武漢大學(xué)2013至2014學(xué)年第一學(xué)期分子生物學(xué)期末考試試題

武漢大學(xué)2013至2014學(xué)年第一學(xué)期分子生物學(xué)期末考試試題《分子生物學(xué)》試卷FinalexamofMolecularBiologyCourse(Spring2014)年級(jí)______

專業(yè)________

學(xué)號(hào)_________

姓名_______成績(jī)______PARTI:

DESCRIPTION(2pointseach)Youranswershoulddescribewhateachitemisandhowitfunctionsinthecell.Diagrams,structureandsequenceinformationshouldbeincludedinyouranswer,asnecessary.1.

Yeastartificialchromosome2.

RNAinterference3.

Proteomics4.

Shine-Dalgarnosequence5.

Alternativesplicing6.

Ribozyme7.

r-dependenttermination8.

RNAediting9.

DNAlesions10.

ProteintargetingPARTII:MULTIPLECHOICES

(1pointseach)Selectthe

onebestanswer

foreachquestion.1.Thecatalyticactivityforpeptidebondformation(thepeptidyltransferaseactivity)islocatedinthe:1)

RNAofthelargeribosomalsubunit.2)

leadersequenceofthemessengerRNA.3)

RNAofthesmallribosomalsubunit.4)

proteinsofthesmallribosomalsubunit.5)

proteinsofthelargeribosomalsubunit.2.Bidirectionalandsemi-conservativearetwotermsthatreferto:1)

transcription.2)

translation.3)

replication.4)

alloftheabove.5)

noneoftheabove.3.Thefactthatmostaminoacidsarespecifiedbymultiplecodonsisknownas:1)

the“wobble”phenomenon.2)

theuniversalityofthegeneticcode.3)

codonbias.4)

theanticodonhypothesis.5)

theredundancyofthegeneticcode.4.RNApolymeraseIistheeukaryoticenzymeresponsiblefor:1)

transcriptionofribosomalRNA.2)

transcriptionoftransferRNAandothersmallRNAspecies.3)

transcriptionofmessengerRNA.4)

initiationofOkazakifragmentsynthesisinDNAreplication.5.RestrictionenzymescancleaveDNAthatiseithersingle-strandedordouble-stranded,aslongasitcontainstheappropriaterecognitionsite.1)True

2)False6.Informationaboutthesequenceofthecodingregionofageneisbestobtainedfrom:1)

aYACclone.2)

agenomicclone.3)

acDNAclone.4)

theprotein.7.Achromatographymethodthatcanbeusedspecificallytopurifyproteinsbasedontheirchargeis:1)

gelfiltrationchromatography.2)

ion-exchangechromatography.3)

DNAaffinitychromatography.4)

antibodyaffinitychromatography.8.AnonsensemutationisachangeintheDNAsequencethatresultsin:1)

asmalldeletionorinsertion.2)

anaminoacidchangeintheproteinencodedbythegene.3)

aprematurestopcodon.4)

alloftheabove.5)

noneoftheabove.9.Aproteincomplexinvolvedindegradationofproteinswithinthecellisknownasthe:1)

ubiquitin/proteasomesystem.2)

molecularchaperone.3)

chaperonin.4)

ribosome.5)

Krebs/TCAcycle.10.___bindstotherepressorandturnonthetranscriptionofthestructuralgenesintheLacoperon.1)

cAMP2)

lactose3)

allolactose4)

CRP11.WhichofthefollowingRNAspeciesisinvolvedindegradationofthemRNAcontainingcomplementarysequence1)

miRNA2)

siRNA3)

tRNA4)

5SRNA5)

U3snRNA12.Thegenomesequencingprojectsareconfirmingthetheorythatgenomesizeisdirectlyproportionaltothenumberofgenescontainedwithinthatgenome.Inotherwords,agenomethatis10timesasbigwillcontainsapproximately10timesasmanyproteincodinggenes.1)True

2)False13.HeLacells,derivedfromahumancervicalcarcinoma,areabletopropagateindefinitelyincultureandarethereforeknownasa(n):1)

tissueculture.2)

tumor.3)

transgeniccellline.4)

immortalizedcellline.14.

E.coli

cellsaresmallerthanyeastcells.1)True

2)False15.WhichofthefollowingdomainsisnotaDNAbindingdomain1)

Proline-richdomains2)

Helix-turnhelixdomains3)

Zincfingerdomains4)

Basicdomains16.Theaminoacyl-tRNAsynthetasesdistinguishbetweenabout40differentshapedtRNAmoleculesinthecells.

1)True

2)FalsePARTIII:SHORTQUESTIONS

(8pointseach)1.

Howdobacterialreplicationstartandaccomplished.Remembertoincludetheproteins/enzymesandimportantDNAsequenceinvolvedinthisprocess.2.

Designexperimentsto

clone

ayeastgeneand

express

thisgeneinyeast.3.

Belowisthemultiplecloningsite(MCS)oftheplasmidvectorpUC18andtheN-terminalandC-terminalsequenceofproteinX.NotethattheMCSconstitutesapartoftheLacZopenreadingframe.SupposethatyouaregoingtoclonetheproteinXgeneintopUC18,sothatyourtargetgeneistranscribedunderthecontrolof

LacZ

promoter,andtranslatedwiththe

LacZ

genetoproduce

afusionprotein.Youarerequestedtouse

BamHIand

PstItotheclone

X

gene,

pleaseaddtheserestrictionsitesonthecorrespondingpositionofthe

Xgene.

Remembertomaintainthereadingframeofthe

X

genewiththe

LacZgene4.

(1)MCSofpUC18EcoRI

SacI

KpnI

SmaI

BamHI

XbaI

SalI

PstIACGAATTCGAGCTCGGTACCCGGGGATCCTCTA

GAGTCG

ACCTGCAGGCATGCAThr

Asn

Ser

Ser

Ser

Val

Pro

Gly

Asp

Pro

Leu

Glu

Ser

Thr

Cys

Arg

His

Ala

(2)N-terminalsequenceof

X

gene.

ATG

ACCCCUCAUAACGGCGAC…

Met

Thr

Pro

His

Asn

Gly

Asp…(3)C-terminalsequenceof

X

gene.…GAUAGUACAGCUGCCAAGTAA

…

Asp

Ser

Thr

Ala

Ala

LysPARTIV:MAJORQUESTIONS

(20

pointseach)1:

Pleasedescribehow

an

mRNAgeneistranscribed,processedandtranslatedinhumancells.Whatarethepossiblemechanismsinregulatingtheexpressionofthisgene?

2(20points):Abacteriumisfoundtometabolizeararesugarproducedbyaplantthatthebacteriagrowon.However,thebacteriapreferglucoseastheenergysource.Theproblemis,ifyouwanttofinishthiscoursewithasatisfiedscore,youmustfigureouttheregulatorymechanismthatthebacteriausedtodeterminethesugarchoice.Thegeneinvolvingintheraresugarmetabolismhasbeenidentifiedas

fun3.Youcanusenorthernblottoanalyzetheexpressionof

fun3

anduseDNAfootprintingtoanalyzethebindingofproteinstothecontrolelementsof

fun3

gene.ThefollowingtableshowstheexperimentalresultsGlucosepresenceRaresugarpresenceLevelsof

fun3RNABindingofproteins

---ProteinAbindstothepromoterregionProteinCbindsupstreamofthepromoter+--ProteinAbindstothepromoterregion-++++ProteinBbindstothepromoterregionProteinCbindsupstreamofthepromoter+++ProteinBbindstothepromoterregionQuestions:1.

Pleaseproposeamechanismtoexplaintheaboveresults.Youshouldfocusonthequestion“Howdoestheexpressionof

fun3

geneistightlyregulatedsothatitisonlyhighlyexpressedwhentheraresugaristheonlycarbonsource”.YoumustanswerwhatproteinsA,BandCare.(8points)2.

HowisproteinAregulated?(2points)(1)

glucoseturnstherepressoron(2)

glucoseturnstherepressoroff(3)

theraresugarturnstherepressoron(4)

theraresugarturnstherepressoroff3.

HowisproteinCregulated?(2points)(1)

glucoseturnstheactivatoron(2)

glucoseturnstheactivatoroff(3)

theraresugarturnstheactivatoron(4)

theraresugarturnstheactivatoroff4.

Howcouldyoumakethebacteriaalwaysusetheraresugarastheenergysourceeveninthepresenceofglucose?(8points)武漢大學(xué)生命科學(xué)學(xué)院2013－2014學(xué)年第一學(xué)期期末考試《分子生物學(xué)》試卷及參考答案FinalexamofMolecularBiologyCourse(Spring2014)寫在參考答案前面的話：?

該課程考試目的是考查學(xué)生對(duì)所學(xué)知識(shí)掌握的情況，除選擇題外，其他題目的答案基本都不是唯一的。你可以從不同的角度去闡明一個(gè)概念。?

公布參考答案的目的：為該課程畫個(gè)句號(hào)，讓學(xué)生過個(gè)安心暑假?

BestwishestoallofyouPARTI:

DESCRIPTION(2pointseach)Youranswershoulddescribewhateachitemisandhowitfunctionsinthecell.Diagrams,structureandsequenceinformationshouldbeincludedinyouranswer,asnecessary.1.

Yeastartificialchromosome:①

酵母人工染色體（0.5point）②

containscomponentsrequiredforreplicationandsegregationofthenaturalyeastchromosome,includingtwotelomericsequences(TEL),onecentromere(CEN)andoneautonomouslyreplicatingsequences(ARS)(0.75point)③

containsgenesactasselectivemarkersinyeastandproperrestrictionsites(0.75point)④

canaccommodategenomicDNAfragmentsofmorethan1Mb(0.5point)2.

RNAinterference:①

RNA干擾

（0.5point）②

aconservedbiologicalresponsetodouble-strandedRNA(1point)③

regulatestheexpressionofprotein-codinggenesthroughsiRNAormiRNA(1point)④

thedsRNAisrestrictedbyDICER,thenRISCmediatesthesiRNAandmiRNArelatedRNAdegradationandtranslationinhibition,respectively.(1point)3.

Proteomics蛋白組學(xué)

（0.5point）②

Thestudyoftheproteomeusingtechniquesofhighresolutionproteinseparationandidentification

(1.5point)④

Thebestseparationmethodistwodimensionalgelelectrophoresis,theindividualproteinspotsarethencutfromthegelandtreatedwithproteasetoproduceasetofpeptidescharacteristicofthatprotein.TheprecisemassesofeachpeptideinthesamplearethendeterminedbyMALDImassspectrometry.Theresultedpeptidemassfingerprintofthatproteinisthencomparedtoadatabasetodeducethefunctionofthatproteinetc.(1.5point)4.

Shine-Dalgarnosequence①

SD

序列

（0.5point）②

Aconservedsequence8-13ntupstreamofthefirstcodontobetranslated(1point).③

ThissequencewasdiscoveredbyShineandDalgarno(0.5point)④

Thesequenceispurine-richandcontainsallorpartof5’-AGGAGGU-3’(0.5point)⑤

Canbase-pairwiththe3’-endofthe16SrRNA(0.5point)5.

Alternativesplicing①

可變剪接

（0.5point）②

ThegenerationofdifferentmaturemRNAsfromaparticulartypeofgenetranscriptbychoosingdifferent5’-and3’-splicesites(2point).6.

Ribozyme①

核酶

（0.5point）②

anRNAmoleculecapableofcatalyzingachemicalreaction(2point).7.

r-dependenttermination①

r-依賴型轉(zhuǎn)錄終止

（0.5point）②

During

bacterialtranscription,someterminatorsitesdonotformstronghairpins,thus

terminationofthetranscriptionbybacterialRNApolymerase

requirestheassistanceofanaccessoryfactorcalled

rho(r)protein

(2point).8.

RNAediting①

RNA編輯

（0.5point）②

Aformof

RNAprocessing

inwhich

nucleotidesequenceoftheprimarytranscript

isaltered

byeitherchanging,insertingordeletingresiduesatthe

specificpoints

alongthemolecule(2point).9.

DNAlesions①

DNA損傷

（0.5point）②

AnalterationtothenormalchemicalorphysicalstructureoftheDNA(2point).③

ThelesionscanleadtocelldeathorDNAmutation(1point).10.

Proteintargeting①

蛋白定位

（0.5point）②

Synthesisofeukaryoticproteinsisusuallyoccurredincytoplasm(0.5point).③

However,manyproteinsneedtobetransportedtospecificcellularlocations,suchasnucleus,mitochondrionorchloroplast,toexerttheirbiologicalfunctions.Thisprocessiscalledproteintargeting

(1point).④

Theultimatecellularlocationofproteinsisoftendeterminedbyspecific,relativeshort,aminoacidsequenceswithintheproteinsthemselves.Thesequenceinsideofaproteindeterminingthecellularlocationoftheproteiniscalledsignalsequence(1point).PARTII:MULTIPLECHOICES

(1pointseach)Selectthe

onebestanswer

foreachquestion.1.Thecatalyticactivityforpeptidebondformation(thepeptidyltransferaseactivity)islocatedinthe:1)

RNAofthelargeribosomalsubunit.2)

leadersequenceofthemessengerRNA.3)

RNAofthesmallribosomalsubunit.4)

proteinsofthesmallribosomalsubunit.5)

proteinsofthelargeribosomalsubunit.2.Bidirectionalandsemi-conservativearetwotermsthatreferto:1)

transcription.2)

translation.3)

replication.4)

alloftheabove.5)

noneoftheabove.3.Thefactthatmostaminoacidsarespecifiedbymultiplecodonsisknownas:1)

the“wobble”phenomenon.2)

theuniversalityofthegeneticcode.3)

codonbias.4)

theanticodonhypothesis.5)

theredundancyofthegeneticcode.4.RNApolymeraseIistheeukaryoticenzymeresponsiblefor:1)

transcriptionofribosomalRNA.2)

transcriptionoftransferRNAandothersmallRNAspecies.3)

transcriptionofmessengerRNA.4)

initiationofOkazakifragmentsynthesisinDNAreplication.5.RestrictionenzymescancleaveDNAthatiseithersingle-strandedordouble-stranded,aslongasitcontainstheappropriaterecognitionsite.1)True

2)False6.Informationaboutthesequenceofthecodingregionofageneisbestobtainedfrom:1)

aYACclone.2)

agenomicclone.3)

acDNAclone.4)

theprotein.7.Achromatographymethodthatcanbeusedspecificallytopurifyproteinsbasedontheirchargeis:1)

gelfiltrationchromatography.2)

ion-exchangechromatography.3)

DNAaffinitychromatography.4)

antibodyaffinitychromatography.8.AnonsensemutationisachangeintheDNAsequencethatresultsin:1)

asmalldeletionorinsertion.2)

anaminoacidchangeintheproteinencodedbythegene.3)

aprematurestopcodon.4)

alloftheabove.5)

noneoftheabove.9.Aproteincomplexinvolvedindegradationofproteinswithinthecellisknownasthe:1)

ubiquitin/proteasomesystem.2)

molecularchaperone.3)

chaperonin.4)

ribosome.5)

Krebs/TCAcycle.10.___bindstotherepressorandturnonthetranscriptionofthestructuralgenesintheLacoperon.1)

cAMP2)

lactose3)

allolactose4)

CRP11.WhichofthefollowingRNAspeciesisinvolvedindegradationofthemRNAcontainingcomplementarysequence1)

miRNA2)

siRNA3)

tRNA4)

5SRNA5)

U3snRNA12.Thegenomesequencingprojectsareconfirmingthetheorythatgenomesizeisdirectlyproportionaltothenumberofgenescontainedwithinthatgenome.Inotherwords,agenomethatis10timesasbigwillcontainsapproximately10timesasmanyproteincodinggenes.1)True

2)False13.HeLacells,derivedfromahumancervicalcarcinoma,areabletopropagateindefinitelyincultureandarethereforeknownasa(n):1)

tissueculture.2)

tumor.3)

transgeniccellline.4)

immortalizedcellline.14.

E.coli

cellsaresmallerthanyeastcells.1)True

2)False15.WhichofthefollowingdomainsisnotaDNAbindingdomain1)

Proline-richdomains2)

Helix-turnhelixdomains3)

Zincfingerdomains4)

Basicdomains16.Theaminoacyl-tRNAsynthetasesdistinguishbetweenabout40differentshapedtRNAmoleculesinthecells.

1)True

2)FalsePARTIII:SHORTQUESTIONS

(8pointseach)1.

Howdobacterialreplicationstartandaccomplished.Remembertoincludetheproteins/enzymesandimportantDNAsequenceinvolvedinthisprocess.Initiation(3points):①

replicationofthebacterialchromosomeistightlycoupledtothegrowthcycle.②

The

E.coli

originiswithinthegeneticlocus

oriC

thatcontainsfour9bpbindingsitesfortheinitiatorproteinDnaA.③

SynthesisofDnaAiscoupledtogrowthratesothatthereplicationofthebacterialchromosomeiscoupledtothegrowthcycle.④

OncethecellularlevelofDnaAreachesacriticallevel,DnaAproteinformsacomplexof30-40moleculesatthe

oriC

DNA,whichfacilitatesmeltingofthree13bpAT-richrepeatsequencestoallowbindingofDnaBprotein.⑤

DnaBprotein

isaDNAhelicasethatutilizestheenergyofATPhydrolysistomeltdsDNA.⑥

ThessDNAcreatedbyDnaBiscoatedwithsingle-strandedbindingprotein(Ssb)toprotectitfrombreakageandtopreventtheDNArenaturing.⑦

TheDNAprimase

thenattachestotheDNAandsynthesizesashortRNAprimer

toinitiatesynthesisoftheleadingstrandofthefirstreplicationfork.Unwinding(1point)①

Forreplicationtoproceedawayfromtheorigin,DNAhelicasesmusttravelalongthetemplatestrandstoopenthedoublehelixforcopying,whichisaccomplishedbythejointeffortsofDnaBandSsb.②

UnwindingcausespositivesupercoilingoftheunwoundDNA;thepositivesupercoilingisrelaxedcontinuouslybytheintroductionoffurthernegativesupercoilsbytypeIItopoisomerasecalledDNAgyrase.Elongation(3points)①

Initiationofthereplicationofthelaggingstrand.

Asthenewlyformedreplicationforkdisplacestheparentallaggingstrand,amobilecomplexcalleda

primosome,whichincludestheDnaBhelicaseandDNAprimase,synthesizesRNAprimersevery1000-2000ntonthelaggingstrand.②

BothleadingandlaggingstrandprimersareelongatedbyDNApolymeraseIIIholoenzyme.

Thismultisubunitcomplexisadimmer,onehalfsynthesizingtheleadingstrandandtheotherthelaggingstrand.③

Laggingstrandsynthesis.

DNApolymeraseIIIholoenzyme:

a

subunit-theactualpolymerase,

e-a3’→5’proofreadingexonuclease.DNApolymeraseIremovesthelaggingstrandprimersandfillsintheresultedgaps.DNAligasemakesthefinalphosphodiesterbondbetweenfragments.Terminationandsegregation(1points)①

Tworeplicationforksmeetattheterminatorsites(terminus)②

tus

geneproductbindstotheterminusandactsasaninhibitorofDnaBhelicase.③

Whenreplicationiscompleted,thetwointerlinkeddaughtercirclesareunlinkedbytopoisomeraseIV.2.

Designexperimentsto

clone

ayeastgeneand

express

thisgeneinyeast.Clone

(4points):①

PCRamplificationofthegeneofinterest,includingtwoproperrestrictionsitesattheendsofthePCRamplifiedDNA②

Chooseacloningvector:

itcould

bearegularcloningvectorthatiscapableofpropagatingin

E.coli,easytoextractfrom

E.coli

andmanipulatedintesttubes.

Itcouldalso

beashuttleandexpressionvectorthatalsocontainstheyeast2m

origintoallowtheplasmidreplicationinyeast,theselectivemarkertoallowtheplasmidcontainingcellsgrow,thepromotertoallowthegeneofinteresttobetranscribed,apoly(A)siteforadditionofpoly(A)tailonthetranscript.Nomatterwhattypeofthevectorischosen,plasmidshallbepreparedfromtheE.coli

cells.③

Restrictiondigestionofbothplasmidandthegeneofinterestwiththesamerestrictionsites.④

Ligationandtransformation⑤

Selectionandidentificationofthetransformantstoobtaintheclonecontainingtherightrecombinantplasmid.Expression

(4points):⑥

Ifashuttleandexpressionvectorischosenasdescribedinstep②,theexpressionprocesswillinclude

1preparationoftherecombinantDNAfrom

E.coli

cells,

2transformationofyeastcellswiththerecombinantDNA,

3selectionofthetransformants,

4growthofthetransformants,

5expressionofthegenebyinductionofthetranscriptionfromthepromoterand

6detectionoftheexpressedRNAand/orprotein.Inductionisnotrequiredifaconstitutivepromoterisused.(4points)⑦

Ifaregularcloningvectorischosenasdescribedinstep②,asubcloneneedtobeobtainedtoallowthegeneofinterestbeingclonedintoashuttleandexpressionvectorsameasdescribedinstep②.Thenexpressionisperformedthesameasdescribedin

⑥.3.

Belowisthemultiplecloningsite(MCS)oftheplasmidvectorpUC18andtheN-terminalandC-terminalsequenceofproteinX.NotethattheMCSconstitutesapartoftheLacZopenreadingframe.SupposethatyouaregoingtoclonetheproteinXgeneintopUC18,sothatyourtargetgeneistranscribedunderthecontrolof

LacZ

promoter,andtranslatedwiththe

LacZ

genetoproduce

afusionprotein.Youarerequestedtouse

BamHIand

PstItotheclone

X

gene,

pleaseaddtheserestrictionsitesonthecorrespondingpositionofthe

Xgene.

Remembertomaintainthereadingframeofthe

X

genewiththe

LacZgene(1)MCSofpUC18EcoRI

SacI

KpnI

SmaI

BamHI

XbaI

SalI

PstIACGAATTCGAGCTCGGTACCCGGGGATCCTCTA

GAGTCG

ACCTGCAGGCATGCAThr

Asn

Ser

Ser

Ser

Val

Pro

Gly

Asp

Pro

Leu

Glu

Ser

Thr

Cys

Arg

His

Ala

(2)N-terminalsequenceof

X

gene.

ATG

ACCCCUCAUAACGGCGAC…

Met

Thr

Pro

His

Asn

Gly

Asp…(3)C-terminalsequenceof

X

gene.…GAUAGUACAGCUGCCAAGTAA

…

Asp

Ser

Thr

Ala

Ala

LysANSWER:

GGATCC

N

ATG

ACCCCUCAUAACGGCGAC(N-terminus)

GAUAGUACAGCUGCCAAGTAACTGCAG

(C-terminus)PARTIV:MAJORQUESTIONS

(20

pointseach)1:

Pleasedescribehow

an

mRNAgeneistranscribed,processedandtranslatedinhumancells.Whatarethepossiblemechanismsinregulatingtheexpressionofthisgene?①

Transcription:

focusingontranscriptioninitiationdescribedonp222ofthetextbook.(5points)②

Processing:

5’capping(additionofam7Gtothe5’endoftheRNApolIItranscriptcatalyzedbymRNAguanyltransferase/cappingenzyme)(1.5point),

intronsplicing

(removalofintronbysplicesomethatcontains5snRNAsandmanyproteins.Firststep-cleavageat5’splicesiteandtheconservedAbeinglinkedtothe5’-endoftheintron,secondstep-3’cleavageandexonligation)(2points),

3’cleavageandpolyadenylation

(First-assemblyoftherecognitioncomplexonthepolyadenylationsite,second-poly(A)polymerase/PAPcleavesthetranscriptatthissite,third-PAPaddsupto250Aresiduestothe3’endofthetranscript)(1.5point).③

Translation:Initiation,elongationandtermination(p276,278)(5points)④

Regulation:Firstly,regulationcanoccuratthetranscriptionlevel,forexample,throughinteractionofthetranscriptionfactorswiththepromoterorUREtoactivateorrepressthetranscription(2.5point).Secondly,regulationcanoccuratthepost-transcriptionlevel,forexample,alternativetranscriptionoralternativepoly(A)site(2.5points).2(20points):Abacteriumisfoundtometabolizeararesugarproducedbyaplantthatthebacteriagrowon.However,thebacteriapreferglucoseastheenergysource.Theproblemis,ifyouwanttofinishthiscoursewithasatisfiedscore,youmustfigureouttheregulatorym