poor MV medium. However, the diploid cells formed from the rightcombinationoftwohaploidstrainscangrowonMV.ByputtingthecellsonMVyoucan select fordiploid cells thathave complementedoneanother, allowing for thecompletionoftheadeninepathway.

c. Hypothesize about how you think your colonieswill look after growing ontheMVmedium.Explain.

2. Makeacopy(replica)oftheYEDplatebytransferringtheoriginalhaploidsandthematingmixtureontoanMVplate.

a. Viewthevideo"ReplicaPlating"foundonourMoodlepage.b. Assembleyourreplica-platingtoolaccordingtotheinstructionsinthevideo

tutorial.c. PressthereplicatoolontoyourYEDyeastplatefromdayone.d. StampthecellsontoyourMVplate.e. LabelthisMVplatewithyourname,thedateand"SelectingDiploids".f. Incubatetheplateat30°Cfortwodays.

3. Makeawet-mountslidefromanyoneoftheparentstrainsprovidedandlookatit

throughthemicroscope.Placeasmalldropofwateronaglassslide,transferaverysmallamountofyeasttothewaterwithasterilewireloop,mixtheyeasttoobtainauniformsuspension,andthenplaceacoverslipoverthediluteculture.

4. Drawasketchofabout10ofthesecellsinyourlabnotebooks.Drawcellsinvariousstagesofthecellcycle(buddingornotbudding)andlabelappropriately.

5. Makeawet-mountslideofthematingmixtureprovidedforyoubyDr.Loomisandlook at it through the microscope. Attempt to find and draw schmoos, diploidzygotes,buddingzygotesandanyothercelltypesyouobserve.Makeyourdrawinginyourlabnotebooks.Labelunbuddedzygoteswith"Z,"buddedzygoteswith"BZ,"andshmooswith"S."

ObservationofaSimpleCrossDay3

1. Obtain your “Selecting Diploids” plate from the incubator. In your lab notebooks,

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makeasketchof theyeastculturesseenon theplate.Label theyeast strains, theculturecolor,thequantityofgrowth(robustorpoor)andthekindofmedium.

Questions:

a. SomematingmixturesarepredictedtogrowwellontheMVplate,whiletheparenthaploidstrainsandothermixturesgrowpoorlyornotatall.Doyourobservationsmatchthesepredictions?

b. Whydothesepredictionsmakesense?Explain.

2. Makeawetmountofcellsfromthematingmixtureonthe“SelectingDiploids”plateandobservetheminthemicroscope.Drawseveralcellsinyourlabnotebooks.

3. Diploid cells can be induced to undergomeiosis and sporulation. Before inducing theyeasttosporulate, it isbettertotransferthediploidcellsbacktoaYEDplate.CellsonYED medium will grow faster than on MV medium and rapidly growing cells willsporulatebetter.Dr.Loomis transferredsomeof thediploids fromtheMVplates toaYED plate and labeled this YED plate as “Prespor”. The “Presporulation” plate wasincubatedat30°Cfor1day.Recordthesestepsinyournotebooks

Bythispointyouhaveseenhalfoftheyeastlifecycle;matingbetweentwohaploidstoformastablediploidcell.Youcannowseetheotherhalfofthelifecyclebysporulatingthediploid,toobtain four haploid spores. To do this you need to transfer the diploid cells to sporulationmedium(YEKAC).YEKACcontainsnonitrogensourceandonlyanonfermentablecarbonsource(acetate). When diploid cells try to grow on YEKAC, they sporulate and go through meiosis.Meiosis produces two important results: the chromosomenumber is reduced fromdiploid tohaploid, and the resulting haploid cells have all possible combinations of the adenine andmating-typegenes

1. Using a sterile toothpick, transfer diploid cells from your “Presporulation” plate to aYEKACplate.LabeltheYEKACplateas“Sporulation”andwithyourinitialsandthedate.

2. Incubatetheplateat30°CuntilMonday.ObservationofaSimpleCrossDay41. Make awet-mount slide of a sample from the “Sporulation” plate and examine itwith a

microscope.Lookforlumpycellsthatappeartohavetwo,three,orfourroundsporesinsideamembrane.Thesearetheasci.Theyshouldallhave fourhaploidspores,butsometimessomeofthesporesdon'tdevelop.Drawasketchofseveralasciinyourlabnotebook.

Questions:

a. WhatcharacteristicofYEKACplatesinducesdiploidyeasttosporulate?b. Whatadvantageisgainedwhenyeastsporulate?

2. Now you will try to isolate and grow individual haploid colonies from the “Sporulation”

culture,completingtheyeastlifecyclefromhaploidtodiploidandbacktohaploid.Obtainasterile toothpick. Scoop up some cells from the YEKAC “Sporulation” plate and streak forsinglecoloniesonaYEDplate.

3. LabeltheYEDplateas“RestorationofHaploids”.4. IncubatetheYEDplateinincubatorforuntilWednesday.

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ObservationofaSimpleCrossDay5(FridayJuly21st)WhenyouputsporesbackontoYEDgrowthmedium,theygerminate,beginbudding,andgrowintocolonies.Sincesomewillbematingtypeaandsomematingtypeα, theymayalsomate.Therefore,thecoloniesthatgrowmaybebothhaploidordiploidcellsandeitherpinkorcream-colored.

1. Lookfordifferentcolorsamongthecolonies.Didyouoranyoneintheclassobservethereemergenceoftheredcolonycolorinsomecolonies?Explainwhythisresultislikely.

2. Drawandlabelasketchofthisplateinyourlabnotebooks.Questions

a. NoneofthehaploidADEmutantstrainscangrowonminimalmedia,yetthediploidproducedbysomematingscangrowonminimalmediawhileotherscan’t.Explainwhythisisso.

b. Basedonyourresults,dothevariousredmutantshavemutations inthesamegeneordifferentgenes?

c. Brieflydescribethelife-cycleofbuddingyeast.