生物文献汇报

生物文献汇报第1页/共20页☞

Tetrahymenathermophilahasbeenproventobeavaluablebiologicalmodelformolecularandgeneticstudiesofeukaryoticcells.Someinvestigationsontheciliatedprotozoanhaveprovidedtheinsightsintothemechanismofribozymes,self-splicingRNA,telomerestructureandfunction,DNAsequencereorganizationandsoon.Compartmentalizationofthegenomeintotwofunctionallydistinctnuclei,thesilentmicronucleusandthetranscriptionallyactivemacronucleus,providesapowerfulmeansforcontrollingtheexpressionoftransgenes.ＢａＣＫＧＲＯＵＮＤ第2页/共20页．T.thermophila,andciliatedprotozoaingeneral,havelongcapturedtheinterestsandimaginationsofgeneticists,becausetheseunicellularorganismsmaintaintwofunctionallydistinctnucleiwithinthesamecytoplasm第3页/共20页Fig.1.ApairofconjugatingTetrahymena,atthecompletionofmeiosisbutbeforenuclearexchange.Oneofthemeioticproductsineachcellwillgeneratetwogametepronucleithatwillbeinvolvedinreciprocalfertilizationacrossthematingjunction.Nucleiarecoloredgreen(withSytox);thecilia(blue)werelabeledusinganantibodythatrecognizesβ-tubulin(courtesyofJoeFrankel,UniversityofIowa,IA,USA);thecortex(magenta)islabeledusinganantiserumthatrecognizesacortically-localizedcalcium-bindingofOsamuNumata,Uniprotein(courtesyversityofTsukuba,Japan).PhotographiscourtesyofEricCole(StOlafCollege,MN,USA).第4页/共20页ThepurposeofthisarticleistoshowcasethetoolsthatareavailableforfunctionalgenomicanalysisinTetrahymena,andtoillustratehowdifferentapproachescanbeharnessedtoaddressawiderangeofbiologicalproblems.AttheheartoftheseapproachesisavarietyofmethodsforefficientDNA

mediatedcelltransformation.Anappreciationofthespectrumofpossibilitiesrequiresabriefreviewoftheunusualgeneticorganizationofthisorganism.Ｐｕｒｐｏｓｅ第5页/共20页Experimentalinvestigationofthegeneofinterest§Overexpression§Antisenseinhibition§Germlineknockoutorgenereplacement§Heterokaryonanalysis§Somaticknockoutorgenereplacement(phenotypic

assortment)§Gene‘knock-in’§Induciblepromotercontent第6页/共20页VersatilityofDNAtransformationmethodsTetrahymenacanbetransformedusinghigh-copy-numberautonomouslyreplicatingrDNAVECTORS,andgenetargetingvectors.ByvaryingthetimingforDNAtransformation,DNAmoleculescanbetargetedtothreedifferenttypesofnuclei(Fig.3):thegermlinemic,thedevelopingmacofconjugatingcells,andthematuremacofvegetativelydividingcells.Thesethreeconditionsallowforthegenerationofdistinctproductsthatcanbeexploitedfordifferentpurposes.Fromapracticalstandpoint,differenttransformationmethodscanbecombinedtofacilitatestructure–functionstudiesofagivengeneproduct.Forexample,germlinetransformationcanbeusedtogenerateahomozygous-nullmutantthatcansubsequentlybetransformedduringdevelopmenttostudyvariantformsofthegene．第7页/共20页ＳｕｍｍａｒｙForwantofspace,wehavenotdiscussedanumberofongoingdevelopmentsinTetrahymenathatwilladvancetheutilityofthisorganismforexperimentalbiologistsfurther.Theseincludeextensivephysicalandgeneticmappingprojects,andthegenerationandlarge-scalesequencingofexpressedsequencetag(EST)libraries.T.thermophilaisahighlydevelopedunicell,belongingtoacladeofancientlineagewhosecellularcomplexityrivalsthatofhighlydifferentiatedtissues.第8页/共20页Itispreciselythiscomplexity,thepresenceoffeaturesthatarecharacteristicofmetazoansbutthatareabsentorlesseasilystudiedinothersimpleeukaryoticsystems,thatmakesTetrahymenasowellsuitedforawiderangeoffundamentalproblemsincludinggermlineorsomaticdifferentiation,programmedDNArearrangements,distributionofmitoticchromosomes,histonemodifications,microtubulediversityandtubulinmodifications,basalbodyduplication,phagocytosis,functionofrRNA,andregulatedpolypeptidesecretion.Thesebiologicalfeaturesandthefunctionalgenomicstoolsdescribedinthisarticlewillgreatlyenhancethevalueofthegenomesequencingproject,currentlyunderadvancedplanningbytheciliateresearchcommunity第9页/共20页ThankYou!第10页/共20页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.第11页/共20页rDNAvectors:VectorsbasedontherDNAreplicon.

BecausetherDNAchromosomeismaintainedat~9000copiesinwild-typecells,rDNAvectorswillsimilarlybepresentathighcopynumber.第12页/共20页

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。第13页/共20页过表达（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