复旦大学细胞生物学课件10细胞核和染色体

1、Chap10 Nucleus and ChromosomesNucleus of a Eukaryotic Cell Nuclear Envelope Nuclear Pore Complex ChromatinNucleolus and Ribosome BiogenesisNuclear Matrix 8.1 The Nucleus of a Eukaryotic CellThe inner nuclear membrane The outer nuclear membrane Surrounded by two concentric membranes 8.1.1 Organizatio

2、n of the Nucleus chromosomesnuclear matrix nucleolinucleoplasman interphase HeLa cell nucleus showing some of the major components of the nucleus 8.1.2 Internal Architecture contrast to the cytoplasm, the nucleus: not individually enclosed by membranesnot visible using conventional light or electron

3、 microscopy techniques revised picture of nuclear structure Nuclear matrix: a proteinaceous scaffold-like networkNucleolus: the center for synthesis and processing of rRNA molecules 8.2 The Nuclear EnvelopeThe separation of a cells genetic material from the surrounding cytoplasm may be single most i

4、mportant feature that distinguishes eukaryotes from prokaryotes. Structure of the nuclear envelope outer nuclear membraneinner nuclear membranenuclear laminaperinuclear spacenuclear pore nuclear laminaFunction of the nuclear envelope a barrier between the nucleus and cytoplasm, as a distinct biochem

5、ical compartment sole channels through the nuclear envelope 8.3 Nuclear Pore ComplexVertebrate: 50100 proteinsDiameter: 120 nm, 125 MDa basketlike apparatus eightfold symmetryComposition of the NPCCytoplasmic ringNuclear ringSpokeCentral plugNuclear envelopes of Xenopus oocytes visualized by field e

6、mission in-lens SEM Cut-away model of the NPC Three-dimensional models of the NPC A structure with eightfold symmetry The NPC consists of an assembly of eight spokes arranged around a central channel The spokes are connected to rings at the nuclear and cytoplasmic surfaces Electron micrograph of NPC

7、 NLSs direct nuclear proteins to the nucleus The nuclear proteins are selective traffic across the nuclear envelope from the cytoplasm to the nucleusThe NLSs include histones, DNA polymerases, RNA polymerases, transcription factors, splicing factors transport through NPC 核孔运输特点被动运输主动运输信号引导双向性Molecul

8、ar traffic through NPC核孔的被动运输Small molecular traffic through the NPC by passive diffusionSmall molecules and some proteins with MW 50 kD pass freely across the nuclear envelope in either direction.Most proteins and RNAs pass through the NPC by an active process in only one direction. 核孔的双向运输mRNA的输出2

9、.1核蛋白运输机制 基本概念核蛋白(nuclear protein)核定位信号(nuclear localization signals ,NLS)核输出信号(nuclear export signals, NES)输入蛋白(importin)输出蛋白(exportin)核定位信号Nuclear localization signals 核蛋白的输入核质蛋白(nucleoplasmin)实验核蛋白输入机理输入蛋白Ran蛋白:小GTPase RanGAP:RanGTP激活蛋白:细胞质中 RCC1：Ran nucleotide-exchange factor1.细胞核中核质素的核定位信号及其作用R

10、eceptors for the NLS transport proteins to the nucleusProtein import through the NPC can be divided into two steps, distinguished by whether they require energy. The first step does not require energy, proteins that contain NLSs bind to the NPC but do not pass through the pore. The second step is an

11、 energy-dependent process that requires GTP hydrolysis. Protein import through NPC Role of the Ran protein in nuclear import 核蛋白输入机理核内RNA与蛋白质输出mRNA的输出异质核糖核蛋白 (heterogeneous ribonucleoprotein, hnRNP)信使RNP(messenger RNP, mRNP)mRNA的输出核内蛋白质输出snRNA的输出Transport of RNA between nucleus and cytoplasm active,

12、 energy-dependent process ribonucleoprotein complexes rather than naked RNAs mRNA的输出snRNA的输出核内蛋白质的输出3 分子伴侣(chaperones)3.1 分子伴侣的发现及种类The term “chaperone was first used by Ron Laskey and his colleagues to describe a protein (nucleoplasmin) that is required for the assembly of nucleosomes from histones

13、 and DNA. Nucleoplasmin binds to histones and mediates their assembly into nucleosomes, but nucleoplasmin itself is not incorporated into the final nucleosome structure.Chaperones thus act as catalysts that facilitate assembly without being part of the assembled complex. 分子伴侣的概念及其特点1991年Ellis等人提出:由不

14、相关类的蛋白质组成的一个家系它们介导其它蛋白质的正确装配但自己不成为最后功能结构中的组分。该概念有以下特点:凡具有“介导”功能的蛋白,都称为分子伴侣,可以是完全不同的蛋白质;作用机理尚不清楚,故用“介导”二字,伸缩性较大；分子伴侣一定不是最终结构的组成部分,但不一定是一个分离的实体;装配的涵意比较广,包括:帮助新生肽的折叠,越膜定位, 亚基组装等。It is important to note that : Chaperones do not convey additional information required for the folding of polypeptides into

15、their correct three-dimensional conformations. The folded conformation of a protein is determined solely by its amino acid sequence. Rather, chaperones catalyze protein folding by assisting the self-assembly process. They appear to function by binding to and stabilizing unfolded or partially folded

16、polypeptides that are intermediates along the pathway leading to the final correctly folded state. 分子伴侣的基本功能分子内伴侣(intromolecular chaperones)分子伴侣的分布从细菌到人,从动物到植物细胞质、线粒体、叶绿体和微体分子伴侣结构上的共同特点家族成员具有高度保守性家族成员结构上具有相似性大部分在体内为组成型表达,在刺激条件下会被进一步诱导。Hsp60的电镜三维镜象照片3.2 functions of chaperones帮助蛋白质折叠和装配蛋白质的转运和定位参与细胞器

17、和细胞核结构的发生应激反应参与信号转导 Action of chaperones during translation Actions of chaperones GroEL,GroES of E.coli in protein foldingSequential actions of Hsp70 and Hsp60 chaperones Action of chaperones in signalingAction of chaperones during protein transport Molecular Chaperones应激反应Molecular chaperones were

18、initially identified as heat-shock proteins, a group of proteins expressed in cells that have been subjected to elevated temperatures or other forms of environmental stress. The heat-shock proteins (abbreviated Hsp), which are highly conserved in both prokaryotic and eukaryotic cells, are thought to

19、 stabilize and facilitate the refolding of proteins that have been partially denatured as a result of exposure to elevated temperature. However, many members of the heat-shock protein family are expressed and have essential cellular functions under normal growth conditions. These proteins serve as m

20、olecular chaperones, which are needed for polypeptide folding and transport under normal conditions as well as in cells subjected to environmental stress.8.4 Chromatin & Chromosomechromatin types Heterochromatin constitutive facultative euchromatin The Functions of chromatinStorage of genetic inform

21、ationPrecise segregation of replicated DNA into two daughter cellsPlatform for transcription, replication, recombination and DNA repairComposition of ChromatinDNA: stable association with histonesHis tone: H1, H2A, H2B, H3, H4 Nonhistone: not as stable as DNA-histone interactionsN terminal tails are

22、subject to covalentmodification-importantfor transcriptionFive major types of histones in calf thymus Histone Mass Residue NoLys (%)Arg (%)H1H2AH2BH3H422 50013 96013 77415 27311 23621512912513510229111610111961314The DNA in chromosomes is highly condensed A scanning electron micrograph of a mitotic

23、chromosome, showing the paired identical chromatids associated along their length and joined tightly at the centromere. Nucleosomes: the lowest level of chromosome organization Nucleosome = a nucleosome core particle + linker DNA + a linker histoneDNA length: 180-200 bpNucleosome core particle = his

24、tone octamer + 146 bp DNANucleosome: the basic units of chromatin structure Kornberg R.(1974): beads on a string 30 nm fiberbeads on a string-nucleosome from interphase nucleusThe organization of chromatin in nucleosomes ?核酸酶超敏感位点(nuclease- supersensitive site)核小体与DNA的复制八聚体的组蛋白进行部分解离;其中(H3-H4)2四聚体在一

25、起,并且在两条子代双链上随机分布;原核小体中的H2A-H2B则是以两个二聚体存在,并相互分离;随机与子代双链上原或新合成的(H3-H4)2四聚体结合组成核小体。 核小体与DNA的转录 即使正在转录的基因仍然有核小体结构,表明转录并不要求整个基因都处于无核小体状态。 Nucleosomes contains DNA wrapped around a protein core of eight histone molecules Nucleosome core particle is released from chromatin by digestion of the linker DNA wi

26、th a nuclease. After dissociation of the isolated nucleosome into its protein core and DNA, the length of the DNA that was wound around the core can be determined. Its length of 146 nucleotide pairs is sufficient to wrap almost twice around the histone core. The high-resolution structure of a nucleo

27、some core particle The nucleosome core particle, as determined by X-ray diffraction analysis, reveals how DNA is tightly wrapped around a disc-shaped histone core, making 1.65 turns in a left-handed coil. K. Luger et al. Nature 1997, 389: 251260Histone depleted metaphase chromosomesChromosomes have

28、several levels of DNA packing Packaging of nucleosomes into the 30-nm chromatin fiber depends on histone H1, which is thought to pull the nucleosomes together into a regular repeating array. Each DNA molecule is packaged into a mitotic chromosome that is 10,000 fold shorter its extended length. Chro

29、matin fibers may be packed according to a zigzag modelzigzag modelThe structure of the 30-nm chromatin fiber may be a combination of these zigzag variations. An interconversion between these three variations may occur through an accordion-like expansion and contraction of the fiber. ProblemsHow the

30、long linear DNA molecules are packaged into compact chromosomes?DNA Packing Eukaryotic DNA is packaged into a set of chromosomesWhy compaction of DNA into chromosome is essential?Simple CalculationHuman: 3109 bp, 23 chromosomes1.02 m/haploid, 2.04 m/cellThe nucleus: 10 m in diameterThe mitotic chrom

31、osome is 1 mIf no compaction, nucleus would be too small to hold all DNA!Compaction of chromatin is cell-stage dependentA. Interphase chromatinB. a mitotic chromosome, which is duplicated alreadyQuestion: How this compaction is achieved?Changes in nucleosome structure allow access to DNA Eukaryotic

32、cells contain chromatin remodeling complexes, protein machines that use the energy of ATP hydrolysis to change the structure of nucleosomes temporarily so that DNA becomes less tightly bound to the histone core. The remodeled state may result from movement of the H2A-H2B dimers in the nucleosome cor

33、e; the H3H4 tetramer is particularly stable and would be difficult to rearrange. Chromatin remodeling complexes alter nucleosome structure The remodeling of nucleosome structure has two important consequences First, it permits ready access to nucleosomal DNA by other proteins in the cell, particular

34、ly those involved in gene expression, DNA replication, and repair. Second, they can catalyze changes in the positions of nucleosomes along DNA; some can even transfer a histone core from one DNA molecule to another. Metaphase ChromosomesMetaphase chromosomes are so highly condensed that their morpho

35、logy can be studied using light microscope.Staining techniques yield characteristic patterns of alternating light and dark chromosome bands.Genes can be localized to specific chromosome bands by in situ hybridization. Human metaphase chromosomes Typical appearance of a metaphase chromosome Scanning

36、electron micrograph of several human metaphase chromosomes showing the paired identical chromatids associated along their length and joined tightly at the centromere.The connections between chromatids consist of a protein called cohesin that contains a number of highly conserved subunits. The sister

37、 chromotids of a mitotic pair each consist of a fiber (30 nm in diameter) compactly folded into the chromosome.染色体的主要结构着丝粒(centromere)主缢痕(Primary constriction) 次缢痕(secondary constriction)动粒(kinetochore)核仁组织区(nucleolar organizing region, NOR)随体(satellite)端粒(telomere)四种不同位置着丝粒的染色体CentromereThe constri

38、cted region of a chromosome that is the position at which the pair of chromatids are held together. The centromeres serve both as the sites of association of sister chromatids and as the attachment sites for microtubules of the mitotic spindle. 着丝粒与动粒Centromere and kinetochoreThe Functions of centro

39、meresRequired for chromosome stabilitySister chromatid pairingMitotic and meiotic spindle attachmentChromosome movementCell cycle checkpoint control 主缢痕与主缢痕Telomeresallow complete replication of the ends of chromosomes protect them from erosion and fusion with other DNA fragments The telomere DNA se

40、quences of a variety of eukaryotes Organism Telomeric repeat sequence Yeasts Saccharomyces cerevisiae Schizosaccharomyces pombeProtozoans Tetrahymena DictyosteliumPlant ArabidopsisMammal Human G13TG25TTACGGGGTT G18AAGGGTTT TTAGGG Telomere signals on chromosomes after FISH with Cy3-labelled (CCCTAAA)

41、3 probe Telomere-mediated chromosome integrity in mammalian cells lacking telomerase or DNA repair factorsA dicentric (Dic) chromosome (pointed arrow) in a human metaphase spread showing telomere signals (red) at the termini and two centromeres (green) along the chromosome arms. 端粒的形成 人工染色体(artifici

42、al chromosome) 人工构建的含有稳定染色体的天然结构序列，即ARS、CEN、TEL序列的微小染色体，可以象天然染色体一样在寄主细胞中稳定复制和遗传，称为人工染色体。DNA结构稳定遗传的功能序列ARS (autonomous replicating sequence)CEN (centromeric sequence) The centromere is a specialized region of the chromosome that plays a critical role in ensuring the correct distribution of duplicated

43、 chromosomes to daughter cells during mitosis TEL(telomeric sequence) The sequences at the ends of eukaryotic chromosomes, called telomeres, play critical roles in chromosome replication and maintenance. 5.3 Giant chromosone多线染色体(polytene chromosome)概念时相：间期存在的组织双翅目昆虫的幼虫组织内, 如唾液腺、气管等。体积也相应增大产生的原因：Pol

44、ytene chromosome灯刷染色体(lampbrush chromosome) 灯刷染色体是卵母细胞进行减数第一 次分裂时,停留在双线期的染色体。 它是一个二价体, 含4条染色单体。它 由轴和侧丝组成,形似灯刷。灯刷染色体8.5 Nucleolus and ribosome biogenesisThe nucleolus is the most obvious structure seen in the nucleus of a eukaryotic cell when viewed in the light microscope.It is the site of rRNA tran

45、scription and processing, and of ribosome assembly. Ultrastructure of nucleolus fibrillar centers, FC dense fibrillar component, DFC granular component, GC nucleolar associated chromatin nucleolar matrixDNA稳定遗传的三种功能位点Electron micrograph of a thin section of a nucleolus in a human fibroblast, showing

46、 its three distinct zones Nucleolar fusion Function of the nucleolus in ribosome and other ribonucleoprotein synthesis The nucleolus is a ribosome production factory, designed to fulfill the need for large-scale production of rRNA and assembly of the ribosomal subunits. In addition to its important

47、role in ribosome biogenesis, the nucleolus is also the site where other RNAs are produced and other RNA-protein complexes are assembled. Nucleolar dynamics The nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. It is demonstrated that the nucleolar prot

48、eome changes significantly over time in response to changes in cellular growth conditions using a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles. The arrangement of rRNA genes The nucleolus is organized around the chromosomal regions tha

49、t contain the genes for the 5.8S, 18S, and 28S rRNA.Ribosomal RNA genes The rRNA transcription unitTranscription of the rRNA genes 18S, 5.8S, 28S rRNARNA pol I, a single unit 5S rRNARNA pol IIIProcessing of pre-rRNAThe 45S pre-rRNA transcript contains external transcribed spacers (ETS) at both ends

50、and internal transcribed spacers (ITS) between the sequences of 18S, 5.8S, and 28S rRNA. The pre-rRNA is processed via a series of cleavages (illustrated for human pre-rRNA) to yield the mature rRNA species. Processing of rRNARibosome AssemblyRibosomal proteins are imported to the nucleolus from cyt

51、oplasm and begin to assemble on pre-rRNA prior to its cleavage. As the pre-rRNA is processed, additional ribosomal proteins and the 5S rRNA assemble to form preribosomal particles. The final steps of maturation follow the export of preribosomal particles to the cytoplasm, yielding the 40S and 60S ri

52、bosomal subunits.Nonhistone proteins Nonhistone chromosomal proteins include a large number of widely diverse structural, enzymatic, and regulatory proteins.非组蛋白的种类与性质序列特异性DNA结合蛋白(sequence-specific DNA-binding protein)。其他蛋白以DNA作为底物的酶作用于组蛋白的一些酶调节基因表达的蛋白因子等非组蛋白的特性: 呈酸性、带负电荷。非组蛋白的功能除了一些酶以外，非组蛋白还具有以下功能参与染色体的构建;参与DNA复制;调控基因的表达。Transcription Factor Motifs Trans-acting factor and cis-acting element反式作用因子(trans-acting factor) They can affect the ex