C2-gene-and-chromosome-分子生物學(xué)雙語課程課件

Chapter2GeneandChromosomeContents1StructureofDNA2Denaturation,renaturationandhybridization4GeneclusterandrepetitivesequenceConceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow72.1StructureofDNA2.1.1primarystructureofDNA

Definition：the

nucletideresiduesequenceofthepolynucleotidechain；

Linkage：3’,5’-phosphodiesterbond；

Backbone：phosphate+

pentose；

Direction：5’→3’；2.1.2secondarystructureofDNADNA

doublehelixX~rayphotographofDNAwithhighquality：DNAspecimensfromdifferentspecieshavethesame

results（constantwidth;3.4nm）；Chargaffrules：theruleofthecompositionofDNAPhysicalchemistrystudiesandacidandalkalititratestudiesonDNAbase；ExperimentalbasisThreedimensionalstructureofDNA10.5bp/turnSugar-Pbackboneisperpendiculartotheplanarbasepairs5’5’3’3’PitchlengthTheWatson-CrickBformDNADeducedbymodelbuilding11?20?Fig8-15chargedPO4--ontheoutsideHydrophobicbasesinsidekeynotesofDNAdoublehelixTwopolynucleotidechainsinaDNAdoublehelix;Alongthesameaxis，twochainsarewoundaroundeachother,resultinginaright-handeddoublehelix;FormsamajorgrooveandaminorgrooveThebaseslieontheinside，thesugar-phosphatebackboneisontheoutside;Thebasesareflatstructure,lyinginpairsperpendiculartotheaxisThediameterofthedoublehelixis2nm;Thereisacompleteturnevery3.4nm,with10bpperturn.2.1.2.1StablefactorsofthedoublehelixBase-stackinginteraction（hydrophobiceffect,themajorfactor）；Hydrogenbondbetweencomplementarybasepairs；electrovalentbond（betweenthenegativechargescarriedonthephosphategroupsandthepositivechargescarriedontheproteinsormetalions）2.1.2.2ConformationpolymorphismofthedoublehelixAlternativedouble-helicalstructuresofDNABaseObliquity

helixriseperbasepair

bpnumberperturn

Helicalsensediameter（nm）

B-form

Z-form

0-119-2090.34nm

0.23nm

0.38nm10

11

12Rhanded

Rhanded

Lhanded2.0-2.37

2.55

1.8-1.84A-formB-form：relativehumidityis92%A-form：relativedevoidofwater(under75%)Z-form：lefthandedhelixH-form：triplehelix2.1.3

TriplexDNA1953,Watson&CrickproposedD.SDNAmodelandfoundmanyredundanthydrogenbondingdonorandreceptorsalongbiggrooves.1957,FelsenfeldproposedT.SDNAconcept

polyA/polyU

polydA/polydT

polyd(AG)/polyd(CT)1983,MirkinS.M.foundplasmidT.SDNAinpH=4.3solutionTriblehelixMajorgroove

Py:Pu:Py1963

HoogsteenTripleHelixDNA2.1.4

TetraplexDNA1958,Poly(G)X-rayphotographRingstructureofhydrogenTetrablehelixDNAFormationcondition:polyG,4(dG)DNASculptureatDisneyland2.1.5DNAsupercoilingSupercoiling:

SupercoilingisthecoilingoftheDNAaxisuponitself。Linkingnumber(L)Twistingnumber(T)纏繞數(shù)Writhingnumber(W)扭曲數(shù)

L=T+WParametersusedtoexpresstopologypropertiesofDNA:PositivesupercoilsNegativesupercoilsDNAisolatedfromcellnegativelysupercoiledby~5turnsper100turnsofthehelix.Lk/Lk=-0.05

PositivesupercoilsNegativesupercoilsRelaxedcoilsTopoisomerasesTopoisomerases:existincelltoregulatethelevelofsupercoilingofDNAmolecules

TypeItopoisomerase:breakonestrandoftheDNA,andchangethelinkingnumberinstepsof±1bypassingtheotherstrandthroughthebreak.TypeIItopoisomerase:breakbothstrandsoftheDNA,andchangethelinkingnumberinstepsof±2bytransferringtheotherdsDNAthroughthebreak.TypeItopoisomeraseTypeIItopoisomeraseContents1StructureofDNA2Denaturation,renaturationandhybridization4GeneclusterandrepetitivesequenceConceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow72.2Denaturation,renaturationandhybridization2.2.1PropertiesofDNA2.2.1.1DenaturationDefinition:anumberofphysicalandchemicalfactorscanleadtothedestructionofdouble-strandedhydrogen-bondedregionsofDNA.thedouble-strandednucleicacidsareconvertedtosinglestrands.DenaturationfactorpH（>11.3或<5.0）Chemicaldenaturation

(urea、methanal)ThermaldenaturationLowionstrengthCharactersofdenaturedDNAbiologicalactivitychanged(evenlost)；viscositydecreased，solubilitydecreased,Hyperchromicity:theabsorbanceofssDNAisgreaterthanthatdsDNA.D.SDNAA260=1.0S.SDNAA260=1.37dNTPsA260=1.60

concentration=50μg/ml：meltingcurveandTm

IncreasedtemperaturecanbringaboutDNAdenaturation;Tm（meltingtemperature):Temperaturewhen50%

DNAdenaturation

Tm

isacharacteristicconstantofDNA94℃2.2.1.2RenaturationDefinition：annealing。D.SDNAS.SDNADenaturation▲▼RenaturationDependsonthecollisionofcomplementaryS.S.DNA2.2.1.3renaturationdynamicsDNA復(fù)性過程遵循二級(jí)反應(yīng)動(dòng)力學(xué)DNA復(fù)性過程中單鏈消失的速度用公式表示：

-dC/dt=kC2C/C0=1/(1+kC0t)其中，C是單位時(shí)間的單鏈DNA的濃度

C0為開始反應(yīng)時(shí)變性解鏈的單鏈DNA濃度，t為復(fù)性時(shí)間K是復(fù)性速度常數(shù)（L/mol·s），k取決于陽離子濃度、溫度、pH值、DNA片段大小。C0t曲線2.2.2hybridizationDefinition：therenaturationofregionsofcomplementaritybetweendifferentnucleicacidstrands(DNAorRNA)Characteristic：sensitive、specificHybridizationofNucleicAcidsRNADNA1DNA2DNASouthernhybridizationNorthernhybridizationJuangRH(2004)BCbasicsProbeSouthernblottingNorthernblottingdotblotinginsituhybridizationWesternblottingHybridizationofNucleicAcidsSouthernblotting電轉(zhuǎn)印法NorthernBlottingdotblottingDenaturedDNAorRNAsampleswereapplieddirectlyonamembraneasadot,thenfollowedbydetectionbyeithernucleotideprobes.insituhybridizationDefinition：atypeofhybridizationthatusesalabeledcomplementaryDNAorRNAstrand(i.e.,probe)tolocalizeaspecificDNAorRNAsequenceinaportionorsectionoftissue(insitu).熒光原位雜交技術(shù)FISH原位聚合酶鏈反應(yīng)IS-PCR基因組原位雜交技術(shù)GISH多彩熒光雜交技術(shù)mFISHFISH&mFISHWesternblottingTheproteinsseparatedfromgelelectrophoresisaretransferredtoamembrane(typicallymitroceluloseorPVDF),wheretheyareprobedusingantibodiesspecifictothetargetprotein.Step1.AntibodyRecognitionoftargetprotein/antigenStep2.SecondaryAntibodyrecognitionofprimaryAbStep3.ColorDevelopmentContents1StructureofDNA2Denaturation,renaturationandhybridization4GeneclusterandrepetitivesequenceConceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow72.3ConceptofgeneHowtodescribegeneticmaterial?

PhysiologicalUnit(1864，HerbertSpencer)

Gemmules

(1868,CharlesRobertDarwin)

Germplasm(1883,Weismann)

Idioplasm(1884,KarlWilhelm)

Pangen(1889,HugodeVries)Mendel’slawsLawofsegregationLawofindependentassortmentGeneticfactor——GregorMendel(1865)Mendelhypothesizedgeneticfactor

(alternativeformsofgenes)determineheredity.1909WilhelmJohannsen

,aDanish

botanist,plantphysiologistandgeneticist.Elemente

der

exakten

Erblichkeitslehre

(TheElementsof

Heredity)

.Itwasinthisbookintroducedtheterm.Gene——Johannsen(1909)JohannsengeneGenotypePhenotype

2.3.1Classicalconceptofgene1910AmericanscientistMorgansuggestedthatgenewasmappedonchromosomes.Morgan

Thegeneisviewedasthefundamentalunitofmutation,changeandfunction.19262.3.2Modernconceptofgene2.3.2.1“Onegene,oneenzyme”hypothesis1941,GWBeadle&ELTatumBeadleTatum

Agenecontainstheinformationforproducingaspecificenzyme.1951,YanofskyOnegeneonepolyeptidechain1953,Watson

&CrickDNAdoublehelixGeneisahereditaryunitconsistingofasequenceofDNAthatoccupiesaspecificlocationonachromosome.2.3.2.2TheoryofCistron(1955,S.Benzer)Cistron——aunitofgeneticfunctiondefinedbyacomplementationtest,whichcorrespondstoanopenreadingframe.BenzerTrans-configurationBbAaAaBb

Cis-configurationComplementationtestrevealswhethertwosimilarmutationsareinthesameordifferentgenes.ComplementationtestbetweenrII－

mutants

RecombinationbetweentwomutationsinthesamegeneTrans-configuration:UnfunctionalFunctionalMutantsitesareindifferentgenes.Mutantsitesareinthesamegene.Cistron——analternativeformsofgenes

Inacistron,therearemanymutantsites(mutons);Inacistron,therearemanycrossingoversites(recons).Ageneisacistron,consistingofmanymutonsandrecons.

2.3.2.3StructuralgeneandregulatorygeneOperontheory(1961,JacobF.&MonodJ.)2.3.2.4Splittinggene1977,Sharp&RobertsR.J.RobertsP.A.SharpNobelPrize1993Intron

Exon

Exon:theregionsoftheDNAthatcontaincodinginformation,whichmakeupofmaturemRNA.

Intron:interveningsequencesbetweenthecodingsequences,theywillalsobetranscribe,butwillberemovedtoyieldthematuremRNA.2.3.2.5Overlappinggene賞花歸去馬如飛，去馬如飛酒力微，

酒力微醒時(shí)已暮，醒時(shí)已暮賞花歸。

蘇東坡Overlappinggenesaredefinedasapairofadjacentgeneswhosecodingregionsarepartiallyoverlapping2.3.2.6PseudogenePseudogenesaredysfunctionalrelativesofgenesthathavelosttheirprotein-codingabilityorareotherwisenolongerexpressedinthecell.2.3.2.7Jumpinggene

Jumpinggene——asegmentofDNAthatcanbecomeintegratedatmanydifferentsitesalongachromosome(especiallyasegmentofbacterialDNAthatcanbetranslocatedasawhole)BarbaraMcClintock1983NobilewinnerAc-Ds系統(tǒng)c)transposableelement

是引起玉米糊粉層花斑不穩(wěn)定現(xiàn)象的遺傳因子

SHBz

Wx

Ac

ci

Bz

Wx

Acchr.9CISHBz

Wx

largecoloredsectorsDsDs

CICI

CI

ci

SH

sh

2.3.2.8Housekeepinggene&LuxurygeneAhousekeepinggeneistypicallyaconstitutivegenethatisrequiredforthemaintenanceofbasiccellularfunction,andareexpressedinallcellsofanorganism.Aluxurygenecodesforspecializedcellproductsandisexpressedabundantly.Theyaretissue-specificororgan-specific,whichmeanstheyarenotexpressedinallcells.Theyarenotconstantlyexpressed,onlywhentheirfunctionisneeded.Examplesofluxurygenesareplasmidsofbacteriaandgenescodingforheat-shockproteins.Contents1StructureofDNA2Denaturation,renaturationandhybridization4Geneclusterandrepetitivesequence

Conceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow72.4GeneclusterandrepetitivesequenceAgenefamilyisasetofseveralsimilargenes,formedbyduplicationofasingleoriginalgene,andgenerallywithsimilarbiochemicalfunctions.GenefamilyGeneclusterAgeneclusterisagroupofcloselyrelatedgenesthatallcodeforthesamefunction,orvariationsonthesamefunction.2.4.1Genecluster2.4.1.1GeneclusterformationGlobingeneGlobingenesareorganizedintwoclusters.Allglobingeneshaveevolvedbyaseriesofduplications,transpositions,andmutationsfromasingleancestralgene.2.4.1.2SequencedifferentiationDivergenceofDNAsequencesdependsonevolutionaryseparation.Eachpointonthegraphrepresentsapairwisecomparison.Replacementsitedivergencesbetweenpairsofβ-globingenesallowthehistoryofthehumanclustertobereconstructed.Thistreeaccountsfortheseparationofclassesofglobingenes.Pesudogenesaredeadendsofevolution.Pesudogenes(ψ):SomeDNAsequencesthatarerelatedtothoseofthefunctionalgenes,butthatcannotbetranslatedintoafunctionalprotein.2.4.1.3PesudogeneManychangeshaveoccurredinaβ-globingenesinceitbecameapseudogene.PseudogenescouldarisebyreversetranscriptionofRNAtogiveduplexDNAsthatbecomeintegratedintothegenome.Ifpseudogenesareevolutionarydeadends,simplyanunwantedaccompanimenttotherearrangementoffunctionalgenes,whyaretheystillpresentinthegenome?Survivedinpresentpopulations,inthepast,anynumberofotherpseudogenesmayhavebeeneliminated.2.4.1.4GeneclusterrearrangmentUnequalcrossing-overrearrangesgeneclusters.Genenumbercanbechangedbyunequalcrossing-over.Thalassemiasresultfromvariousdeletionsinthea-globingenecluster.

2.4.2RepetitivesequenceRepetitivesequence——DNAsequencewithhighcopynumbers.replicationslippageunequalcrossingover2.4.2.1UniquesequenceAgenomeisonlyonecopyorveryfewcopiesofDNAsequences.

Generallybythecodingsequenceandintervalsequences.2.4.2.2moderatelyrepetitivesequencesize：0.1~1kb(lessthan10kb)copies：10~10000includinggeneclusterandmanytransposon(sometimesviewedasselfishDNA)functions：donottranscribe,maybeplaysomeroleingeneexpression2.4.2.3highlyrepetitivesequencetypically<100bp,thousandsofcopies,oftenorganizedaslongtandemrepeats；AfractionofthissortiscalledsatelliteDNA.mainlynearthecentromeres(inheterochromatin)；maybehassomestructuralfunctioninthechromosome.Thisfunctioncouldbeconnectedwiththeprocessofchromosome

segregation；SatelliteDNAContents1StructureofDNA2Denaturation,renaturationandhybridization4GeneclusterandrepetitivesequenceConceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow72.5Chromosomeandnucleosome2.5.1Chromosome2.5.1.1prokaryoticchromosomeProcaryoticcellE.

coliTobaccoMosaicvirus2.5.1.2EukaryoticchromosomeStructureofthenucleusAnimalcellTheeukaryoticcellstructureismorecomplexthanprokaryotic；Componentofeukaryoticchromosome:DNA,RNA,Histone,NonhistoneschromatineuchromatinheterochromatinSpecialPatternofEukaryoticchromosomelampbrushchromosomemultinemechromosomecentromeretelomereAtelomereisaregionofrepetitivenucleotidesequencesattheendofachromosome,whichprotectstheendofthechromosomefromdeteriorationorfromfusionwithneighboringchromosomes.Repeatmotif:

5’-T1-4-A0-1-G1-8-3’2.5.2nucleosomeElectronmicrographofDrosophilamelanogasterchromatinafterswellingrevealsthepresenceofnucleosomesas"beadsonastring".2.5.2.1Subunitofnucleosome

Anucleosomecontains~200bpofDNA,associatedwithahistone

octamerthatconsistsoftwocopieseachofH2A,H2B,H3,andH4.2.5.2.2histone

octamer2.5.2.3pathwayofnucleosome6nucleosomesperturn2.5.2.3PackageofEukaryoticgenomeCompactionratio=8000FromDNAtoChromosomeContents1StructureofDNA2Denaturation,renaturationandhybridization4GeneclusterandrepetitivesequenceConceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow72.6GenomeGenome:allDNAsequencesinacell;acompletegeneticsequenceononesetofchromosomes.

MaximumC-value:

thequantityofDNAinthe(haploid)genome.

Minimum