上海高校产学研合作的主要障碍及政策性建议课件.ppt

1、Chap 8 Nucleus and Chromosomes Nucleus of a Eukaryotic Cell Nuclear Envelope Nuclear Pore Complex Chromatin and Chromosome Nucleolus and Ribosome Biogenesis Nuclear Matrix 2nd Edition8.1 The Nucleus of a Eukaryotic CellThe inner nuclear membrane The outer nuclear membrane Surrounded by two concentri

2、c membranes 8.1.1 Organization 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 con

3、ventional light or electron microscopy techniques Revised picture of nuclear structure Nuclear matrix:a proteinaceous scaffold-like network Nucleolus:the center for synthesis and processing of rRNA molecules 8.2 The Nuclear Envelope The separation of a cells genetic material from the surrounding cyt

4、oplasm may be single most important feature that distinguishes eukaryotes from prokaryotes.Structure of the nuclear envelope outer nuclear membrane inner nuclear membrane nuclear lamina perinuclear space nuclear pore Nuclear laminaFunction of the nuclear envelope a barrier between the nucleus and cy

5、toplasm,as a distinct biochemical compartment sole channels through the nuclear envelope 8.3 Nuclear Pore Complex Vertebrate:50 100 proteins Diameter:120 nm,125 MDa basketlike apparatus eightfold symmetryComposition of the NPC Cytoplasmic ring Nuclear ring Spoke Central plugNuclear envelopes of Xeno

6、pus oocytes visualized by field emission 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 su

7、rfaces Electron micrograph of NPC The nuclear pore are the sites through which materials pass between the nucleus and cytoplasm Small 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

8、 in only one directionFunction of the NPCNLSs 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 thro

9、ugh NPC The NLSs of T antigen and nucleoplasmin Molecular traffic through NPCReceptors 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 N

10、LSs bind to the NPC but do not pass through the pore.The second step is an energy-dependent process that requires GTP hydrolysis.Protein import through NPC Role of the Ran protein in nuclear import Transport of RNA between nucleus and cytoplasm Active,energy-dependent process Ribonucleoprotein compl

11、exes rather than naked RNAs 8.4 Chromatin&Chromosome Chromatin typesHeterochromatin Constitutive Facultative Euchromatin The Functions of chromatin Storage of genetic information Precise segregation of replicated DNA into two daughter cells Platform for transcription,replication,recombination and DN

12、A repairComposition of Chromatin DNA:stable association with histones His tone:H1,H2A,H2B,H3,H4 Nonhistone:not as stable as DNA-histone interactionsN terminal tails aresubject to covalentmodification-importantfor transcriptionFive major types of histones in calf thymus Histone Mass Residue NoLys(%)A

13、rg(%)H1H2AH2BH3H422 50013 96013 77415 27311 23621512912513510229111610111961314The DNA in chromosomes is highly condensed A scanning electron micrograph of a mitotic chromosome,showing the paired identical chromatids associated along their length and joined tightly at the centromere.Nucleosomes:the

14、lowest level of chromosome organization Nucleosome=a nucleosome core particle+linker DNA+a linker histone DNA length:180-200 bp Nucleosome core particle=histone octamer+146 bp DNANucleosome:the basic units of chromatin structure Kornberg R.(1974):beads on a string 30 nm fiberbeads on a string-nucleo

15、some from interphase nucleus 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 with a nuclease.After dissociation of the isolated nucleosome into its protein core and DNA,the length of

16、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 nucleosome core particle The nucleosome core particle,as determined by X-ray diffraction analysis,reveals how DNA

17、is tightly wrapped around a disc-shaped histone core,making 1.65 turns in a left-handed coil.K.Luger et al.Nature 1997,389:251 260Histone depleted metaphase chromosomesChromosomes have several levels of DNA packing Packaging of nucleosomes into the 30-nm chromatin fiber depends on histone H1,which i

18、s 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.zigzag model The structure of the 30-nm chromatin fiber may be a combination of these zigzag variations.An interconversion

19、 between these three variations may occur through an accordion-like expansion and contraction of the fiber.Chromatin fibers may be packed according to a zigzag modelProblems How the long linear DNA molecules are packaged into compact chromosomes?DNA Packing Eukaryotic DNA is packaged into a set of c

20、hromosomes Why compaction of DNA into chromosome is essential?Simple Calculation Human:3 109 bp,23 chromosomes 1.02 m/haploid,2.04 m/cell The nucleus:10 m in diameter The mitotic chromosome is 1 m If no compaction,nucleus would be too small to hold all DNA!Compaction of chromatin is cell-stage depen

21、dentA.Interphase chromatinB.a mitotic chromosome,which is duplicated alreadyQuestion:How this compaction is achieved?Changes in nucleosome structure allow access to DNA Eukaryotic cells contain chromatin remodeling complexes,protein machines that use the energy of ATP hydrolysis to change the struct

22、ure 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 core;the H3H4 tetramer is particularly stable and would be difficult to rearrange.Chromatin remodeling complexes alter nucleo

23、some structure The remodeling of nucleosome structure has two important consequences First,it permits ready access to nucleosomal DNA by other proteins in the cell,particularly those involved in gene expression,DNA replication,and repair.Second,they can catalyze changes in the positions of nucleosom

24、es along DNA;some can even transfer a histone core from one DNA molecule to another.Metaphase Chromosomes Metaphase chromosomes are so highly condensed that their morphology can be studied using light microscope.Staining techniques yield characteristic patterns of alternating light and dark chromoso

25、me bands.Genes can be localized to specific chromosome bands by in situ hybridization.Human metaphase chromosomes Typical appearance of a metaphase chromosome Scanning electron micrograph of several human metaphase chromosomes showing the paired identical chromatids associated along their length and

26、 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 chromotids of a mitotic pair each consist of a fiber(30 nm in diameter)compactly folded into the chromosome.Three functional element

27、s are required for replication and stable inheritance of chromosome Centromere Telomeres Replication origins Centromere The constricted 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 chrom

28、atids and as the attachment sites for microtubules of the mitotic spindle.Three conserved regions can be identified by the sequence homologies between yeast CEN elements The Functions of centromeres Required for chromosome stability Sister chromatid pairing Mitotic and meiotic spindle attachment Chr

29、omosome movement Cell cycle checkpoint control Telomeres Allow complete replication of the ends of chromosomes Protect them from erosion and fusion with other DNA fragments lfor the discovery of how chromosomes are protected by telomeres and the enzyme telomerase Elizabeth Blackburn Carol Greider Ja

30、ck SzostakThe telomere DNA sequences of a variety of eukaryotes Organism Telomeric repeat sequence Yeasts Saccharomyces cerevisiae Schizosaccharomyces pombeProtozoans Tetrahymena DictyosteliumPlant ArabidopsisMammal Human G1 3TG2 5TTACGGGGTT G1 8AAGGGTTT TTAGGG A typical telomere has a simple repeat

31、ing structure with a G-T-rich strand that extends beyond the C-A-rich strand.The G-tail is generated by a limited degradation of the C-A-rich strand Telomere signals on chromosomes after FISH with Cy3-labelled(CCCTAAA)3 probe Telomere-mediated chromosome integrity in mammalian cells lacking telomera

32、se 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.8.5 Nucleolus and ribosome biogenesis The nucleolus is the most obvious structure seen in the nucleus of a euka

33、ryotic cell when viewed in the light microscope.It is the site of rRNA transcription and processing,and of ribosome assembly.Ultrastructure of nucleolus fibrillar centers,FC dense fibrillar component,DFC granular component,GC nucleolar associated chromatin nucleolar matrixElectron micrograph of a th

34、in section of a nucleolus in a human fibroblast,showing 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

35、 the ribosomal subunits.In addition to its important 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 res

36、ponses.It is demonstrated that the nucleolar proteome 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

37、 is organized around the chromosomal regions that 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-rRNA The 45S pre-rRNA transcript contains exte

38、rnal transcribed spacers(ETS)at both ends 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 Assembly Ribosomal proteins are

39、 imported to the nucleolus from cytoplasm 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 cytopl

40、asm,yielding the 40S and 60S ribosomal subunits.8.6 Nuclear matrix The protein network in the nucleus is called the nuclear matrix,a proteinaceous scaffold-like network that permeates the cell.It is composed of actin and numerous other protein components that have not been fully characterized,includ

41、ing components of the chromosomal scaffold that rearranges and condenses to form metaphase chromosomes during mitosis.Terms or abbreviation Nuclear matrix or nuclear skeleton SARs:scaffold-associated regions MARs:matrix-associated regions Histone depleted metaphase chromosomesSARs Regions of the chr

42、omosomes with sequences specific for topoisomerase,HMG protein,and H1 binding Found only in untranscribed regions of chromosomes Spaced along the chromosomes,with the intervening regions containing one or more genes?Highly AT rich(65%)and several hundred bp long DNA binds to the protein matrix Summa

43、ry The nucleus is the largest structure in the eukaryotic cell.It consists of DNA,proteins,and RNA,and plays a vital role in:Protein synthesis The passage of genetic information from one generation to the next Summary Nuclear envelope encloses the nucleus.It consists of two layers of membrane.Nuclea

44、r pores are found at points of contact between the inner and outer membranes.Chromatin is the collective name for the long strands of DNA and associated proteins.Summary Nucleoli are extremely dense structures in the nuclei and are highly active in rRNA synthesis.The nuclear matrix consists of DNA,nucleoproteins,and structural proteins.