考試分子生物學lecture7

Levelsofgeneexpressioncontrolineukaryotes

Transcription

Precursor‐RNAprocessing

mRNAtransport

Translation

RNAdegradation

Proteindegradation

RNAProcessingControl

Regulatestheproductionofmature‐RNAfrom

precursor‐RNA:

1.Choiceofalternativepoly(A)site

toproducedifferentpre‐mRNAmolecules

2.Choiceofalternativesplicingsite

toproducedifferentfunctionalmRNAs

RNATransportControl

spliceosomeretentionmodel

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Spliceosome(thesplicingcomplexesformedbythe

associationofseveralsnRNPsboundtothepre‐mRNA)

assemblycompeteswithnuclearexport

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Aftersplicingprocess,intronisassociatedwithsnRNPs

beforedegradation

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The

methylated5’capisnecessaryformRNAtobe

exportedtothecytoplasm

TranslationalTimingControl

maskedmRNAs

Deadenylation:inthe3’UTRofmRNA,upstreamof

AAUAAAsequence,thereisaconsensussequence

(AU)‐richelement(ARE)as

(UUUUUAU).

deadenylationsignal

Polyadenylation:toactivateastoredmRNAinthis

class,thissignal(AREelement)isrecognizedbya

polyadenylationenzymeandadd~150As

Samesignalfordeadenylationandpolyadenylationincytoplasm

RNAdegradationcontrol

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Deadenylation‐dependentdecaypathway

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poly(A)tailaredeadenylateduntilthetailaretooshort(5‐15As)

tobindPAB(polyAbindingprotein)

Then5’capisremoved(decaping)(DCP1)

5’‐to‐3’exonucleasedegradationAllareenzyme‐catalyzedprocesses

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–

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Deadenylation‐independentdecaypathway

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–

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Yeastdcp1mutantiscapabletodegrademRNA

Decapingwithoutbeingdeadenylated

Rapiddegradationby5’‐to‐3’exonuclease,orbecleaved

internallyw/obeingdeadenylated,andthenbrokendownfurther

Proteindegradationcontrol

E1:ubiquitin-activatingenzyme

E2:ubiquitin-conjugatingenzyme

E3:ubiquitin-proteinligases

AproteintaggedfordestructionoftenrequiresseveralmoleculesofUbiquitin.

GeneRegulationinDevelopment

andDifferentiation

‐‐‐‐long‐termgeneregulation

DevelopmentandDifferentiation

Keypoints

Whataredevelopmentanddifferentiation?

Doesthegenomeremainconstantduring

development,oristherealossofDNA?

Exampleofdifferentialgeneactivityduring

development‐hemoglobin

Howdogenerearrangementsgenerateantibodydiversity?

DevelopmentandDifferentiation

Complexeukaryoteshavemanydifferentcells,

tissuesandorganswithspecializedfunctions,

yetallcellsofthesameorganismhavethe

samegenotype

Duringanorganism’sdevelopment,cellsthat

aregeneticallyidenticalbecomephysiologicalandphenotypicallydifferentiatedintermsoftheirstructureandfunction

DevelopmentandDifferentiation

Developmentistheprocessofregulated

growththatresultsfromthegenome’s

interactionwithcytoplasmandthecellularexternalenvironmentandinvolvesaprogrammedsequenceofcellular‐level

phenotypiceventsthataretypicallyirreversible

Thetotalofthephenotypicchangesconstitutes

thelifecycleofanorganism

DevelopmentandDifferentiation

Developmentalpotential‐describestherange

ofdifferentcelltypesitCANbecome.

Totipotencyiscommoninplants,butis

uncommoninanimalsafterthe2‐4cellstage.

Asdevelopmentprogresses,thedevelopmental

potentialofmostindividualcellsdecreasesuntil

theirfateisdetermined.

DevelopmentandDifferentiation

Thedeterminationofdifferentcelltypes(cell

fates)involvesprogressiverestrictionsintheir

developmentalpotentials.

Whenacell“chooses”aparticularfate,itis

saidtobedetermined,althoughitstill"looks"

justlikeitsundeterminedneighbors.

Determinationimpliesastablechange‐the

fateofdeterminedcellsdoesnotchange.

Mechanismsofcellulardetermination

Insomecases,determinationresultsfromtheasymmetricsegregationofcellulardeterminants.

Mechanismsofcellulardetermination

However,inmostcases,determinationistheresultofinductivesignalingbetweencells.

DevelopmentandDifferentiation

Differentiation,themostspectacularaspect

ofdevelopment,involvestheformationof

differenttypesofcells,tissuesandorgansthroughtheprocessesofspecificregulationofgeneexpression

Differentiatedcellshavethecharacteristic

structuralandfunctionalproperties

DevelopmentandDifferentiation

Differentiationresultsfromdifferentialgeneexpression:ahighlyprogrammedpatternofgeneactivationandgenerepression

HowMuchDNA

CodesforProtein

Eukaryoticgenomes

Unique‐sequenceDNA–onetoafewcopies

RepetitiveDNA–manycopies

ModeratelyrepetitiveDNA–afewto105copies

HighlyrepetitiveDNA–105to107copies

DispersedrepeatedDNA(InterspersedrepeatedDNA)

Shortinterspersedrepeatedsequence(SINEs):100‐500bp

Longinterspersedrepeatedsequence(LINEs):≥5000bp

TandemlyrepeatedDNA

Shorttandemrepeat(STR,microsatellite):2‐4bp

Variablenumberoftandemrepeats(VNTR,minisatellite):5‐10bp

HUMANGENOME

GenomicActivityinMulticellularEukaryotes

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20to40percentofthegenomicDNAishighly

repetitiveDNA(doesnotencodeprotein)

Theremaining60to80percentoftheDNAisdistributedbetweenmoderatelyrepetitiveand

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unique-sequence

DNA

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Atanyonetimeamaximumofabout6%of

theuniquesequenceDNAistranscriptionallyactiveandproducingprotein(seaurchin)

ConstancyofDNAintheGenomeDuringDevelopment

Regenerationofcarrotplantsfrommaturesinglecells

Cloning

animals

Differentialgeneactivityamongtissuesandduringdevelopment

Hemoglobintypesandhumandevelopment

Immunogeneticsandchromosomerearrangementsduringdevelopment

AssemblyofantibodygenesfromgenesegmentsduringBcelldevelopment

ConstancyofDNAintheGenomic

DuringDevelopment

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Whetherdevelopmentanddifferentiation

involvealossofgeneticinformation(i.e.,ofDNA),orwhethertheseprocessesinvolveaprogrammedsequenceofgeneactivationandrepressionwithaconstantgenome,thatis,agenomethatisthesameinalladultcellsasitisinthezygote?

RegenerationofCarrotPlantsfrom

MatureSingleCells

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1950s,FrederickSteward

DissociatephloemtissueofacarrotSeparatethecells

Culturenewcarrotplantsfromthosecellsusingplanttissueculturetechniques

Cloningofamaturecarrotplantfromacellofamaturecarrot

DNAcontentofacellremainsconstantduringdevelopmentanddifferentiation

CloningAnimals

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Annucleusfromaskincellofanadultfroginjected

intoanenucleatedeggcoulddirectdevelopmenttothetadpolestage

Cloningofasleep,thenmice,cattleandmonkeyAdifferentiatedadultsomaticnucleushasallthe

geneticinformationnecessarytodirectdevelopment

againfromthestart

Thenucleusissaidtobetotipotentortodemonstratetotipotency

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RepresentationofWilmot’ssheepcloningexperiment,whichshowedthetotalpotencyofthenucleusofadifferentiated,adultcell

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步驟一：從一只６歲芬蘭多塞特白面母綿羊（Ａ）的乳腺中取出乳腺細胞，將其放入低濃度的營養(yǎng)培養(yǎng)液中，細胞逐漸停止分裂，此細胞稱之為“供體細胞”；

步驟二：從一頭蘇格蘭黑面母綿羊（Ｂ）的卵巢中取出未受精的卵細胞，并立即將細胞核除去，留下一個無核的卵細胞，此細胞稱之為“受體細胞”；

步驟三：利用電脈沖方法，使供體細胞和受體細胞融合，最后形成“融合細胞”。從而形成“胚胎細胞”；

步驟四：將胚胎細胞轉移到另一只蘇格蘭黑面母綿羊（Ｃ）的子宮內，胚胎細胞進一步分化和發(fā)育，最后形成小綿羊--多莉。

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Cloning101

Keynote

Thepreciseregulationofactivationandrepressionofspecificgenesisresponsiblefortheprocessofdevelopmentanddifferentiation.

DevelopmentanddifferentiationresultfromdifferentialgeneactivityofagenomethatcontainsaconstantamountofDNA,fromthezygotestagetothematureorganismstage.

ConstancyofDNAintheGenomeDuringDevelopment

Regenerationofcarrotplantsfrommaturesinglecells

Cloning

animals

Differentialgeneactivityamongtissuesandduringdevelopment

Hemoglobintypesandhumandevelopment

Immunogeneticsandchromosomerearrangementsduringdevelopment

AssemblyofantibodygenesfromgenesegmentsduringBcelldevelopment

DifferentialGeneActivityAmong

TissuesandDuringDevelopment

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Specializedcelltypehavedifferentcell

morphology,forexample,nervecellsareclearlydifferentfromintestinalepithelialcells

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SincetheamountofDNAtypicallyremains

constantduringdevelopment,thesimplesthypothesisisthatthephenotypicdifferencesbetweendifferentcelltypesreflectdifferentialgeneactivity

HemoglobinTypesandHumanDevelopment

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HumanadulthemoglobinHb-Aisatetrameric

proteinmadeupoftwoαandβpolypeptides.Eachtypeofpolypeptideiscodedbyaseparategene,αglobinandβglobin.

Structureofhumanhemoglobin

Red--αsubunitsBlue–βsubunits

Green–ironcontaininghemegroups

PDBID:1GZX

Molecularorganizationofthehuman‐

globinand‐globingenes

HemoglobinTypesandHumanDevelopment

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HemoglobinHb-Aisonlyonetypeof

hemoglobinfoundinhumans.

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Thereareseveralgenescodeforαandβ-like

globinpolypeptides,whichformdifferenttypesofhemoglobinatdifferenttimesduringhumandevelopment

Comparisonofsynthesisofdifferentglobin

chainsatgivenstagesofdevelopment

Linkagemapsofhumanglobingeneclusters

Sicklecellanemia

Classicalsicklecellanemiaisadevastatingdiseasethatafflictsasmanyas1in64blacksinAfricaandfrom1in200to1in400blacksintheUnitedStates

SicklecellanemiaiscausedbyanAtoTtransversioninthesixthcodonofthehumanβ-globingene(GAGGTG)resultinginaglutamicacidtovalinesubstitutionintheprotein

Itcausedeoxygenatedhemoglobintoaggregate,resultinginalterationintheredbloodcellsshapeandblockbloodflow

Effortsaremadetoupregulateγ-globin(fetalformofhemoglobin)orδ-globin(preventionofβ-globinaggregation)toalleviatesdiseasesymptoms

SickleCellAnemia

SickleCellDisease

ConstancyofDNAintheGenomeDuringDevelopment

Regenerationofcarrotplantsfrommaturesinglecells

Cloning

animals

Differentialgeneactivityamongtissuesandduringdevelopment

Hemoglobintypesandhumandevelopment

Immunogeneticsandchromosomerearrangementsduringdevelopment

AssemblyofantibodygenesfromgenesegmentsduringBcelldevelopment

ImmunogeneticsandChromosome

RearrangementsDuringDevelopment

Thecellsresponsibleforimmunespecificityare

lymphocytes,specificallyTcellsandBcells.

Blymphocytesdevelopintheadultbonemarrow

Whenactivatedbyanantigen,Bcellsdevelopintoplasmacellsthatmakeantibodies,thatis,specializedproteinscalledimmunoglobulins(Igs)

Antibodymoleculesareinsertedintheplasmamembraneoftheplasmacells,andtheyarealsoreleasedintothebloodandlymph,wheretheyareresponsibleforthehumoralimmuneresponses.

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ImmunogeneticsandChromosome

ReaarangementsDuringDevelopment

Forthehumoralresponsesystem,thereisapopulationofBcells,eachofwhichcanrecognizeasingleantigen.

AparticularBcellrecognizesanantigenbecausetheBcellhasmadeantibodymolecules,whichareattachedtotheoutermembraneofthecellandactasreceptormolecules.

Anygivencellmakesonlyonespecifickindofantibodytowardonespecificantigen.

ImmunogeneticsandChromosome

ReaarangementsDuringDevelopment

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Inmammals,therearefivemajorclassesof

antibodies:IgA,IgD,IgE,IgGandIgM.

TheyhavedifferentHchains:α,δ,ε,γandμ,respectively

TwotypeofLchainsarefound:κandλ.BothLchaintypesarefoundinallIgclasses.

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IgGantibodymolecule

AssemblyofAntibodyGenesfromGene

SegmentsDuringBCellDevelopment

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Mammalmayproduce106to108differentantibodies

Theseantibodiestheoreticallywouldrequire103to104differentLchainsand103to104differentHchains

Theproblemisthatthehumangenomecontainonly~105gene

Howthediversityofantibodiesbegenerated?

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VariabilityinLandHchainsresultsfromparticularDNArearrangementthatoccurduringBcelldevelopment

AssemblyofAntibodyGenesfromGene

SegmentsDuringBCellDevelopment

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TheserearrangementsinvolvethejoiningofdifferentgenesegmentstoformagenethatistranscribedtoproduceanIgchain,theprocessiscalledsomaticrecombination

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Thepermutationsofrecombinationareresponsiblelargelyforthediversityofantibodystructure.

Productionofkappa()lightchaingeneinmouse

L-leadersequence

J-joiningsegments

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350Vk*4Jk=1400

FurtherdiversityresultsfromimprecisejoiningoftheVksegments

andJkgene

LightChainGene