Characterization of a Chinese Hamster Ovary Cell Mutant Having a Mutation in Elongation Factor-2 英文参考文献.docVIP

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Characterization of a Chinese Hamster Ovary Cell Mutant Having a Mutation in Elongation Factor-2 英文参考文献.doc

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Characterization of a Chinese Hamster Ovary Cell Mutant Having a Mutation in Elongation Factor-2 英文参考文献

CharacterizationofaChineseHamsterOvaryCellMutant HavingaMutationinElongationFactor-2 PradeepK.Gupta,ShihuiLiu,StephenH.Leppla* LaboratoryofBacterialDiseases,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth,Bethesda,Maryland,UnitedStatesofAmerica Abstract Retroviralinsertionalmutagenesisprovidesaneffectiveforwardgeneticmethodforidentifyinggenesinvolvedinessential cellularpathways.AChinesehamsterovarycelllinemutantresistanttoseveralbacterialADP-ribosylatingwasobtainedby thisapproach.ThetoxinsusedcatalyzeADP-ribosylationofeukaryoticelongationfactor2(eEF-2),blockproteinsynthesis, andcausecelldeath.Strikingly,intheCHOPR328mutantcells,theeEF-2substrateoftheseADP-ribosylatingtoxinswas foundtobemodified,butthecellsremainedviable.Asystematicstudyofthesecellsrevealedthepresenceofastructural mutation in one allele of the eEF-2 gene. This mutation, Gly717Arg, is close to His715, the residue that is modified to become diphthamide. This Arg substitution prevents diphthamide biosynthesis at His715, rendering the mutated eEF-2 non-responsivetoADP-ribosylatingtoxins,whilehavingnoapparenteffectonproteinsynthesis.Thus,CHOPR328cellsare heterozygous,havingwildtypeandmutanteEF-2alleles,withthelatterallowingthecellstosurviveeveninthepresenceof ADP-ribosylatingtoxins.Here,wereportthecomprehensivecharacterizationofthesecells. Citation:GuptaPK,LiuS,LepplaSH(2010)CharacterizationofaChineseHamsterOvaryCellMutantHavingaMutationinElongationFactor-2.PLoSONE5(2): e9078.doi:10.1371/journal.pone.0009078 Editor:Dong-YanJin,UniversityofHongKong,HongKong ReceivedNovember10,2009;AcceptedJanuary11,2010;PublishedFebruary5,2010 Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsPublicDomaindeclarationwhichstipulatesthat,onceplacedinthepublic domain,thisworkmaybefreelyreproduced,distributed,transmitted,modified,builtupon,orotherwiseusedbyanyoneforanylawfulpurpose. Funding:ThisresearchwassupportedbytheIntramuralResearchProgramoftheNationalInstitutesofHealth(NIH)