大学分子生物学经典双语C2geneandchromosome省公开课金奖全国赛课一等奖微课获奖

Chapter2GeneandChromosome1/130Contents1StructureofDNA2Denaturation,renaturationandhybridization4GeneclusterandrepetitivesequenceConceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow72/1302.1StructureofDNA2.1.1primarystructureofDNADefinition：thenucletideresiduesequenceofthepolynucleotidechain；

Linkage：3’,5’-phosphodiesterbond；

Backbone：phosphate+

pentose；

Direction：5’→3’；3/1302.1.2secondarystructureofDNADNA

doublehelix4/130X~rayphotographofDNAwithhighquality：DNAspecimensfromdifferentspecieshavethesame

results（constantwidth;3.4nm）；Chargaffrules：theruleofthecompositionofDNAPhysicalchemistrystudiesandacidandalkalititratestudiesonDNAbase；Experimentalbasis5/130ThreedimensionalstructureofDNA6/13010.5bp/turnSugar-Pbackboneisperpendiculartotheplanarbasepairs5’5’3’3’PitchlengthTheWatson-CrickBformDNADeducedbymodelbuilding11Å20ÅFig8-15chargedPO4--ontheoutsideHydrophobicbasesinside7/130keynotesofDNAdoublehelixTwopolynucleotidechainsinaDNAdoublehelix;Alongthesameaxis，twochainsarewoundaroundeachother,resultinginaright-handeddoublehelix;Formsamajorgrooveandaminorgroove8/130Thebaseslieontheinside，thesugar-phosphatebackboneisontheoutside;Thebasesareflatstructure,lyinginpairsperpendiculartotheaxis9/130Thediameterofthedoublehelixis2nm;Thereisacompleteturnevery3.4nm,with10bpperturn.10/1302.1.2.1StablefactorsofthedoublehelixBase-stackinginteraction（hydrophobiceffect,themajorfactor）；Hydrogenbondbetweencomplementarybasepairs；electrovalentbond（betweenthenegativechargescarriedonthephosphategroupsandthepositivechargescarriedontheproteinsormetalions）11/1302.1.2.2Conformationpolymorphismofthedoublehelix12/130Alternativedouble-helicalstructuresofDNA13/130BaseObliquity

helixriseperbasepair

bpnumberperturn

Helicalsensediameter（nm）

B-form

Z-form

0-1

19-20

9

0.34nm

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：triplehelix14/1302.1.3

TriplexDNA1953,Watson&CrickproposedD.SDNAmodelandfoundmanyredundanthydrogenbondingdonorandreceptorsalongbiggrooves.1957,FelsenfeldproposedT.SDNAconceptpolyA/polyUpolydA/polydTpolyd(AG)/polyd(CT)1983,MirkinS.M.foundplasmidT.SDNAinpH=4.3solutionTriblehelixMajorgroove

Py:Pu:Py1963Hoogsteen15/130TripleHelixDNA16/1302.1.4

TetraplexDNA1958,Poly(G)X-rayphotographRingstructureofhydrogenTetrablehelixDNAFormationcondition:polyG,4(dG)17/130DNASculptureatDisneyland18/1302.1.5DNAsupercoilingSupercoiling:

SupercoilingisthecoilingoftheDNAaxisuponitself。19/13020/130Linkingnumber(L)Twistingnumber(T)缠绕数Writhingnumber(W)扭曲数

L=T+WParametersusedtoexpresstopologypropertiesofDNA:21/130PositivesupercoilsNegativesupercoilsDNAisolatedfromcellnegativelysupercoiledby~5turnsper100turnsofthehelix.

Lk/Lk=-0.0522/130

PositivesupercoilsNegativesupercoilsRelaxedcoils23/130TopoisomerasesTopoisomerases:existincelltoregulatethelevelofsupercoilingofDNAmolecules

TypeItopoisomerase:breakonestrandoftheDNA,andchangethelinkingnumberinstepsof±1bypassingtheotherstrandthroughthebreak.TypeIItopoisomerase:breakbothstrandsoftheDNA,andchangethelinkingnumberinstepsof±2bytransferringtheotherdsDNAthroughthebreak.24/130TypeItopoisomerase25/130TypeIItopoisomerase26/130Contents1StructureofDNA2Denaturation,renaturationandhybridization4GeneclusterandrepetitivesequenceConceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow727/1302.2Denaturation,renaturationandhybridization2.2.1PropertiesofDNA2.2.1.1DenaturationDefinition:anumberofphysicalandchemicalfactorscanleadtothedestructionofdouble-strandedhydrogen-bondedregionsofDNA.thedouble-strandednucleicacidsareconvertedtosinglestrands.28/130DenaturationfactorpH（>11.3或<5.0）Chemicaldenaturation

(urea、methanal)ThermaldenaturationLowionstrength29/130CharactersofdenaturedDNAbiologicalactivitychanged(evenlost)；viscositydecreased，solubilitydecreased,Hyperchromicity:theabsorbanceofssDNAisgreaterthanthatdsDNA.D.SDNAA260=1.0S.SDNAA260=1.37dNTPsA260=1.60

concentration=50μg/ml：30/130meltingcurveandTm

IncreasedtemperaturecanbringaboutDNAdenaturation;Tm（meltingtemperature):Temperaturewhen50%

DNAdenaturationTm

isacharacteristicconstantofDNA94℃31/1302.2.1.2RenaturationDefinition：annealing。D.SDNAS.SDNADenaturation▲▼RenaturationDependsonthecollisionofcomplementaryS.S.DNA32/1302.2.1.3renaturationdynamicsDNA复性过程遵照二级反应动力学DNA复性过程中单链消失速度用公式表示：

-dC/dt=kC2C/C0=1/(1+kC0t)其中，C是单位时间单链DNA浓度

C0为开始反应时变性解链单链DNA浓度，t为复性时间K是复性速度常数（L/mol·s），k取决于阳离子浓度、温度、pH值、DNA片段大小。C0t曲线33/1302.2.2hybridizationDefinition：therenaturationofregionsofcomplementaritybetweendifferentnucleicacidstrands(DNAorRNA)Characteristic：sensitive、specific34/130HybridizationofNucleicAcidsRNADNA1DNA2DNASouthernhybridizationNorthernhybridizationJuangRH()BCbasicsProbe35/13036/130SouthernblottingNorthernblottingdotblotinginsituhybridizationWesternblottingHybridizationofNucleicAcids37/130Southernblotting38/13039/130电转印法40/130NorthernBlotting41/130dotblottingDenaturedDNAorRNAsampleswereapplieddirectlyonamembraneasadot,thenfollowedbydetectionbyeithernucleotideprobes.42/130insituhybridizationDefinition：atypeofhybridizationthatusesalabeledcomplementaryDNAorRNAstrand(i.e.,probe)tolocalizeaspecificDNAorRNAsequenceinaportionorsectionoftissue(insitu).43/130荧光原位杂交技术FISH原位聚合酶链反应IS-PCR基因组原位杂交技术GISH多彩荧光杂交技术mFISH44/130FISH&mFISH45/130WesternblottingTheproteinsseparatedfromgelelectrophoresisaretransferredtoamembrane(typicallymitroceluloseorPVDF),wheretheyareprobedusingantibodiesspecifictothetargetprotein.Step1.AntibodyRecognitionoftargetprotein/antigenStep2.SecondaryAntibodyrecognitionofprimaryAbStep3.ColorDevelopment46/130Contents1StructureofDNA2Denaturation,renaturationandhybridization4GeneclusterandrepetitivesequenceConceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow747/1302.3ConceptofgeneHowtodescribegeneticmaterial?

PhysiologicalUnit(1864，HerbertSpencer)

Gemmules(1868,CharlesRobertDarwin)

Germplasm(1883,Weismann)

Idioplasm(1884,KarlWilhelm)

Pangen(1889,HugodeVries)48/130Mendel’slawsLawofsegregationLawofindependentassortmentGeneticfactor——GregorMendel(1865)Mendelhypothesizedgeneticfactor

(alternativeformsofgenes)determineheredity.49/1301909WilhelmJohannsen

,aDanishbotanist,plantphysiologistandgeneticist.ElementederexaktenErblichkeitslehre(TheElementsof

Heredity)

.Itwasinthisbookintroducedtheterm.Gene——Johannsen(1909)JohannsengeneGenotypePhenotype

50/1302.3.1Classicalconceptofgene1910AmericanscientistMorgansuggestedthatgenewasmappedonchromosomes.Morgan

Thegeneisviewedasthefundamentalunitofmutation,changeandfunction.192651/1302.3.2Modernconceptofgene2.3.2.1“Onegene,oneenzyme”hypothesis1941,GWBeadle&ELTatumBeadleTatum

Agenecontainstheinformationforproducingaspecificenzyme.52/13053/1301951,YanofskyOnegeneonepolyeptidechain54/13055/1301953,Watson

&CrickDNAdoublehelixGeneisahereditaryunitconsistingofasequenceofDNAthatoccupiesaspecificlocationonachromosome.56/1302.3.2.2TheoryofCistron(1955,S.Benzer)Cistron——aunitofgeneticfunctiondefinedbyacomplementationtest,whichcorrespondstoanopenreadingframe.Benzer57/130Trans-configurationBbAaAaBbCis-configurationComplementationtestrevealswhethertwosimilarmutationsareinthesameordifferentgenes.58/13059/13060/130ComplementationtestbetweenrII－

mutants

61/130Recombinationbetweentwomutationsinthesamegene62/13063/130Trans-configuration:UnfunctionalFunctionalMutantsitesareindifferentgenes.Mutantsitesareinthesamegene.64/130Cistron——analternativeformsofgenes

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

65/1302.3.2.3StructuralgeneandregulatorygeneOperontheory(1961,JacobF.&MonodJ.)66/1302.3.2.4Splittinggene1977,Sharp&RobertsR.J.RobertsP.A.SharpNobelPrize1993IntronExon67/130Exon:theregionsoftheDNAthatcontaincodinginformation,whichmakeupofmaturemRNA.

Intron:interveningsequencesbetweenthecodingsequences,theywillalsobetranscribe,butwillberemovedtoyieldthematuremRNA.68/13069/1302.3.2.5Overlappinggene赏花归去马如飞，去马如飞酒力微，

酒力微醒时已暮，醒时已暮赏花归。

苏东坡Overlappinggenesaredefinedasapairofadjacentgeneswhosecodingregionsarepartiallyoverlapping70/13071/1302.3.2.6PseudogenePseudogenesaredysfunctionalrelativesofgenesthathavelosttheirprotein-codingabilityorareotherwisenolongerexpressedinthecell.72/1302.3.2.7Jumpinggene

Jumpinggene——asegmentofDNAthatcanbecomeintegratedatmanydifferentsitesalongachromosome(especiallyasegmentofbacterialDNAthatcanbetranslocatedasawhole)73/130BarbaraMcClintock1983Nobilewinner74/130Ac-Ds系统75/130c)transposableelement

是引发玉米糊粉层花斑不稳定现象遗传因子

SHBzWx

Ac

ciBzWx

Acchr.9CISHBzWx

largecoloredsectorsDsDs

CICI

CIci

SH

sh76/1302.3.2.8Housekeepinggene&LuxurygeneAhousekeepinggeneistypicallyaconstitutivegenethatisrequiredforthemaintenanceofbasiccellularfunction,andareexpressedinallcellsofanorganism.Aluxurygenecodesforspecializedcellproductsandisexpressedabundantly.Theyaretissue-specificororgan-specific,whichmeanstheyarenotexpressedinallcells.Theyarenotconstantlyexpressed,onlywhentheirfunctionisneeded.Examplesofluxurygenesareplasmidsofbacteriaandgenescodingforheat-shockproteins.77/130Contents1StructureofDNA2Denaturation,renaturationandhybridization4Geneclusterandrepetitivesequence

Conceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow778/1302.4GeneclusterandrepetitivesequenceAgenefamilyisasetofseveralsimilargenes,formedbyduplicationofasingleoriginalgene,andgenerallywithsimilarbiochemicalfunctions.GenefamilyGeneclusterAgeneclusterisagroupofcloselyrelatedgenesthatallcodeforthesamefunction,orvariationsonthesamefunction.79/1302.4.1Genecluster2.4.1.1GeneclusterformationGlobingene80/130Globingenesareorganizedintwoclusters.81/130Allglobingeneshaveevolvedbyaseriesofduplications,transpositions,andmutationsfromasingleancestralgene.82/1302.4.1.2SequencedifferentiationDivergenceofDNAsequencesdependsonevolutionaryseparation.Eachpointonthegraphrepresentsapairwisecomparison.83/130Replacementsitedivergencesbetweenpairsofβ-globingenesallowthehistoryofthehumanclustertobereconstructed.Thistreeaccountsfortheseparationofclassesofglobingenes.84/130Pesudogenesaredeadendsofevolution.Pesudogenes(ψ):SomeDNAsequencesthatarerelatedtothoseofthefunctionalgenes,butthatcannotbetranslatedintoafunctionalprotein.2.4.1.3Pesudogene85/130Manychangeshaveoccurredinaβ-globingenesinceitbecameapseudogene.86/130PseudogenescouldarisebyreversetranscriptionofRNAtogiveduplexDNAsthatbecomeintegratedintothegenome.87/130Ifpseudogenesareevolutionarydeadends,simplyanunwantedaccompanimenttotherearrangementoffunctionalgenes,whyaretheystillpresentinthegenome?Survivedinpresentpopulations,inthepast,anynumberofotherpseudogenesmayhavebeeneliminated.88/1302.4.1.4GeneclusterrearrangmentUnequalcrossing-overrearrangesgeneclusters.89/130Genenumbercanbechangedbyunequalcrossing-over.90/130Thalassemiasresultfromvariousdeletionsinthea-globingenecluster.

91/1302.4.2RepetitivesequenceRepetitivesequence——DNAsequencewithhighcopynumbers.replicationslippage92/130unequalcrossingover93/1302.4.2.1UniquesequenceAgenomeisonlyonecopyorveryfewcopiesofDNAsequences.

Generallybythecodingsequenceandintervalsequences.94/1302.4.2.2moderatelyrepetitivesequencesize：0.1~1kb(lessthan10kb)copies：10~10000includinggeneclusterandmanytransposon(sometimesviewedasselfishDNA)functions：donottranscribe,maybeplaysomeroleingeneexpression95/1302.4.2.3highlyrepetitivesequencetypically<100bp,thousandsofcopies,oftenorganizedaslongtandemrepeats；AfractionofthissortiscalledsatelliteDNA.mainlynearthecentromeres(inheterochromatin)；maybehassomestructuralfunctioninthechromosome.Thisfunctioncouldbeconnectedwiththeprocessofchromosome

segregation；96/130SatelliteDNA97/13098/130Contents1StructureofDNA2Denaturation,renaturationandhybridization4GeneclusterandrepetitivesequenceConceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow799/1302.5Chromosomeandnucleosome2.5.1Chromosome100/1302.5.1.1prokaryoticchromosomeProcaryoticcell101/130E.

coliTobaccoMosaicvirus102/1302.5.1.2EukaryoticchromosomeStructureofthenucleusAnimalcell103/130Theeukaryoticcellstructureismorecomplexthanprokaryotic；Componentofeukaryoticchromosome:DNA,RNA,Histone,Nonhistones104/130chromatineuchromatinheterochromatin105/130SpecialPatternofEukaryoticchromosomelampbrushchromosomemultinemechromosome106/130centromere107/130telomereAtelomereisaregionofrepetitivenucleotidesequencesattheendofachromosome,whichprotectstheendofthechromosomefromdeteriorationorfromfusionwithneighboringchromosomes.Repeatmotif:

5’-T1-4-A0-1-G1-8-3’108/1302.5.2nucleosomeElectronmicrographofDrosophilamelanogasterchromatinafterswellingrevealsthepresenceofnucleosomesas"beadsonastring".109/1302.5.2.1SubunitofnucleosomeAnucleosomecontains~200bpofDNA,associatedwithahistoneoctamerthatconsistsoftwocopieseachofH2A,H2B,H3,andH4.110/1302.5.2.2histoneoctamer111/130112/1302.5.2.3pathwayofnucleosome6nucleosomesperturn113/1302.5.2.3PackageofEukaryoticgenome114/130Compactionratio=8000FromDNAtoChromosome115/130Contents1StructureofDNA2Denaturation,renaturationandhybridization4GeneclusterandrepetitivesequenceConceptofgene3Chromosomeandnucleosome5Genome6Geneticinformationflow7116/1302.6GenomeGenome:allDNAsequencesinacell;acompletegeneticsequenceononesetofchromosomes.117/130

MaximumC-value:

thequantityofDNAinthe(haploid)genome.

MinimumC-value:

theminimumgenom