分子生物學(xué)課件第七章part1

1000多種EULEC分別識(shí)別不同底物，靈活地降解各種不同的蛋白，協(xié)調(diào)各種生理活動(dòng)被EULEC識(shí)別的N-endaamarkeroftargetingprotein

Met,Gly,Ala,Ser,Thr,Val>20hrs(穩(wěn)定因子)

Ile,Glu~30’(不穩(wěn)定因子)Gln

~10’

Leu,Phe,Asn,Lys~3’(極不穩(wěn)定因子)Arg~2’Pro~7’N-endaaoftargetingproteinhalf—life-決定與Ubiquitin發(fā)生isopeptidebond的難易程度-決定被E3識(shí)讀結(jié)合難易程度-N端氨基酸種類對蛋白質(zhì)壽命的決定性，從原核到真核生物高度一致泛素/26S蛋白酶體通路與植物激素應(yīng)答植物抗病反應(yīng)植物光形態(tài)建成高等植物有性生殖植物環(huán)境脅迫抗性

MolecularBiologyGibberellinSignalingPathwayade-repressiblesystemmoderatedbyDELLA-pdegradationinducedbyGA●InduceseedgerminationGerminatingpeaseedGerminatingmaizeseeds

GAscontrolplantgrowthanddevelopment●PromotestemelongationandfruitsdevelopingDwarfpeas-GA+GASeedlessgrapes-GA+GA

GreenRevolution

第一次綠色革命；

糧食作物增產(chǎn)20%NormanE.Borlaug

Directorofwheatprogram(CIMMYT)1970NobelPeacePrizeGreenRevolutionGeneSemi-dwarfricecultivarscontainingrice‘G.R’genesd1GreenRevolutionGeneSemi-dwarfmaizecultivarscontainingthe‘G.R’gened8

HighlyconservedinG.R.gene

Arabidopsis:

(RGA,GAI,RGL1,RGL2,RGL3)

maize:(d8),wheat(Rht)

rice:(SLR1),barley(SLN1)

grape:(VvGAI1),Brassicarapa(BrRGA1)phosphotyrosinebindingdomainTheDELLAsubfamilyofGRASproteinsChokebackgrowth●AconservedanduniqueN-terminaldomain,namedDELLA,afteraDELLAmotif

●WildtypeDELLAproteinslocalizeincellnucleianddisappearbyGAtreatmentphosphotyrosinebindingdomain●DELLAdomain-mutatedareresistant(G.R.gene)toGA-induceddegradation(GA-insensitive)

ByusingimmunoblotanalysisandGFPfusionproteinanalysis,itisshownthat

GAinducesrapiddegradationofthewildtypeDELLAproteins,suchasGFP-At-RGA

GFP-Os-SLR1

FromSilverstoneetal.,PlantCell,2001,13:1555-1566ThedisappearanceofGFP-RGAproteinonGAtreatment-GA+GAGFP-(rga-Δ17)proteinsareresistanttoGA-induceddegradation+H2O+GA3(rga-Δ17hasthesame17AAdeletionintheDELLAregionasingai-1)GFP-RGAGFP-(rga-Δ17)GFP-(rga-Δ17)FromDilletal.,ProcNatlAcadSciUSA,2001,98:14162-14167SchemeofthefunctionaldomainsofSLR1forGAsignalingpathwayPolyS/T/VFromItohetal.,PlantCell,2002,14:57-70AmodelofGAinduceddegradationofDELLAproteinsviaE3ubiquitinligaseenzymecomplex(EULEC)FromItohetal.,TrendsPlantSci,2003,8:492-497DELLADELLA

Involvementoftheubiquitin-proteasomepathwayindegradationofDELLAproteinsInresponsetoGA,DELLAproteinsarerapidlydegradedbytheubiquitin-proteasomepathwayUbiquitylatingenzymeE3complexNegativeG.R.gene

？)6.3.真核生物基因表達(dá)與調(diào)控的特殊模式

以positivecontrol為主的基因表達(dá)調(diào)控真核生物染色質(zhì)的狀態(tài)對基因表達(dá)的調(diào)控m5C與基因表達(dá)的相關(guān)轉(zhuǎn)錄后多種方式的加工調(diào)節(jié)個(gè)體發(fā)育的階段調(diào)控真核生物基因表達(dá)調(diào)控的特異性6.3.1.mRNA的結(jié)構(gòu)對翻譯水平的調(diào)控Leadingseq.(5’-Untranslationregion,UTR)Cap具有增譯作用Cap+poly(A)具有增譯的協(xié)同效應(yīng)Cap對eIF4b的相對濃度的選擇

Shatkin(1976)---m7GpppN(Cap)---AUGflankedseq.effect5’L.S存在AUG對第一個(gè)AUG密碼的起譯產(chǎn)生負(fù)控效應(yīng)5’------AUG-------AUG-NNN--

L.S錯(cuò)誤起譯改變讀框降低速率Effectofmultiplereplicasofthetranslationinitiationsiteimmunoprecipitatdproinsulin-containingpolypeptidesthefarthestupstreamAUGcodonwasusedwhichisagainconsistentwiththescanningmodelKozak(1987)699種脊椎動(dòng)物，植物，酵母和原生動(dòng)物----A-3NNAUGG+4----具有促譯性，普遍性A-3CCAUGG+4也是核糖體小亞基對翻譯密碼的掃描信號(hào)---AUGflankedconsensusseq(-3A,+4G)

KozakmodelKozak(1986)Ratpre－proinsulingeneunderthecontrolofSV40promoter

mutagenized

Ntat--3and+4positionintroductionthemintomonkeycell

Effectsofsinglebasechangesinpositions-3(A)and+4(G)RatpreproinsulingeneunderthecontrolofananSV40promoterKozakCell44(31)Jan1986mutationmutationKozakmodel[35S]-Met---5’UTR二級結(jié)構(gòu)的影響5‘端高度結(jié)構(gòu)化的mRNA是核糖體進(jìn)入的位點(diǎn)并通過富集核糖體和起始因子促進(jìn)基因的表達(dá)Hairpin(Stem-loop)順式阻扼40ssubunitofribosome遷移G/C多，阻扼強(qiáng)G/C近AUG,阻扼強(qiáng)capChloramphenicolAcetylTransferase氯霉素乙酰轉(zhuǎn)移酶Higherstability62kca/mollowerstability30kca/mol1)30-kcalstemlooponly12ntdownstreamofthecapstronglyinhibitstranslation,

becauseitinterfereswithbindingofthe40Sribosomeandfactorsatthecap.

2)62-kcalstemloopplaced71ntdownstreamofthecapcompletelyblockedappearanceoftheCATprotein.Why?6.3.2染色質(zhì)重建對基因表達(dá)的控制表觀遺傳及其分子機(jī)制

EpigeneticandMolecularMechanismEpigeneticinheritence:Theabilityofdifferentstates,whichmayhavedifferentphenotypeconsequences,tobeinheritedwithoutanychangeintheDNAsequence.在DNA序列沒有發(fā)生任何改變的情況下，不同表型效應(yīng)具有可以遺傳的能力（W>wineuchromatin）w

WwRedeye

（W(nearheterochromatin)besilenced

WwWwwhiteeye

PositioneffectofvariegationinDrosophilaeyes

EpigeneticphenomenaSpoteye

CellularMemory:ArabidopsisVernalizationEpigeneticphenomenaOneXchromosomeisinactivedatrandomparentalimprintingBCDAAllelicinteraction(paramutation)-Position-effectvariegation-InactivationofchromosomeX-Cell-typeconversion-Allelicinteraction(paramutation)-Transgenesilencing-ParentalimprintingCellmemory:VernalizationHeterosis??？Epigeneticphenomena染色質(zhì)結(jié)構(gòu)的調(diào)整核小體的松弛組蛋白的乙酰化組蛋白的甲基化沉默子….染色質(zhì)重建

ChromatinRemodelingEpigeneticandMolecularMechanism

BasicunitofchromatinNucleosome(ν-body~200bp)Histone1+linkerDNAof20-60bp

(diff.Fromdiff.creature)2×H2AH2BH3H4Octamer+coreDNAof146bp1.75cycle6.3.2.1染色質(zhì)結(jié)構(gòu)6nm11nm30nm10nmfibreHeterochromatinEuchromatin組蛋白中富含Lys+,Arg.+

量大，進(jìn)化保守，與DNA無專一性結(jié)合區(qū)N端外露Arg＋與DNA14個(gè)小溝結(jié)合富含Lys＋的N端（特別是H4）能保證Histone間的穩(wěn)定性和與DNA的結(jié)合N端磷酸化、乙?；饶芙档徒M蛋白正電荷，使組蛋白間以及與DNA的結(jié)合狀態(tài)發(fā)生松弛Histoneacetyltransferase(HAT)

Histonedeacetylase(HDAC)Histonemethyltransferase(HMT)LysArgLysSerLysHistoneCodon

Histonecode組蛋白一個(gè)位點(diǎn)的修飾可以激活或抑制另一個(gè)位點(diǎn)的修飾，這種信號(hào)的組合可以寓意染色質(zhì)的類型與性質(zhì)。這種組合也被稱為組蛋白密碼6.3.2.2chromatin狀況與基因表達(dá)相關(guān)EuchromatingeneonHeterochromatingeneoffHeterochromatin；ConstitutiveHeterochromatin:

組成型異染色質(zhì)除DNA復(fù)制以外，一直處于高度致密的固縮狀態(tài)，DNA從不轉(zhuǎn)錄（高度重復(fù)序列，著絲點(diǎn)，端粒等）FacultativeHeterochromatin:

特異型異染色質(zhì)有時(shí)處于異染色質(zhì)狀態(tài)，有時(shí)為常染色質(zhì)狀態(tài)轉(zhuǎn)錄活化區(qū)/非轉(zhuǎn)錄活化區(qū)chromatin的結(jié)構(gòu)差異

DNaseS

Nucleosomeincomplete

NoHistone1inlinkerDNAintranscriptionalactivatedregion表達(dá)非?；钴S的rDNA

區(qū)無核小體結(jié)構(gòu)凡被Trans-factor結(jié)合的區(qū)域均能阻止nucleosome再形成

H2A、H2B,、H3、H4acetylated降低組蛋白的正電荷組蛋白解聚ν-body松弛80%H3,H4乙?；∷徕c（去乙?；敢种苿〩elacell基因高效表達(dá)Pazin＆Kadonaga1997Cell.89:325-328

從酵母中分離出組蛋白去乙?；窤／B

以及相應(yīng)的催化組分HDAC1／RPD3

HDAC1／RPD3基因突變導(dǎo)致H3/H4超乙酰化引起基因高效表達(dá)OveracetylationModelforparticipationofhistonedeacetylaseintranscriptionrepressionhistonedeacetylasecooperatorHDAC1

NucleosomerelaxedNucleosomereconstructedHistonemethylationcausesinactivechromatinHeterochromatinprotein1分離得到與甲基化DNA結(jié)合抑制轉(zhuǎn)錄的MeCP2JonesP.L.1998.MethylatedDNAandMeCP2recurithistonedeacetylasetorepresstranscription.NatureGenet.19:187-191組蛋白去乙?；窤(dHAT-A)dHAT-A的催化因子(SIN3)MeCP2(m5C結(jié)合蛋白)complexMeCP2SIN3dHAT-AMeCP2與m5C結(jié)合阻止TFII與DNA結(jié)合Histone去乙?；饔?重建核小體抑制基因表達(dá)HistonemethylationcausesinactivechromatinHeterochromatinprotein1HP1maypropagateheterochromatinInactiveChromatin6.3.2.3.m5C對基因表達(dá)的調(diào)控a)m5C是真核生物DNA中的主要修飾成分多發(fā)生在CpG序列中,不同細(xì)胞間m5C相差甚大胚胎細(xì)胞與特化體細(xì)胞相差106b)m5C對基因轉(zhuǎn)錄抑制的表現(xiàn)

DNA大溝內(nèi)的m5C影響與T.F.間氫鍵的形成

DNA大溝內(nèi)的m5C導(dǎo)致空間擁擠,破壞構(gòu)象的平衡使B-DNA

Z-DNA

T.F.結(jié)合空間的改變

染色質(zhì)重建降低轉(zhuǎn)錄因子與DNA的結(jié)合GenomeandgeneimprintinEukaryots一定組織和細(xì)胞中，某些基因在DNA水平上的表達(dá)程度及表達(dá)時(shí)空受到一種“后成修飾，表觀修飾”（epigeneticmodification相同的DNA結(jié)構(gòu)，不同的表型）機(jī)制控制。使僅來自雙親中某一親本的基因得以表達(dá)。這一現(xiàn)象也稱為“基因組印記”DNA的甲基化、異染色質(zhì)化是“imprint”的重要機(jī)制染色體區(qū)域性印記中相鄰印記基因具有“相反”的印記作用模式

父本基因印記母本基因印記合子及幼胚期，DNA去甲基化程度高，geneimprint被抑制，表現(xiàn)了細(xì)胞的“全能性”Genome/geneimprint表現(xiàn)的特點(diǎn)

Genomeimprint具有染色體區(qū)域性或整體性印記現(xiàn)象具有組織細(xì)胞的特異性基因印記的發(fā)生常與該基因的編碼區(qū)，啟動(dòng)子區(qū)及其上下游的CpG序列甲基化相關(guān)Genome/geneimprint表現(xiàn)的特點(diǎn)印記效應(yīng)會(huì)因去甲基化而消失

(水稻白葉枯病成株抗性？)OneXchromosomeisinactivedatrandomTwoXchro.Euchromatinin16dayprecursorcellOneXchro.inactivedatrandomVariegation

Prader-willi癥(PWS)：肌肉無力、矮小、神經(jīng)疾病

Argelmen癥（Ag）：平衡性差、過分興奮與發(fā)笑

共同的病因：15q11-q13缺失，基因型相同子女的父父染色體母母病癥：AgPWS母本基因印記父本基因印記

被異染色質(zhì)化的X染色體常染色質(zhì)化

導(dǎo)致雌性哺乳動(dòng)物宮頸癌，胃癌雙重遺傳病因：缺失/印記3typesofmodificationaffectchromatin甲基化的CpG序列可招募組蛋白去乙?；?，抑制基因的表達(dá)。Vernalisation植物低溫春化現(xiàn)象的分子機(jī)理AfterVernalization

FLC

Leafy

MADS

FloweringFLCmethylationN5-C的春化效應(yīng)WithoutVernalization

FLC

Leafy

MADS

Flowering低溫春化處理消除對MADSBox基因表達(dá)的負(fù)控制效應(yīng)Vernalization低溫春化－－－引起脫乙?；?、甲基化修飾

－－－導(dǎo)致異染色質(zhì)化RegulationofthefloralrepressorFLCby

histonedeacetylation(FLD)yesNoNoACACACACACACACACACACACACACACACACACACACACmut.

-Chromatinstructureandremodeling -DNAmethylation -Histonemodification(Ac&Me) -RelationshipbetweenDNAMeandHistoneMe -Creationofrepressivedomain

-

ncRNA -Heterochromatinformation

Chromatinmodification&remodelingastheepigeneticbasis

No-codingRNAncRNA

看家RNA

rRNA，tRNA，UsnRNA，telomereRNA...

調(diào)節(jié)RNAmicroRNA（miRNA），siRNA，

anti-senseRNA...WhatismicroRNA?MicroRNAsareanewlyidentifiedclassofsmallsingle-strandednon-codingRNAs;MicroRNAsregulatetheirtargetgenesviatwomainmechanisms:targetmRNAcleavage

translationalrepression.ThecharactersofmiRNALength:18-25nt;Single-strand;Conservedamongspecies;EncodedbyinternalDNAsequences,whicharelocatedinthewholegenome,mostlyintheintervalregionsofcodinggenes,centromeres,or,occasionallyintheintrons;Temporallyandtissue-specificallyexpressed

ThebiogenesisofmiRNAReview:GeogreSMack,NatureBiotech.,2007dsRNA由Dicer加工成21Nt±siRNA，在RDRP的作用下可以擴(kuò)增，siRNA與靶mRNA來源與同一基因60－100nt，3‘具有2Nt突起莖環(huán)結(jié)構(gòu)的pre－miRNA，由Dicer加工成一條雙鏈的21±m(xù)iRNA，與靶mRNA來自不同的基因

siRNAmiRNA來源于mRNA，病毒RNA，轉(zhuǎn)座子，外源RNA來源于基因組內(nèi)的microDNA、剪切的intron...（內(nèi)源性）與靶基因可不完全配對，因此可調(diào)控若干不同的基因在近緣物種間具有較高的保守性與靶基因完全配對，專一性強(qiáng)不同內(nèi)源siRNA具有較大的差異與mRNA結(jié)合，或剪切mRNA，抑制翻譯，沉默基因表達(dá)(特別是轉(zhuǎn)錄因子)。進(jìn)入RISC體系，在轉(zhuǎn)錄后水平上沉默基因表達(dá)進(jìn)入RISC體系降解靶mRNA，轉(zhuǎn)錄后水平上沉默基因表達(dá)，RNAiworksbygeneratingsiRNADicerRISC置換Lin14蛋白調(diào)節(jié)秀麗新線蟲的幼蟲發(fā)育，但受lin4基因的控制Lin4編碼22Nt的microRNA,與Lin14mRNA3‘端非翻譯區(qū)出現(xiàn)7次重復(fù)的10Nt序列互補(bǔ)，抑制Lin14蛋白的翻譯

commonmechanismformicroRNA,RNAiandheterochromatinformation6.3.4.2.ProgrammedCellDeath(PCD)anddevelopment2002NobelPrizeJohnSulstonUSASydneyBrennerJapan

田中耕一

H.RobertHorvitzSwitzerland

細(xì)胞的“生”發(fā)育、分裂、繁殖、分化、成熟細(xì)胞的“死”….?---高等生物的誕生—發(fā)育—成長—死亡即壽命是生物固有的特征---死亡是生物體不可缺少的生命內(nèi)容是生物借以存活的需要并貫穿壽命的全部周期中PCDgene的發(fā)現(xiàn)與克隆人類長壽癌癥發(fā)生提高產(chǎn)量……---細(xì)胞死亡的發(fā)生及過程是在長期進(jìn)化中已形成的遺傳程序（PCD）這一程序表達(dá)的失控個(gè)體發(fā)育受阻、病變、畸形、蹼趾、有尾…..癌癥（白血?。?--細(xì)胞周期變短,增殖過快,pcd—失控,細(xì)胞死亡速率變低細(xì)胞增殖與細(xì)胞死亡平衡失調(diào)癌癥的發(fā)生---皮膚,指(趾)甲(細(xì)胞從有生命經(jīng)PCD向無生命的轉(zhuǎn)化)pcd—無皮，無鱗，無爪失去保護(hù)與防御功能---pcd—

白細(xì)胞不分化，不死亡，不代換41天51天在細(xì)胞自身遺傳程序的控制下，有關(guān)基因的正常表達(dá)，細(xì)胞裂解成apoptoticbodies,逐漸死亡的現(xiàn)象a)PCD的概念細(xì)胞的死亡；

necrosis(accidentalcelldeath)壞死外因細(xì)胞急速死亡，病變死亡

ProgrammedCellDeath(PCD)或Apoptosis

凋亡細(xì)胞程序性死亡Apoptosis細(xì)胞變園變小與鄰近細(xì)胞脫離胞漿濃縮染色質(zhì)凝集成月牙狀內(nèi)質(zhì)網(wǎng)擴(kuò)張成泡狀并與膜融合PCD的細(xì)胞學(xué)特征膜內(nèi)陷，細(xì)胞呈沸騰運(yùn)動(dòng)，細(xì)胞自行解體成若干有膜包圍，內(nèi)容物不外溢的凋亡小體被巨噬細(xì)胞吞噬核內(nèi)DNA被核酸酶在核小體單位間降解電泳檢測呈

ladderof180-200bpapoptoticbodiesApoptosis:

"active"celldeath;"cellsuicide"Necrosis:"accidental"celldeath;"cellmurder"imagesofnucleifromapoptoticandnecroticcells●

促進(jìn)PCD的基因Ces(celldeathspecification)細(xì)胞凋亡特異基因p53抑癌基因Caspase（半胱氨酸酶家族，蛋白降解途徑）….●

抑制PCD的基因LAP(inhibitorofapoptosis)凋亡抑制劑基因Cystatin….b)與PCD相關(guān)的基因Caspase,p53pathwaycystatinBI-1植物的PCD表現(xiàn)與機(jī)制植物抗病的過敏性反應(yīng)HypersensitiveResponsePCD枯斑的形成花藥的發(fā)育（CMS？）木質(zhì)部的分化絨氈層的退化禾本科植物胚乳的發(fā)育缺氧條件下誘導(dǎo)的玉米根部通氣組織的形成與根冠的形成具有與動(dòng)物細(xì)胞相似的細(xì)胞學(xué)表型與生化特征植物PCD的相關(guān)基因與機(jī)制半胱氨酸酶家族（Caspase）衰老葉片，果實(shí)，花器發(fā)育中木質(zhì)部的形成…Caspase乙烯在PCD中起重要作用

缺氧條件下，乙烯誘導(dǎo)玉米根部胞內(nèi)Ca++增加氣生組織的形成小麥胚乳形成中，乙烯調(diào)節(jié)DNA降解

acd1,acd2（acceleratedcelldeath）基因

Acd負(fù)控制ArabidopsisHR誘導(dǎo)的

PCD過程---植物抗病的PCD表現(xiàn)

Acd

(AcceleratesCellDeath)

Lsd(LesionsStimulatingDiseaseResistance)ProgrammedCellDeath

Neg.controlConstitutivePathogenPlantPCD負(fù)調(diào)控關(guān)閉PCD系統(tǒng)表達(dá)OxidativeburstH2O2,-OH,O-22

升高NADPH—oxidaseAcd/Lsdrepressor失活枯斑停止擴(kuò)大高濃度H2O2

消失枯斑形成限制病原物擴(kuò)展H2O2inducedcytoplasmshrinkage,atypicalmarkerofplantPCD.在發(fā)育進(jìn)程中，PCD何時(shí)啟動(dòng)？如何決定某一細(xì)胞執(zhí)行PCD?機(jī)體內(nèi)是否存在真正殺手基因（killergene）?Killer只在PCD細(xì)胞中表達(dá)！其產(chǎn)物可使細(xì)胞走向死亡！PCD的信號(hào)，傳遞？

Prok.與Euk.基因結(jié)構(gòu)及表達(dá)的差異

RepetitivegeneOverlappinggeneRetro-transposon

Non-codingregion(function?)

SplittinggeneEukaryoteProkaryoteDNA，RNA,protein

DNA+histonechromatinNakedDNA

andchromatinRemodeling

Enhancer&SilencerAttenuater

Monocistron

polycistron(operon)

m5C

geneoff

acetylationphosphorylationmathylation

glucosylationT.FactiveformEukaryoteProkaryote

Post-transcriptionTranscription&translation

RNAprocessingSynchronizely

Housekeepinggene(constitutive)Basicpromoter+T.F.Luxurygene(inducible)多種組合的Trans-Factor

MultiplegenefamilyClustergene

QuantitativeTraitloci

Positivecontrol