Presentation is loading. Please wait.

Presentation is loading. Please wait.

Welcome Each of You to My Molecular Biology Class.

Similar presentations

Presentation on theme: "Welcome Each of You to My Molecular Biology Class."— Presentation transcript:

1 Welcome Each of You to My Molecular Biology Class

2 Molecular Biology of the Gene, 5/E --- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 3/011/05

3 Part II: Maintenance of the Genome Dedicated to the structure of DNA and the processes that propagate, maintain and alter it from one cell generation to the next

4 Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome Ch 8: The replication of DNA Ch 9: The mutability and repair of DNA Ch 10: Homologous recombination at the molecular level Ch 11: Site-specific recombination and transposition of DNA 3/11/05

5 CHAPTER 7: Chromosomes, chromatin, and the nucleosome

6 Consider the structure of DNA within the cell, and the biological relevance of the structure.

7 Nucleus: 细胞核 ; Nucleolus: 核仁 Nucleoid: 类核 Mitosis: 有丝分裂; Meiosis :减数分裂 interphase :分裂间期 Histone: 组蛋白; Nucleosome: 核小体 Chromotasome: 染色小体 Chromosome: 染色体; Chromatin: 染色质; eu-; hetero- Centromere ( 中心粒) Telomere (端粒) Repetitive DNA ( 重复 DNA) Tandem gene cluster (串联基因 簇) Vocabulary

8 OUTLINE  Chromosome sequence & diversity  Chromosome duplication & segregation  The nucleosome  Higher-order chromatin structure  Regulation of chromatin structure  Nucleosome assembly

9 D1 Prokaryotic chromosome ( 原核 染色体 ) Prokaryotic and eukaryotic chromosome structure

10 1.A single closed-circular DNA, 4.6 X 10 6 bp 2.The DNA packaged into a region known as Nucleoid ( 类核 ) that contains high concentration of DNA (up to 30-50 mg/ml) as well as all proteins associated with DNA. 3.Continuous replication (no cell cycle) 4.Attach to a part of cell membrane by ? D1 Prokaryotic chromosome structure D1-1 The E. coli chromosome

11 D1 Prokaryotic chromosome structure Nucleoid ( 类核, 拟核 ) – Bacterial chromosome 细菌染色体

12 D1-2 DNA domains/loops 1.Observed under electron microscope 2.50-100 domains or loops per E. coli chromosome, with 50-100 kb/loop 3.The ends of loops are constrained ( 束缚 ) by 4.Are loops dynamic or static? What do you think, why? Could you design experiments to prove it? D1 Prokaryotic chromosome structure

13 Domain/ Loop Basic protein Supercoiled DNA Member binding proteins? D1 Prokaryotic chromosome structure

14 Supercoiled domain Non-supercoiled domain Protein-membrane core or scaffold D4 Genome Complexity

15 D1-3 Supercoiling of the genome 1.E. coli chromosome as a whole is negatively supercoiled 2.Individual domains may be supercoiled independently (topological independent) because the protein-membrane scaffold may prevent DNA rotation. Oops ! Direct biochemical evidence is lacking for different level of supercoiling in different domains. Do you want to try an experiment? D1 Prokaryotic chromosome structure back

16 D1-4 DNA-binding proteins Histone-like proteins essential for DNA packaging to stabilize and constrain the supercoiling. HU: a small basic dimeric ( 碱性双体 ) protein, non-specific binding to DNA, most abundant. H-NS (protein H1): neutral monomeric ( 中性 单体 ), partially non-specific binding Site-specific DNA binding proteins important for organization of DNA domains (RNA polymerases, IHF etc). D1 Prokaryotic chromosome structure back

17 Organelle genomes: circular, multiple copies 1.ctDNA: chloroplast ( 叶绿体 ) DNA, 140kb in plants, and <200kb in lower eukaryotes. Similar to cyanobacteria ( 兰细菌 ) 2.mtDNA: mitochondrial ( 线粒体 ) DNA, 16.5 kb in mammals, 80 kb in yeast, >100kb in plant. Similar to  -purple bacteria, Rickettsia) mtDNA Supplementary 1:

18 Range of genome size found in different organism phyla. Supplementary 2:

19 Prokaryotic and eukaryotic chromosome structure D2. Chromatin Structure ( 染色质结构 )

20 A highly organized complex of DNA and protein (nucleoprotein complex), which makes up the eukaryotic chromosomes ( 染色体 ). >50% of the mass is protein D2-1. Chromatin ( 染色质 ) D2 Chromatin structure Solving the packing problem: chromatin The length of a chromosomal DNA can be up to several centimeters (cm), but the the diameter of nucleus is about 1-10  m. (10 4 -fold of condensation) The DNA concentration in nucleus is about 200 mg/ml

21 Chromosome (condensed) Chromatin structure enables the chromosomes to alter their compactness as the cell progress the cell cycle. Chromatin (diffused) Interphase Mitosis D2 Chromatin structure

22 1.The major protein components of chromatin 2.Four families of core histone: H2A, H2B, H3 and H4. An additional non-core histone H1 3.Small, 10 kDa for core histones and 23 kDa for H1. 4.Basic (rich in lysine and arginine) and tightly binds to DNA D2-2&3. Histones ( 组蛋白 ) and Nucleosomes ( 核小体 ) D2 Chromatin structure

23 “Members of the same histone class are very highly conserved between unrelated species, but there is not much similarity in sequence between the different histone class (page 50 of your text book). ” What does this fact suggest to you? D2 Chromatin structure

24 Histone octamer ( 组蛋白八聚体 ) Top view Side view Nucleosome core D2 Chromatin structure

25 Nucleosome core 146 bp, 1.8 superhelical turn Chromatosome 166 bp, 2 superhelical turn DNA Histone octamer Histone H1 D2 Chromatin structure

26 D2-4. The role of H1 D2 Chromatin structure 23 kDa, located outside of nucleosome core, binds to DNA more loosely Less conserved in its sequence (What does this suggests?) 1.Stabilizes the point at which DNA enters and leaves the nucleosome core. 2.C- tail of H1: stabilizes the DNA between the nucleosome cores.

27 D2-5. Linker DNA D2 Chromatin structure How was the linker identified? The additional DNA required to make up the 200 bp nucleosomal repeat, ~55 bp

28 Linker DNA 100 bp average 55 bp “Beads on a string” structure Nucleosome Histone H1 Nucleosome repeat: Core + linker DNA 200 bp

29 D2-6. The 30 nm fiber - Steps to make it D2 Chromatin structure

30 Nucleosome ( 核小体 ) is the basic structural subunit of chromatin, consisting of ~200 bp of DNA and an octamer of histone proteins. DNA + Histone octamer ( 组蛋白八聚体 )  Nucleosome core ( 核小体核心 146bp) + H1  Chromatosome ( 染色小体 166bp) + linker DNA  Nucleosome ( 核小体 ) (~200 bp of DNA) Step 1: form a Nucleosome ( 核小体 ) D2 Chromatin structure

31 Linker DNA 100 bp average 55 bp Step 2: “Beads on a string” structure Nucleosome Histone H1 Nucleosome repeat: Core + linker DNA 200 bp D2 Chromatin structure

32 The 30 nm fiber Higher ordered Left-handed helix Six nucloesomes per turn 30 nm fiber (30nm in diameter) Solenoid ( 螺线管) D2 Chromatin structure Step 3: 30 nm fiber

33 Nuclear matrix ( 核基质 ), protein complex 30 nm fiber 300 nm Step 4: looped domain structure Highest level of chromatin organization D2 Chromatin structure

34 Supplementary 1: Nucleosomes and micrococcal nuclease treatment

35 Steps from DNA to chromosome Supplementary 2:

36 Prokaryotic and eukaryotic chromosome structure D3Eukaryotic chromosome ( 真核染色体 )

37 Prokaryotic and eukaryotic chromosome structure D3-1 Cell cycle ( 细胞周期 ) ( 增补内容 )

38 Cell cycle Interphase 间期 : G1 + S + G2 (G0) M phase (mitosis 有丝分裂 ): D3 Eukaryotic chromosome structure

39 D3 Eukaryotic Chromosomal Structure M phase includes: prophase ( 前期 ), metaphase ( 中期 ), anaphase ( 后期 ), telophase ( 末期 ) Cell cycle D3 Eukaryotic chromosome structure

40 Mitotic chromosome D3 Eukaryotic chromosome structure

41 D3-2 Mitotic chromosome ( 有丝分裂期的染色体 ) D3 Eukaryotic chromosome structure m ore condensed than chromatin, and most highly condensed at mitosis

42 Centromere 中心粒 Telomere Mitotic chromosome at metaphase Nuclear matrix 核基质 Loops of 30nm fiber Sister chromatid 姊妹染色单体 Chromatid 染 色单体 D3 Eukaryotic chromosome structure

43 1.The region where two chromatids ( 姊 妹染色体) are joined 2.The sites of attachment to the mitotic spindle ( 纺锤体 ) via kinetochore ( 动原 体 ) 3.Centromere DNA: D3 Eukaryotic chromosome structure The centromere ( 中心粒 ) AT-rich (88bp) Yeast:

44 Mitotic chromosome D3 Eukaryotic Chromosomal Structure Mitotic spindle D3 Eukaryotic chromosome structure

45 Mitotic chromosome - centromere Yeast centromere Mammalian cells: much longer, flanked by satellite DNA ( 卫星 DNA) D3 Eukaryotic chromosome structure

46 1.Specialized DNA sequences which form the ends of the linear DNA of the eukaryotic chromosome 2.Contains up to hundreds copies of a short repeated sequence (5’-TTAGGG-3’ in human) 3.Synthesized by the enzyme telomerase (a ribonucleoprotein) independent of normal DNA replication. 4.The telomeric DNA forms a special secondary structure to protect the chromosomal ends from degradation The Telomere ( 端粒 ) D3 Eukaryotic chromosome structure

47 Telomere & Telomerase Repeat sequence: Tetrahymena- TTGGGG; human- TTAGGG D3 Eukaryotic Chromosomal Structure D3 Eukaryotic chromosome structure

48 A loop structure forms at the end of chromosomal DNA Telomere: structure D3 Eukaryotic chromosome structure

49 D3-3 Interphase chromosomes ( 间期的染色体结构 ) Prokaryotic and eukaryotic chromosome structure

50 Interphase chromosomes: chromatin ( 染色质 ) Heterochromatin ( 异染色质 ) 1.Highly condensed 2.Transcriptionally inactive 3.Can be the repeated satellite DNA close to the centromeres, and sometimes a whole chromosome (e.g. one X chromosome in mammals) D3 Eukaryotic chromosome structure

51 Euchromatin ( 常染色质 ) : chromatin other than heterochromatin. 1.More diffused and not visible 2.The region where transcription takes place 3.Not homogenous, only a portion (~10%) euchromatin is transcriptionally active where the 30nm fiber has been dissociated to “beads on a string” structure and parts of these regions may be depleted of nucleosome. D3 Eukaryotic chromosome structure

52 DNase I hypersensitivity: How to map the regions of transcriptionally active chromaatin ? D3 Eukaryotic chromosome structure

53 Euchromatin CpG methylation ( 甲基化 ): CpG island (CpG 岛 ) Methylation of C-5 in the cytosine base of 5’-CG-3’ Occurs in mammalian cells Signaling the appropriate level of chromosomal packing at the sites of expressed genes CpG methylation is associated with transcriptionally inactive regions of chromatin Islands of unmethylated CpG are coincident with regions of DNase I hypersensitivity “Islands”: surround the promoters of housekeeping genes. Responsible for epigenetic ( 表位遗传) and may also to RNA silencing (RNA 沉默 ) D3 Eukaryotic chromosome structure

54 Euchromatin D3 Eukaryotic chromosome structure

55 Fig. 3. CpG islands and the promoters of housekeeping genes D3 Eukaryotic chromosome structure

56 Transcription & nucleosome formation Supplementary

57 Prokaryotic and eukaryotic chromosome structure D3-4 Histone variants and modification ( 组蛋白的变体和修饰) The major mechanisms for the condensing and decondensing of chromatin operate directly through the histone proteins which carry out the packaging.

58 Short-term changes in chromosome packing modulated by chemical modification of histone proteins Actively transcribed chromatin: via acetylation ( 乙酰化 ) of lysine residues in the N-terminal regions of the core histones. Condensation of chromosomes at mitosis: by the phosphorylation ( 磷酸化 ) of histone H1. D3 Eukaryotic chromosome structure

59 Longer term differences in chromatin condensation: associated with changes due to stages in development ( 发育 ) and different tissue types. Utilization of alternative histone variants, H5 replacing H1 in some very inactive chromatin. D3 Eukaryotic chromosome structure

60 Prokaryotic and eukaryotic chromosome structure D4 Genome Complexity ( 基因组的复杂性 )

61 D4-1 Genome ( 基因组 ) & genetic complexity ( 遗传复杂性 ) ( 增补内容 ) D4 Genome Complexity

62 Range of genome size in different phyla D4 Genome Complexity 植物 动物 真菌 等 真菌 等 细菌

63 D4 Genome Complexity Genome & genes ( 基因组和基因的关系) Genome: all DNA sequences in a cell Genes: a stretch of continuous DNA sequence encoding a protein or RNA C-value is the quantity of DNA in the genome (per haploid set of chromosomes). C-value paradox (C 值矛盾 ) refers to the lack of a correlation between genome size and genetic complexity

64 D4-2 Reassociation kinetics ( 重新结合动力学 ) D4 Genome Complexity 1.Genomic DNA extraction  2.Sonication or shearing to a uniform size (x 100-1000 bp)  3. Thermal Denaturation  4. Re-annealing 5. Measure & plotting the re-annealing process will yield kinetics

65 Measuring methods : Spectroscopy/UV absorption hydroxyapatite ( 羟 [ 基 ] 磷灰石 ) chromotography D4 Genome Complexity Highly repetitive DNA moderately repetitive DNA unique DNA Human E. coli

66 D4-3: non-coding sequence DNA sequence that does not code for protein or RNA, including 1.Introns (unique sequence) in genes 2.DNA consisting of multiple repeats, can be tandemly repeated sequences ( 串联重复序列) (e.g. satellite DNA) or interspersed repeats ( 分 散重复 ) (e.g. Alu element) etc. D4 Genome Complexity

67 D4-4 Unique sequence DNA ( 单一序列 DNA) D4 Genome Complexity The slowest to reassociate Corresponds to coding regions of genes occurring in one or a few copies/haploid genome All the DNA in E. coli genome has a unique sequence. Why E. coli DNA associate fast?

68 Tandem gene clusters ( 串联基因簇) : (1) moderately repetitive DNA consists of a number of types of repeated sequence. (2) genes whose products are required in unusually large quantities, e.g. there are 10-10000 copies of rDNA encoding 45S precursor and X100 copies of histone genes. D4 Genome Complexity D4-5 Repetitive DNA ( 重复序列 DNA)

69 Dispersed repetitive DNA ( 离散重复序列) 1.Moderately repetitive (x100- x1000 copies) 2.Scattered throughout the genome Human Alu elements: 300bp, 300 000 –500 000 copies of 80-90% identity Human L1 element Alu + L1= ~ 10% of human genome. Functions of these repetitive DNA : largely unknown D4 Genome Complexity

70 Satellite DNA ( 卫星 DNA, simple sequence) : - Highly repetitive DNA (>10 6 ). - very short (2 to 20-30bp, mini- or micro-), in tandem arrays - concentrated near the centromeres and forms a large part of heterochromatin. - as separate band in buoyant density gradient - no function found, except a possible role in kinetochore binding - Minisatellite repeats are the basis of the DNA fingerprinting techniques ( 指纹印迹 ), Why? D4 Genome Complexity

71 Satellite DNA Mouse genome DNA 30% GC in satellite DNA D4 Genome Complexity CsCl centrifugation

72 5’ – ATAAACTATAAACTATAAACT – 3’ 3’ – TATTTGATATTTGATATTTGA – 5’ Drosophila satellite DNA repeat (several million copies) n ACAAACT, 1.1x10 7 bp, 25% genome ATAAACT, 3.6x10 6 bp, 8% genome ACAAATT, 3.6x10 6 bp, 8% genome AATATAG, cryptic Satellites comprise more than 40% of the genome D4 Genome Complexity

73 Genes in Drosophila genome Supplementary

74 Summary 1.Prokaryotic chromosome: closed-circular DNA, domains/loops, negatively supercoiled, HU & H-NS 2.Eukaryotic chromatin: Histones (octamer: H2A, H2B, H3, H4)+146bp DNA > Nucleosomes + H1 > chromatosome + Linker DNA > beads on string > 30nm fiber > fiber loop + nuclear matrix > highly ordered chromatin > > > chromosome 3.Eukaryotic chromosome structure: centromere, kinetochore, telomere, hetero- or euchromatin, CpG island and methylation 4.Genome complexity: noncoding DNA, unique sequence, repetitive DNA, satellite DNA

75 Homework (on the CD) 1.See the animations for DNA topology, Topoisomerase, as well as Ribozyme Structure and Activity. Answering the questions in “applying your knowledge” is required. 2.Play the structural tutorial “Introduction to the DNA structure” to better understand DNA structure 3.Finish all the critical thinking exercise

Download ppt "Welcome Each of You to My Molecular Biology Class."

Similar presentations

Ads by Google