細(xì)胞學(xué)第十章 DNA遺傳重組課件

第十章DNA遺傳重組一、DNA遺傳重組的種類(lèi)

所有生物DNA都是重組體（recombinant）。

1.同源重組（homologousrecombination）:RecombinationinvolvingreactionbetweenhomologoussequencesofDNAiscalledgeneralizedorhomologousrecombination.Ineukaryotes,itoccursatmeiosis,usuallybothinmales(duringspermatogenesis)andfemales(duringoogenesis).Werecallthatithappensatthe"fourstrand"stageofmeiosis,andinvolvesonlytwoofthefourstrands.

細(xì)胞學(xué)第十章DNA遺傳重組2.轉(zhuǎn)座重組（transpositionrecombination）:Anothertypeofeventsponsorsrecombinationbetweenspecificpairsofsequences,andhasbeenbestcharacterizedinprokaryotes.

Transpositionprovidesameansbywhichcertainelementsmovefromonechromosomallocationtoanother.ThemechanismsinvolvedintranspositiondependuponbreakageandreunionofDNAstrands,andthusarerelatedtotheprocessesofrecombination.細(xì)胞學(xué)第十章DNA遺傳重組3.位點(diǎn)專一性重組（site-specificrecombination）:AdifferenttypeofeventallowsoneDNAsequencetobeinsertedintoanotherwithoutrelianceonsequencehomology.

Site-specificrecombinationisresponsiblefortheintegrationofphagegenomesintothebacterialchromosome.TherecombinationeventinvolvesspecificsequencesofthephageDNAandbacterialDNA,whichincludeashortstretchofhomology.Theenzymesinvolvedinthiseventactonlyontheparticularpairoftargetsequences.Relatedreactionsareresponsibleforinvertingspecificregionsofthebacterialchromosome.細(xì)胞學(xué)第十章DNA遺傳重組4.異常重組（illegitimaterecombination）:AnothertypeofrecombinationisusedbyRNAviruses,inwhichthepolymeraseswitchesfromonetemplatetoanotherwhileitissynthesizingRNA.Asaresult,thenewlysynthesizedmoleculejoinssequenceinformationfromtwodifferentparents.Thistypeofmechanismforrecombinationiscalledcopychoice,andisdiscussedbrieflyin16Retrovirusesandretroposons.ItisnotusedforrecombinationbetweenduplexDNAsubstrates.細(xì)胞學(xué)第十章DNA遺傳重組Figure14.1Recombinationoccursduringthefirstmeioticprophase.Thestagesofprophasearedefinedbytheappearanceofthechromosomes,eachofwhichconsistsoftworeplicas(sisterchromatids),althoughtheduplicatedstatebecomesvisibleonlyattheend.Themolecularinteractionsofanyindividualcrossing-overeventinvolvetwoofthefourduplexDNAs.

（一）同源重組細(xì)胞學(xué)第十章DNA遺傳重組Figure14.2RecombinationbetweentwopairedduplexDNAscouldinvolvereciprocalsingle-strandexchange,branchmigration,andnicking.

細(xì)胞學(xué)第十章DNA遺傳重組Figure14.3Branchmigrationcanoccurineitherdirectionwhenanunpairedsinglestranddisplacesapairedstrand.

細(xì)胞學(xué)第十章DNA遺傳重組Figure14.4ResolutionofaHollidayjunctioncangenerateparentalorrecombinantduplexes,dependingonwhichstrandsarenicked.BothtypesofproducthavearegionofheteroduplexDNA.細(xì)胞學(xué)第十章DNA遺傳重組Figure14.5Recombinationisinitiatedbyadouble-strandbreak,followedbyformationofsingle-stranded3’ends,oneofwhichmigratestoahomologousduplex.

細(xì)胞學(xué)第十章DNA遺傳重組Figure15.8Reciprocalrecombinationbetweendirectrepeatsexcisesthematerialbetweenthem;eachproductofrecombinationhasonecopyofthedirectrepeat.

細(xì)胞學(xué)第十章DNA遺傳重組Figure15.9Reciprocalrecombinationbetweeninvertedrepeatsinvertstheregionbetweenthem.

細(xì)胞學(xué)第十章DNA遺傳重組（二）轉(zhuǎn)座重組（transpositionrecombination）

1.轉(zhuǎn)座子的類(lèi)型:

在原核生物中已發(fā)現(xiàn)兩種類(lèi)型的轉(zhuǎn)座子，簡(jiǎn)單轉(zhuǎn)座子（simpletransposon）和復(fù)合轉(zhuǎn)座子（compositetransposon），前者稱為插入序列（insertionsequence，IS）。復(fù)合轉(zhuǎn)座子除了轉(zhuǎn)座過(guò)程必需的基因外還含有一到幾個(gè)基因，它們常有可鑒定的遺傳標(biāo)記如抗菌素抗性或毒素生成等。細(xì)胞學(xué)第十章DNA遺傳重組表2.幾種復(fù)合轉(zhuǎn)座子的特征元件長(zhǎng)度bp遺傳標(biāo)記末端組件組件取向組件間關(guān)系組件功能Tn92500canRIS1正向可能相同可能有功能Tn109300tetRIS10R/IS101反向2.5％差異有功能/無(wú)Tn55700kanRIS50R/IS501反向1bp變化有/無(wú)功能Tn9033100kanRIS903反向完全相同兩者都有功能表1.某些插入序列的參數(shù)元件長(zhǎng)度bp末端反向重復(fù)bp靶位點(diǎn)反向重復(fù)bp靶位點(diǎn)選擇ISI768239隨意IS21327415熱點(diǎn)IS414281811或12AAAN20TTTIS51195164熱點(diǎn)IS10R1329229NGCTNAGCAIS50R153199熱點(diǎn)IS9031057189未知細(xì)胞學(xué)第十章DNA遺傳重組2.轉(zhuǎn)座子結(jié)構(gòu)特征：①兩端有20~40bp的反向重復(fù)序列（invertedrepetitivesequence，IR）；②具有編碼轉(zhuǎn)座酶（transposase）的基因，這種酶催化轉(zhuǎn)座子插入新的位置。以Pelement為例進(jìn)行說(shuō)明。

P因子大小為2907bp，稱為完整P因子(intactPelement，IPE)。小于2.9kb的P因子，稱為缺陷P因子(defectivePelement，DPE)，是由IPE通過(guò)不同方式的內(nèi)部切除形成的。細(xì)胞學(xué)第十章DNA遺傳重組3.轉(zhuǎn)座子的轉(zhuǎn)座機(jī)制

1）復(fù)制型轉(zhuǎn)座在復(fù)制型轉(zhuǎn)座（replicativetransposition）中，轉(zhuǎn)座子作為可移動(dòng)的元件被復(fù)制，一個(gè)拷貝保留在供體的原來(lái)部位不變；另一個(gè)拷貝則插入到受體的位點(diǎn)上。復(fù)制型轉(zhuǎn)座需要兩種酶：轉(zhuǎn)座酶作用于原來(lái)轉(zhuǎn)座子的兩個(gè)末端，解離酶（resolvase）作用于復(fù)制后的拷貝上。

2）非復(fù)制型轉(zhuǎn)座在非復(fù)制型轉(zhuǎn)座（nonreplicativetransposition）中，轉(zhuǎn)座子直接從供體位點(diǎn)移動(dòng)到受體位點(diǎn)處，但在供體位點(diǎn)上留下一個(gè)裂口，其結(jié)果是使供體基因組受到輕微的損傷（在具有多拷貝的細(xì)菌中可以忍受）或致死。還有一種可能，就是宿主修復(fù)系統(tǒng)能識(shí)別雙鏈斷裂處，并加以修復(fù)。非復(fù)制型轉(zhuǎn)座只需要轉(zhuǎn)座酶。細(xì)胞學(xué)第十章DNA遺傳重組3）保守型轉(zhuǎn)座保守型轉(zhuǎn)座（conservativetransposition）是另一種非復(fù)制型轉(zhuǎn)座。轉(zhuǎn)座過(guò)程中，轉(zhuǎn)座子從供體位點(diǎn)上切除，通過(guò)一系列反應(yīng)插入到靶位點(diǎn)上，其中每個(gè)核苷酸鍵都保留下來(lái)。這種保守型轉(zhuǎn)座反應(yīng)過(guò)程正好與λ整合機(jī)制相似，而且這種轉(zhuǎn)座子的轉(zhuǎn)座酶與λ整合酶家族有關(guān)。利用這種機(jī)制不但可以介導(dǎo)轉(zhuǎn)座子自身的轉(zhuǎn)移，而且也可以介導(dǎo)供體DNA從一個(gè)細(xì)菌轉(zhuǎn)移到另一個(gè)細(xì)菌中。所以，把保守型轉(zhuǎn)座的元件稱作附加體（episone）更為合適。

實(shí)際上，有些轉(zhuǎn)座子僅使用一條途徑進(jìn)行轉(zhuǎn)座；而有的則可能使用兩種途徑。細(xì)胞學(xué)第十章DNA遺傳重組Figure15.10Transpositionisinitiatedbynickingthetransposonendsandtargetsiteandjoiningthenickedendsintoastrandtransfercomplex.

細(xì)胞學(xué)第十章DNA遺傳重組Figure15.13Mutranspositiongeneratesacrossoverstructure,whichisconvertedbyreplicationintoacointegrate.

復(fù)制型：細(xì)胞學(xué)第十章DNA遺傳重組Figure15.12Transpositionmayfuseadonorandrecipientrepliconintoacointegrate.Resolutionreleasestworeplicons,eachcontainingacopyofthetransposon.

非復(fù)制型：細(xì)胞學(xué)第十章DNA遺傳重組Figure15.14Nonreplicativetranspositionresultswhenacrossoverstructureisreleasedbynicking.ThisinsertsthetransposonintothetargetDNA,flankedbythedirectrepeatsofthetarget,andthedonorisleftwithadouble-strandbreak.

細(xì)胞學(xué)第十章DNA遺傳重組4.轉(zhuǎn)座子的某些遺傳學(xué)效應(yīng)

1）插入位置上出現(xiàn)新的基因；

2）造成插入位置上出現(xiàn)靶DNA的少數(shù)核苷酸對(duì)的重復(fù)；

3）轉(zhuǎn)座后原來(lái)位置上保持原有的轉(zhuǎn)座子；

4）引起染色體的畸變；

5）切除。細(xì)胞學(xué)第十章DNA遺傳重組5.真核生物的轉(zhuǎn)座子及其應(yīng)用:Figure15.27Hybriddysgenesisisasymmetrical;itisinducedbyPmalexMfemalecrosses,butnotbyMmalexPfemalecrosses.細(xì)胞學(xué)第十章DNA遺傳重組P轉(zhuǎn)座子:Ingermlinetissues,anadditionalsplicingeventoccurstoremoveintron3.ThisconnectsallfouropenreadingframesintoanmRNAthatistranslatedtogenerateaproteinof87kD.Thisproteinisthetransposase.Two