1、第十章 细胞核与染色体 chapter 10 Nucleus and Chromosome,主讲人：高志芹,2,Introduction,Double-membrane-enclosed organelleSeparates the genetic material from the surrounding cytoplasm.Principal feature that distinguishes eukaryotes from prokaryotes,3,Size: Nuclei( sing. nucleus) range in size from about 1 m to more th
2、an 10 m in diameter.Number: Most cells only contain one nucleus. -skeletal muscle cells have many nuclei -mature red blood cells have none. Appearance:Nuclei vary in appearance according to cell type and organism,Introduction,4,Nucleus, 复数nuclei,细胞核的概念 The largest and most important organelle Regula
3、tion center of cell heredity and metabolism. 细胞核是真核细胞内最大、最重要的细胞器，是细胞遗传与代谢的调控中心。,5,The Nucleus Structure,The interphase nucleus is a highly organized structure that consists of the nuclear envelope, nucleolus, chromatin, and nucleoplasm, etc.,6,Nucleus Structure,Nuclear envelope(核被膜),Nucleolus(核仁),Ch
4、romatin(染色质),Nuclear matrix(核基质),7,Nucleus structure,Nuclear envelope（核被膜） (nuclear membrane- is a double membrane surrounding the nucleus and its fluid. Chromatin（染色质）-genetic materialNucleolus（核仁）- dark-staining structure in the nucleus that plays a role in making ribosomes Nucleoplasm(核基质)- is a
5、highly viscous liquid that surrounds the chromatin and nucleoli (sing.nucleous),8,Nucleus Structure,Cell in the interohase,9,Nucleus Structure,10,NPR=Vn/(Vc-Vn),核质比(nucleoplasmic ratio ,NPR),11,Main contents,Section 1 nuclear envelope and nuclear pore complex Section 2 chromatin Section 3 chromatin
6、structure and gene activation Section 4 chromosome Section 5 nucleolus Section 6 nuclear matrix,12,Section 1 nuclear envelope and nuclear pore complex 第一节 核被膜与核孔复合体,Nuclear envelope(核被膜) Structure and composition of nuclear envelope Disassembly and assembly of the nuclear envelope Nuclear pore compl
7、ex（核孔复合体） Model of NPC Study about NPC Functions of NPC,13,Structure and composition of nuclear envelope,14,Structure of nuclear envelope,15,Structure of nuclear envelope,inner membrane and outer membrane.The outer membrane is continuous with the RERPerinuclear space is continuous with the RER lumen
8、.The inner membrane has a protein lining called the nuclear lamina, which binds to chromatin and other nuclear components. The nuclear membrane contains nuclear pores, which provide selective transport into and out of the nucleus.,16,(二)核被膜在细胞周期中的崩解与组装 Breakdown and reformation of nuclear envelope d
9、uring cell cycle,17,Nuclear Lamina during cell division:,They may play a role in assembly and disassembly before and after mitosis. After they are phosphorylated, this triggers the disassembly of the lamina and causes the nuclear envelope to break up into vesicles. Dephosphorylation reverses this an
10、d allows the lamina & nuclear envelope to reform.,18,二、核孔复合体 The Nuclear pore complex,The nuclear envelope in all eucaryotes, from yeasts to humans, is perforated by thousands of tiny holes called nuclear pores It is lined by an intricate protein structure called the nuclear pore complex. 核孔是指内外核膜融合
11、形成的圆环孔状结构，是由蛋白质构成的复杂结构，故称核孔复合体。,19,二、核孔复合体 The Nuclear pore complex,Generally, the more active the nucleus is in transcription, the greater the number of pore complexes. The nuclear envelope of a typical mammalian cell contains 3000-4000 complexes. 核孔数目与细胞代谢活跃程度有关。 活跃细胞可达60个/m2(如非洲爪蟾卵母细胞)； 不活跃细胞只有13
12、个/m2； 典型哺乳动物细胞30004000个核孔/细胞核。,Electron micrograph showing nuclear pores,Many nuclear pores (arrows) are visible in this freeze-fracture preparation of the nuclear envelope,21,Nuclear pore complex (NPC),22,“fish-trap”Model,Dhree-dimensional model of the nuclear pore complex,23,Cytoplasmic ring (胞质环)
13、 Nuclear ring (核质环)column subunit (柱状亚单位) Spoke (辐) luminal subunit (腔内亚单位)annular subunit (环带亚单位) Central plug (中央栓 transporter),“fish-trap”Model,24,Component of NPC 核孔复合体的成分,Each nuclear pore has an estimated a total mass of about 125 MDa Each nuclear pore is thought to be composed of more than 10
14、00 different proteins, arranged with a striking octagonal symmetry.,25,介导核孔复合体与核被膜的连接，将核孔复合体锚定在“孔膜区”，从而为核孔复合体装配提供一 个起始位点。 在内、外核膜融合形成核孔中起重要作用。 在核孔复合体的核质交换活动中起一定作用。,gp210：结构性跨膜蛋白,26,p62： 功能性的核孔复合体蛋白，具有两个功能结构域,疏水性N端区：可能在核孔复合体功能活动中直接参与核质交换。 C端区：可能通过与其它核孔复合体蛋白相互作用，从而将p62分子稳定到核孔复合体上，为其N端进行核质交换活动提供支持。,27,三
15、、核孔复合体的功能 Functions of NPC,The nuclear pore complex (NPC) acts as a selective gate that mediates the bidirectional transport of macromolecules between the cytoplasm and the nucleus of eukaryotic cells. The nuclear pore is an hydrophilic channel,28,三、核孔复合体的功能 Functions of NPC,Molecules enter and exit
16、 the nucleus through NPC,29,Import Proteins,Many proteins are required to function in the nucleus: histones, ribosomal proteins, DNA and RNA Polymerases, transcription factors, RNA-processing proteins, etc. 组蛋白， DNA复制、转录所需的各种酶，核糖体蛋白等。,30,Export material,the ribosomal subunits containing both rRNA an
17、d proteins messenger RNA (mRNA) molecules (accompanied by proteins) transfer RNA (tRNA) molecules (also accompanied by proteins) transcription factors that are returned to the cytosol to await reuse,31,Molecules enter and exit the nucleus through nuclear pore complex,Bidirectional traffic,32,三、核孔复合体
18、的功能 Functions of NPC,P314, Fig10-4,33,1.通过核孔复合体的被动运输 Passive transport through NPC,Channel is 9 nm in diameter 15 nm longResults from injection: 60 Kd: cannot enterPore dilates to 26 nm,Gold particles coated with nuclear proteins,34,Passive transport through NPC,passive: Molecules with molecular mas
19、s less than 60 Kd diffuse passively through nuclear pore complexes, estimated to have diameters of approximately 9 nm .,35,2. 核孔复合体的主动运输 Active transport through NPC,Basic character of ative transport 主动运输的基本特征 Nucleoplasmin/ karyophilic protein 亲核蛋白 NLS(nuclear locolization signal) 核定位信号 Import pro
20、cess of karyophilic protein 亲核蛋白的核输入,36,Basic character of ative transport through NPC主动运输的基本特征,Limited to the particle size (受颗粒大小的限制)。 energy dependent(需要消耗能量） a signal dependent and carrier-mediated process （是一个信号识别与载体介导的过程） Biodirectional（具有双向性）。,37,核质蛋白(nucleoplasmin)是一种亲核蛋白质(karyophilic protei
21、n)，指在细胞质合成，输入到细胞核中发挥作用的一类蛋白质。 all nucleoplasmin synthesize in cytoplasam but needed in nucleus.,核质蛋白(nucleoplasmin)的核内运输,38,Discovery of NLS 核定位信号发现的实验,39,小 结,核质蛋白,单纯尾部能入核,完整蛋白质能入核,单纯头部不能入核,Site of NLS?,40,Short amino acid sequencein in karyophilic protein, to determine the import to nucleus of prot
22、ein. NLS是存在于亲核蛋白内的一些短的氨基酸序列片段，能够定向亲核蛋白通过核孔复合体进入细胞核。 NLS与信号肽(signal peptide)的区别,NLS(nuclear locolization signal) 核定位信号,41,Nuclear Localization Signal (NLS),The first nuclear localization signal was discovered by Alan Smith and his colleagues in 1984 The first NLS to be discovered is in SV40 T antigen
23、 :- pro lys lys lys arg lys val ,42,Nuclear localization signals,In normal cells the antigen is made in the cytoplasm and is imported to the nucleus where it is concentrated 10 fold more than in the cytoplasm. During a mutagenesis study of the SV-40 virus, a class of mutants were isolated that had a
24、 higher concentration of T-antigen in the cytoplasm than in the nucleus.,43,Main characteristics of NLS,NLSs consist of one or two short sequences that are rich in lysine and arginine (positively charged). Position of NLS in protein is not important except needs to be on surface. The precise sequenc
25、e can vary.,44,Transport of large proteins into nucleus needs nuclear localization signal (NLS) NLS are present only in nuclear proteins,Function of NLS NLS的作用,45,Process of nucleoplasmin import to nucleus 核质蛋白的入核过程,The protein with a classical basic amino acid-rich nuclear localization sequence (NL
26、S) is recognized by importin , which forms a complex with importin .含NLS结构的蛋白质被importin ，识别，并与形成复合体,46,Process of nucleoplasmin import to nucleus 核质蛋白的入核过程,Importin binds to the cytoplasmic filaments of the nuclear pore complex, bringing the target protein to the nuclear pore.Importin 介导复合体与NPC的胞质纤丝
27、结合。 The protein and importin are then translocated through the nuclear pore complex in a second.转运复合体瞬间将物质通过NPC运入细胞核。,47,Process of nucleoplasmin import to nucleus 核质蛋白的入核过程,Ran-GTP is converted to Ran-GDP by the enzyme Ran-GAP in cytosol. Ran-GDP diffuses back to the nucleus. Enzyme Ran-GEF re-form
28、s Ran-GDP to Ran-GTP in the nucleus.,Ran-GTP displaces the cargo from the receptor.Ran-GTP与转运复合体结合，释放“货物”。 Ran-GTP - importin complex moves back to the cytoplasm.,48,亲核蛋白与NLS受体形成转运复合物。 转运复合物与核孔复合体的胞质纤维结合。 转运复合物通过核孔复合体构象改变从胞质面转移到核质面。 转运复合物在核质面与Ran-GTP结合，导致复合物解离，亲核蛋白释放。 受体的亚基与Ran返回胞质， Ran-GTP水解生成Ran-G
29、DP并与受体解离， Ran-GDP返回核内再转换成Ran-GTP状态。,Process of nucleoplasmin import to nucleus 核质蛋白的入核过程,49,The compartmentalization of Ran-GDP and Ran-GTP.,Ran-GTP is converted to Ran-GDP by the enzyme Ran-GAP in cytosol. Ran-GDP diffuses back to the nucleus. Enzyme Ran-GEF re-forms Ran-GDP to Ran-GTP in the nucle
30、us.,50,Export(核输出),RNA moves through nuclear pore as complex of ribonucleoprotein (RNP) . Nuclear export receptors(NER) do not bind directly to RNA, they bind proteins bound to the RNA. Protein component of RNP contains a nuclear export signal (NES) that is recognised by export proteins,51,Export(核输
31、出),RNA的出核转运是RNA-蛋白质复合体RNP的形式转运， 核输出受体与RNP中的蛋白质结合， RNP中的蛋白质含有出核输出信号。,52,Export(核输出),细胞核侧，核输出受体（nuclear export receptor） 、 Ran-GTP与货物同时结合； 细胞质侧， Ran-GTP因为Ran-GAP而水解成Ran-GDP, 与nuclear export receptor分离，因而货物也与受体分离。,53,Export(核输出),RNA聚合酶I转录的rRNA分子：以RNP的形式离开细胞核，需要能量； RNA聚合酶III转录的5s rRNA与 tRNA的核输出由蛋白质介导；,5
32、4,mRNA的出核转运,转录和新生mRNA前体(pre-mRNA)在核内加工成为成熟的mRNA才能被转运出核 5端的m7GpppG“帽子”结构对mRNA的出核转运是必要的 有极性，其5端在前，3端在后,55,Section2 chromatin(染色质),Definition of chromatin 染色质的概念: 指间期细胞核内由DNA、组蛋白、非组蛋白及少量RNA组成的线性复合结构, 是间期细胞遗传物质存在的形式。在分裂期凝集成染色体。 Chromatin: genetic substance complex present in interphase cells, consisting
33、 of DNAs, histones, nonhistones and RNAs. They condense to form chromosomes during eukaryotic cell division.,56,Section2 chromatin(染色质),Components of chromatin染色质的化学组成: DNA, Histones Non-histone proteins RNA,57,chromatin plays important roles in cell living 一、染色质是细胞生命活动的基础,the ratio of histons to DN
34、A is 1:1 the ratio of non-histons to DNA is 0.6:1 the ratio of RNA to DNA is 0.1:1,58,二、染色质DNA,染色体与DNA Each unreplicated chromosome contains a single, continuous DNA molecule(每条未复制的染色体由1个DNA分子组成) （一） Genome（基因组） One copy of all the genetic information of an organism. 某一物种的细胞内储存于单倍体染色体中的遗传信息即构成该种的基因组
35、。,59,Genome size-Tab10-2,Genome size generally increases with an organisms complexity. Some amphibian and plants have larger genome size than human. 物种的C值与其进化复杂性之间无严格对应关系。C值和生物结构或组成的复杂性不一致的现象称为C值悖论(C-value paradox)。,Species Genome size SV40 5 103bp E.coli 4.6106bp Yeast 2107bp Fruit fly 2108bp Human
36、 3109bp,60,(二) 染色质DNA类型,Protein-coding sequences（蛋白质编码序列） 编码rRNA, tRNA, snRNA和组蛋白的基因 repeated DNA(重复序列DNA) unassorted Interspersed DNA(未分类的间隔DNA),61,Protein-coding sequences 蛋白质编码序列,25-50% of the protein-coding genes are solitary(唯一的) genes（单一基因）. Others belong to gene family（基因家族）, which encode pro
37、teins with similar but nonidentical amino acid sequences. -like globin gene family:Contains 5 functional genes Tandemly repeated genes（串联重复基因） encode rRNAs, tRNAs, and Histones (Moderately repeated) Tandemly repeated genes encode identical or nearly identical proteins or functional RNAs.,62,repeated
38、 DNA(重复序列DNA),Simple-sequence DNA（简单序列DNA） Character-Highly repeated , Noncoding Types: Satellite DNA: Centromeres, Telomeres (105107bp) Microsatellite DNA:102105bp at each site Minisatellite DNA: 10100bp at each site Interspersed moderately repeated DNA (散在重复DNA),63,DNA fingerprinting depends on di
39、fferences in length of minisatellite or microsatellite DNA,基因组DNA中含有高变区，是一系列可变数目串连重复序列，具有高度多态性 ,不同高变区的重复单位内有一段序列是近似的，被称为核心序列，以此做为探针对DNA分子进行限制性片断长度多态性分析，可检测出许多高变区，产生相应的谱带，所得到的图谱具有高度个体特异性，如同人类指纹一样的高度专一，所以被称为DNA指纹图谱。,Review,Definitiion:nucleoplasmin(karyophilic protein); NLS; chromatin; Genome outline
40、of the discussion in last class: Structrue of NPC Main characteristics of NLS Process of nucleoplasmin import to nucleus. Components of chromatin,65,Interspersed moderately repeated DNA (散在重复DNA),Interspersed repeated DNA makes up 25-40% of most mammalian genomes. SINEs(short interspersed elements短散
41、在重复元件)：长度少于500bp（ Alu family (Alu基因家族 ）。 LINEs( long interspersed elements长散在重复元件):长度多于1000bp。,Interspersed moderately repeated DNA (散在重复DNA),短散在重复序列： 长度105 如人Alu序列长散在重复序列： 长度1000bp(可达7Kb)， 拷贝数104-105， 如人Kpn,67,Alu family (Alu基因家族 ):,Alu家族是灵长类基因组特有的含量丰富的短散在重复序列,在基因组中的拷贝数已经超过了100万, 每个拷贝长度约300bp，大约平均每
42、隔4Kb左右就有一个Alu序列，占人类DNA的10%. Alu family Interspersed repetitive DNA sequences that occur nearly a million times in the human genome. About 300bp long/unit, accounting for about 10% of the human DNA.,Alu 基因家族的结构,69,三、染色质蛋白质(Chromatin protein),Two types of Chromatin proteins Histones (组蛋白) Non-histone
43、proteins(非组蛋白),p322,70,（一）Histones（组蛋白）,Small（种类少）, well-defined（研究清楚）, basic（碱性）, positively charged（正电荷）. The most abundant proteins associated with eukaryotic DNA.是细胞核内含量最丰富的蛋白质，与DNA结合。 Chromatin contains five types of histones: H1(or H5), H2A, H2B, H3 and H4.染色质含5种组蛋白。 Rich in positively charged
44、 basic amino acids, which interact with the negtively charged phosphate groups in DNA.富含带正电荷的氨基酸，与带负电荷的DNA结合。 The amino acid sequences of histones H2A, H2B, H3, H4 are remarkably similar among distantly species. 组蛋白H2A, H2B, H3, H4在远亲物种间高度相似。,71,（二） Non-histone proteins -sequence specific DNA bindin
45、g protein,In chromatin, those proteins which remain after the histones have been removed, are classified as non-histone proteins. Most are acidic protein. 染色体中组蛋白以外的成分称非组蛋白。绝大部分非组蛋白呈酸性，因此也称酸性蛋白质或剩余蛋白质。,72,（二） Non-histone proteins sequence specific DNA binding protein,1. Character of non-histone prot
46、eins Diverse多样性: Structural结构的, enzymatic酶的, regulatory调控的。 sequence specific DNA binding protein序列特异性DNA结合蛋白。 Complexity fuction功能复杂性: regulate gene expression, form high level of chromosom. Mostly not well characterized.对多数非组蛋白的作用仍不清楚。,73,（二） Non-histone proteins sequence specific DNA binding prot
47、ein,sequence specific DNA binding protein 螺旋/转角/螺旋（Helix-turn-helix,HTH）基序 锌指(Zinc finger)基序 亮氨酸拉链(Leucine-Zipper)基序 螺旋-环-螺旋(Helix-loop-Helix，HLHt)基序 HMG框结构模式（HMG box motif）,74,四、染色质的基本结构单位-核小体 The fundamental unit of chromatin- nucleosome,DNA and proteins are organised in a highly regular pattern t
48、o form nucleosomes and chromatin. The fundamental unit of chromatin is the nucleosome.染色质的基本结构单位是核小体 The lowest level of Chromosome organization. 染色体组装的一级结构。 When Chromatin is isolated in low salt, see “beads on a string”低盐分离染色质可见“串珠样”结构。,75,Evidence 1: Electron micrographs of chromatin fibers,beads
49、 on a string,76,Evidence 1: Electron micrographs of chromatin fibers,Isolated from interphase nucleus: 30nm thick,Chromatin unpacked, show the uncleosome,77,Evidence 2: Nuclease digestion (Rat liver chromatin),78,Gel electrophoresis after removal of chromatin proteins,Analyzed by electron microscopy,The basic repeat unit, containing an average of 200bp of DNA associated with a protein particle, is the nucleosome,
