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Analysisofstructuralandfunctionalchangestothe

Saccharomycescerevisia
ekinesinrelated
motorprotein,Cin8p,inmitoticspindleassemblyandmicrotubulelocomotion
HumzaA.Siddiqui
CollegeofChemistry/Letters&Sciences,DepartmentofMolecularCellBiology
UniversityofCalifornia,Berkeley
Berkeley,CA,USA
Abstract:
TheKinesin5motorprotein,Cin8p,isanevolutionarilyconservedbiomoleculeacross
eukaryotesinvolvedinavarietyofcytoskeletalandmitoticspindleassemblydynamics,including
processivemovementalongmicrotubules,chromosomepositioning,centrosomeseparation,anaphase
spindleelongation,andthegenerationofparadoxicalforcesnecessarytoprovidespindletensionduring
SPhase[
1,2,3
].PreviousstudieshaveattemptedtoidentifytheproteomicregionsofCin8pthatmight
accountforitsfunctionality,buthaveyieldedinconclusivedataaboutthesequenceelementsthat
accountforitslocomotiveproperties[
4
].Therefore,thefocusoftheresearchhereinistobetter
characterizeCin8pandinvestigatethefunctionalnatureofthreemutantvariants,truncatedatvarying
levelsattheCterminal,relativetothewildtype(WT)Cin8pORF.Thesemutants,Cin8p955,
Cin8p871,andCin8p590,arerestrictionenzymecleavedproductsofthe
Saccharomycescerevisiae
gene
CIN8
thatarealreadyclonedandinsertedintoeitherbacterialstrain,BLR(DE3)pGroRIL,oryeast
strains,MS2922orDDY904mCherryaspartofourpreliminaryexperiment.Tobetterinvestigatethe
relationshipbetweenstructureandfunctionboth
invitro
and
invivo
foreachwildtypeandmutant
Cin8p,thisinvestigationwilltrackthesubcellularlocalizationofthemotorproteinusingGFPand
mCherrytagged tubulin,analysetheMichaelisMentenkineticparametersofpurifiedCin8pusinga
BradfordandMalachiteGreenATPaseassay,scorethetheeffectofCincreasin,Benomyl,Sorbitol,
and/or6azauraciladditivesonCin8pphenotype,andgaugethemetabolicactivityofCin8pusing
RTPCRtoanalysemRNAtranscriptionlevels.Ifsuccessful,theresultsofthisprojectwillhavefar
reachinginfluenceinthedesignofefficientpharmaceuticaldeliveryagents,anticancertherapies,and
novellaboratorytracingmethods.


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22723345
BackgroundandSignificance:
Theearlieststudiesofpartiallypurifiedkinesindatebackto1985whenRonaldD.Valeet.al
revealedthemotorproteinscapabilityofformingahighaffinitycomplexwith[taxolprepared]
microtubules[
1
].Alaterexperimentalstudy,publishedin1992,ofthekinesin5/BimCsuperfamilyof
motorproteinsbyDavidM.Roofet.alidentifiedtwonew
S.cerevisiae
genes,
KIP1
and
KIP2
using
PCRprimersdesignedtoamplifyhighlyconservedregionsofthekinesinmotordomain.[
2
].Usinga
geneticscreenformutationsthatmake
KIP1
essential,Roofet.alidentifiedkinesinrelated
CIN8
,and
soonthereafterdiscovered,viatheduplicatedyetunseparatedmicrotubuleorganizingcentersof
arrestedcells,thateither
KIP1
or
CIN8
wererequiredformitoticspindleassemblydynamics[
2
].This
includedchromosomepositioning,centrosomeseparation,anaphasespindleelongation,andgeneration
oftheoutwardforcenecessarytoactuponthepolesofmicrotubules,oppositetheantagonisticforce
mediatedby
KAR3
[
3
].
Despiteanunderstandingofthefunctionalroleofthekinesin5familyinacell,muchisstill
unknownaboutthesequenceelementsthatareimportantfortheformationofthetetrameric
complexes[
4
].Additionally,ithasnotbeenprovenifthehomotetramericformoftheproteinis
essentialfornormalcellularfunction[
4
]orifcertainsecondarystructuresorsequenceelements
accountforfunctionality,spindlelocalization,andmultimerization[
4
]EmilyR.Hildebrandtet.al
attemptedtoinvestigateaseriesof
S.cerevisiae
Cin8ptruncationsandinternaldeletionstoidentifythe
structuralelementsnecessaryforWTCin8pfunction[
4
]anddiscoveredallvariantsofCin8pthatare
functional
invivo
formtetramericcomplexes.Moreso,Hildebrandtconcludedthatthefirstcoiledcoil
domainintheCin8pstalkwasfoundtoberequiredfordimerizationandthatsequenceelementsfound
laterinthestalkwererequiredfortetramerization[
4
].ItwasalsodiscoveredthatdimericCin8pfound
nonfunctional
invivo
canstillbindtomicrotubules,suggestingactivityrequiresmorethanjustthe
microtubulebinding[
5
].Assuch,thisinvestigationseekstoanswerthemostimmediateandcentral


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biologicalquestionsurroundingCin8pWhatstructuralfeaturesofthiskinesin5motorprotein
accountforitsfunctionality?Tothisextent,thisstudyhopestoelucidatetherelationshipdifferent
CterminaltruncationsofCin8phaveforproteomicactivityrelativetothewildtype.
PreliminarydatagatheredconsistedofcloningeachdifferenttruncationofCin8p
invitro
with
theBLR(DE3)pGroRILbacterialstrainandexpressionvectorpET24b(+)and
invivo
withtheMS2922
and/orDDY904mCherryyeaststrainsandshuttlevectors,pCIN8GFPCFUSandpCIN8 GFP[
6
].
OneMS2922strainwillreceivetheexpressionvectorpCIN8GFPCFUSwhileanotherwilluptake
thepCIN8 GFPasacontrolvector.TheDDY904mCherrywillreceivethepCIN8GFPCFUS.Itis
expectedthatstudyingpET24b(+)inBLR(DE3)pGroRIL,
invitro
,willtestforproteinexpressionin
microtubuleand/orinhibitorpresenceandallowforpurificationofWTandvariantsviastandard
biochemicaltechniquesincludingaBradfordassay,MalachiteGreenATPaseassay,SDSPAGE
Electrophoresis,WesternBlotting,andAffinityChromatographyinaproteinbiochemistryexperiment
[
6
].ItisexpectedthatstudyingpCIN8GFPCFUSandpCIN8 GFPinMS2922willelucidatethe
roleofGFPfusioninfunctionalactivityandallowtestingforcomplementationofendogenousCin8p
inaphenotypicanalysisexperiment[
6
].Lastly,itisexpectedthatstudyingpCIN8GFPCFUSin
DDY904mCherrywillrevealsubcellularlocalizationinformationviafluorescencemicroscopy[
6
].All
oftheseexperimentswill,together,bestcharacterizethestructureandmetabolicactivityofCin8p,
hopefullyelucidating,intheprocess,whichsequenceelementsgiverisetofunctionality.
Thekinesin5proteinslocomotiveabilityandcentralroleinmitoticcelldivisionsuggests
importantpharmaceuticalbenefits,novelanticancertherapies,anduniquebiochemicaltagging
proceduresthatcanbeofpotentialvaluetomodernmedicine.Therefore,investigatingitsproperties
both
invitro
and
invivo
iscritical.Byinvestigatingtheframeworkoutlinedhereforwhichstructuresin
Cin8pillicitwhichfunctionalresponses,highlyspecializedkinesinmotorproteinscanbedeveloped
tangentiallywithnewdrugstoachievethesegoals.


HumzaSiddiqui
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PreliminaryResults:
A)Assays
CurrentresearchprogresshasbeenfocuseduponcreatingLBandYPDrichnutrientplatesfor
BLR(DE3)pGroRIL/DH5 orMS2922/DDY904mCherryrespectively,formingbaselineBradfordor
MalachiteGreenATPaseassaysforproteinconcentrationandactivitydetermination,cloning
WTCin8p(1038)anditstruncatedvariants(955,871,590),andbecomingfamiliarwiththegenomic
andproteomicdatabasesthatwillbenecessarytoutilizeinordertointerpretexperimentalresults.
Therearegenerallytwotypesofassays:proteinassays,whichmeasureproteinconcentration,
andenzymeassaysthatmeasureproteinfunction.Thelatterisespeciallyimportant,forourpurposes,
becauseenzymeassaysmeasureenzymaticactivity( mol/min)andspecificactivity( mol/min mg).
Eachoftheseparametersrevealimportantdataaboutaproteinssteadystatedynamicsandprocessivity.
MichaelisMentenmodelscanbesubsequentlyutilizedtoanalysekineticparameterssotogaugethe
purityofaproteinofinterestmethodsthatwillbesurelyemployedhere.FortheWTCin8pand
truncatedvariants,aBradfordproteinassaytoquantifyproteinconcentrationandaMalachiteGreen
ATPaseassaytomeasureCin8pactivityasCterminalregionsaretruncatedwillbeused.Bothare
continuous,colorimetricassaysthatrelyonabsorbancemeasurementsofaspectrophotometerandthe
BeerLambertLaw.Itisimperativethatonlyalinearrange(0.95>Absorbance>0.05ofeachassayis
usedsoaccuratemeasurementsaretakenandoutliersareavoided.
InaBradfordassay,proteinquantificationisdeterminedbyrelatingtheabsorbanceofan
unknowntotheabsorbanceofaknownsolution.Itisquickandsensitive,butdestructiveandsensitive
tolipids.TotesttheeffectivenessofaBovineSerumAlbumin(BSA)standardcurveforlaterCin8p
measurements,apreliminaryexperimentdeterminingtheproteinconcentrationofanotherunknown
proteinwasperformedfirst,resultinginthedata(
Table1,Table2
)below.TheBradfordassayutilizes
CoomassieBrilliantBlueG250,ared/browndyeat max=465nm,butadeepbluehue( max=


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595nm)whenboundtoaminoacidsF,W,Y,R,HasaresultofananionicchargeatneutralpHoreven
aneutralchargeatacidicpH[
5
].Theincreaseinabsorptionat595nmisproportionaltotheamountof
proteinboundthroughhydrophobicandionicinteractions.Threetrialswereruntotesttheeffectiveness
ofaBSAstandardcurveinquantifyingthemysteryproteinconcentration.Eachrxnhadatotalvolume
of1mL,with800 LofBradfordReagentandappropriatevolumesofddH2OandBSAorUnknown
eachwasanalysedat595nmina
Genesys20Spectrophotometer
.
Rxn

1mg/mL
BSA
(L)

10

15

20

Unknown
(
L)

Bradford
Reagent
(
L)

800

800

800

800

800

800

800

800

ddH2O(
L)

200

195

190

185

180

195

195

195

Table1:BradfordAssayProtocol[
6
]
Rxn

Absorbance
Trail1

0.001

.401

.625

.788

.997

.757

Absorbance
Trial2

.352

.575

.762

.990

.728

Absorbance
Trial3

.382

.550

.754

.953

.737

Mean(
x
)

.3783

.5833

.768

.9800

.7407

Standard
Deviation
(
x
)

.0247

.0381

.0177

.0236

.0148

Table2:A595nmReadingsofBradfordAssayProtocolandStatisticalAnalyses(
x
,x
)


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22723345
Absorbanceat595nm=m(concentration)+b
y=39.793(x)+.18,R=.999
wherey=absorbancereadingat595nmandx=concentration(mg/mL)

A595=39.793C+.18,wheny=.7407,x=.0141mg/mL(dilutionfactor=200fold)=
2.82mg/mL
2.82mg/mL=(molesofaminoacid/111g)(protein/590aminoacids)(2.82g/L)=
.00004306M

Figure1:KnownConcentrationsofBovineSerumAlbumin(mg/mL)vs.A595nmreadings

y=39.793(x)+.18,R^2=.9994
x=ConcentrationrelativetostandardBSA(mg/mL)
y=Absorbanceat595nmpeak
Concentration(mg/mL)ofunknown:.0141mg/mLwhenabsorbanceis.7407
Using5mLofunknownin1000mLtotalisaneffectivedilutionof200xtherefore
.0141(200)=2.82mg/mL(originalunknownconcentration)
Giventhattheactualconcentrationoftheunknownproteinis3mg/mL
PercentError=(ActualTheoretical/Theoretical)(100%)=(2.823/3)(100%)=
6.1%error
Afterthreetrialsandstatistical/graphicalanalysesofthedataobtained,itiscleartheBradford
assaychecksoutasaneffectivemeansofanalysingproteinconcentrations.Itwillbeused,confidently,
tomeasureWTCin8pandtruncatedvariantconcentrationsinactualCin8ptesting.Thepreliminary
studyrevealeda6.1%error,whichconfirmsthereliabilityoftheexperimentallygatheredvaluesand
andtheeffectivenessoftheBradfordassay.


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Cin8p,likeotherkinesin5motorproteins,functionsincoordinationwithATPhydrolysisand
conformationalchangesinthemotorheaddomaintogenerateprocessivemovementalong
microtubules(MT)in80
stepstowardsapositivepole[
6
].Oncebound,ATPaserateofactivityis
enhancedseveralhundredtoseveralthousandfold[
6
].
TomeasureCin8pactivityafterpurification,a
MTactivatedkinesinATPaseassaywillberequired.TotesttheeffectivenessoftheMalachiteGreen
ATPaseassayforthispurpose,apreliminaryexperimenthasalreadybeenrunanalysingATP
hydrolysisactivityforadifferentcommercialkinesinwithandwithoutmicrotubules.MalachiteGreen
isapHresponsivechromophore.Resonanceandprotonationallowsforavarietyoffluorescenceand,
assuch,makesforaneffectivedye[
6
].TheMalachiteGreenATPaseassayusedmeasuredthefree
phosphate,releasedfromATPhydrolysis,thatcomplexedwithmolybdateionsandthenwiththe
MalachiteGreenatlowpHtocauseashiftinthe maxofthedye.Measuringthis shiftyielded
quantifiabledata(
Table4,Table6
)abouttheenzymeandspecificactivityofthecommercialkinesin
(
Table5,Table7
)inthepresenceorabsenceofMTand/orATP.Amechanisticdemonstrationofthe
MalachiteGreenATPaseassayandadeterminationoftheworkingconcentrationsofthereagentsused
isalsooffered.
Theextinctioncoefficient, ,usedtoconvertabsorbancetoconcentrationwasgatheredby
preparingastandardcurveusing(mono)1mMSodiumphosphateasasourceoffree,inorganic
phosphate[
6
].Aseriesofsampleswithvarious[Pi](
Table3
),wereusedtoestablishtherelationship
between[Pi]andtheA650intheMalachiteGreenATPaseassay.Individualandgroupextinction
coefficientvalueswerecalculatedtoderive
Tables4and6
respectively.Standarddeviationsforeach
epsilonvalueisgivenandotherstatisticalanalysesispresentedbelowaswell.
Giventhedatapresented,itiscleartheMalachiteGreenATPaseassayisaneffectivemeansof
quantifyingproteinfunctionality.Likehypothesized,thepresenceofmicrotubulestructuresstimulated
theactivityofcommercialkinesinbyactivatingthemechanochemicalATPasenatureofkinesinand


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providingacellularsurfaceforthemotorproteintoperformbiophysicalworkupon.Thedatasupports
thisasKcatincreasesinthepresenceofATP,MT,andATP+MTthereforesuggestingVmaxreaches
diffusioncontrolandcatalyticefficiencymuchfaster(turnovernumbersareextremelyhigh)inthis
environmentalconditions.Cin8pwillbemodeledsimilarlytothecommercialkinesinthesuccessof
thispreliminarystudyisonlyprooftheMalachiteGreenATPaseassaywillworkforCin8pstudies.
[Pi]
M

A650

.25

.024

.50

.048

1.0

.105

2.5

.269

5.0

.569

7.5

.883

10.0

1.198

12.5

1.515

15

1.813

20

2.407

25

2.673

50

2.890

Table3:A650ReadingsofVariousConcentrationsofFreeInorganicPhosphate(
M)
FromNaH2PO4

Absorbance=(concentration)(extinctioncoefficient)(pathlength)
ExtinctionCoefficient=(Absorbance)/(Concentration)(PathLength),similartom= y/ x
A650=.1214C.0166,whereextinctioncoefficient=.1214
/M cmor121.4permMpercm
Tocalculateextinctioncoefficientfortheentireclass,weaverageevery dataasfollows

= valueforallgroups/(totalnumberofgroups)

Individualepsilonvalue:
121.4permMpercmwithastd.deviationof16.6permMpercm
Groupepsilonvalue:
106permMpercmwithastd.deviationof14.2permMpercm[
6
]


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Figure2:FreePhosphateConcentrationofSodiumphosphatevs.A650nmreadings(outliersincluded)

Figure3:FreePhosphateConcentrationofSodiumphosphatevs.A650nmreadings(outliersexcluded)


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Rxn

A650

Pi(mol)

Enzymatic
Activity
(mol/min)

Kinesin
(mg)

Specific
Activity
(mol/min
mg)

Blank

MT

0.002

0.00001652892 0.00000082644

ATP

0.033

0.00027272727 0.00001363636

MT+ATP

0.031

0.00025619834 0.00001280991

Kinesin

0.006

0.00004958677 0.00000247933

0.000835

0.00293

Kinesin+MT

0.009

0.00007438016 0.00000371900

0.000835

0.0044

Kinesin+
ATP

0.033

0.00027272727 0.00001363636

0.000835

0.016

0.686
0.00566942148 0.00028347107
0.000835
Table4:MalachiteGreenATPaseAssayActivity(individualepsilonvalue)

0.335

Kinesin+MT
+ATP

SpecificActivity
(U/mg)

Enzyme
( Mol)

Kcat
(/min)

Kinesin

0.00293

.0000119

246.22

Kinesin+MT

0.0044

.0000119

1344.57

Kinesin+ATP

0.016

.0000119

369.74

Kinesin+MT+ATP

0.335

.0000119

28151.26

Table5:SpecificActivityandTurnoverNumberofKinesin(individualepsilonvalue)


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Rxn

A650

Pi(mol)

Enzymatic
Activity
(mol/min)

Kinesin
(mg)

Specific
Activity
(mol/min
mg)

Blank

MT

0.002

0.00001886792 0.00000094339

ATP

0.033

0.00031132075 0.00001556603

MT+ATP

0.031

0.00029245283 0.00001462264

Kinesin

0.006

0.00005660377 0.00000283018

0.000835

0.00338944752

Kinesin+MT

0.009

0.00008490566 0.00000424528

0.000835

0.00508417128

Kinesin+
ATP

0.033

0.00031132075 0.00001556603

0.000835

0.01864196136

0.686
0.00647169811 0.00032358490
0.000835
Table6:
MalachiteGreenATPaseAssayActivity(groupepsilonvalue)

0.3875268331

Kinesin+MT
+ATP

SpecificActivity
(U/mg)

Enzyme
( Mol)

Kcat
(/min)

Kinesin

0.00338944752

0.0000119

284.8275227

Kinesin+MT

0.00508417128

0.0000119

427.241284

Kinesin+ATP

0.01864196136

0.0000119

1566.551375

Kinesin+MT+ATP

0.3875268331

0.0000119

32565.28009

Table7:SpecificActivityandTurnoverNumberofKinesin(groupepsilonvalue)
WorkingReagentCalculations

1)(50mMMgCl2)(17.5L)=x(175L),thereforex=5mM[MgCl2]
2)(150mMPipes)(17.5L)=x(175L),thereforex=15mM[Pipes]
3)(.2mg/mL)(15L)=x(175L),thereforex=.0171mg/mL[MT]
4)(.167g/mL)(5L)=x(175L),thereforex=.00477g/mL[commercialkinesin]
5)(2mM)(20L)=x(175L),thereforex=.2286mM[ATP]
6)(.067*10mg/mL)(333.33mL)=x(500mL),thereforex=.44666mg/mL
(.4466622mg/mL)(687.5L)=x(1000L),thereforex=.307mg/ml[MalachiteGreen]
7)(5*10mg/mL)(166.67mL)=x(500mL),thereforex=16.667mg/mL
(16.667)(687.5L)=x(1000L),thereforex=11.458mg/mL[Molybdate]
8)(34*10mg/mL)(137.5L)=x(1000L),thereforex=46.75mg/mL[SodiumCitrate]


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B)Cloning

Thethreemutant
cin8
genesgiverisetoCin8pwithCterminaltruncationsthatcleaveatthe
955,871,and590aminoacid.Thesetruncationswerechosenbasedonthefindingsofthepaper,

HomotetramericFormofCin8p,aSaccharomycescerevisiaeKinesin5Motor,isEssentialforItsin
VivoFunction.
Hildebrandtet.alconstructedsimilarmutantstoclonealongsideWTCin8ptostudy

invivo
function,dominance,immunolocalization,coimmunoprecipitationandhydrodynamics[
4
].
LiketheHildebrandtpaper,the
CIN8
truncationanddeletion
alleleswereconstructedby
restrictionenzymecleavageand
religationandwerechosenfor
theirassessedabilitytofunction
asthesolesourceofkinesin5
activityandcellviabilityas
deemedbytheHildebrandt
paper[
4
].
Asdepictedon
(
Figures4*[TOP]and5*
[BOTTOM]
)ontheleft,the
kinesinmotorproteinis
comprisedofsecondary
elementsthatformtwoNterminalmotordomains(aminoacids74513),aneck,ahinge,severalcoils,
astalktaillinker,(aminoacids534935)acoiledcoiltail,andaglobulartail(aminoacids9461038)
[
4
].
Itisstillunclearwhichsequenceelements,domains,andgeneralstructuresaccountforthe
functionalityofthemotorproteinand,assuch,thetruncationscreatedinthisstudyattemptto


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22723345
investigatethespecificpropertiesHildebrandtet.alcouldnotincludingtheeffectdifferent
combinationsof helical,coileddomainsinthestalkhaveonactivity(nocoileddomainsorNLSin
the590truncation,coileddomains13butnoNLSinthe871truncationandcoileddomains14butno
NLSinthe955truncation).Hildebrandtfailstoutilizeavarietyofbiochemicalmethodsthatcan
quantifyenergeticsandkineticsofthekinesin5motorproteinthesebiochemicalmethodswillbe
appliedinourinvestigation.
Itcanalreadybeassumedthattheevolutionarilyconservedmotordomainisnecessaryfor
functionalityasitparticipatesinATPhydrolysisandprocessivemovement.GiventheHildebrandt
paper,itishypothesizedthat,attheveryleast,thefirstcoiledcoildomainandthelastpartofthestalk
arenecessaryfordimerizationandtetramerizationrespectively[
4
].Itisstillunknownwhatsequence
elementsintheseregionsareessentialfordimerization/tetramerizationorifthehomotetrameric
configurationisabsolutelyrequiredforfunctionality[
4
]Hildebrandtdiscoveredactivityduringmitosis
mightbehaltedfortheCin8p590evenifmicrotubulebindingandATPaseactivityisstillfunctional
[
4,5
]Thiscomplexityinstructurefunctionrelationshipswillbefurtherinvestigated,
invivo
and
in
vitro
,astheWTCin8panditstruncatedvariantsareanalysedfortheirpropertieswithrespectto
variablestestingfunctionality,multimerization,subcellularlocalization,phenotypicbehavior,and
transcriptionalexpressioninthisexperiment.BycomparingthethreemutantstoCin8pORF,itis
possibletoelucidatethemechanismbywhichstructureandfunctionoperate.
Firstandforemost,theCin8pORFanditstruncatedvariantsmustbecloned.Thecloning
strategyutilizedinthispreliminaryexperimentinvolvedPolymeraseChainReaction(PCR),product
purification,digestion,enzymaticpurification,gelelectrophoresis,ligation,transformationwithDH5
/BLR(DE3)pGroRIL/MS2922/DDY904mCherry,andscreening/selection[
6,7
].DuringthePCRstep,
theWT
CIN8
and
cin8
mutantswereamplifiedviaaNewEnglandBioLabsDNAPhusionPolymerase
protocolandthenpurifiedwiththeQiagenQuickKit/SpinKit.Designedprimerscreatedoverhangs


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thatwereligatedtotheappropriateplasmidvectorsviaT4DNAligaseafterutilizingCalfIntestinal
PhosphatasetolimitintramolecularreligationafterdigestionwithBamHIandHindIIIrestriction
enzymesoneachvectorsMultipleCloningSites(MCS)region.Theseenzymaticsiteswerechosenfor
theirsinglecutsintheMCSandtheirabsenceintheinsertPCRproduct.Transformationofthe
appropriatevectorsintobacterial(DH5 orBLR(DE3)pGroRIL)oryeaststrains(MS2922orDDY904
mCherry)wascompletedbychemicaltransformationand/orelectroporation(
Table11
).
Table8,
Table9,andTable10
summarizethethebacterial/yeaststrainsutilizedforcloningandthevectors
usedforinsertionofWT
CIN8
ORFortruncatedvariantsinthecontextofpropertiesandpurposesof
thisproposedexperiment.
Thesuccessofourcloningwasanalysedviaa0.8%agarosegelelectrophoresisandconfirmed
byDNAsequencingattheUCBerkeleySequencingFacility.Sequenceswereloadeddirectlyinto
sequencecomparisonBLASTNdatabasetoanalyseagainst
CIN8
sequenceattheSaccharomyces
GenomeDatabase(SGD).Sequenceidentitieswere>99%inallcases.
Figures8andFigure9
are
UVvisualizedgelimagesofWT
CIN8
andthethreemutant
cin8
variants.
Figure8
confirmsthe
successofPCRandthepurificationofproductDNAfragmentsand
Figure9
confirmsthesuccessof
enzymaticpurificationpostdigestion.Comparingeachbandtothe5 Lof1Kbladderaddedtothe
lastwellallowsonetoanalyzethemass(ng)andsize(bp/Kb)ofeachDNAfragment.Giventhesegel
images,itisclearthattheWTCin8p(1038),Cin8p955,Cin8p871,andCin8p590havebeencloned
successfullysubsequentanalysisofDH5 withallthreeplasmidvectorsfollowingtransformation
revealstheefficiencyofthetransformationproceduresbeingused.Thisdataandtheappearanceof
coloniesonAmp100andKan50platesfollowingtransformationofpET24b(+)into
BLR(DE3)pGroRIL,pCIN8GFPCFUSandpCIN8 GFPintoMS2922andpCIN8GFPCFUSinto
DDY904mCherryrespectivelyisevidenceofthesuccessofcloning.


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Itishypothesizedthatthepresenceofmicrotubulestructureswillstimulatethe
mechanochemicalATPasenatureofkinesin5andprovideacellularsurfaceforthemotorproteinto
performbiophysicalworkupon.Furthermore,weexpectKcattoincreaseinthepresenceofATP,MT,
andATP+MTthereforesuggestingVmaxreachesdiffusioncontrolandcatalyticefficiencymuch
fasterinsuchenvironmentalconditions.Turnovernumberscanbeexpectedtobequitehighinthese
cases.Itcanalsobeexpected,giventheresultsoftheHildebrandtpaperdiscussedearlier,that
Cin8pORFanditstruncatedvariantsshouldallbeabletodimerizeandengageinmotoractivitywith
themicrotubuleeachmutantandthewildtypestillpossesthefirst coiledcoildomainandmotor
headsnecessaryfortheseactions[
4
].
Itcanalsobehypothesizedthatsubcellularlocalizationwouldrevealfluorescentactivityinthe
nucleusoftheyeastkinesinwheremitoticspindleactivityislikelytobefoundfollowingtheseGFP
taggedfusedproteinswillrevealifmitoticactivityisstillplausibleinthewakeoftruncations
pinpointingthesourceoffunctionalkinesin5activity.ItishypothesizedthattheCin8p590willbe
unlikelytoundergomitosisduetoitsfailuretetramize.Allothermutantsandthewildtypewillstillbe
abletotetramizegiventheresultsoftheHildebrandtstudy[
4
].Degreeofmitoticactivitywillrevealto
whichextentaparticulartruncationplaysaroleinfunctionalactivity.Allofthesehypotheseswillbe
testedusingthemethodsoutlinedinResearchDesignandMethods.


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BacterialStrains

Properties

Purpose

BLR(DE3)pGroRIL

DerivativeofE.colistrain,
BL21(DE3)
Contains
prophagethat
carriesgeneforT7phage
RNApolunderlactosepromoter
andoperator
Transformedw/pGroRIL
plasmid,inducedw/Arabinose

IPTGinducessynthesisofT7
RNApolinculturesoto
transcribegenesthatareunder
thecontrolofaT7phagelate
genepromoter
pGroRILforproductionof
raretRNAArgU,IleY,LeuW
Synth.pGroEL/pGroES

DH5

DerivativeofE.coliK12
endA1mt.andhsdR17mt.
DH5 recAmt.
WThsdMgene

invitro
analysisofCin8p
inactivatesplasmiddegrading
endonuc.,methylatesDNA,
elim.homologousrecomb.

Table8:PropertiesandPurposesofBacterialStrains[
6
]
YeastStrains

Properties

Purpose

MS2922

containsaninactivatedgene
foronekinesincin8 :: LEU2
containsatemperature
sensitivemutantin
KIP1
gene
(Kip1101^ts)
ura352mutation

Replacementof
CIN8
allows
forexpressionofmutantcin8
Inactivationof
CIN8
allows
KIP1
tobecomeactivewitha
temperaturesensitive
phenotype
Willbeusedtotestforuptake
ofpCIN8GFPCFUSandp
CIN8GFPasvectorscontains
aWTURA3thatmustbe
expressedsotosynthesizethe
uracilnecessaryforitsgrowth
onSCURA
Willbeusedtotesttheeffects
ofGFPfusiononCin8pmotor
protein

invivo
analysisofCin8p

DDY904mCherry

mCherrytaggedtubulin

Subcellularlocalizationof
microtubulesandmotor
proteins
UptakeofpCIN8GFPCFUS

invivo
analysisofCin8p

Table9:PropertiesandPurposesofYeastStrains[
6
]


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Plasmid/Vectors

Properties

Purpose

pET24b(+)

Novagen
Cterminal6xHIStag
Kanamycinresistancegenefor
selectioninbacteria

Expressionin
BLR(DE3)pGroRILand
DH5
Proteinbiochemistry,
characterizingCin8p
invitro

pCIN8GFPCFUS

CterminalGFPtag
CIN8
promoter
Ampicillinresistancegenefor
selectioninbacterialstrains
containsURA3genefor
selectioninyeaststrains

ExpressioninMS2922and
DDY904mCherry
ExpressioninDH5
Subcellularlocalizationvia
fluorescencemicroscopy
Analysingmovementand
locationofCin8pandeffectsof
GFPfusiononactivity
invivo

pCIN8 GFP

CIN8
promoter
Ampicillinresistancegenefor
selectioninbacterialstrains
containsURA3genefor
selectioninyeaststrains

ExpressioninMS2922
ExpressioninDH5
Replacementofendogenous
Cin8p(complementation)
ControlforGFPfusion
analysis
invivo
andphenotypic
analysisundervariousstress
conditions
invivo

Table10:PropertiesandPurposesofExpression/ShuttleVectors[
6
]

Figure6:PlasmidMapsofpET24b(+)andpCIN8GFPCFUS/pCIN8 GFP
LeBlanc,I.,Wilson,R.(2015)
GeneralBiochemistryandMolecularBiologyLaboratoryProtocol
Guide.
DepartmentofMolecularandCellBiology,UniversityofCalifornia,Berkeley,CA


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22723345

Figure7:SchematicDiagramofPlasmidMapPurposesandEndCloningInsert+VectorProduct
LeBlanc,I.,Wilson,R.(2015)
GeneralBiochemistryandMolecularBiologyLaboratoryProtocol
Guide.
DepartmentofMolecularandCellBiology,UniversityofCalifornia,Berkeley,CA

TransformationProcedure

Properties

*ChemicalTransformationofBacteria
Strain:DH5
AssociatedVector(s):pET24b(+),
pCIN8GFPCFUS,pCIN8 GFP

Uptakethroughvoltagegatedmembrane
channelsintoantibioticsensitive,competent
bacterialcells.Cellstreatedwithpolyvalent
cationssuchasCaCl2,allowsbindingofDNAto
cellsurfaceandpermeabilizescellwall
Resultsininflowofextracellularmediumand
exogenousvector+insert

*ChemicalTransformationofYeast
Strain:MS2922
AssociateVector(s):pCIN8GFPCFUS,
pCIN8 GFP

Strain:DDY904mCherry
AssociatedVector(s):pCIN8GFPCFUS

Uptakethroughvoltagegatedmembrane
channelsintoantibioticsensitive,competent
yeastcells.Li+treatmentofcellsviaLiOAcand
DMSOtoincreasecellpermeability
IncubationwithPEGtoenhanceDNAbinding
capacitytocellsurface
ssDNA(salmonsperm)ascotransportcarrier
withdesiredplasmid.Willsaturatenonspecific
bindingsitesandprotectvector+insertfrom
nucleaseactivity

*Electroporation
Strain:BLR(DE3)pGroRIL
AssociatedVector(s):pET24b(+)

Electrocompetentcellswashedwithdeionized
wateranditscellmembraneissubjectedtoan
electricfield(2500V/cm)
temporaryporeformationenablesinflowof
extracellularmediumandexogenousvector+
insert.Mustremainsaltfree

Table11:ExperimentalTransformationProceduresandProperties[
6
]


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Eachgelshouldberead,fromlefttoright,assuch:
1

PCR
neg.
control

3
PCR
sample

PCR
sample
purified

PCR
pGFPC
pET24 p GFP
sample
FUS, dig&pur dig&pur
dig&pur dig&pur

9
1kb
ladder

Table12:0.8%AgaroseGelElectrophoresisWellProtocol[
6
]

Figure8:PostPCRProductPurificationImagesforWT
CIN8ORF
andMutant
cin8
Constructs
TopLeft:Cin8pORF,BottomLeft:Cin8p590,TopRight:Cin8p871,BottomRight:Cin8p955
Source:
MolecularCellBiology110LBCoursesWebsite

Figure9:PostDigestion,EnzymePurificationImagesforWT
CIN8
ORFandMutant
cin8
Constructs
TopLeft:Cin8pORF,BottomLeft:Cin8p590,TopRight:Cin8p871,BottomRight:Cin8p955
Source:
MolecularCellBiology110LBCoursesWebsite


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C)BioinformaticsandGeneExpression
Kinesinareinvolvedinanumberofbiochemicalandmechanochemicalactivitiesincluding
transportofcellularcargohousedinCOPI,COPIIand/orclathrinvesicles[
8
]andinspindleassembly
andelongationduringmitosis[
6
].Mitosisisacellulardivisionprocessthatis,inoneformoranother,
conservedamongstalleukaryotesandrequiresagreatdealofspeed.Becauseofthenecessityfor
speed,itcanbeexpectedthatalargeamountofkinesincanbegenerallyfoundactiveinoneorganism.
Theevolutionarilyconservedprocessofmitosisamongstalleukaryotesandfunctionallyconserved
processofcellulardivisionamongstallorganismsexplainsforwhykinesinsaresocloselyrelated
betweenorganisms.ThisisverifiedbytheSaccharomycesGenomeDatabase,wheregenessuchas
KIP1
,
CIK1
,
STU2
,
EG5
,and
CIN8
,canbeanalysedtorevealconservedsequences.Relatedproteins,
consequently,shareconserveddomainsandphenotypicfunction.Uponfurtherresearchinthepaper

TheForkheadTranscriptionFactorHCM1RegulatesChromosomeSegregationGenesandFillsthe
SPhaseGapintheTranscriptionalCircuitryoftheCellCycle
byPramillaet.al,itisclearthereare
phylogeneticallyconservedsequenceswithinthepromotersofgenes,suchas
CIN8
and
KIP1
,whose
transcriptspeakduringSPhase[
9
].Uponinspectionofthegenes 30geneexpressiondataset,itis
cleartoseethereisapositiveandhighlevelofexpressionattime20<t<60,correspondingto
SPhasepeaktime.BothgeneexpressionprofilesforCin8pandKip1pareperiodicamongfavedata
setsand
CIN8
hasaweightedaveragepeakat37%ofthecellcyclewhereas
KIP1
hasapeakat40%of
thecellcycle(relativetoM/G1boundarybeingat0%minimumexpression).Thereisalsoapeakat
80<t<100forbothgeneexpressionprofilescorrelatingtoanSPhasethereaswellaccordingto
SGD.ThepeakrunsfromStotheG2/Mboundary.Thesepeaksmatchwhatisknownaboutthe
kinesinmotorproteinsactivityastheyaremostactiveintheassemblyofthemitoticspindleand
centrosomeduplication(SPhase).


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ResearchDesignandMethods:
Thebiologicalquestionbeingposed,atitsmostbasallevel,isWhatstructuralfeaturesof
Cin8p,akinesin5motorprotein,accountforitsfunctionality?In
HomotetramericformofCin8p,a
Saccharomycescerevisiaekinesin5motor,isessentialforitsinvivofunction
,Hildebrandtet.al
developedresearchstrategiesthatprovedthefirst coiledcoildomainwasessentialfordimerization
andthatsequenceelementsfoundlaterinthestalkwererequiredfortetramerization[
4
].Hildebrandt
foundnonfunctional,dimericCin8pstillboundtomicrotubulessuggestingthemotorproteinsactivity
requiresmorethanacellularplatformforittoperformworkupon[
4
].
TheresearchdesignandmethodsemployedinthisinvestigationseekstoexploreCin8pwith
respecttocharacteristicsHildebrandtet.aldidnotanalyseandbuilduponfurtheringthescientific
communitysunderstandingofwhatsequenceelements,specifically,determinethemotoractivityor
lackthereofofCin8p.Theeffectsofthisinvestigativestudyislimitlessifsuccessful,itwillbe
possibletodeveloppharmaceuticaldrugsthatmimickinesinlocomotivebehavior,betterdeliveringthe
agentofinteresttotargetcells.Itwillbepossibletocreatenovelanticancertherapies.Pinpointingthe
sourceofkinesinmotoractivitysuggestsanabilitytonegatively/positivelyregulategeneexpression
andturningoffthegenesabilitytoassembleaspindleformitosiswilleffectivelyhaltproteomic
activityandkeepcellsfromdividingandpotentiallymetastasizing.Moreso,kinesinslocomotive
propertiesmakeitaneffectivemoleculetotagwithGFPorsimilarfluorescentproteinsototrack
cellularcargomovementthroughacell,effectivelyenablingavarietyofnovelmeansoftracing
research.

ThesuccessfulcloningofCin8pORFandtruncatedvariantsinthepreliminaryinvestigation
hascreatedconstructsthatcanbecharacterizedandusedtoanswertheseopenendedquestions.Four
experimentswillberuntoaddressquestionsofCin8psubcellularlocalization,characterization,
phenotypes,andtranscriptionalpotential.


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A)SubcellularLocalization
TobestunderstandthestructureofCin8panditsfunction,itisbesttofirstanalysethe
environmentwhereCin8pisfoundandhowitsenvironmentmayaffectitsactivity.Itisexpectedthat
thewildtypeCin8pandmutantCin8pwillbefoundwithinthenucleusornucleolusifapplicable
involvedinmitoticactivityorthroughoutthecytoplasminteractingwithmicrotubulestodeliver
cellularcargotodifferentregionsofthecelliffunctional.Ifnonfunctional,weexpecttoseeimmobile
kinesinmotorproteinsormotorproteinsinvolvedinexclusivelymicrotubuleactivity.Totestthis
hypothesis,livecellimagingfluorescencemicroscopywillbeutilized.Thekeyadvantagetothis
particulartypeoffluorescencemicroscopyisthatitusesGreenFluorescentProtein(GFP)isolatedfrom
the
Aequoreavictoria
togenerateagreenfluorescentsignal(excitationpeak=395nmemissionpeak
=509nm)tovisualizeaproteinfusedtoit[
6
].Inrealtime,mitoticspindleassemblyandkinesin
dynamicscanbeobservedastheproteinwillcontinuetolocalizeasitwouldnormallywithouttheGFP
asGFPdoesnotnormallyinterferewithproteomicactivity.Still,forgoodexperimentaldesign
purposes,acontrolwillbeestablishedanalysingpCIN8GFPCFUSandpCIN8 GFPintheMS2922
yeaststraintoensurelocalizationisnotaltered.Thefluorescencewillbeobservedwiththe
pCIN8GFPCFUSmutantintheDDY904mCherryyeaststrain.Thetruncatedvariantisfusedwith
GFPfortrackingsowhentheCin8pmutantistranscribedandtranslated,theGFPgeneinthesame
readingframeisalsotranscribed,translate,and,undoubtedly,activated.TheDDY904mCherrystrain
usedinthisexperimentcontainsmCherry,aRedFluorescentProtein(RFP)derivative,fusedtoan
tubulingene(
TUB1
)sotobettervisualizethelocationandappearanceofthemitoticspindle[
6
].The
redfluorescenceoftheDDY904mCherrywillbeusedasabackgroundreferencetothegreen
fluorescenceofthepCIN8GFPCFUS.


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B)EnzymeExperiment
Afteranalysingsubcellularlocalizationbylivecellimagingfluorescencemicroscopy,itwillbe
necessarytobeginanalysingthekineticandthermodynamicparametersofthewildtypeCin8pandits
truncatedvariantssotobetterunderstandiftheproteinofinterest(beitwildtypeormutant)isviable
andtocharacterizethefunctionalityoftheprotein.
GiventhattheBLR(DE3)pGroRILstraincontainsa prophagethatcarriesageneforT7
PhageRNApol,eachconstructwillhaveitsexpressioninducedbyaddingIPTGsototranscribegenes
thatareunderthecontrolofaT7phagelategenepromoter[
6
].Thisexperimentwillalsorequire
collectingtimepointsofbacteriaculturesanddisruptingthecellstoreleaseproteinsthatwillberunon
twoSDSPAGEgels.Tojudgethepurityofproteinpreparation,CoomassieBluewillbeusedasa
generaldetectionmarker.InpuresamplesofCin8p,onlyonebandshouldtheoreticallyappearasitwill
representthemajorsubunitoftheprotein.CoomassieBluewill,however,seealloftheproteins
releasedfromthecell.TheotherSDSPAGEgelwillbesubjectedtoaWesternBlot.Thenitrocellulose
orPVDFmembranewillbeprobedwithaprimaryantibodyspecificfor6xHIStags,washed,andthen
probedwithasecondarypolyclonalantibodyspecificfortheantimouse[
6
].Thesecondaryantibodyis
coupledwithHRPproteinwhichwillreactwithchloronaphtholtoproduceanamplifiedsignaldetected
colorimetrically[
6
].Thissignalrevealstheproteinslocationonthegel.FollowingCoomassieBlue
stainingandtheWesternBlot,furtherSDSproteingelswillbecreatedtotestforsolubilityofCin8p.
ThepurificationofCin8pORFanditstruncatedvariants,aftersolubility,willbethenextstep.
ThisensuresthatfutureBradfordassaysandMalachiteGreenATPaseassayswillyieldresultsthatbest
characterizeCin8pbehavior
invitro.
Thedeterminedstructuresofkinesinswillallowtheinvestigator
topredictwhataccountsfordifferencesinsolubility,function,andpropertiesbetweenCin8pORFand
itsmutants.Summatively,eachinvestigation(Cin8pORFandtruncatedvariants)willrequire5gin
20mLofBLR(DE3)pGroRILbacterialcelllysate[
6
].Collectiontubeswillbeprechilledandchilled


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lysisbuffer,W1/3/4buffer,W2buffer,andelutionbufferwillbepouredthroughanaffinitycolumn,at
theappropriatetimes,followingbatchbinding(6xHIStagsonCterminalbindingtoNiNTAon
agarosebeads)toyieldlysateflowthroughandwashes/elutionfractions[
6
].Thisprocesswillcontinue
untilrinsinghasremovedcorrespondingproteinsoffofresin.Bytheendofpurification,45 Laliquots
ofcrudeextract,lysateflowthrough,specificwashandallelutionfractionswillbecollectedfor
runninggels(CoomassieStainingandWesternBlot)and25 Laliquotswillbecollectedfora
MalachiteGreenATPaseassay[
6
].Thegelsshouldconfirmthattheeluatesfromtheaffinitycolumn
arecomprisedofpureCin8p.Duringpurification,aBradfordassaywillberunconcomitantlywith
purificationtomeasureproteinconcentrationofallflowthrough,washes,andelutionfractions.These
measuredproteinconcentrationswillbedeterminedviaaBSAderivedstandardcurve.Elution
fractionsthatcontainthebulkoftheproteinwillbepooledtogethersoaconsistentproteinsampleis
usedfortheMalachiteGreenATPaseassayandfurthercharacterizationexperiments.
Furtherstudiesonretentionofnativeactivitywillbenecessaryasexpressionin
BLR(dE3)pGroRIL(abacterialstrainforaeukaryoticprotein)doesnotutilizeposttranslational
modifications[
6
].Moreso,itwillbenecessarytoanalyseifthe6xHIStaginterfereswithactivity[
6
].
Assuch,aheatinactivationstability/activityassaywillbeperformedtorevealcluesaboutthe
requirementsnecessaryforCin8pactivation.Thiswillallowthepurificationbufferandstorage
conditionstobealteredtooptimizeactivity.Theadditivesusedinclude10%glycerol,50mM
potassiumacetate,5mMdithiothreitol,and0.01%TritonX100[
6
].Alinearityassaywilltestthe
biochemicalbehaviorofthepurifiedCin8pasafunctionoftime(fixedenzymeamount)andasa
functionofenzymeconcentration(fixedincubationtime)[
6
].Finally,MichaelisMentenkineticswill
beemployedtobettercharacterizethekineticparametersofCin8pwithandwithoutinhibition.Enzyme
concentrationwillbeheldconstanttoensuretheKmderivedisameasureoftheinitialrateofATP
hydrolysisasonlyafunctionofATPconcentration[
6
].ThiswillhelptoanalysehowwellCin8p


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mutantsinteractwithATPandhowfastithydrolyzesATPatitsmaximumspeedcomparedto

Cin8pORFprotein[
6
].Therelevant,specificformulaeinclude V = V max[S]/Km + [S] and
1/V = (1/[S] K m/V max) + 1/V max .Asmentionedearlier,wewillalsoexaminekineticparameters
withrespecttoinhibitorsandhowtheymightaffectinitialrateofATPhydrolysisassubstrate
concentrationisvariedandinhibitorconcentrationiskeptconstant.Theinhibitorsusedwillbe
STritylLcysteineandADP[
6
].Forthoroughness,allkineticplotswillberecorded
(MichaelisMenten,LineweaverBurk,andEadieHofstee).


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C)InVivoPhenotypes
ThesesetofexperimentswillbettertestthecapacityofWTCin8pORFanditstruncated
mutantstosuppresstheKip1101temperaturesensitivealleleandrescuegrowthat37degreesCelsius
(thenonpermissivetemperature),examinetheeffectofGFPfusiononCin8pactivityinthe
pCIN8GFPCFUS,andtheeffectofvariousadditivesonthephenotypeoftheWTCin8pORFand
truncatedvariants[
6
].TheadditivesaddedtotheSCURAagarplatesincludeCincreasin,Benomyl,
Sorbitol,and6azauracil[
6
].Itishopedthatutilizingtheseadditiveswillaidtodeterminetheeffect
eachhasonthefunctionofthecytoskeletonofthecelland,inparlayingfashion,thephenotypeofthe
Cin8pconstructswhensubjectedtoexternalpressure.Itisalreadyknownthatcellularintegritywillbe
compromisedwiththepresenceoftheadditivesbutthisexperimentwillservetoexaminetowhat
degreethecytoskeletoniscompromisedviaascoringsystem.Theseexperimentswillallrequire
duplicateplatingandtestingforthevariableofinterest.Dilutionswillbeperformedwhennecessary.
D)Transcriptionof
CIN8
MethodsemployedforanalysingRNAlevelsincludeRTPCRaftergrowthandtreatmentof
yeastcellsandRNAisolationandpurificationviaRNeasyfromQiagen.TheintegrityoftherRNAin
sampleswillbeanalysedviaa0.8%gelelectrophoresis.RTPCRwillbeperformedusingQiagen
OneStepRTPCRkittodetermineconcentrationandvariationofsynthesisofRNAsfromthe
CIN8
geneanditsmutantsunderdefinedgrowthconditions.[
6
].ThiswillconvertRNAintocDNAthatcan
beresolvedonanagarosegeltocomparebandintensitiesofORFwithandwithoutadditivesand
deletionmutantstoORF.TheamountofcDNAisarepresentationofthe
cin8
or
CIN8
transcript.Itis
hopedthat,byrunningthisexperiment,inferencescanbemadeaboutthemetabolicactivityofthe
Cin8pmutantsinthecellrelativetothewildtypeandwhetherthereisanenhancedtranscript
concentrationgivenoptimalculturesand/oradditives.Thesoledrawbackofthismethodisinits
quantificationbycomparinggelbands,quantificationisaresultofaneyeballmethod[
6
].


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References

1. Vale,RonaldD.,ThomasS.Reese,andMichaelP.Sheetz.(1985)Identificationofanovel
forcegeneratingprotein,kinesin,involvedinmicrotubulebasedmotility.
Cell
42
.1,3950.
2. Hoyt,M.A.,He,L.I.N.G.,Totis,L.,&Saunders,W.S.(1993).Lossoffunctionof
Saccharomycescerevisiae

kinesinrelatedCIN8andKIP1issuppressedbyKAR3motor
domainmutations.
Genetics
,
135
.1,3544.
3. Roof,D.M.,Meluh,P.B.,&Rose,M.D.(1992).Kinesinrelatedproteinsrequiredfor
assemblyofthemitoticspindle.
TheJournalofCellBiology
118
.1,95108.
4. Hildebrandt,E.R.,Gheber,L.,Kingsbury,T.,&Hoyt,M.A.(2006).Homotetramericformof
Cin8p,aSaccharomycescerevisiaekinesin5motor,isessentialforitsinvivofunction.
JournalofBiologicalChemistry
,
281
.36,2600426013.
5. Kapitein,L.C.,Kwok,B.H.,Weinger,J.S.,Schmidt,C.F.,Kapoor,T.M.,&Peterman,E.J.
(2008).Microtubulecrosslinkingtriggersthedirectionalmotilityofkinesin5.
TheJournalof
CellBiology
,
182
.3,421428.
6. LeBlanc,I.,Wilson,R.(2015)
GeneralBiochemistryandMolecularBiologyLaboratory
ProtocolGuide.
DepartmentofMolecularandCellBiology,UniversityofCalifornia,
Berkeley,Berkeley,CA
7. Russel,P.J.(2005).iGenetics:AMolecularApproach.PearsonEducation,SanFrancisco,CA.
ISBN:0805346651
8. LippincottSchwartz,J.,Cole,N.B.,Marotta,A.,Conrad,P.A.,&Bloom,G.S.(1995).
KinesinisthemotorformicrotubulemediatedGolgitoERmembranetraffic.
TheJournalof
CellBiology
,
128
.3,293306.


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9. Pramila,T.,Wu,W.,Miles,S.,Noble,W.S.,&Breeden,L.L.(2006).TheForkhead
transcriptionfactorHcm1regulateschromosomesegregationgenesandfillstheSphasegapin
thetranscriptionalcircuitryofthecellcycle.
Genes&Development
,
20
.16,22662278.

AsteriskedFiguresCitation
*LeBlanc,I.,Wilson,R.(2015)
GeneralBiochemistryandMolecularBiologyLaboratoryProtocol
Guide.
DepartmentofMolecularandCellBiology,UniversityofCalifornia,Berkeley,Berkeley,CA

NoteonPreliminaryResults&ResearchDesignandMethods
TheconceptualbasisforthePreliminaryResults,andthemethods/experimentsusedtherein,are
methods/experimentsemployedin
GeneralBiochemistryandMolecularBiologyLaboratoryProtocol
Guide
byIsabelleLeBlancandRossWilson.TheconceptualbasisfortheResearchDesignand
Methods,andthemethods/experimentsusedtherein,aremethods/experimentsemployedin
General
BiochemistryandMolecularBiologyLaboratoryProtocolGuide
byIsabelleLeBlancandRoss
Wilson.Allactualexperiments/methodsdescribedinthisproposalareadaptedfromthislabmanual
and,assuch,shouldbereferredtointhecarryingoutofsaidmethods/experimentstobestcharacterize
Cin8p.Methodsandexperimentsinthisproposalareuniversallyusedinlaboratoriesforthepurposes
outlinedinthisproposalsocitationsfortheseinstrumentaltechniqueshavebeenignored.If
explanationsformethodsandexperimentsinthisproposalrequireacitationwherethereisnt,the
methods/experimentsshouldbeinterpretedasbeingcitedfromthissource:

6.LeBlanc,I.,Wilson,R.(2015)
GeneralBiochemistryandMolecularBiologyLaboratoryProtocol
Guide.
DepartmentofMolecularandCellBiology,UniversityofCalifornia,Berkeley,Berkeley,CA