1、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,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 Chromo
2、some: 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,OUTLINE,Chromosome sequence & diversity Chromosome duplication & segregation The nucleosome Higher-order chromatin structure Regulation of chromatin structure Nucleosome
3、 assembly,D1 Prokaryotic chromosome (原核 染色体),Prokaryotic and eukaryotic chromosome structure,A single closed-circular DNA, 4.6 X 106bp 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. Contin
4、uous replication (no cell cycle) Attach to a part of cell membrane by ?,D1 Prokaryotic chromosome structure,D1-1 The E. coli chromosome,D1 Prokaryotic chromosome structure,Nucleoid (类核,拟核) Bacterial chromosome 细菌染色体,D1-2 DNA domains/loops,Observed under electron microscope 50-100 domains or loops pe
5、r E. coli chromosome, with 50-100 kb/loop The ends of loops are constrained (束缚) by Are loops dynamic or static? What do you think, why? Could you design experiments to prove it?,D1 Prokaryotic chromosome structure,Domain/ Loop,Basic protein,Supercoiled DNA,Member binding proteins?,D1 Prokaryotic ch
6、romosome structure,An unrestrained supercoil in the DNA path creates tension, but no tension is transmitted along DNA when a supercoil is restrained by protein binding,D1 Prokaryotic chromosome structure,Supercoiled domain,Non-supercoiled domain,Protein-membrane core or scaffold,D4 Genome Complexity
7、,D1-3 Supercoiling of the genome,E. coli chromosome as a whole is negatively supercoiled 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 su
8、percoiling in different domains. Do you want to try an experiment?,D1 Prokaryotic chromosome structure,back,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,
9、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,Organelle genomes: circular, multiple copies ctDNA: chloroplast
10、 (叶绿体) DNA, 140kb in plants, and 100kb in plant. Similar to -purple bacteria, Rickettsia),mtDNA,Supplementary 1:,Range of genome size found in different organism phyla.,Supplementary 2:,Prokaryotic and eukaryotic chromosome structure,D2. Chromatin Structure(染色质结构),A highly organized complex of DNA a
11、nd 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,Chromosome (condensed),Chromatin structure enables the chromosomes to alter their compactness as the cell progress the cell cycle.,Chromatin (d
12、iffused),Interphase,Mitosis,D2 Chromatin structure,The major protein components of chromatin Four families of core histone: H2A, H2B, H3 and H4. An additional non-core histone H1 Small, 10 kDa for core histones and 23 kDa for H1. Basic (rich in lysine and arginine) and tightly binds to DNA,D2-2&3. H
13、istones (组蛋白) and Nucleosomes (核小体),D2 Chromatin structure,“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 Ch
14、romatin structure,Histone octamer (组蛋白八聚体),Top view,Side view,Nucleosome core,D2 Chromatin structure,Nucleosome core 146 bp, 1.8 superhelical turn,Chromatosome 166 bp, 2 superhelical turn,DNA,Histone octamer,Histone H1,D2 Chromatin structure,D2-4. The role of H1,D2 Chromatin structure,23 kDa, locate
15、d outside of nucleosome core, binds to DNA more loosely Less conserved in its sequence (What does this suggests?),Stabilizes the point at which DNA enters and leaves the nucleosome core. C- tail of H1: stabilizes the DNA between the nucleosome cores.,D2-5. Linker DNA,D2 Chromatin structure,How was t
16、he linker identified?,The additional DNA required to make up the 200 bp nucleosomal repeat, 55 bp,Linker DNA100 bp average 55 bp,“Beads on a string” structure,Nucleosome,Histone H1,Nucleosome repeat: Core + linker DNA 200 bp,D2-6. The 30 nm fiber - Steps to make it,D2 Chromatin structure,Nucleosome
17、(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 s
18、tructure,Linker DNA100 bp average 55 bp,Step 2: “Beads on a string” structure,Nucleosome,Histone H1,Nucleosome repeat: Core + linker DNA 200 bp,D2 Chromatin structure,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
19、structure,Step 3: 30 nm fiber,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,Supplementary 1:,Nucleosomes and micrococcal nuclease treatment,Steps from DNA to chromosome,Supplementary 2:,Prokaryo
20、tic and eukaryotic chromosome structure,D3 Eukaryotic chromosome (真核染色体),Prokaryotic and eukaryotic chromosome structure,D3-1 Cell cycle (细胞周期) (增补内容),Cell cycle,Interphase 间期: G1 + S + G2 (G0) M phase (mitosis 有丝分裂):,D3 Eukaryotic chromosome structure,D3 Eukaryotic Chromosomal Structure,M phase inc
21、ludes: prophase (前期), metaphase (中期), anaphase (后期), telophase (末期),Cell cycle,D3 Eukaryotic chromosome structure,Mitotic chromosome,D3 Eukaryotic chromosome structure,D3-2 Mitotic chromosome (有丝分裂期的染色体),D3 Eukaryotic chromosome structure,more condensed than chromatin, and most highly condensed at m
22、itosis,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,The region where two chromatids (姊妹染色体）are joined The sites of attachment to the mitotic spindle (纺锤体) via kinetochore (动原体)
23、 Centromere DNA:,D3 Eukaryotic chromosome structure,The centromere (中心粒),AT-rich (88bp),Yeast:,Mitotic chromosome,D3 Eukaryotic Chromosomal Structure,Mitotic spindle,D3 Eukaryotic chromosome structure,Mitotic chromosome - centromere,Yeast centromere,Mammalian cells: much longer, flanked by satellite
24、 DNA (卫星DNA),D3 Eukaryotic chromosome structure,Specialized DNA sequences which form the ends of the linear DNA of the eukaryotic chromosome Contains up to hundreds copies of a short repeated sequence (5-TTAGGG-3 in human) Synthesized by the enzyme telomerase (a ribonucleoprotein) independent of nor
25、mal DNA replication. The telomeric DNA forms a special secondary structure to protect the chromosomal ends from degradation,The Telomere (端粒),D3 Eukaryotic chromosome structure,Telomere & Telomerase,Repeat sequence: Tetrahymena- TTGGGG; human- TTAGGG,D3 Eukaryotic Chromosomal Structure,D3 Eukaryotic
26、 chromosome structure,A loop structure forms at the end of chromosomal DNA,Telomere: structure,D3 Eukaryotic chromosome structure,D3-3 Interphase chromosomes(间期的染色体结构),Prokaryotic and eukaryotic chromosome structure,Interphase chromosomes: chromatin (染色质),Heterochromatin (异染色质)Highly condensed Trans
27、criptionally inactiveCan 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,Euchromatin (常染色质) : chromatin other than heterochromatin. More diffused and not visible The region where transcript
28、ion takes place 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,DNase I hypersensitivity: How to map th
29、e regions of transcriptionally active chromaatin ?,D3 Eukaryotic chromosome structure,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 cellsSignaling the appropriate level of chromosomal packing at the sites of expressed gene
30、s CpG methylation is associated with transcriptionally inactive regions of chromatinIslands 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 E
31、ukaryotic chromosome structure,Euchromatin,D3 Eukaryotic chromosome structure,Fig. 3. CpG islands and the promoters of housekeeping genes,D3 Eukaryotic chromosome structure,Transcription & nucleosome formation,Supplementary,Prokaryotic and eukaryotic chromosome structure,D3-4 Histone variants and mo
32、dification (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.,Short-term changes in chromosome packing modulated by chemical modification of histone proteins Actively transcribed chromatin: vi
33、a 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,Longer term differences in chromatin condensation: associated with changes due to stages in de
34、velopment (发育) and different tissue types. Utilization of alternative histone variants, H5 replacing H1 in some very inactive chromatin.,D3 Eukaryotic chromosome structure,Prokaryotic and eukaryotic chromosome structure,D4 Genome Complexity (基因组的复杂性),D4-1 Genome (基因组) & genetic complexity (遗传复杂性)(增补
35、内容),D4 Genome Complexity,Range of genome size in different phyla,D4 Genome Complexity,植物,动物,真菌 等,细菌,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 (
36、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,D4-2 Reassociation kinetics (重新结合动力学),D4 Genome Complexity,Genomic DNA extraction Sonication or shearing to a uniform size (x 100-1000 bp) 3. Thermal Denaturation 4.
37、 Re-annealing 5. Measure & plotting the re-annealing process will yield kinetics,Measuring methods： Spectroscopy/UV absorption hydroxyapatite (羟基磷灰石)chromotography,D4 Genome Complexity,D4-3: non-coding sequence,DNA sequence that does not code for protein or RNA, including Introns (unique sequence) i
38、n genes 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,D4-4 Unique sequence DNA (单一序列DNA),D4 Genome Complexity,The slowest to reassociate Corresponds to coding regions of
39、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?,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 unusuall
40、y 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),Dispersed repetitive DNA (离散重复序列） Moderately repetitive (x100- x1000 copies) Scattered throughout the genome Human Alu elements: 300b
41、p, 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,Satellite DNA (卫星DNA, simple sequence) : Highly repetitive DNA (106).very short (2 to 20-30bp, mini- or micro-), in tandem arraysconcent
42、rated near the centromeres and forms a large part of heterochromatin.as separate band in buoyant density gradientno function found, except a possible role in kinetochore bindingMinisatellite repeats are the basis of the DNA fingerprinting techniques (指纹印迹), Why?,D4 Genome Complexity,Satellite DNA,Mo
43、use genome DNA,30% GC in satellite DNA,D4 Genome Complexity,CsCl centrifugation,5 ATAAACTATAAACTATAAACT 3 3 TATTTGATATTTGATATTTGA 5,Drosophila satellite DNA repeat (several million copies),n,ACAAACT, 1.1x107 bp, 25% genome ATAAACT, 3.6x106 bp, 8% genome ACAAATT, 3.6x106 bp, 8% genome AATATAG, crypti
44、c Satellites comprise more than 40% of the genome,D4 Genome Complexity,Genes in Drosophila genome,Supplementary,Summary Prokaryotic chromosome: closed-circular DNA, domains/loops, negatively supercoiled, HU & H-NS Eukaryotic chromatin: Histones (octamer: H2A, H2B, H3, H4)+146bp DNA Nucleosomes + H1
45、chromatosome + Linker DNA beads on string 30nm fiber fiber loop + nuclear matrix highly ordered chromatin chromosome Eukaryotic chromosome structure: centromere, kinetochore, telomere, hetero- or euchromatin, CpG island and methylation Genome complexity: noncoding DNA, unique sequence, repetitive DN
46、A, satellite DNA,Homework (on the CD),See the animations for DNA topology, Topoisomerase, as well as Ribozyme Structure and Activity. Answering the questions in “applying your knowledge” is required. Play the structural tutorial “Introduction to the DNA structure” to better understand DNA structure Finish all the critical thinking exercise,
