Identification and purification of young macronuclear anlagen from conjugating cells of Tetrahymena thermophila☆

[摘要] Populationsoftotalnucleicollectedfromgrowing,starved,andconjugatingcellsofTetrahymenathermophilahavebeenanalyzedbyflowmicrofluorometry.Intheseanalyses,twoparameters,size(lightscatter)andDNAcontent(fluorescenceafterstainingwithethidiumbromide),areevaluatedonindividualnuclei.Withthistechnique,wehaveidentifiedmacro-andmicronucleifrombothgrowingandstarvedcellsandshownthatthesenucleihaveDNAcontentsconsistentwithpreviouslypublishedreports.Whentotalnucleiareanalyzedfromconjugatingcellsinearlystagesofmacronuclearanlagenformation(9–10hraftermixingoppositematingtypes),onemajornewpopulationofnucleiisobservedwhichwasnotseenpreviouslyingrowingorstarvedcells.Thesenucleihaveasizeinbetweenthatofmacro-andmicronucleiandaDNAcontentverysimilartothatof4Cmicronuclei.Wehavealsoshownthatthisclassofnucleibecomesevidentpreciselyasmatingcellsentermacronuclearanlagenstage.Inlaterstagesofconjugation(16hr),membersofthis4CclassdisappearandapopulationofnucleiwithaDNAcontentcorrespondingto8Careobserved.Wesuggestthattheselarge4and8Cnucleirepresentyoungmacronuclearanlagenintheprocessoftransformationfrommicro-intomacronuclei.Wehavealsoexploitedtheuniquesizeanddensitypropertiesofmacronuclearanlagentopurifythesenucleifromconjugatingcells.Followingsedimentationatunitgravity,anlagenbehaveasnucleiintermediateinsizebetweenmacro-andmicronuclei.Thisisconsistentwiththefactthatanteriormicronucleiswellconsiderablyinsizefollowingthesecondpostzygoticdivisionandapproachthatofmacronuclei.Afterdensitycontrifugation,anlagenbehaveasnucleilessdensethanbothmacro-andmicronuclei.ThisisnotsurprisingsinceyounganlagenhaveaDNAcontentsimilartothatofmicronuclei(2–4C),butareconsiderablylarger.Microscopically,anlagenarerelativelyunstainedwithmethylgreenandaredarkerusingphasecontrastthaneithermacro-ormicronuclei.Thepurityand“developmentalage”ofouranlagenpreparationshavebeenmonitoredbyflowmicrofluorometry.Routinely,fractionscontaining80–90%of2–4Canlagen(3–4×107)canbeobtainedbyeither1gsedimentationordensitycentrifugation.Asby-productsofthesetwoprocedures,purifiedpopulationsofmacro-andmicronucleiarealsoobtained.Theseprocedureswillallowbiochemicalcharacterizationstobemadeofhowacondensed,transcriptionallyinactive,germinalnucleusisconvertedintoanextended,transcriptionallyactive,somaticnucleus.

[学科分类] 生物科学（综合）