CHAPTER4

DNAandRNA:MoleculesofheredityDNAisaverylong,threadlikemacromoleculemadeupofalargenumberofdNt(脫氧核糖核苷酸),eachcomposedofabase,asugar,andaphosphategroupthebaseofDNAmoleculescarrygeneticinformationsugarandphosphategroupsperformastructuralroleDNAisthegeneticmaterialDNAdoublehelixthecomplementarynatureofthetwochains,eachisatemplatefortheotherinDNAreplicationDNApolymerases聚合酶replicateDNAbytakinginstructionsfromDNAtemplatesthegenesofallcellsandmanyvirusesaremadeofDNAsomevirusesuseRNAastheirgeneticmaterialDNAconsistsof4kindsofbasesjoinedtoasugar-phosphatebackboneDNAisapolymerofdNtunitsNt(nucleotide)核苷酸consistsofnitrogenousbase(purine嘌呤:adenine腺,guanine鳥orpyrimidine嘧啶:thymine胸腺,cytosine胞),sugar(deoxyribose),andoneormorephosphategroupsNs(nucleoside)核苷consistsofpurineorpyrimidinebasebondedtosugar4NsinDNAare:deoxyadenosine,deoxyguanosine,deoxythymidine,anddeoxycytidineIndNs,N-9ofpurineorN-1ofpyrimidineisattatchedtoC-1ofdeoxyriboseThe3’-hydroxylofthesugarmoietyofonedeoxyribonucleotideisjoinedtothe5’-hydroxyloftheadjacentsugarbyaphosphodiesterbridge磷酸二酯鍵transformationofPneumococci肺炎球菌byDNArevealedthatgenesaremadeofDNAApneumococcusisnormallysurroundedbyaslimy,glisteningpolysaccharidecapsule莢膜thisouterlayerisessentialforthepathogenicity致病力ofthebacterium光滑型肺炎球菌外包一層粘性發(fā)光的多糖莢膜是其致病的必要成分，無(wú)莢膜的粗糙型因缺乏一種合成多糖所必要的酶而無(wú)法合成莢膜whichcausespneumoniainhumansandothersusceptiblemammalsmutantsdevoidofapolysaccharidecoatarenotpathogenicnormalbacteriumisreferredtoastheSform(菌落光滑)whereasmutantswithoutcapsulesarecalledRforms(lackinganenzymeneededforthesynthesisofcapsularpolysaccharide)anonpathogenicRmutantcouldbetransformedintothepathogenicSform供體細(xì)胞的DNA進(jìn)入受體細(xì)胞而導(dǎo)入新的遺傳信息,使非致病的R突變型轉(zhuǎn)化成致病的S型miceisinjectedwithamixtureofliveRandheat-killedSpneumococcithismixturewaslethaltothemice,whereaseitherliveRorheat-killedSpneumococcialonewerenotthebloodofthedeadmicecontainedliveSpneumococcitheheat-killedSpneumococcihadsomehowtransformedliveRintoliveS.thischangewaspermanent:thetransformedpneumococciyieldedpathogenicprogenyoftheSformthisR→StransformationcanoccurinvitroOswaldAveryandotherspublishedtheirdiscovery“anucleicacidofthedeoxyribosetypeisthefundamentalunitofthetransformingprincipleofpneumococcustypeⅢ”

Theexperimentalbasisfortheirconclusionwas對(duì)純化的,高度活性的(引起轉(zhuǎn)化的)物質(zhì)進(jìn)行分析:elementalchemicalanalysisagreedcloselywiththatcalculatedforDNAoptical,ultracentrifugal,diffusive擴(kuò)散,andelectrophoreticpropertiesofthepurifiedmaterialwerelikethoseofDNAtherewasnolossoftransformingactivityuponextractionofproteinorlipidthepolypeptide-cleavingenzymestrypsinandchymotrypsin胰凝乳蛋白酶didnotaffecttransformingactivityribonuclease核糖核酸酶hadnoeffectonthetransformingprincipletransformingactivitywaslostfollowingtheadditionofdeoxyribonuclease脫氧核糖核酸酶

thisworkisalandmarkinthedevelopmentofbiochemistryuntil1944,itwasgenerallyassumedthatchromosomalproteinscarrygeneticinformationandthatDNAplaysasecondaryroleitwasshatteredbythefindingthatpurifiedDNAhasgeneticspecificityFurthersupportforthegeneticroleofDNAcamefromthestudiesofavirusthatinfectsthebacteriumE.coliT2bacteriophage噬菌體consistsofacoreofDNAsurroundedbyaproteincoatDNA內(nèi)芯+蛋白外殼TheresultsoftheexperimentsofA.HersheyandM.Chasewere:mostofthephageDNAwasfoundinthebacteriamostofthephageproteinwasfoundinthesupernatant上清液theblender搗碎器treatmenthadalmostnoeffectonthecompetence感受態(tài)oftheinfectedbacteriatoproduceprogenyvirus病毒外殼（蛋白）對(duì)（細(xì)菌）細(xì)胞內(nèi)噬菌體生長(zhǎng)沒(méi)有作用additionalexperimentsledtotheconclusionaphysicalseparationofthephageT2intogeneticandnon-geneticpartsispossible…Thesulfur-containingproteinofrestingphageparticlesisconfined限于toaprotectivecoatthatisresponsiblefortheadsorptionofbacteria附著細(xì)菌,andfunctionsasaninstrumentfortheinjectionofthephageDNAintothecell.TheDNAhassomefunction.inagivenspeciestheDNAcontentisthesameforallcellsthathaveadiploidsetofchromosomes二倍體.Haploid單倍體cellswerefoundtohavehalfasmuchDNAThediscoveryoftheDNAdoublehelixbyWastonandCrickrevolutionizedbiologyWastonandCrick’smodelofDNA:twohelicalpolynucleotidechainsarecoiledaroundacommonaxis.Thechainsruninoppositedirections反平行雙螺旋thePurandPyrbasesareontheinsideofthehelix,whereasthephosphateanddeoxyriboseunitsareontheoutside.Theplanesofthebasesareperpendiculartothehelixaxis.Thoseofthesugarsarenearlyatrightanglestothoseofthebases.堿基在內(nèi),磷酸核糖骨架在外,堿基平面與對(duì)稱軸(即與骨架)垂直Thediameterofthehelixis20A.Adjacentbasesareseparatedby3.4Aalongthehelixaxisandrelatedbyarotationof36degrees.Hence,thehelicalstructurerepeatsafter10residuesoneachchain,thatis,atintervalsof34A.Thetwochainsareheldtogetherbyhydrogenbondsbetweenpairsofbases.AisalwayspairedwithT;GwithC.Thesequenceofbasesalongapolynucleotidechainisnotrestrictedinanyway.Theprecisesequenceofbasescarriesthegeneticinformation.遺傳信息是由堿基的精確順序決定的

themostimportantaspectoftheDNAdoublehelixisthespecificityofthepairingofthebases.A-T;G-C(becauseofstericandhydrogen-bondingfactors)（DNA結(jié)構(gòu)和功能的精髓）堿基配對(duì)的特異性：空間限制因素和氫鍵成鍵性質(zhì)thestericrestrictionisimposedbytheregularhelicalnatureofthesugar-phosphatebackboneofeachpolynucleotidechaintheglycosidicbonds糖苷鍵thatareattachedtoabondedpairofbasesareverynearly10.8AApurine-pyrimidinebasepairfitsperfectlyinthisspaceThebasepairingisfurtherrestrictedbyhydrogen-bondingrequirementsAcannotpairwithCbecausetherewouldbetwohydrogensnearoneofthebondingpositionsandnoneattheotherLikewise，GcannotpairwithTIncontrast,AformstwohydrogenbondswithT，GformsthreewithCtheearlierstudiesfoundthattheratiosofAtoTandofGtoCwerenearly1.0inallspeciesstudiedtheabove-mentionedistheBformofDNA.DNAcanassumedifferenthelicalforms，suchasA-DNA(由B型經(jīng)脫水形成)andZ-DNA(左手雙螺旋).Undermostphysiologicconditions,mostoftheDNAisintheWaston-CrickBformthecomplementarychainsactastemplatesforeachotherinDNAreplicationDNAreplicationissemiconservativeoneofthestrandsofeachdaughterDNAmoleculeisnewlysynthesized，whereastheotherispassedonunchangedfromtheparentDNAmoleculethedistributionof14Nand15Nwasrevealedbythenewlydevelopedtechniqueofdensity-gradientequilibriumsedimentation密度梯度沉降平衡Theabsenceof15NDNAindicatedthatparentalDNAwasnotpreservedasanintactunitonreplication.(byMeselsonandStahl)把細(xì)菌從15N介質(zhì)轉(zhuǎn)移到14N介質(zhì)中后，每隔一段時(shí)間取樣以提取其中的DNA，再用密度梯度技術(shù)分析。thedoublehelixcanbereversiblymelted解鏈thetwostrandsofaDNAhelixreadilycomeapartwhenthehydrogenbondsbetweenitspairedbasesaredisruptedbyheatingasolutionofDNAorbyaddingacidoralkalitoionizeitsbases

theunwindingofthedoublehelixiscalledmeltingbecauseitoccursabruptlyatacertaintemperaturethemeltingtemperature（Tm）isdefinedasthetemperatureatwhichhalfthehelicalstructureislost解鏈溫度:螺旋-線圈轉(zhuǎn)變的中點(diǎn)theabruptnessofthetransitionindicatesthattheDNAdoublehelixisahighlycooperative協(xié)同structure,heldtogetherbymanyreinforcingbondsitisstabilizedbythestackingofbases堿基堆積力aswellasbybasepairing配對(duì)堿基themeltingofDNAisreadilymonitoredbymeasuringitsabsorbanceoflightatwavelength260nm.Theunstackingofthebasepairsresultsinincreasedabsorbance,aneffectcalledhyperchromism增色性themeltingtemperatureofaDNAmoleculedependsmarkedlyonitsbasecomposition.DNArichinG-CbasepairshaveahigherTmthanthosehavinganabundanceofA-Tbasepairsinaddition，adjacentG-CbasepairsinteractmorestronglywithoneanotherthandoadjacentA-Tbasepairs.HencetheA-T-richregionsofDNAarethefirsttomeltthedoublehelixismeltedinvivobytheactionofspecificproteins（DnaAprotein）separatedcomplementarystrandsofDNAspontaneouslyreassociatetoformadoublehelixwhenthetemperatureisloweredbelowTm.Thisrenaturation復(fù)性processissometimescalledannealing退火DNAmoleculesareverylongDNAmoleculesmustbeverylongtoencodethelargenumberofproteinspresentineventhesimplestcellsE．colichromosomeisasinglemoleculeofdouble-helicalDNAconsistingof4millionbasepairsItsmassis2.6x109daltons(4x106x600)ithasahighlyasymmetricshapewhentakenoutofthecelllength:14x106A(1.4mm);diameter:20AthelargestchromosomeofDrosophilamelanogaster(黑腹果蠅)containsasingleDNAmoleculeof6.2x107basepairs,whichhasalengthof2.1cm(6.2x107x3.4÷108)suchhighlyasymmetricmoleculesareverysusceptibletocleavagebyshearingforcesthefollowingcomparisonsemphasizetheremarkablelengthandasymmetryofDNAmoleculespolyomavirus多瘤病毒:5100basepairs,lengthof1.7μmhemoglobin:diameterof65Aasroughlysphericalcollagen:lengthof3000A(oneofthelongestproteins)ManyDNAmoleculesarecircularandsupercoiledintactDNAmoleculesfrommanysourcesarecircularthegene-linkagemapofE.coliiscircularthetermcircularreferstothecontinuityoftheDNAchain,nottoitsgeometricalformDNAmoleculesinvivonecessarilyhaveaverycompactshapeThelengthofE.colichromosomeisabout1000timesaslongasitsgreatestdiameterE.coli染色體長(zhǎng)度(如完全伸展)是E.coli最大徑向(真實(shí))長(zhǎng)度的1000倍DNAfromtheT7bactreriophageislinear.DNAmoleculesofsomeviruses(λbacteriophage)interconvertbetweenlinearandcircularforms.Thelinearformispresentinsidethevirusparticle,whereasthecircularformispresentinthehostcellAnewpropertyappearsintheconversionofalinearDNAduplexintoaclosedcircularmolecule.TheaxisofthedoublehelixcanitselfbetwistedtoformasuperhelixAcircularDNAwithoutanysuperhelicalturnsisknownasarelaxedmolecule.SupercoilingisbiologicallyimportantforasupercoiledDNAhasamorecompactshapethanitsrelaxedcounterpart(packagingofDNAinthecell)supercoilingaffectsthecapacityofthedoublehelixtounwind,andtherebyaffectsitsinteractionwithothermoleculesDNAisreplicatedbypolymerasesthattakeinstructionsfromtemplatesDNApolymeraseⅠisa103-kdsinglepolypeptidechain.Itcatalyzesthestep-by-stepadditionofdNtunitstoaDNAchainDNApolymeraseⅠrequiresthefollowingcomponentstosynthesizeachainofDNAdATP,dGTP,dTTP,dCTP(activatedprecursors),andMg2+aprimer引物chainwithafree3’-OHgroupaDNAtemplate(single-ordoublestranded)Thechain-elongationreactioncatalyzedbyDNApolymeraseisanucleophilicattackofthe3’-OHterminusoftheprimerontheinnermostphosphorusatomofadNTP引物3’-OH端對(duì)加入的那個(gè)dNTP最靠里的(即α)磷原子的親核進(jìn)攻ElongationoftheDNAchainproceedsinthe5’→3’directionDNApolymerasecatalyzestheformationofaphosphodiesterbondonlyifthebaseontheingnucleotideiscomplementarytothebaseonthetemplatestrandThus,DNApolymeraseisatemplate-directedenzymeAnotherstrikingfeatureofDNApolymeraseⅠisthatitcorrectsmistakesinDNAbyremovingmismatchednucleotidesDNA聚合酶:1.是以模板為指導(dǎo)的酶2.它還具有糾錯(cuò)的功能,即能除去錯(cuò)配的(脫氧核糖)核苷酸ThesepropertiesofDNApolymeraseⅠcontributetotheremarkablyhighfidelity(保真度)ofDNAreplication,whichhasanerrorrateoflessthan10－8perbasepairsomeviruseshavesingle-strandedDNAduringpartoftheircycleNotallDNAisdouble-strandedDNAinφX174,asmallvirusthatinfectsE.coli,issingle-strandeditsbaseratiosdonotconformtotherule:A=T;G=CitssolutionismuchlessviscousthanasolutionofthesameconcentrationofE.coliDNA.Itshydrodynamicpropertiesarelikethoseofarandomlycoiledpolymer(theDNAdoublehelixbehavesasaquiterigidrod)1.單鏈DNA粘度較低2.動(dòng)力學(xué)性質(zhì)與隨機(jī)卷曲的聚合物相似,但不同于雙鏈DNA(剛性桿狀)3.它的堿基上的氨基易與甲醛反應(yīng),而雙鏈DNA中堿基上的氨基不易與外來(lái)試劑接觸.theaminogroupsofitsbasesreactreadilywithformaldehyde甲醛(thebasesindouble-helicalDNAarevirtuallyinaccessibletothisreagent)

Thefindingofthissingle-strandedDNAraiseddoubtsconcerningtheuniversalityofthesemiconservativereplicativescheme.ButφX174DNAissingle-strandedforonlyapartofthelifecycleofthevirus.infectedE.colicellscontainadouble-strandedformofφX174DNAthisdouble-helicalDNAiscalledthereplicateformofφX174DNA單鏈的φX174噬菌體在感染(進(jìn)入)E.coli后,以雙鏈形式存在,即“復(fù)制型”thegeneralityoftheWatson-Crickschemeforreplicationwasreinforcedbythefindingofthisdouble-strandedviralDNAintermediatethegeneofsomevirusesaremadeofRNAGenesinallprokaryoticandeukaryoticorganismsaremadeofDNA.Inviruses,genesmadeofeitherDNAorRNARNA,likeDNA,isalong,unbranchedpolymerconsistingofNtjoinedby3’→5’phosphodiesterbondsThecovalentstructureofRNAdiffersfromthatofDNAin:thesugarunitsinRNAareribosesratherthandeoxyribose.Ribosecontainsa2’-hydroxylgroupnotpresentindeoxyribose核糖-脫氧核糖(無(wú)2’-羥基)oneofthefourmajorbasesinRNAisuracil(U)insteadofthymine(T)尿嘧啶-胸腺嘧啶RNAmoleculescanbesingle-strandedordouble-strandedRNAcannotformadoublehelixoftheB-DNAtypebecauseofstericinterferencebythe2’-hydroxylgroupsofitsriboseunitsRNAcanadoptamodifieddouble-helicalform(likeA-DNA)TMV(TobaccoMosaicVirus)isoneofthebest-characterizedRNAvirusesItconsistsofasinglestrandofRNAsurroundedbyaproteincoatTheproteincanbeseparatedfromtheRNAbytreatmentoftheviruswithphenol苯酚theisolatedviralRNAisinfective,whereastheviralproteinisnotAfterinfection,theprogenyvirusalwaysco