PDF-Proc.Natl.Acad.Sci.USAVol.95,pp.10564±10569,September1998StructureofI

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IfromFlavobacteriumokeanokoitesisamemberoftheunusualtypeIIsclassofrestrictionendonucleasesthatrecognizeaspecificDNAsequenceandcleavenonspecificallyashortdistanceawayfromthatsequence1Ibindstheco

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Proc.Natl.Acad.Sci.USAVol.95,pp.10564±10569,September1998StructureofI: Transcript

IfromFlavobacteriumokeanokoitesisamemberoftheunusualtypeIIsclassofrestrictionendonucleasesthatrecognizeaspecificDNAsequenceandcleavenonspecificallyashortdistanceawayfromthatsequence1Ibindstheco. Proc.Natl.Acad.Sci.USA90(1993)10391days.Whenthecellswereforamicroinjectionexperiment,coverslipswerefirstmarkedwithadiamond-tippentofacilitatethelocalizationandvisualizationofmicroinjectedcells.After8- Proc.Natl.Acad.Sci.USA93(1996)9293exonjunctionsinthespy-1andspy-2mutants,genomicDNAwassubjectedtoPCRandtheproductsweresequenceddirectly.RESULTSANDDISCUSSIONIsolationofspy-4.Thespy-4mutantwasisolatedby Proc.Natl.Acad.Sci.USA90(1993)solvedinapyrogenicwaterandthesolutionwasfilteredthrougha0.22-,mcelluloseacetatefilter.Thelaminarinfiltratewaslyophilizedandresuspendedtoaconcentrationof1.47mg/ml.Lipopoly Proc.Natl.Acad.Sci.USA93(1996)Table1.PNDsdefinedbyautoimmuneantibodiesNeurologicsyndromeParaneoplasticnamePhenotypeTumorAntibodyCerebellardegenerationPan-cerebellardysfunction(Purkinjecell)TypicalBrea Proc.Natl.Acad.Sci.USA92(1995)7141FRETefficiencyRNAbulgeA7AgFIG.2.KinkingofDNAandRNAduplexesbybasebulgesisrevealedbyFRET.Thedistancebetweendonor(D)andacceptor(A)fluorophoresattachedtothe5'terminiofthe Proc.Natl.Acad.Sci.USA75(1978)RESULTSWhennormalhumanglobinDNAwasdigestedwithEcoRIandprobedwithamixtureofa-and(3-globincDNAs,fivemajorfragmentswereseen(Fig.1).Thelargestfragment,about21kilobases(kb)inl Proc.Natl.Acad.Sci.USA93(1996)11529BothLandL*canbeprotonatedonLys-156,butthepKaforprotonationisconsiderablylowerforL*thanforL;pKL*iswhilepKLis10.OneimportanttenetofthismodelisthattheIndsmutationsincre Proc.Natl.Acad.Sci.USA90(1993)2031dues,0.98forLexA-C52containingasinglecysteine,and0againforLexA-C52aftermodificationwith4-azidophenacylbromide,suggestingthatessentiallyallthecysteinesidechainshaverea 6148Biochemistry:LittleandHarperTable1.BacterialstrainsemployedSource,reference,StrainRelevantgenotypeorderivationAB1157recA+lexA+sup-37(15)DM49recA+lexA3sup-37(12)DM511recA+IexA3ts1-1sup-37(16)DM1187 Proc.Natl.Acad.Sci.USA90(1993)pHP45QC(13)bySmaIdigestionandgelpurification.pIB415isaderivativeofpIB410(ofIncPreplicon)containingvirAA63/240,avirAderivativelacking178aminoacidsintheperiplasmicdomain(14 Genetics:Slupskaetal.Table1.ComparisonofmutAandmutCwithmutA,CStrainLac+revertantsper(CC105)108cells,no.+2.3 TheNunproteinofbacteriophageHK022isamemberofthearginine-richmotiffamilyofRNAbindingproteins,whichincludesthephageNtranscriptionantiterminationproteinandtheHIVTatandRevproteins(Fig.1;refs.1±3).Nunbind Proc.Natl.Acad.Sci.USA82(1985)6415fragmentwasisolatedfroma0.8%agarosegelbyusingtheapparatusforpreparativegelelectrophoresis.DNASequencing.DNAsequencingusingdideoxynucleo-tideswasperformedasdescribedby Proc.Natl.Acad.Sci.USA93(1996)2583multiplexamplificationasdescribedabove.Theonlydiffer-encebetweenthesetwoproceduresisthatsomePprimersusedinMGSmaycontainsingle-basemismatchestothetemplatestoconvertthe

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