RNA and protein synthesis during zoospore differentiation in synchronized cultures of Blastocladiella☆

[摘要] TechniquesaredescribedforproducingthehighlysynchronizeddifferentiationofBlastocladiellazoosporesinliquidculture.Undertheseconditions,theplantsformdischargepapillaeat1712hours,nuclearcapformationandzoosporecleavageoccurjustbefore19hours,andzoosporedischargeiscompleteby1912hours.Thepatternsofchangeindryweight,RNA,DNA,andproteinwereexaminedbeforeandaftertheinductionofdifferentiationattheendoftheexponentialgrowthphase.Afterchangingfromtherichgrowthmediumtoadilutesaltsmedium,allsyntheticratesdecrease,andnetincreaseinDNA,RNA,dryweight,andproteinceasesat1612,1612,17,and1712hours,respectively.SignificantRNAdegradationoccursafter1612hoursresultinginafinal35%lossinthetotalamount.IncorporationofexternallyaddedC14-uracilintoRNAfallstoaverylowvalueby17hours,whileC14-leucineincorporationintoproteinreachesamaximumbetween1612and1712hours,followedbyasteadydecline.Pulselabelinganddensitygradientanalysisofthewhole-cellRNAdemonstratedthatbefore1612hourstransfer-RNAanda“heavy”RNAfractionofhighspecificactivitywererapidlylabeled,followedbylabelingintheribosomalpeaks.NouracilenteredRNAduringa10-minutepulseofplantsat,orafter,17hours.ThecessationofmajorRNAsynthesisafter1612hourswasaccompaniedbyasignificantdecreaseinapparenturacilpoolsize.Inanexperimentwherethepoolswereheavilylabeledbefore16hours,asmallamountofuracilincorporationcouldbedetectedafter17hours,buttheextensiveRNAdegradationandfailureofexogenousuraciltoenterthepoolspreventedareliableestimateoftheactualquantityofnewRNAproduced.However,whenthewhole-cellRNAwasrandomlylabeledduringexponentialgrowthandexaminedduringdifferentiation,noevidencecouldbeobtainedforsignificantturnoverofthisRNAafter1612hours,despitealargelossinthetotalamount.Theseresultsprovideddirectevidencefortheconservationofpreexisting,exponential-phaseribosomesbyaggregationtoformthezoosporenuclearcap.Indirectevidencefortherequirementofmessenger-RNAsynthesisduringsporedifferentiationwasobtainedbytheuseofactinomycinD,puromycin,andp-fluorophenylalanine(PFP).Puromycinat100μg/mlwasnoteffectiveinreducingleucineincorporationanddidnotinhibitpapillaformation,butdidpreventzoosporecleavage.Thepapillaeformedinthepresenceofpuromycinwere,however,multipleandabnormal.ActinomycinD(25μg/ml)andtheaminoacidanalogPFP(0.005M)wereeffectiveinhibitorsfortheincorporationofuracilandleucine,respectively.Bothcausedinhibitionofpapillaeonlyifaddedtoplants30minutesormorebeforethesewereformed.Bothalsopreventedsporecleavageonlywhenaddedtoplants1hourbeforethisevent.Fromtheiridenticalpatternsofinhibition,itwasconcludedthatpapillaformationandcleavageprobablyrequiretheproductionofoneormoreshort-livedmessenger-RNA's30minutesand1hour,respectively,beforetheyoccur.

[学科分类] 生物科学（综合）