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1、Functionalgenomics:thecomingofageforTetrahymenathermophilaAaronP.Turkewitz,EduardoOriasandGeoffreyKapler☞Tetrahymenathermophilahasbeenproventobeavaluablebiologicalmodelformolecularandgeneticstudiesofeukaryoticcells.Someinvestigationsontheciliatedprotozoanhaveprovidedthein
2、sightsintothemechanismofribozymes,self-splicingRNA,telomerestructureandfunction,DNAsequencereorganizationandsoon.Compartmentalizationofthegenomeintotwofunctionallydistinctnuclei,thesilentmicronucleusandthetranscriptionallyactivemacronucleus,providesapowerfulmeansforcontro
3、llingtheexpressionoftransgenes.BaCKGROUND.T.thermophila,andciliatedprotozoaingeneral,havelongcapturedtheinterestsandimaginationsofgeneticists,becausetheseunicellularorganismsmaintaintwofunctionallydistinctnucleiwithinthesamecytoplasmFig.1.ApairofconjugatingTetrahymena,att
4、hecompletionofmeiosisbutbeforenuclearexchange.Oneofthemeioticproductsineachcellwillgeneratetwogametepronucleithatwillbeinvolvedinreciprocalfertilizationacrossthematingjunction.Nucleiarecoloredgreen(withSytox);thecilia(blue)werelabeledusinganantibodythatrecognizesβ-tubulin
5、(courtesyofJoeFrankel,UniversityofIowa,IA,USA);thecortex(magenta)islabeledusinganantiserumthatrecognizesacortically-localizedcalcium-bindingofOsamuNumata,Uniprotein(courtesyversityofTsukuba,Japan).PhotographiscourtesyofEricCole(StOlafCollege,MN,USA).Thepurposeofthisarticl
6、eistoshowcasethetoolsthatareavailableforfunctionalgenomicanalysisinTetrahymena,andtoillustratehowdifferentapproachescanbeharnessedtoaddressawiderangeofbiologicalproblems.AttheheartoftheseapproachesisavarietyofmethodsforefficientDNAmediatedcelltransformation.Anappreciation
7、ofthespectrumofpossibilitiesrequiresabriefreviewoftheunusualgeneticorganizationofthisorganism.PurposeExperimentalinvestigationofthegeneofinterest§Overexpression§Antisenseinhibition§Germlineknockoutorgenereplacement§Heterokaryonanalysis§Somaticknockoutorgenereplacement(phe
8、notypicassortment)§Gene‘knock-in’§InduciblepromotercontentVersatilityofDNAtransformationmethodsT