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Chapter5ExploringGenes&GenomesRecombinantDNAtechnologyhasrevolutionizedbiochemistrysinceitcameintobeinginthe1970s.Thegeneticendowmentoforganismscannowbepreciselychangedindesignedways.RecombinantDNAtechnologyisafruitofseveraldecadesofbasicresearchonDNA,RNA,andvirusesandbenefitsalot,includinghumangenomesequencing.Thistechnologydepends,first,onenzymesthatcancut,join,andreplicateDNAandreversetranscribeRNA.Restrictionenzymes:cut;DNAligases:join;base-pairing:DNAcombination;virusesorplasmids:deliverandreplicatethroughtheirownreplicateoriginorincorporationofforeignDNAsintothehostgenome.Newinsights(forexample):•Theregulationofgeneexpressionincanceranddevelopmentandtheevolutionaryhistoryofproteinsaswellasorganisms.•Newproteinscanbecreatedbyalteringgenesinspecificwaystoprovidedetailedviewsintoproteinfunction.•Clinicallyusefulproteins,suchashormones,arenowsynthesizedbyrecombinantDNAtechniques.•Cropsarebeinggeneratedtoresistpestsandharshconditions.Alltheseeffortsofferusnewopportunitiestoimproveourlifegreatly.Processessuchasdevelopmentfromacaterpillarintoabutterflyinvolvedramaticchangesinpatternsofgeneexpression.TheexpressionlevelsofthousandsofgenescanbemonitoredthroughtheuseofDNAarrays.Atright,aGeneChiprevealstheexpressionlevelsofmorethan12,000humangenes;thebrightnessofeachspotindicatestheexpressionlevelofthecorrespondinggene.[(Left)RogerHart/Rainbow.(Right)GeneChipcourtesyofAffymetrix.]Therapidprogressinbiotechnologyindeeditsveryexistenceisaresultofarelativelyfewtechniques.1.Restriction-enzymeanalysis.2.Blottingtechniques.3.DNAsequencing.4.Solid-phasesynthesisofnucleicacids.5.Thepolymerasechainreaction(PCR).6.Computer.Restrictionenzymes,alsocalledrestrictionendonucleases,recognizespecificbasesequencesindouble-helicalDNAandcleave,atspecificplaces,bothstrandsofaduplexcontainingtherecognizedsequences.Tobiochemists,theseexquisitelyprecisescalpelsaremarvelousgiftsofnature.Theyareindispensableforanalyzingchromosomestructure,sequencingverylongDNAmolecules,isolatinggenes,andcreatingnewDNAmoleculesthatcanbecloned.WernerArberandHamiltonSmithdiscoveredrestrictionenzymes,andDanielNathanspioneeredtheiruseinthelate1960s.Restrictionenzymesarefoundinawidevarietyofprokaryotes.TheirbiologicalroleistocleaveforeignDNAmolecules.Thecell'sownDNAisnotdegraded,becausethesitesrecognizedbyitsownrestrictionenzymesaremethylated.Manyrestrictionenzymesrecognizespecificsequencesoffourtoeightbasepairsandhydrolyzeaphosphodiesterbondineachstrandinthisregion.Astrikingcharacteristicofthesecleavagesitesisthattheyalmostalwayspossesstwofoldrotationalsymmetry.Inotherwords,therecognizedsequenceispalindromic,oraninvertedrepeat,andthecleavagesitesaresymmetricallypositioned.Forexample,thesequencerecognizedbyarestrictionenzymefromStreptomycesachromogenesis:Morethan100restrictionenzymeshavebeenpurifiedandcharacterized.Theirnamesconsistofathree-letterabbreviationforthehostorganism(e.g.,EcoforEscherichiacoli,HinforHaemophilusinfluenzae,HaeforHaemophilusaegyptius)followedbyastraindesignation(ifneeded)andaromannumeral(ifmorethanonerestrictionenzymefromthesamestrainhasbeenidentified).ThespecificitiesofseveraloftheseenzymesareshowninFigure5.1.Notethatthecutsmaybestaggeredoreven.Figure5.1.SpecificitiesofSomeRestrictionEndonucleases.Thebase-pairsequencesthatarerecognizedbytheseenzymescontainatwofoldaxisofsymmetry.Thetwostrandsintheseregionsarerelatedbya180-degreerotationabouttheaxismarkedbythegreensymbol.Thecleavagesitesaredenotedbyredarrows.Theabbreviatednameofeachrestrictionenzymeisgivenattherightofthesequencethatitrecognizes.SmalldifferencesbetweenrelatedDNAmoleculescanbereadilydetectedbecausetheirrestrictionfragmentscanbeseparatedanddisplayedbygelelectrophoresis.TheelectrophoreticmobilityofaDNAfragmentisinverselyproportionaltothelogarithmofthenumberofbasepairs,uptoacertainlimit.Polyacrylamidegelsareusedtoseparatefragmentscontainingaboutasmanyas1000basepairs,whereasmoreporousagarosegelsareusedtoresolvemixturesoflargerfragments(aboutasmanyas20kb).Animportantfeatureofthesegelsistheirhighresolvingpower.•Incertainkindsofgels,fragmentsdifferinginlengthbyjustonenucleotideofseveralhundredcanbedistinguished.•Moreover,entirechromosomescontainingmillionsofnucleotidescanbeseparatedonagarosegelsbyapplyingpulsedelectricfields(pulsed-fieldgelelectro-phoresis,PFGE)indifferentdirections.ThistechniquedependsonthedifferentialstretchingandrelaxingoflargeDNAmoleculesasanelectricfieldisturnedoffandonatshortintervals.DetectionBandsorspotsofradioactiveDNAingelscanbevisualizedbyautoradiography.Alternatively,agelcanbestainedwithethidiumbromide,whichfluorescesanintenseorangewhenboundtodouble-helicalDNAmolecule.Abandcontainingonly50ngofDNAcanbereadilyseen.Figure5.2.GelElectrophoresisPatternofaRestrictionDigest.ThisgelshowsthefragmentsproducedbycleavingSV40DNAwitheachofthreerestrictionenzymes.Thesefragmentsweremadefluorescentbystainingthegelwithethidiumbromide.[CourtesyofDr.JeffreySklar.]Amixtureofrestrictionfragmentsisseparatedbyelectrophoresisthroughanagarosegel,denaturedtoformsingle-strandedDNA