cloning HIV-1 co-receptor

cloning HIV-1 co-receptor -...

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Unformatted text preview: 10.6),andpeptideatconcentrationsupto100puM (finalpH,8.3).Afterincubationfor30minatroom temperature, reaction mixtures were filtered by meansofadouble-filtersystem(34)withBA85nitro- celluloseandNA45DEAEmembranes(Schleicher& Schuell).Allexperimentsweredoneatleastintripli- cate.DatawerequantitatedwithaPhosphorlmager (MolecularDynamics). 11.Although thepeptides bind slightlylesstightlyto DNA intheabsenceofMg2+(because100to200 mM NaCIcansubstitutefor10mM Mg2+),thisre- quirementisnotaspecificdivalentioneffect. 12. 0. N.Voloshinand R.D.Camerini-Otero, unpub- lisheddata. 13.T.Koller,E.DiCapua,A.Stasiak,inMechanismsof DNA ReplicationandRecombination,N.Cozzarelli, Eds.(Liss,NewYork,1983),pp.723-729. 14.X.YuandE.H.Egelman,J.Mol.Biol.232,1(1993); T.Ogawa,X.Yu,A.Shinohara,E.H.Egelman,Sci- ence259,1896(1993). 15.A.M.MaxamandW.Gilbert,MethodsEnzymol.65, 499(1980). 16. Peptide-DNA complexes were obtained as de- scribed(10),exceptthatthevolumewas100or140 Rl. Inthelatterinstance,40 pRl wereusedforfilter bindingandtherestwastreatedwithPP.Forbinding ofRecAproteintoBS-S1,2.7puMRecAand0.5p.M 32P-oligonucleotidewereincubated inavolumeof 100p,1containing25mM tris-HCI(pH7.5),10mM MgCI2,20mM NaCl,0.4 mM dithiothreitol(DTT),0.5 mM EDTA,0.3mM ATPyS,1.1mM adenosine5'- diphosphate(ADP),andBSA(10,g/ml)for30minat 37C.Complexesformedweresubjectedto0.5mM PPfor1minatroomtemperature.Reactionswere terminatedbyadditionofdimethylsulfatestopsolu- tion(15)followedbyethanolprecipitation.Aftertreat- mentwith1M pyrrolidine(95C,20min),thesam- pleswereseparatedona20% ureagel(Sequagel, NationalDiagnostic).ReactivityofssDNAinthecom- plexwithRecAislessthanthatinthecomplexeswith thepeptidesbecauseofthepresenceofDTT,which isaquencherofthePP modification, intheRecA reaction. 17. Inthisassay,thebindingofRecAtodsDNA [inthe presenceofeither0.3mM ATPySplus1.1mM ADP (22),or0.3mM ATPyS]doesnotinduceahyperre- activityofthethyminestoPP(12). 18.AlltheCD spectraweremeasuredonaJasco720 Spectropolarimeter.Theinstrument was calibrated with a 0.06% ammonium (+)-d1o-camphorsulfate solution,whichgenerates aCD intensityof190.4x 10-3 degreesat290.4nm. A baselineof10mM NaH2PO4bufferwassubtractedoutasbackground. Cellsof0.20-,0.50-,or1.00-mm pathlengthwere used and were maintained at22 + 1C.The re- sponseandbandwidthfordatacollectionwere2.0s and 1.0nm, respectively.Spectrawere smoothed withtheuseofSavitsky-Golaysmoothfunctionand are presented inmolar residue ellipticity(degrees M-1 cm-1)orasrawdata(10-3degrees). 19. R.W.Woody,inThePeptides:Analysis,Synthesis, Biology, V.J.Hruby, Ed. (Academic Press, New York,1985),vol.7,pp.15-114. 20. J.Kansy,B.Clack,D.Gray,J.Biomol.Struct.Dyn. 3,1079(1986). 21. L.Wang, 0.N.Voloshin,R.D.Camerini-Otero,un- publisheddata....
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This note was uploaded on 07/12/2011 for the course BIO 620 taught by Professor Hardy during the Spring '11 term at University of Florida.

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cloning HIV-1 co-receptor -...

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