生物文獻(xiàn)匯報(bào)課件

Functionalgenomics:thecomingofageforTetrahymenathermophilaAaronP.Turkewitz,EduardoOriasandGeoffreyKapler?

Tetrahymenathermophilahasbeenproventobeavaluablebiologicalmodelformolecularandgeneticstudiesofeukaryoticcells.Someinvestigationsontheciliatedprotozoanhaveprovidedtheinsightsintothemechanismofribozymes,self-splicingRNA,telomerestructureandfunction,DNAsequencereorganizationandsoon.Compartmentalizationofthegenomeintotwofunctionallydistinctnuclei,thesilentmicronucleusandthetranscriptionallyactivemacronucleus,providesapowerfulmeansforcontrollingtheexpressionoftransgenes.ＢａＣＫＧＲＯＵＮＤ．T.thermophila,andciliatedprotozoaingeneral,havelongcapturedtheinterestsandimaginationsofgeneticists,becausetheseunicellularorganismsmaintaintwofunctionallydistinctnucleiwithinthesamecytoplasmThepurposeofthisarticleistoshowcasethetoolsthatareavailableforfunctionalgenomicanalysisinTetrahymena,andtoillustratehowdifferentapproachescanbeharnessedtoaddressawiderangeofbiologicalproblems.AttheheartoftheseapproachesisavarietyofmethodsforefficientDNA

mediatedcelltransformation.Anappreciationofthespectrumofpossibilitiesrequiresabriefreviewoftheunusualgeneticorganizationofthisorganism.ＰｕｒｐｏｓｅExperimentalinvestigationofthegeneofinterest§Overexpression§Antisenseinhibition§Germlineknockoutorgenereplacement§Heterokaryonanalysis§Somaticknockoutorgenereplacement(phenotypic

assortment)§Gene‘knock-in’§InduciblepromotercontentVersatilityofDNAtransformationmethodsTetrahymenacanbetransformedusinghigh-copy-numberautonomouslyreplicatingrDNAVECTORS,andgenetargetingvectors.ByvaryingthetimingforDNAtransformation,DNAmoleculescanbetargetedtothreedifferenttypesofnuclei(Fig.3):thegermlinemic,thedevelopingmacofconjugatingcells,andthematuremacofvegetativelydividingcells.Thesethreeconditionsallowforthegenerationofdistinctproductsthatcanbeexploitedfordifferentpurposes.Fromapracticalstandpoint,differenttransformationmethodscanbecombinedtofacilitatestructure–functionstudiesofagivengeneproduct.Forexample,germlinetransformationcanbeusedtogenerateahomozygous-nullmutantthatcansubsequentlybetransformedduringdevelopmenttostudyvariantformsofthegene．ＳｕｍｍａｒｙForwantofspace,wehavenotdiscussedanumberofongoingdevelopmentsinTetrahymenathatwilladvancetheutilityofthisorganismforexperimentalbiologistsfurther.Theseincludeextensivephysicalandgeneticmappingprojects,andthegenerationandlarge-scalesequencingofexpressedsequencetag(EST)libraries.T.thermophilaisahighlydevelopedunicell,belongingtoacladeofancientlineagewhosecellularcomplexityrivalsthatofhighlydifferentiatedtissues.ThankYou!Fig.3.Phenotypicassortmentillustratedduringtwocellcycles.ThetopcirclerepresentsaheterozygousG1macronucleus(mac)generatedeitherafteracrossorafterDNA-mediatedtransformationfollowedbyintegrativerecombination.Forclarity,themacshowsfourallelecopies(insteadof45):threearewildtypeandthefourthismutant;forexample,aknockoutallelewherethegeneisdisruptedbyinsertionofaneomycin-resistancecassette(red).ThefusedovalsrepresentmacsundergoingdivisionafterDNAreplication.Notethatallelecopiesarepartitionedrandomlyateachmacdivision.Ifneitherallelehasselectiveadvantage,assortantspureforeitheralleleareultimatelygenerated.Inthepresenceofneomycin,vegetativedescendantsthatacquire(bychancealone)ahigherfractionofmaccopieswiththedisruptedallelewillbeselectedfor;ifthedisruptedgeneisessential,bothalleleswillbemaintainedbybalancedselection.

rDNA(rRNA-encodingDNA）§rDNA是大核形成過(guò)程中由單一拷貝的小核rDNA復(fù)制而成的，游離于大核染色體外的回文二聚體；§同源性很高，沒有外源基因的插入，是基因工程的良好材料；§

rDNA位于核內(nèi)自主復(fù)制的DNA上，長(zhǎng)度約為21kb；§

rDNA不同的遺傳等位基因表現(xiàn)出不同的復(fù)制特點(diǎn):

當(dāng)不同的rDNA等位基因進(jìn)行比較時(shí)，序列的差異會(huì)顯示出來(lái)，這些重復(fù)的保守序列主要位于5’-非轉(zhuǎn)錄區(qū)（5’-NTS）的復(fù)制原點(diǎn)，因此，這些序列被推測(cè)是復(fù)制的陽(yáng)性調(diào)控因子。過(guò)表達(dá)（Overexpression）：本來(lái)基因表達(dá)是受到各種內(nèi)外信號(hào)的精確調(diào)控的，一旦這種調(diào)控機(jī)制的任何一個(gè)環(huán)節(jié)出現(xiàn)問(wèn)題就會(huì)失控?；虻谋磉_(dá)過(guò)程就可能進(jìn)入失控狀態(tài)，就有可能表現(xiàn)為表達(dá)過(guò)度，即該基因被過(guò)度的轉(zhuǎn)錄、翻譯，最終基因表達(dá)產(chǎn)物超過(guò)正常水平。反義抑制（Antisenseinhibition）

：主要相對(duì)于RNA來(lái)說(shuō)，DNA分為正義鏈、反義鏈，相對(duì)轉(zhuǎn)錄出來(lái)的RNA也分為正義RNA、反義RNA。反義RNA是一種能與mRNA分子互補(bǔ)的RNA,可特異抑制某一mRNA的加工、翻譯以及DNA的復(fù)制,特異阻斷該基因的表達(dá)?；蚯贸腔虼虬屑夹g(shù)的一種，類似于基因的同源重組。指外源DNA與受體細(xì)胞基因組中序列相同或相近的基因發(fā)生同源重組，從而代替受體細(xì)胞基因組中的相同/相似的基因序列，整合入受體細(xì)胞的基因組中。此法可產(chǎn)生精確的基因突變，也可正確糾正機(jī)體的基因突變。