CHAPTER6AnatomyAndFunctionOfAGene:DissectionThroughMutationMutation:heritablechangesinbasesequencethataffectphenotye.Forwardmutation:changesthewildtypealleleofagenetoadifferentallele.Reversemutation:causesanovelmutantalleletorevertbacktowildtype.

Concepts:

Examplesofmutation1

Four-wingedfruitfly

Examplesofmutation2XerodermaPigmentosum(XP)canresultfromatleast9genesXPA,XPB,XPC,XPD,XPF,XPGrequiredforexcisionrepairHumanrequireatleast17polypeptides

1.MutationclassificationPointmutation(a)frame-shiftmutation(b,c,d,e)

I.MUTATION(1)Mutationsaffectingphenotype

occurveryrarely.

(2)Differentgenesmutateat

differentrates.

(3)Therateofforwardmutationis

higherthantherateofreverse.

Ratesofspontaneousmutation1943,bacterialresistancearisesfrommutationsthatexistbeforeexposuretobacteriophage.(NobelPrizein1969)

TheLuria-DelbrueckfluctuationexperimentMutation:ARandom,NonadaptiveProcess

Mutation:UsuallyaRandom,NonadaptiveProcess1952,JoshuaandEstherLederberg

2.Mutagens

Anyphysicalorchemicalagentthatraisesthefrequencyofmutationsabovethespontaneousrate(1)PhysicalmutagensExposuretoXraysincreasesthemutationratesinDrosophila(H.J.Muller,1927)

PyrimidinephotoproductsofUVirradiationCytosinehydrate(水合胞嘧啶)Thymine-dimer(胸腺嘧啶二聚體)

MutationsInducedbyRadiation

RelationshipbetweenirradiationdosageandmutationfrequencyinDrosophila

(2)Chemicalmutagens

Baseanalogs(堿基類似物)

Hydroxylatingagent(羥化劑)Alkylatingagents(烷化劑)Deaminatingagents(脫氨劑)Intercalatingagent(嵌入劑)

MutagensandMutationsSomePotentChemicalMutagens

1.5-BU,baseanalog,A:TG:C,transition2.HNO3,deaminatingagent,A:TG:C,transition3.Proflavin,acridine,intercalatingagent,additionordeletion,frameshiftmutation

4.EMSorEES,alkylatingagent,G:CA:T,transition5.HA(NH2OH),hydroxylatingagent,G:CA:T,transition

3.TheMolecularBasisOfMutationa.TautomericformsofthefourcommonbasesinDNAketo(酮式)---enol(烯醇式)amino(氨式)---imino(亞氨式)

Tautomericshiftsb.TheeffectsoftautomericshiftsinthenucleotidesinDNA

MutationsInducedbyChemicalsc.TypesofpointmutationsthatoccurinDNA

5-bromouracil:basepairing

i.Base-pairingbetween5-bromouracil

Themutageniceffectsof5-bromouracil

5-bromouracil:amutagenInducesmutationsbyoxidativedeaminationofthebasesinDNA

ii.Nitrousacidhypoxanthine(次黃嘌呤)uracil(尿嘧啶)xanthine(黃嘌呤)Intercalationofproflavin（原黃素）intotheDNAdoublehelix

iii.ProflavinProposedmutageniceffect

iv.AlkylatingagentandHAethylguanine(乙基鳥嘌呤)hydroxylaminocytosine(羥氨基胞嘧啶)ScreeningChemicals

forMutagenicity:

Bacteria:SalmonellatyphimuriumAuxotrophs

his-

4.TheAmesTest5.Unequalcrossing-overandtransposable

elements(TE)causeDNAchanges

6.DNArepairmechanisms

a.ProofreadingDNApolymerase3’-5’exonuclease

DNAphotolyase

photoreactionb.Light-dependentrepair

Baseexcisionrepair:glycosylaseAPendonucleasephosphodiesteraseDNApolymeraseDNAligase

c.ExcisionRepair

ExcisionRepair

RepairofDNAbythenucleotideexcisionpathwayinE.coli---excisionnucleaseB,C(uvrB,uvrCgene)andDNApolymerase,DNAligase

NucleotideexcisionrepairmutH,mutL,mutS,mutU(uvrD)MutSrecognitionproteinMutHGATC-specificendonuclease(1000nt)

d.MismatchRepair(recombinationrepair)---RecA,DNApolymeraseIligase

e.PostreplicationRepair

f.TheSOSRepairSOSRepressedSOSInducedTheError-freerepair

ProofreadingLight-DependentRepaironlyinbacteriaExcisionRepairMismatchRepairPostreplicationRepairTheError-pronerepair

SOSRepair

DNARepair1.TheComplementationTestcis---couplingtrans---repulsion

II.MUTATIONANDGENESTRUCTURE

Thecis-transpositioneffectcisheterozygote-wildtypetransheterozygote-mutantThecistest

Thecistest:wild-typeThetranstest

Thetranstest:themutantphenotype

Thecis-transtestIfthetransheterozygotehasthemutantphenotype,thenthetwomutationsareinthesameunitoffunction,thesamegene.Ifthetransheterozygotehasthewild-typephenotype,thenthetwomutationsareintwodifferentunitsoffunction,twodifferentgenes.Thegene(cistron)isdefinedastheunitoffunctionbythecomplementation.

mutant1mutant2mutant3wildtype

Iftwohomologouschromosomeinanindividualeachcarriesamutationrecessivetowildtype,anormalphenotypewillresultifthemutationsareinthedifferentgenes.

Complementationtest:ComplementationtestingofDrosophilaeyecolormutations

2.Mutationandrecombination

Priorto1940,thegenewas(1)Theunitoffunction(2)Theunitofstructurea.Byrecombination???b.Bymutation???Gene:Three-in-Oneconcept

Lozenge(菱形眼),lzs-spectacle,lzg-glassyLozengelocusontheXChromosomeofDrosophila(ClarenceP.Oliver,1940)

DiscoveryofRecombinationwithintheGeneCrossoverinlozengelocus(Green)

SeymourBenzer(1921-2007)

SeymourBenzerreasonedthatifrecombinationcanoccurnotonlybetweengenes,butwithinageneaswell,crossoversbetweenhomologouschromosomecarryingdifferentmutationsknowntobeinthesamegenecouldintheorygenerateawildtypeallele.

BacteriophageT4

PhenotypicpropertiesofrII-mutants

Acustomizedcomplementationtest

betweenrII-mutantsofbacteriophageT4

ComplementationtestofbacteriophageT4Detectingrecombinationbetween

twomutationsinthesamegene

RecombinationtestofbacteriophageT4Ageneconsistsofdifferentpartsthatcaneachmutate;recombinationbetweendifferentmutablesitesinthesamegenecangenerateanormal,wildtypeallele;andageneperformsitsnormalfunctiononlyifallitscomponentsarewildtype.

Conclusion:cistron

BacteriophageT4rIIgenesIII.MUTATIONANDGENEFUNCTIONAlkaptonuria:aninbornerrorofmetabolism.

BeadleandTatum(1945):NobelPrizein1958

TheOneGene,OneEnzymeHypothesis

Aminoacids,peptideandp