北京大學(xué)細(xì)胞生物學(xué)課件-8xibaohe

1、Nucleus and ChromosomesChapter 8 Learning ObjectivesA 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-memb

2、rane nuclear envelope surrounds the nucleusB. The nuclear envelope consists consists of two membranes by a perinuclear space.The 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; Provid

3、es a structure 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 disa

4、ssembly 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 t

5、hose bound 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 complexBidirecti

6、onal traffic 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 b

7、y injecting various sizes of colloidal gold particles and examined by electron microscopy.10 nm in diameter60kd globular proteinAble to enter the nucleus Active transportTransport of large proteins into nucleus needs nuclear localization signal (NLS)NLS are present only in nuclear proteins A typical

8、 NLS contains several 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 cytosolNuclear import and exportNuclear import receptors bind NL

9、S and NucleoporinsThe Ran 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

10、 signal (NES) Reference: Cell 92: 327, 19982. Eukaryotes package DNA in Chromatin and chromosomesChromosomes 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 divisio

11、n, these fibers condense and fold into larger, compact structureA. The complexity of eukaryotic genomesGenome: One copy of all the genetic information of an organism. Genome size generally increases with an organisms complexity.If each nucleotide pair is 1 mm,then the human genome would extend 3200

12、km, far enough to stretch across the center 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.DNA sequencing is being applied to whole genomes as well as to ge

13、nes Early: E.coli, 6 years to complete its 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 si

14、milar but nonidentical amino acid sequences.-like 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 g

15、reat cellular demand for their transcripts.5S rRNA 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 f

16、ingerprinting depends on differences in length of minisatellite 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 pa

17、ckaging Nucleosomes are the basic unit of chromatin structureEach 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

18、micrographs of chromatin fibersIsolated from interphase nucleus: 30nm thickChromatin 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

19、200bp of DNA associated with a protein particle, is the nucleosomeEvidence: (3)X-ray diffraction studies3-D of nucleosomeNature 389:251, 1997Structural organization of the nucleosome A histone octamer forms the nucleosome coreHistone octamer:(H2A-H2B)-(H3-H4)-(H3-H4)-(H2A-H2B)Where is the histone H1

20、?H1 molecules are associated with the linker region. 146+1550bp linker DNA200bpDNA: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

21、most abundant proteins associated with eukaryotic DNARich in positively charged 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 tai

22、ls: Ac, P and/or Met.Localization of nucleosome is affected by two factors:Sequence-specific DNA binding proteinsDNA bending C. Nucleosomes are packed together to form chromatin fibers and chromosomesScaffold radial loop structure model Nonhistone proteins provide a structural scaffold for long chro

23、matin loops2nm DNA 11nm nucleosome 30nm fiber Loop Metaphase chromosomeEvidence for Scaffold Radial Loop ModelLoopD. Euchromatin and HeterochromatinEuchromatin The possibility of transcription; The types of chromosomal structure 30-nm fibers and looped domains; Light-staining, less condensed; Pulse

24、labeling with 3H-uridine: Transcriptional activityHeterochromatin: Dark-staining, condensed 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:

25、 Constitutive & facultativeCompacted state at all time: CentromereInactivated at certain phase of 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

26、 cat: a mosaic patchesMale Or ?femaleThe position effects of hetrochromatinyeast;Fluit fly.3. Chromosome number,size,and shape at metaphase are species specificChromatidsKaryotypeBindingHuman mitotic chromosomes and karyotypeA. Main structures of chromosome Centromere & KinetochoreCentromere: Highly

27、 repeated DNA+KinetochoreThe Centromere and Kinetochore: serve as a site for the attachment of spindle 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,NORsate

28、llitetelomerecentromereA typical mitotic chromosome at metaphase Three functional elements are required for replication and stable inheritance of chromosomesAutonomously replicating sequences (ARS)ARS act as an origin of replicationCentromeresTransfected Leu- cell by Plasmid with sequenceProgeny of

29、transfected cellGrowth without LeuMitotic segregationConclusionTelomeres:FunctionsFor the complete replication of chromosomeProtect the chromosomes from nuclease influencePrevent the ends of chromosomes from fusing Tetrahymena FISH (Human Probe ): TTAGGGAnti-RAP1 antibody:Yeast cellTelomere, telomer

30、ase and cellular aging, cancer cellTelomerase is found in germ cells, not in somatic cells.The telomere length 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 hu

31、man tumors contain an active telomerase.Yeast Artificial Chromosome (YAC) Giant chromosomesPolytene chromosomesEndomitosisChromocenterBands and interbandsLuxury genesHouse-keeping genesPuffs in plolytene chromosome: Gene activityPuffs: transcriptionally active chromatinLabeled with anti-RNA polymerase II antibodyIf labeled with 3H-U, thenLampbrush chromos