抑制粟酒裂殖酵母中過程表達編碼tRNA3’末端加工酶基因Trz2毒性的基因的篩選.pdf

1、符號說明符號說明英文縮寫英文全稱中文全稱aaaminoacid氨基酸BSAbovineserumalbumin牛血清白蛋白DAPI4’，6diamidino2phenylindole4、6二脒基2苯基吲哚EDTAEthyleneDiamineTetraaceticAcid乙二胺四乙酸GeneDBS.pombeGenomeDatabaseS.pombe基因組數(shù)據(jù)庫GFPgreenfluescentprotein綠色熒光蛋白EGFPenha

2、ncedgreenfluescentprotein加強型綠色熒光蛋白ntNucleotide核苷酸PBSPhosphateBufferedSaline磷酸鹽緩沖液PCRpolymerasechainreaction聚合酶鏈式反應(yīng)PEGpolyethyleneglycol聚乙二醇pretRNAprecursortRNA前體tRNARNasePRibonucleaseP核糖核酸酶PSDSsodiumdodecylsulfate十一燒基硫酸納

3、SpTrzlpS.pombetRNaseZu粟酒裂殖酵母tRNaseZL1蛋白SpTrz2pS.pombetRNaseZL2粟酒裂殖酵母tRNaseZL2蛋白TrisTris(hydroxymethyl)aminomethane三輕甲基氨基甲烷tRNaseZtRNA3endonucleasetRNA3末端加工核酸內(nèi)切酶IABSTRACTABSTRACTAsanadaptermoleculebetweennucleonicandprote

4、instRNAhasacentralroleinbiology.The3CCAtripletoftRNAisaprerequisiteftRNAaminoacylation.ButineukaryotesmanyeubacteriasomearchaeatheirtRNAgenesnotencodeCCAendonucleasetRNaseZcatalyzesthegenerationofthemature3!endoftRNAprec

5、urssthroughspecificendonucleolyticcleavageprovidingthesubstratefCCAadditionbytRNAnucleotidyltransferase.tRNaseZenzymesbelongtothesuperfaxnilyofmetalloPlactamases(MBL).Thissuperfamilycontainsfivehighlyconservedmotifsinclu

6、dingmotifIIalsocalledhistidinemotif(HxHxDHxisanyhyhobicresidues)aacteristicoftheMBLsuperfamily.Thesearehighlyconservedinactivemembersofthesuperfamilyandparticipateinmetalcodinationandthehydrolysisreaction.tRNaseZcT1ispre

7、sentinashtfm(tRNaseZ)alongfm(tRNaseZ).ThehumantRNaseZgeneELAC2wasinitiallyfoundasaprostatecancersusceptibilitygeneSchizosacomycespombecontainstwotRNaseZLgenesdesignatedsptrzlandsptrz2Wecertificatedthatthetwogenesbothwere

8、essentialgenesweshowedthatthelocalizationthefunctionofthegenesptrzlinourlab5whichshowedthatthefunctionofthegenesptrz2.5.pombewasusedasamodelganismtoinvestigatethefunctionsofitstRNaseZ(SpTrz2p)inthisstudyfpromotingtheunde

9、rstandingofthediseasewhichcausedbythe3endprocessingdefectofpretRNA.Themajfindingswerelistedasfollows:1.Highleveloverexpressionofsptrzlhadnodetectablephenotypes.Incontraststrongoverexpressionofsptrz2waslethalinwildtypecel

10、lsresultsinmphologicalabnmalitiesincludingswollenroundcellsdemonstratingthatthecrectexpressionlevelofsptrz2wascritical.2.WeintroducedmutationsontheconservedmotifII(HxHxDH)usingoverlapPCRbasedmutagenesismethodwefounditthe

11、sametothewildtype.Itshowedthatthedeathstrongoverexpressionofsptrz2hadnoconnectwiththemetalcodinationthehydrolysisofthehistidinemotif.3.ScaningthecDNAlibraryoffissionyeastwhichcalledSPLE1，Wefoundsomecidatesactlgas2seemedi