Download The Making of the Fittest: Natural Selection and Adaptation

Lesson
The Making of the
TheFittest:
Making of the Fittest
Natural
Selection and Adaptation
Natural Selection and Adaptation
Educator MaterialsLESSON
TEACHERMATERIALS
INTRODUCTIONTOTHEMOLECULARGENETICSOFTHE
COLORMUTATIONSINROCKPOCKETMICE
OVERVIEW
TheselessonsserveasanextensiontotheHowardHughesMedicalInstituteshortfilmTheMakingoftheFittest:
NaturalSelectionandAdaptation.Studentswilltranscribeandtranslateportionsofthewild-typeandmutantrock
pocketmouseMc1rgene.BycomparingDNAsequences,studentsidentifythelocationsandtypesofmutations
responsibleforthecoat-colorchangedescribedinthefilm.Studentswillansweraseriesofquestionstoexplain
howachangeinaminoacidsequenceaffectsthefunctionalityoftheMC1Rprotein,andhowthatchangemight
directlyaffectthecoatcoloroftherockpocketmousepopulationsandthesurvivalofthatpopulation.
KEYCONCEPTSANDLEARNINGOBJECTIVES
•
•
Amutationisarandomchangetoanorganism’sDNAsequence.
Mostmutationshavenoeffectontraits,butsomemutationsaffecttheexpressionofageneand/orthe
geneproduct.
• Theenvironmentcontributestodeterminingwhetheramutationisadvantageous,deleterious,orneutral.
• Naturalselectionpreservesfavorabletraits.
• Variation,selection,andtimefueltheprocessofevolution.
• Boththetypeofthemutationanditslocationdeterminewhetherornotitwillhaveaneffecton
phenotype(advancedversiononly).
Studentswillbeableto:
• transcribeandtranslateaDNAsequence.
• connectDNAchangestophenotype.
• analyzeandorganizedata.
CURRICULUMCONNECTIONS
Curriculum
NGSS(April2013)
CommonCore(2010)
APBiology(2012–13)
IBBiology(2009)
Standards
HS-LS1-1,HS-LS3-1,HS-LS3-2,HS-LS4-2,HS-LS4-4,HS-LS4-5
HS.LS1.A,HS.LS4.B,HS.LS4.C
CCSS.ELA-Literacy.RST.9-10.3,CCSS.ELA-Literacy.RST.9-10.4,
CCSS.ELA-Literacy.RST.9-10.7
1.A.1,1.A.2,3.A.1,3.C.1,4.B.1
3.5,4.1,4.3,5.4,7.3,7.4,D.2,G.1
KEYTERMS
evolution,naturalselection,variation,trait,mutation,adaptation
TIMEREQUIREMENT
Theseactivitiesaredesignedforone50-minuteclassperiodincludingshowingthefilm.Additionaltimeforthe
analysisquestionsmightberequiredforhomeworkdependingonstudents’pace.Bothactivitiescoverthesame
material,butactivity2hasadditionalmaterialcoveringintracellular,transmembrane,andextracellulardomains.
www.BioInteractive.org
PublishedFebruary,2017
Page1of8 Lesson
The
Making of the Fittest:
The Making of the Fittest
Natural
Selectionand
andAdaptation
Adaptation
Natural Selection
LESSON
Educator
Materials
TEACHERMATERIALS
SUGGESTEDAUDIENCE
Activity1:Theactivityisdesignedforhighschoolbiology(primarilyfirst-yearbiology,bothregularandhonors).
Activity2:TheactivityisdesignedforAPandIBhighschoolbiologyandintroductorycollegebiology.
PRIORKNOWLEDGE
Studentsshouldbefamiliarwiththedefinitionsof“gene”and“protein.”Theyshouldalsobecomfortablewith
basicmoleculargenetics,includingafamiliaritywiththeprocessesoftranscriptionandtranslation.Finally,
studentsshouldunderstandthataprotein’saminoacidsequencedeterminesitsstructure,whichdeterminesits
function.
MATERIALS
Studentswillneed:
geneticcodechart(providedinstudentmaterials)
blue,red,andgreencoloredpencils
TEACHINGTIPS
•
•
Ifyoudonothaveaccesstoacolorprintertoprintthechartonpage2ofthisdocument,youshould
comparestudents’worktohowthechartsbelowappearonyourcomputerscreen.
Youcouldassignanalysisquestionsashomeworktoreducetheamountofclasstimerequiredforthis
lesson.
ANSWERKEYACTIVITY1
GENETABLE1:WILD-TYPEMC1RGENE(LIGHTCOAT-COLORPHENOTYPE)
DNA
mRNA
Amino
Acid
015
TTG
AAC
AGG
UCC
TGG
ACC
GCG
CGC
TGT
ACA
CCG
GGC
CAA
GUU
022
GGA
CCU
Asn
Ser
Thr
Arg
Thr
Gly
Val
Pro
DNA
mRNA
Amino
Acid
105
CGG
GCC
GAC
CUG
CGG
GCC
TGG
ACC
GCC
CGG
CAC
GUG
TGA
ACU
112
CAC
GUG
Ala
Leu
Ala
Thr
Arg
Val
Thr
Val
DNA
mRNA
Amino
Acid
154
TCA
AGU
TAA
AUU
CAC
GUG
TGT
ACA
GAC
CUG
GGG
CCC
GCC
CGG
161
CGA
GCU
Ser
Ile
Val
Thr
Leu
Pro
Arg
Ala
DNA
mRNA
Amino
Acid
209
GTG
CAC
TAC
AUG
GAA
CUU
212
CGT
GCA
His
Met
Leu
Ala
www.BioInteractive.org
PublishedFebruary,2017
Page2of8 Lesson
The
Making of the Fittest:
The Making of the Fittest
Natural
Selectionand
andAdaptation
Adaptation
Natural Selection
LESSON
Educator
Materials
TEACHERMATERIALS
DNA
mRNA
Amino
Acid
230
GAA
CUU
CAG
GUC
GTG
CAC
GTT
CAA
CCA
GGU
AAG
UUC
GCT
CGA
237
GAG
CUC
Leu
Val
His
Gln
Gly
Phe
Arg
Leu
GENETABLE2:MUTANTMC1RGENE(DARKCOAT-COLORPHENOTYPE)
DNA
mRNA
Amino
Acid
015
TTG
AAC
AGG
UCC
TGG
ACC
ACG
UGC
TGT
ACA
CCG
GGC
CAA
GUU
022
GGA
CCU
Asn
Ser
Thr
Cys
Thr
Gly
Val
Pro
DNA
mRNA
Amino
Acid
105
CGG
GCC
GAC
CUG
CGG
GCC
TGG
ACC
ACC
UGG
CAC
GUG
TGA
ACU
112
CAC
GUG
Ala
Leu
Ala
Thr
Trp
Val
Thr
Val
DNA
mRNA
Amino
Acid
154
TCA
AGU
TAA
AUU
CAC
GUG
TGT
ACA
GAC
CUG
GGG
CCC
ACC
UGG
161
CGA
GCU
Ser
Ile
Val
Thr
Leu
Pro
Trp
Ala
209
GTG
CAC
TAC
AUG
GAG
CUC
212
CGT
GCA
His
Met
Leu
Ala
DNA
mRNA
Amino
Acid
DNA
mRNA
Amino
Acid
www.BioInteractive.org
230
GAA
CUU
CAG
GUC
GTG
CAC
GTG
CAC
CCA
GGU
AAG
UUC
GCT
CGA
237
GAG
CUC
Leu
Val
His
His
Gly
Phe
Arg
Leu
PublishedFebruary,2017
Page3of8 Lesson
The
Making of the Fittest:
The Making of the Fittest
Natural
Selectionand
andAdaptation
Adaptation
Natural Selection
LESSON
Educator
Materials
TEACHERMATERIALS
QUESTIONSANSWERKEY
1.Ingeneexpression,whichenzymeisresponsiblefortranscribingtheDNAsequenceintomRNAbyadding
complementaryRNAnucleotides?RNApolymerase
2.Inaeukaryoticcell,wheredoestranscriptionoccur?Inthenucleus
3.Describetheprocessoftranslation.
TranslationistheprocessofturninginstructionsfrommRNAintochainsofaminoacids.Itoccursinthe
cytoplasmwiththehelpofribosomesandtRNA.
4.Inaeukaryoticcell,whatmainorganelleisinvolvedintranslation?Ribosome
5.ExplaintherelationshipbetweenDNAsequence,aminoacidsequence,andproteinstructureandfunction.
StudentsmaysimplyrelateDNAsequencetoaminoacidsequence,andaminoacidsequencetothethreedimensionalshapeoftheprotein.Anexampleofastudentresponsemaybe:“DNAsequenceprovidesthecode
fortheaminoacidsequence.Theaminoacidsequencedeterminesthestructureoftheprotein,whichaffects
thefunctionoftheprotein.”
6.TheMc1rgenecodesforthemelanocortin1receptor(MC1R)protein.
7.IftheMC1Rproteinis317aminoacidslong,whyarethere954basepairsinthecodingregionofthegene?
EachoftheaminoacidshasacorrespondingmRNAcodonandDNAtripletconsistingofathree-basesequence.A
proteinthathas317aminoacidsthereforehasaDNAbasesequenceconsistingof951basepairs(317×3=951
basepairs).Thethreeadditionalbasepairscorrespondtoastopcodonforwhichthereisnocomplementary
aminoacid.Thestopcodonsignalstheterminationoftranslation.
8.OfthefivemutationsyouidentifiedintheMc1rgene,howmanyare:
5substitutions;0insertions;0deletions
9.OfthefivemutationsyouidentifiedintheMc1rgene,howmanyare:
1silent;4missense;0nonsense
10.Micewiththewild-type(nonmutant)Mc1rgenehavelight-coloredfur.Whichpigmentisresponsibleforthis
coloration?Pheomelanin
11.Usingtheinformationintheintroductiononmutationsandyourknowledgeofproteins,developahypothesis
toexplainhowthechangesintheMC1Rprotein’saminoacidsequencemightaffectitsfunction.
Sampleanswer:ThefourmissensemutationsintheMc1rgenechangetheaminoacidsequenceoftheMC1R
protein,whichchangesthestructureoftheprotein.Thechangeinproteinstructurewillaffecttheprotein
function.
12.Explainhowsilentmutationsaffectthestructureandfunctionoftheprotein.
Silentmutationsdonotchangetheaminoacid,andthereforewillnotchangethestructureoftheprotein.
Becauseaprotein’sstructureisrelatedtoitsfunction,silentmutationsdonotaffectthefunctionofthe
protein.
13.Usingtheinformationprovidedintheintroductionunder“MC1RGene,”explainhowthemutantMC1Rprotein
directlyaffectsarockpocketmouse’scoatcolor.
TheaminoacidchangesintheMC1Rproteinmaychangethestructureandfunctionoftheprotein.Thisleads
toincreasedproductionofeumelanin,whichresultsinthedarkcolor.
14.Mutationsareasourceofgeneticvariation.Inthefilm,Dr.Carrollsaysthatmutationsoccurrandomly.What
doesthatmean?
Sampleanswer:Itmeansthatmutationsdonotoccurforapurposeorforanypredeterminedresult.
15.Itisacommonmisconceptionthat“allmutationsarebad.”Usetheexampleofrockpocketmicetoexplain
whythisisnottrue.Inyouranswer,explainhowthedarkcoatcolormutationcanbeanadvantagetosomemice
andadisadvantagetoothers.
Sampleanswer:Mutationscanresultinnewtraits.Theselectiveadvantageprovidedbyatraitdependsonthe
environment.Forexample,onalightsubstrate,individualswithdark-coloredcoatswouldbeatadisadvantage
www.BioInteractive.org
PublishedFebruary,2017
Page4of8 Lesson
The
Making of the Fittest:
The Making of the Fittest
Natural
Selectionand
andAdaptation
Adaptation
Natural Selection
LESSON
Educator
Materials
TEACHERMATERIALS
becausetheywouldstandoutmorethanindividualswithlight-coloredcoats,makingthemeasierforpredators
tospot.However,inthedarklavaflowhabitat,thosesamedark-coloredindividualswouldhaveaselective
advantagebecausetheywouldbebettercamouflagedthanlight-coloredindividuals.So,thestatementthat“all
mutationsarebad”isincorrect,becausetherearedifferentselectivepressuresonthetraitsproducedby
mutationsdependingonthehabitat.Therearealsosilentmutationsthatdonotchangetheresultingprotein;
theseareneutral,neithergoodnorbad.
16.Useyourunderstandingofevolutionandtheinformationinthefilmtoexplainhowthedark-coloredmutation
cametobesocommoninsomepopulationsofrockpocketmice.Bespecific.
Sampleanswer:Thedark-coloredmousehasaselectiveadvantageinahabitatsuchasthePinacatelavaflow,
whichhasadark-coloredsubstrate.Sincerockpocketmicereproducequicklyandoften,thefrequencyofthis
favoredtraitwouldspreadrapidlythroughthepopulation.Anylight-coloredmiceinthedark-coloredhabitat
wouldbeataselectivedisadvantage,thusdecreasingtheirgenefrequencyinfuturegenerations.Inthisway,
favorabletraitsaccumulateandincreaseinfrequency—justasDarwinexplained.
ANSWERKEYACTIVITY2
GENETABLES
024
GAG
CUC
His
Leu
GTG
CAC
024
GAG
CUC
His
Leu
WILD-TYPEMC1RGENE(LIGHT-COLOREDCOATPHENOTYPE) 105
DNA
CGG
GAC
CGG
TGG
GCC
CAC
TGA
CAC
mRNA
GCC
CUG
GCC
ACC
CGG
GUG
ACU
GUG
CAT
GUA
114
GTC
CAG
Amino
Acid
Val
Gln
CAT
GUA
114
GTC
CAG
Val
Gln
015
TTG
AAC
Asn
MUTANTMC1RGENE(DARK-COLOREDCOATPHENOTYPE)
AGG
TGG
ACG
TGT
CCG
CAA
GGA
UCC
ACC
UGC
ACA
GGC
GUU
CCU
Ser
Thr
Cys
Thr
Gly
Val
Pro
GTG
CAC
DNA
mRNA
Amino
Acid
WILD-TYPEMC1RGENE(LIGHT-COLOREDCOATPHENOTYPE) 015
DNA
TTG
AGG
TGG
GCG
TGT
CCG
CAA
GGA
mRNA
AAC
UCC
ACC
CGC
ACA
GGC
GUU
CCU
Amino
Asn
Ser
Thr
Arg
Thr
Gly
Val
Pro
Acid
EXTRACELLULARDOMAINI
DNA
mRNA
Amino
Acid
105
CGG
GCC
Ala
www.BioInteractive.org
Leu
Ala
Thr
Arg
Val
Thr
Val
MUTANTMC1RGENE(DARK-COLOREDCOATPHENOTYPE)
GAC
CGG
TGG
ACC
CAC
TGA
CAC
CUG
GCC
ACC
UGG
GUG
ACU
GUG
Leu
Ala
Thr
Trp
Val
Thr
Val
PublishedFebruary,2017
Page5of8 Ala
EXTRACELLULARDOMAINIII
Lesson
The
Making of the Fittest:
The Making of the Fittest
Natural
Selectionand
andAdaptation
Adaptation
Natural Selection
LESSON
Educator
Materials
TEACHERMATERIALS
DNA
mRNA
Amino
Acid
163
ACC
UGG
Arg
Trp
MUTANTMC1RGENE(DARK-COLOREDCOATPHENOTYPE)
154
TCA
TAA
CAC
TGT
GAC
GGG
ACC
CGA
AGU
AUU
GUG
ACA
CUG
CCC
UGG
GCU
GCC
CGG
163
ACC
UGG
Ser
Arg
Trp
Val
Thr
Leu
Pro
Trp
Ala
GCC
CGG
Ile
WILD-TYPEMC1RGENE(LIGHT-COLOREDCOATPHENOTYPE) 154
DNA
TCA
TAA
CAC
TGT
GAC
GGG
GCC
CGA
mRNA
AGU
AUU
GUG
ACA
CUG
CCC
CGG
GCU
Amino
Ser
Ile
Val
Thr
Leu
Pro
Arg
Ala
Acid
INTRACELLULARDOMAINI
230
GAA
CUU
Leu
www.BioInteractive.org
MUTANTMC1RGENE(DARK-COLOREDCOATPHENOTYPE)
CAG
GTG
GTG
CCA
AAG
GCT
GAG
GUC
CAC
CAC
GGU
UUC
CGA
CUC
Val
His
His
Gly
Phe
Arg
Leu
TTT
AAA
239
CCG
GGC
Lys
Gly
TTT
AAA
239
CCG
GGC
Lys
Gly
PublishedFebruary,2017
Page6of8 WILD-TYPEMC1RGENE(LIGHT-COLOREDCOATPHENOTYPE) 230
DNA
GAA
CAG
GTG
GTT
CCA
AAG
GCT
GAG
mRNA
CUU
GUC
CAC
CAA
GGU
UUC
CGA
CUC
Amino
Leu
Val
His
Gln
Gly
Phe
Arg
Leu
Acid
DNA
mRNA
Amino
Acid
MUTANTMC1RGENE(DARK-COLOREDCOAT
PHENOTYPE)
208
212
DNA
CAC
GTG
TAC
GAG
CGT
mRNA GUG
CAC
AUG
CUC
GCA
Amino
Val
His
Met
Leu
Ala
Acid
INTRACELLULARDOMAINIII
WILD-TYPEMC1RGENE(LIGHT-COLOREDCOATPHENOTYPE)
208
212
DNA
CAC
GTG
TAC
GAA
CGT
mRNA
GUG
CAC
AUG
CUU
GCA
Amino
Val
His
Met
Leu
Ala
Acid
TRANSMEMBRANEV
Lesson
The
Making of the Fittest:
The Making of the Fittest
Natural
Selectionand
andAdaptation
Adaptation
Natural Selection
LESSON
Educator
Materials
TEACHERMATERIALS
QUESTIONSANSWERKEY
1.Usingtheaminoacidlocationnumbersprovidedabovethefirstandlastcolumnofeachtable,listthelocations
ofthefiveaminoacidsthatcontainamutation.
Theaminoacidlocationsare018,109,160,211,and233.
2.OfthefivemutationsyouidentifiedintheMc1rgene,howmanyarethefollowing:
5substitutions,0insertions,0deletions
3.OfthefivemutationsyouidentifiedintheMc1rgene,howmanyarethefollowing:
1silent,4missense,0nonsense
4. a.Whichfouraminoacidlocations(seeQuestion1above)containthemissensemutations?
Theaminoacidsare018,109,160,and233.
b.ExplainthelinkbetweenDNAsequenceandproteinstructureandfunction.
StudentsmaysimplyrelateDNAsequencetoaminoacidsequence,andaminoacidsequencetothethreedimensionalshapeoftheprotein.More-advancedstudentsshouldbeabletolinkthemutationtoachangein
theprotein’sprimarystructure,whichaffectsotherlevelsofstructure(secondaryandtertiary).Allstudent
responsesshoulddemonstrateanunderstandingofthelinkbetweenDNAandthesequenceofaminoacids
thatdeterminesthestructure,andthereforefunction,ofaprotein.
5.Usingtheinformationonmutationsintheintroductionandyourknowledgeofproteins,developahypothesisto
explainhowthechangesintheMC1Rprotein’saminoacidsequencemightaffectitsfunction.
StudentsmightsuggestthatsincethefourmissensemutationsintheMc1rgenechangetheaminoacid
sequenceoftheMC1Rprotein,theproteinwillnotfunctionproperly,asaprotein’sfunctionisdeterminedby
itsstructure.
6.Manyproteins,includingMC1R,containseveralstructuraldomainsthatcanfoldandfunctionindependently
fromtherest.ThedomainnameswereprovidedforeachportionofDNAsequenceyoutranslatedearlier.Answer
thefollowingquestions.
a.WhereistheMC1Rproteinfound,andwhatisitsfunction?Bespecific.
Itisareceptorproteinembeddedinthemembraneofmelanocytes.Itisspecializedforpigmentproduction.
b.WhichproteindomainscontainthefourMc1rmissensemutations?(Refertothegenetablesyoucompleted
earlier.)
Themutationat018isinExtracellularDomainI,themutationat109isinExtracellularDomainIII,themutation
at160isinIntracellularDomainI,andthemutationat233isinIntracellularDomainIII.
c.Define“extracellular.”
Extracellularmeans“somethingoutsideofacell.”
d.Define“intracellular.”
Intracellularmeans“somethinginsideofacell.”
e.Whyisitsignificantthatthefourmissensemutationsarefoundintheextracellularandintracellulardomainsof
theprotein?Explainyouranswer.(Hint:ThinkaboutMC1R’sfunction.)
Specificanswerswillvary,butstudentsshouldhavetheideathataproteinthatspansacellmembranehasa
portionthatprojectsoutofthecell(extracellular)andaportionthatprojectsintothecell(intracellular).This
typeofreceptorproteinusuallyfunctionsineithercelltransportorcellsignaling.Changesinthestructureof
extracellularandintracellularportionscanchangethefunctionoftheproteininthesignalingpathwayorthe
transportmechanism.(Note:Seethelesson“TheBiochemistryandCellSignalingPathwayoftheMc1rGene”
formoredetailonthisconcept.)
www.BioInteractive.org
PublishedFebruary,2017
Page7of8 Lesson
The
Making of the Fittest:
The Making of the Fittest
Natural
Selectionand
andAdaptation
Adaptation
Natural Selection
LESSON
Educator
Materials
TEACHERMATERIALS
7.UsingtheinformationontheMc1rgeneintheintroductionandyourknowledgeofproteins,developa
hypothesistoexplainhowthechangeinMC1Rproteinfunctionmightdirectlyaffectarockpocketmouse’scoat
color.Bespecificandconsiderboththelight-coloredanddark-coloredphenotypes.
Specificanswerswillvary,butstudentsshouldsuggestthatthenormalMC1Rreceptorproteinwillproduce
relativeamountsofeumelaninandpheomelaninthatwillresultinthelightcoatcolor.Inaddition,thedarkcoloredmousepopulationcontainsthemutantMc1rgene,whichresultsinadifferentreceptorprotein.This
changeinstructuremightleadtoincreasedproductionofeumelanin,whichresultsinthedarkcolor.
8.Explainwhythemutationataminoacidlocation211isnotassignificantastheotherfourmutations.
Itisasilentmutation,sotheaminoacidinthatpositiondoesnotchange,nordoesthestructureofthespecific
domain.Thisisimportantbecauseaprotein’sstructurerelatestoitsfunction.Nochangeinthestructure
suggeststhatthereisnochangeinthefunctionofthisparticulardomainoftheprotein.
9.Mutationsareasourceofgeneticvariation.Inthefilm,Dr.SeanCarrollsaysthatmutationsoccurrandomly.
Whatdoesthismean?
Sampleanswer:“Itmeansthatmutationsdonotoccurforapurposeorforanypredeterminedresult.”
10.Itisacommonmisconceptionthat“allmutationsarebad.”Usetheexampleofrockpocketmicetoexplain
whythisstatementisnottrue.Inyouranswer,explainhowthedarkcoat-colormutationcanbeanadvantageto
somemiceandadisadvantagetoothers.
Sampleanswer:“Mutationscanresultinnewtraits.Theselectiveadvantageprovidedbyatraitdependsonthe
environment.Forexample,onalightsubstrate,individualswithdark-coloredcoatswouldbeatadisadvantage
becausetheywouldstandoutmorethanindividualswithlight-coloredcoats,makingthemeasierforpredators
tospot.However,inthedarklavaflowhabitat,thosesamedark-coloredindividualswouldhaveaselective
advantagebecausetheywouldbebettercamouflagedthanlight-coloredindividuals.Sothestatementthat“all
mutationsarebad”isincorrect,becausetherearedifferentselectivepressuresonthetraitsproducedby
mutationsdependingonthehabitat.Therearealsosilentmutationsthatdonotchangetheresultingprotein;
theseareneutral,neithergoodnorbad.”
11.Useyourunderstandingofevolutionandtheinformationinthefilmtoexplainhowthedark-coloredmutation
cametobesocommoninsomepopulationsofrockpocketmice.Bespecific.
Sampleanswer:“Thedark-coloredmousehasaselectiveadvantageinahabitatsuchasthePinacatelavaflow,
whichhasadark-coloredsubstrate.Sincerockpocketmicereproducequicklyandoften,thefrequencyofthis
favoredtraitwouldspreadrapidlythroughthepopulation.Anylight-coloredmiceinthedark-coloredhabitat
wouldbeataselectivedisadvantage,thusdecreasingtheirgenefrequencyinfuturegenerations.Inthisway,
favorabletraitsaccumulateandincreaseinfrequency—justasCharlesDarwinexplained.”
AUTHOR
AnnBrokaw
APBiologyTeacher
RockyRiverHighSchool
RockyRiver,Ohio
www.BioInteractive.org
PublishedFebruary,2017
Page8of8