《生物文献汇报》PPT课件.ppt

Functionalgenomics:thecomingofageforTetrahymenathermophilaAaronP.Turkewitz,EduardoOriasandGeoffreyKapler,Tetrahymenathermophilahasbeenproventobeavaluablebiologicalmodelformolecularandgeneticstudiesofeukaryoticcells.Someinvestigationsontheciliatedprotozoanhaveprovidedtheinsightsintothemechanismofribozymes,self-splicingRNA,telomerestructureandfunction,DNAsequencereorganizationandsoon.Compartmentalizationofthegenomeintotwofunctionallydistinctnuclei,thesilentmicronucleusandthetranscriptionallyactivemacronucleus,providesapowerfulmeansforcontrollingtheexpressionoftransgenes.,T.thermophila,andciliatedprotozoaingeneral,havelongcapturedtheinterestsandimaginationsofgeneticists,becausetheseunicellularorganismsmaintaintwofunctionallydistinctnucleiwithinthesamecytoplasm,Fig.1.ApairofconjugatingTetrahymena,atthecompletionofmeiosisbutbeforenuclearexchange.Oneofthemeioticproductsineachcellwillgeneratetwogametepronucleithatwillbeinvolvedinreciprocalfertilizationacrossthematingjunction.Nucleiarecoloredgreen(withSytox);thecilia(blue)werelabeledusinganantibodythatrecognizes-tubulin(courtesyofJoeFrankel,UniversityofIowa,IA,USA);thecortex(magenta)islabeledusinganantiserumthatrecognizesacortically-localizedcalcium-bindingofOsamuNumata,Uniprotein(courtesyversityofTsukuba,Japan).PhotographiscourtesyofEricCole(StOlafCollege,MN,USA).,ThepurposeofthisarticleistoshowcasethetoolsthatareavailableforfunctionalgenomicanalysisinTetrahymena,andtoillustratehowdifferentapproachescanbeharnessedtoaddressawiderangeofbiologicalproblems.AttheheartoftheseapproachesisavarietyofmethodsforefficientDNAmediatedcelltransformation.Anappreciationofthespectrumofpossibilitiesrequiresabriefreviewoftheunusualgeneticorganizationofthisorganism.,ExperimentalinvestigationofthegeneofinterestOverexpressionAntisenseinhibitionGermlineknockoutorgenereplacementHeterokaryonanalysisSomaticknockoutorgenereplacement(phenotypicassortment)Geneknock-inInduciblepromoter,content,VersatilityofDNAtransformationmethodsTetrahymenacanbetransformedusinghigh-copy-numberautonomouslyreplicatingrDNAVECTORS,andgenetargetingvectors.ByvaryingthetimingforDNAtransformation,DNAmoleculescanbetargetedtothreedifferenttypesofnuclei(Fig.3):thegermlinemic,thedevelopingmacofconjugatingcells,andthematuremacofvegetativelydividingcells.Thesethreeconditionsallowforthegenerationofdistinctproductsthatcanbeexploitedfordifferentpurposes.Fromapracticalstandpoint,differenttransformationmethodscanbecombinedtofacilitatestructurefunctionstudiesofagivengeneproduct.Forexample,germlinetransformationcanbeusedtogenerateahomozygous-nullmutantthatcansubsequentlybetransformedduringdevelopmenttostudyvariantformsofthegene,Forwantofspace,wehavenotdiscussedanumberofongoingdevelopmentsinTetrahymenathatwilladvancetheutilityofthisorganismforexperimentalbiologistsfurther.Theseincludeextensivephysicalandgeneticmappingprojects,andthegenerationandlarge-scalesequencingofexpressedsequencetag(EST)libraries.T.thermophilaisahighlydevelopedunicell,belongingtoacladeofancientlineagewhosecellularcomplexityrivalsthatofhighlydifferentiatedtissues.,Itispreciselythiscomplexity,thepresenceoffeaturesthatarecharacteristicofmetazoansbutthatareabsentorlesseasilystudiedinothersimpleeukaryoticsystems,thatmakesTetrahymenasowellsuitedforawiderangeoffundamentalproblemsincludinggermlineorsomaticdifferentiation,programmedDNArearrangements,distributionofmitoticchromosomes,histonemodifications,microtubulediversityandtubulinmodifications,basalbodyduplication,phagocytosis,functionofrRNA,andregulatedpolypeptidesecretion.Thesebiologicalfeaturesandthefunctionalgenomicstoolsdescribedinthisarticlewillgreatlyenhancethevalueofthegenomesequencingproject,currentlyunderadvancedplanningbytheciliateresearchcommunity,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.,rDNAvectors:VectorsbasedontherDNAreplicon.BecausetherDNAchromosomeismaintainedat9000copiesinwild-typecells,rDNAvectorswillsimilarlybepresentathighcopynumber.,rDNA(rRNA-encodingDNA）,rDNA是大核形成过程中由单一拷贝的小核rDNA复制而成的，游离于大核染色体外的回文二聚体；同源性很高，没有外源基因的插入，是基因工程的良好材料；rDNA位于核内自主复制的DNA上，长度约为21kb；rDNA不同的遗传等位基因表现出不同的复制特点:当不同的rDNA等位基因进行比较时，序列的差异会显示出来，这些重复的保守序列主要位于5-非转录区（5-NTS）的复制原点，因此，这些序列被推测是复制的阳性调控因子。,过表达（Overexpression）：本来基因表达是受到各种内外信号的精确调控的，一旦这种调控机制的任何一个环节出现问题就会失控。基因的表达过程就可能进入失控状态，就有可能表现为表达过度，即该基因被过度的转录、翻译，最终基因表达产物超过正常水平。,反义抑制（Antisenseinhibition）：主要相对于RNA来说，DNA分为正义链、反义链，相对转录出来的RNA也分为正义RNA、反义RNA。反义RNA是一种能与mRNA分子互补的RNA,可特异抑制某一mRNA的加工、翻译以及DNA的复制,特异阻断该基因的表达。,基因敲除是基因打靶技术的一种，类似于基因的同源重组。指外源DNA与受体细胞基因组中序列相同或相近的基因发生同源重组，从而代替受体细胞基因组中的相同/相