1、Nucleus and ChromosomesChapter 8 Learning Objectives(1)A typical nonmitotic nucleus includes several major components.(2)Nuclear envelope and nuclear pore complex.(3)The components of chromatin and packaging of chromosome.(4)Nucleolus.1.The nucleus:Nuclear envelope and NPCA.Structure:Double-membrane
2、 nuclear envelope surrounds the nucleusB.The nuclear envelope consists consists of two membranes by a perinuclear space.vThe inner surface of the nuclear envelope is lined by the nuclear laminaThe nuclear lamina supports the nuclear envelope:Gives shape and stability of nuclear envelope;Provides a s
3、tructure link between chromatin and nuclear envelopeThe nuclear lamina is composed of lamins.The integrity of the nuclear lamina is regulated by phosphorylation and dephosphorylation.Breakdown and reformation of nuclear envelope during mitosisThe phosphorylation of the lamins triggers the disassembl
4、y of the nuclear lamina,which in turn cause the nuclear envelope to break up into vesicles.Dephosphorylation of the lamins helps reverse the process.Because the new nuclear envelope is so closely applied to the surface of the chromosomes,it excludes all of the proteins in the cell except those bound
5、 to the chromosomes.So nuclear localization signals are not cleaved off after transport into the nucleus.Old nuclear envelope reforms new nuclear envelope.C.Nuclear pore complex(NPC)Cytoplasmic RingNuclear RingD.Molecules enter and exit the nucleus through nuclear pore complexBidirectional trafficv
6、Passive transportpassively diffuse3000-4000 NPC/cell(mammalian);To import about 106 histone/3 mins.(DNA-sythesizing cell)=100 histone/min/NPCEach NPC contains one or more open aqueous channels:9 nm in diameter and 15 nm longThe effective size of these channels has been determined by injecting variou
7、s sizes of colloidal gold particles and examined by electron microscopy.10 nm in diameter60kd globular proteinAble to enter the nucleusv Active transportTransport of large proteins into nucleus needs nuclear localization signal(NLS)NLS are present only in nuclear proteins A typical NLS contains seve
8、ral consecutive basic amino acids(the positively charged Aa:Lys,Arg)Normal pyruvate kinase:in cytosolChimeric pyruvate kinase containing SV40 NLS:in nucleusWild-type:T-antigen in nucleusMutant-type:T-antigen in cytosolvNuclear import and exportNuclear import receptors bind NLS and NucleoporinsThe Ra
9、n GTPase drives directional transport through NPCThe compartmentalization of Ran-GDP and Ran-GTP.A model for how GTP hydrolysis by Ran provides directionality for nuclear transportNuclear export works like nuclear import,but in reverse hnRNP proteins contain a nuclear-export signal(NES)Reference:Cel
10、l 92:327,19982.Eukaryotes package DNA in Chromatin and chromosomesqChromosomes exist in different states throughout the life of a cellChromatin:(Interphase)Fibers,10-30nm in diameter,Dispersed through the nucleus.DNA+Proteins+RNA.Chromosomes:(M phase)Cell division,these fibers condense and fold into
11、 larger,compact structureA.The complexity of eukaryotic genomesvGenome:One copy of all the genetic information of an organism.vGenome size generally increases with an organisms complexity.If each nucleotide pair is 1 mm,then the human genome would extend 3200 km,far enough to stretch across the cent
12、er of Africa,the site of our human origins.Species Genome size SV40 5 103bpE.coli 4.6106bpYeast 2107bp Fruit fly 2108bpHuman 3109bp Some amphibian and plants have larger genome size than human.vDNA sequencing is being applied to whole genomes as well as to genes Early:E.coli,6 years to complete its
13、sequence.If do as so:Almost 6000 years to sequence the.Each DNA molecule that forms a linear chromosome.Protein-coding sequences(Nonrepeated)25-50%of the protein-coding genes are solitary genes.Others belong to gene family,which encode proteins with similar but nonidentical amino acid sequences.-lik
14、e globin gene family:Contains 5 functional genesTandemly repeated genes encode rRNAs,tRNAs,and Histones(Moderately repeated)Tandemly repeated genes encode identical or nearly identical proteins or functional RNAs.These genes are needed to meet the great cellular demand for their transcripts.5S rRNA
15、and pre-rRNA genes:More than 100 copy/Genome.Tandemly repeated DNA:Simple-sequence,Highly repeated,Noncoding Satellite DNA:Centromeres,Telomeres(105107bp)Microsatellite DNA:102105bp at each siteMinisatellite DNA:10100bp at each site DNA fingerprinting depends on differences in length of minisatellit
16、e or microsatellite DNAUnequal exchange during meiosis.Interspersed moderately repeated DNAInterspersed repeated DNA makes up 25-40%of most mammalian genomes.Alu family:300bp long/unit,accounting for about 10%of the human DNAB.DNA packagingv Nucleosomes are the basic unit of chromatin structureEach
17、human cell contains about 2 m of DNA within nucleus if stretched end-to-end,yet the nucleus of a human cell itself is only about 6 um in diameter.Compaction ratio=nearly 10000-fold.(Chromosome 22:DNA 1.5cm2um)Evidence:(1)Electron micrographs of chromatin fibersIsolated from interphase nucleus:30nm t
18、hickChromatin unpacked,show the unclesomeEvidence:(2)Nuclease digestion(Rat liver chromatin)Gel electrophoresis after removal of chromatin proteinsAnalyzed by electron microscopyThe basic repeat unit,containing an average of 200bp of DNA associated with a protein particle,is the nucleosomeEvidence:(
19、3)X-ray diffraction studies3-D of nucleosomeNature 389:251,1997Structural organization of the nucleosomev A histone octamer forms the nucleosome coreHistone octamer:(H2A-H2B)-(H3-H4)-(H3-H4)-(H2A-H2B)Where is the histone H1?H1 molecules are associated with the linker region.146+1550bp linker DNA200b
20、pDNA:Linker DNA:15-50bpNucleosomal DNA:146bp to wrap 1.65 times around the histone core.Structure of nucleosomeDigested briefly:H1+Octamer+200bpDNADigested longer:Octamer+146bpH1 is releasedLinkerCore particleHistones:The most abundant proteins associated with eukaryotic DNARich in positively charge
21、d basic amino acids,which interact with the negtively charged phosphate groups in DNAThe amino acid sequences of histones H2A,H2B,H3,H4 are remarkably similar among distantly speciesCovalent modification of histone tails:Ac,P and/or Met.Localization of nucleosome is affected by two factors:Sequence-
22、specific DNA binding proteinsDNA bending C.Nucleosomes are packed together to form chromatin fibers and chromosomesvScaffold radial loop structure model Nonhistone proteins provide a structural scaffold for long chromatin loopsv2nm DNA 11nm nucleosome 30nm fiber Loop Metaphase chromosomeEvidence for
23、 Scaffold Radial Loop ModelLoopD.Euchromatin and HeterochromatinvEuchromatin The possibility of transcription;The types of chromosomal structure 30-nm fibers and looped domains;Light-staining,less condensed;Pulse labeling with 3H-uridine:Transcriptional activityvHeterochromatin:Dark-staining,condens
24、ed chromatin;No transcriptional activity;There are position effects;in a typical mammalian cell,approximately 10%of the genome is packaged into heterochromatins forming CEN and TEL Divided into two classes:Constitutive&facultativeCompacted state at all time:CentromereInactivated at certain phase of
25、lifeExample of facultative heterochromatin:Random inactivation of X chromosome in different cells during early embryonic development creates a mosaic of tissue patches.Barr body in a womans cellA calico cat:a mosaic patchesMale Or?female The position effects of hetrochromatinA.yeast;B.Fluit fly.3.Ch
26、romosome number,size,and shape at metaphase are species specificChromatidsKaryotypeBindingHuman mitotic chromosomes and karyotypeA.Main structures of chromosomev Centromere&KinetochoreCentromere:Highly repeated DNA+KinetochorevThe Centromere and Kinetochore:serve as a site for the attachment of spin
27、dle microtubules during mitosis and meiosisThe structure of a human CENThe plasticity of human CEN formation(MBC pp228)Human chromosome No.14 secondary constriction nucleolar organizing region,NORsatellitetelomerecentromereA typical mitotic chromosome at metaphasev Three functional elements are requ
28、ired for replication and stable inheritance of chromosomesAutonomously replicating sequences(ARS)ARS act as an origin of replicationCentromeresTransfected Leu-cell by Plasmid with sequenceProgeny of transfected cellGrowth without LeuMitotic segregationConclusionTelomeres:FunctionsFor the complete re
29、plication of chromosomeProtect the chromosomes from nuclease influencePrevent the ends of chromosomes from fusing Tetrahymena FISH(Human Probe):TTAGGGAnti-RAP1 antibody:Yeast cellTelomere,telomerase and cellular aging,cancer cellTelomerase is found in germ cells,not in somatic cells.The telomere len
30、gth of adult is shorter than that of younger.Telomere shortening is thought to activate a suicide program.So,telomere shortening plays a key role in protecting the body from cancer.90%of human tumors contain an active telomerase.Yeast Artificial Chromosome(YAC)v Giant chromosomesPolytene chromosomes
31、EndomitosisChromocenterBands and interbandsLuxury genesHouse-keeping genesPuffs in plolytene chromosome:Gene activityPuffs:transcriptionally active chromatinLabeled with anti-RNA polymerase II antibodyIf labeled with 3H-U,thenvLampbrush chromosomesIn growing amphibian oocyteStay in meiosis IStructur
32、eFunctions:Transcript into RNA,storage4.NucleolusA.StructureFibrillar centersDense fibrillar componentGranular componentB.Functions of nucleolus:Ribosomal Biogenesisv Ribosomal Biogenesis:pre-rRNA synthesisProcessAssemblyFC.DFCDFC.GCCytosolDirectional processv Characteristics of RNA transcriptionNOR
33、s in human chromosomes:1314152122Code for(in eukaryotes):18s,28s,5.8s rRNAvrRNA genes are tandemly arrayed in genome:Christmas-Like tree when transcriptedSynthesis and processing of rRNA precursor General structure of eukaryotic pre-rRNA transcription units pre-rRNA transcription unitsCleavage of pr
34、e-rRNA as RNP particlesMammaliaSynthesis and processing of 5s rRNA5s rRNAs are encoded by a large number genes(Human,2000)5s rRNA gene are located outside the nucleolus.5s rRNAs are transcibed by RNA poly III.The 3 end of 5s rRNA is removed during processing.v The assembly of ribosomesv The nucleolus disappears during mitosisNucleolar fusion of human fibroblast in cultrue
