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 GeneticsPart II: Maintenance of the GenomePart III: Expression of the GenomePart IV: RegulationPart V: Methods3/011/05
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 nucleosomeCh 8: The replication of DNACh 9: The mutability and repair of DNACh 10: Homologous recombination at the molecular levelCh 11: Site-specific recombination and transposition of DNA3/11/05
5 CHAPTER 7: Chromosomes, chromatin, and the nucleosome
6 Consider the structure of DNA within the cell, and the biological relevance of the structure.
9 Prokaryotic chromosome Prokaryotic and eukaryotic chromosome structureD1Prokaryotic chromosome(原核 染色体)
10 D1-1 The E. coli chromosome D1 Prokaryotic chromosome structureD1-1 The E. coli chromosomeA single closed-circular DNA, 4.6 X 106bpThe DNA packaged into a region known as Nucleoid (类核) that contains high concentration of DNA (up to mg/ml) as well as all proteins associated with DNA.Continuous replication (no cell cycle)Attach to a part of cell membrane by ?
12 D1-2 DNA domains/loops Observed under electron microscope D1 Prokaryotic chromosome structureD1-2 DNA domains/loopsObserved under electron microscopedomains or loops per E. coli chromosome, with kb/loopThe ends of loops are constrained (束缚) byAre loops dynamic or static? What do you think, why? Could you design experiments to prove it?
13 Domain/ Loop Basic protein Supercoiled DNA Member binding proteins? D1 Prokaryotic chromosome structureDomain/LoopBasic proteinMember binding proteins?Supercoiled DNA
15 D1-3 Supercoiling of the genome D1 Prokaryotic chromosome structureD1-3 Supercoiling of the genomeE. coli chromosome as a whole is negatively supercoiledIndividual 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?back
16 D1-4 DNA-binding proteins D1 Prokaryotic chromosome structureD1-4 DNA-binding proteinsbackHistone-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 bindingSite-specific DNA binding proteins important for organization of DNA domains (RNA polymerases, IHF etc).
17 circular, multiple copies Supplementary 1:Organelle genomes:circular, multiple copiesctDNA: chloroplast (叶绿体) DNA, 140kb in plants, and <200kb in lower eukaryotes. Similar to cyanobacteria (兰细菌)mtDNA: mitochondrial (线粒体) DNA, 16.5 kb in mammals, 80 kb in yeast, >100kb in plant. Similar to -purple bacteria, Rickettsia)mtDNA
18 Range of genome size found in different organism phyla. Supplementary 2:Range of genome size found in different organism phyla.
20 D2 Chromatin structureD2-1. Chromatin (染色质)A highly organized complex of DNA and protein (nucleoprotein complex), which makes up the eukaryotic chromosomes (染色体). >50% of the mass is proteinSolving the packing problem: chromatinThe length of a chromosomal DNA can be up to several centimeters (cm), but the the diameter of nucleus is about 1-10 mm. (104-fold of condensation)The DNA concentration in nucleus is about 200 mg/ml
21 D2 Chromatin structureChromatin structure enables the chromosomes to alter their compactness as the cell progress the cell cycle.MitosisInterphaseChromatin(diffused)Chromosome(condensed)
22 D2-2&3. Histones (组蛋白) and Nucleosomes (核小体) D2 Chromatin structureD2-2&3. Histones (组蛋白) and Nucleosomes (核小体)The major protein components of chromatinFour families of core histone: H2A, H2B, H3 and H4. An additional non-core histone H1Small, 10 kDa for core histones and 23 kDa for H1.Basic (rich in lysine and arginine) and tightly binds to DNA
23 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?
26 D2 Chromatin structureD2-4. The role of H1Stabilizes the point at which DNA enters and leaves the nucleosome core.C- tail of H1: stabilizes the DNA between the nucleosome cores.23 kDa, located outside of nucleosome core, binds to DNA more looselyLess conserved in its sequence (What does this suggests?)
27 D2 Chromatin structureD2-5. Linker DNAThe additional DNA required to make up the 200 bp nucleosomal repeat, ~55 bpHow was the linker identified?
28 “Beads on a string” structure Nucleosome repeat:Core + linker DNA200 bpHistone H1NucleosomeLinker DNA<10 to > 100 bpaverage 55 bp
29 D2-6. The 30 nm fiber - Steps to make it D2 Chromatin structureD2-6. The 30 nm fiber- Steps to make it
30 Step 1: form a Nucleosome (核小体) D2 Chromatin structureStep 1: form a Nucleosome (核小体)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)Nucleosome (核小体) is the basic structural subunit of chromatin, consisting of ~200 bp of DNA and an octamer of histone proteins.
31 “Beads on a string” structure D2 Chromatin structureStep 2:“Beads on a string” structureNucleosome repeat:Core + linker DNA200 bpHistone H1NucleosomeLinker DNA<10 to > 100 bpaverage 55 bp
41 more condensed than chromatin, and most highly condensed at mitosis D3 Eukaryotic chromosome structureD3-2 Mitotic chromosome(有丝分裂期的染色体)more condensed than chromatin, and most highly condensed at mitosis
42 Mitotic chromosome at metaphase D3 Eukaryotic chromosome structureTelomereSister chromatid姊妹染色单体Centromere中心粒Mitotic chromosome at metaphaseNuclear matrix核基质Loops of 30nm fiberChromatid 染色单体
43 The centromere (中心粒) Yeast: D3 Eukaryotic chromosome structureThe centromere (中心粒)The region where two chromatids (姊妹染色体）are joinedThe sites of attachment to the mitotic spindle (纺锤体) via kinetochore (动原体)Centromere DNA:Yeast:AT-rich (88bp)
45 Mammalian cells: much longer, flanked by satellite DNA (卫星DNA) D3 Eukaryotic chromosome structureMitotic chromosome - centromereYeast centromereMammalian cells: much longer, flanked by satellite DNA (卫星DNA)
46 D3 Eukaryotic chromosome structure The Telomere (端粒)Specialized DNA sequences which form the ends of the linear DNA of the eukaryotic chromosomeContains up to hundreds copies of a short repeated sequence (5’-TTAGGG-3’ in human)Synthesized by the enzyme telomerase (a ribonucleoprotein) independent of normal DNA replication.The telomeric DNA forms a special secondary structure to protect the chromosomal ends from degradation
50 Interphase chromosomes: chromatin (染色质) D3 Eukaryotic chromosome structureInterphase chromosomes: chromatin (染色质)Heterochromatin (异染色质)Highly condensedTranscriptionally inactiveCan be the repeated satellite DNA close to the centromeres, and sometimes a whole chromosome (e.g. one X chromosome in mammals)
51 Euchromatin (常染色质) : chromatin other than heterochromatin. D3 Eukaryotic chromosome structureEuchromatin (常染色质) : chromatin other than heterochromatin.More diffused and not visibleThe region where transcription takes placeNot 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.
52 D3 Eukaryotic chromosome structure DNase I hypersensitivity: How to map the regions of transcriptionally active chromaatin ?
53 Euchromatin CpG methylation (甲基化): CpG island (CpG 岛) D3 Eukaryotic chromosome structureEuchromatinCpG 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 genesCpG 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)
57 D3-4 Histone variants and modification Prokaryotic and eukaryotic chromosome structureD3-4 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 D3 Eukaryotic chromosome structure Short-term changes in chromosome packing modulated by chemical modification of histone proteinsActively 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.
59 D3 Eukaryotic chromosome structure 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.
62 Range of genome size in different phyla D4 Genome ComplexityRange of genome size in different phyla植物动物真菌等细菌
63 Genome & genes (基因组和基因的关系） Genome: all DNA sequences in a cell D4 Genome ComplexityGenome & genes(基因组和基因的关系）Genome: all DNA sequences in a cellGenes: a stretch of continuous DNA sequence encoding a protein or RNAC-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 D4 Genome ComplexityD4-2 Reassociation kinetics(重新结合动力学)Genomic DNA extraction Sonication or shearing to a uniform size(x bp) 3. Thermal Denaturation 4. Re-annealing5. Measure & plotting the re-annealing process will yield kinetics
66 D4-3: non-coding sequence D4 Genome ComplexityD4-3: non-coding sequenceDNA sequence that does not code for protein or RNA, includingIntrons (unique sequence) in genesDNA 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.
67 D4-4 Unique sequence DNA (单一序列DNA) The slowest to reassociate D4 Genome ComplexityD4-4 Unique sequence DNA(单一序列DNA)The slowest to reassociateCorresponds to coding regions of genes occurring in one or a few copies/haploid genomeAll the DNA in E. coli genome has a unique sequence. Why E. coli DNA associate fast?
68 D4-5 Repetitive DNA (重复序列DNA) D4 Genome ComplexityD4-5 Repetitive DNA(重复序列DNA)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 copies of rDNA encoding 45S precursor and X100 copies of histone genes.
69 Alu + L1= ~ 10% of human genome. D4 Genome ComplexityDispersed repetitive DNA (离散重复序列）Moderately repetitive (x100- x1000 copies)Scattered throughout the genomeHuman Alu elements: 300bp, – copies of 80-90% identityHuman L1 elementAlu + L1= ~ 10% of human genome.Functions of these repetitive DNA：largely unknown
70 Satellite DNA (卫星DNA, simple sequence) : D4 Genome ComplexitySatellite DNA (卫星DNA, simple sequence) :Highly repetitive DNA (>106).very short (2 to 20-30bp, mini- or micro-), in tandem arraysconcentrated 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?
71 Satellite DNA Mouse genome DNA 30% GC in satellite DNA D4 Genome ComplexitySatellite DNAMouse genome DNA30% GC in satellite DNACsCl centrifugation
72 Drosophila satellite DNA repeat (several million copies) D4 Genome Complexity5’ – ATAAACTATAAACTATAAACT – 3’3’ – TATTTGATATTTGATATTTGA – 5’nACAAACT, 1.1x107 bp, 25% genomeATAAACT, 3.6x106 bp, 8% genomeACAAATT, 3.6x106 bp, 8% genomeAATATAG, crypticSatellites comprise more than 40% of the genomeDrosophila satellite DNA repeat(several million copies)
73 Genes in Drosophila genome SupplementaryGenes in Drosophila genome
74 SummaryProkaryotic chromosome: closed-circular DNA, domains/loops, negatively supercoiled, HU & H-NSEukaryotic 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 > > > chromosomeEukaryotic chromosome structure: centromere, kinetochore, telomere, hetero- or euchromatin, CpG island and methylationGenome complexity: noncoding DNA, unique sequence, repetitive DNA, satellite DNA
75 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 structureFinish all the critical thinking exercise