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
Vocabulary
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）

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

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

10. D1-1 The E. coli chromosome
D1 Prokaryotic chromosome structure
D1-1 The E. coli chromosome
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 mg/ml) as well as all proteins associated with DNA.
Continuous replication (no cell cycle)
Attach to a part of cell membrane by ?

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

12. D1-2 DNA domains/loops Observed under electron microscope
D1 Prokaryotic chromosome structure
D1-2 DNA domains/loops
Observed under electron microscope
domains or loops per E. coli chromosome, with 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?

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

14. Non-supercoiled domain
D4 Genome Complexity
D1 Prokaryotic chromosome structure
Supercoiled domain
Non-supercoiled domain
Protein-membrane core or scaffold

15. D1-3 Supercoiling of the genome
D1 Prokaryotic chromosome structure
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 supercoiling in different domains. Do you want to try an experiment?
back

16. D1-4 DNA-binding proteins
D1 Prokaryotic chromosome structure
D1-4 DNA-binding proteins
back
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).

17. circular, multiple copies
Supplementary 1:
Organelle genomes:
circular, multiple copies
ctDNA: 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.

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

20. D2 Chromatin structure
D2-1. 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 protein
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 mm. (104-fold of condensation)
The DNA concentration in nucleus is about 200 mg/ml

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

22. D2-2&3. Histones (组蛋白) and Nucleosomes (核小体)
D2 Chromatin structure
D2-2&3. Histones (组蛋白) and Nucleosomes (核小体)
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

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?

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

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

26. D2 Chromatin structure
D2-4. The role of H1
Stabilizes 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 loosely
Less conserved in its sequence (What does this suggests?)

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

28. “Beads on a string” structure
Nucleosome repeat:
Core + linker DNA
200 bp
Histone H1
Nucleosome
Linker DNA
<10 to > 100 bp
average 55 bp

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

30. Step 1: form a Nucleosome (核小体)
D2 Chromatin structure
Step 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 structure
Step 2:
“Beads on a string” structure
Nucleosome repeat:
Core + linker DNA
200 bp
Histone H1
Nucleosome
Linker DNA
<10 to > 100 bp
average 55 bp

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

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

34. Nucleosomes and micrococcal nuclease treatment
Supplementary 1:
Nucleosomes and micrococcal nuclease treatment

35. Steps from DNA to chromosome
Supplementary 2:
Steps from DNA to chromosome

36. D3 Eukaryotic chromosome (真核染色体)
Prokaryotic and eukaryotic chromosome structure
D3 Eukaryotic chromosome (真核染色体)

37. D3-1 Cell cycle (细胞周期) (增补内容)
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
Cell cycle
Interphase 间期: G1 + S + G2 (G0)
M phase (mitosis 有丝分裂):

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

40. D3 Eukaryotic chromosome structure
Mitotic chromosome

41. more condensed than chromatin, and most highly condensed at mitosis
D3 Eukaryotic chromosome structure
D3-2 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 structure
Telomere
Sister chromatid
姊妹染色单体
Centromere
中心粒
Mitotic chromosome at metaphase
Nuclear matrix
核基质
Loops of 30nm fiber
Chromatid 染色单体

43. The centromere (中心粒) Yeast:
D3 Eukaryotic chromosome structure
The centromere (中心粒)
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)
Centromere DNA:
Yeast:
AT-rich (88bp)

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

45. Mammalian cells: much longer, flanked by satellite DNA (卫星DNA)
D3 Eukaryotic chromosome structure
Mitotic chromosome - centromere
Yeast centromere
Mammalian 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 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 normal DNA replication.
The telomeric DNA forms a special secondary structure to protect the chromosomal ends from degradation

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

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

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

50. Interphase chromosomes: chromatin (染色质)
D3 Eukaryotic chromosome structure
Interphase chromosomes: chromatin (染色质)
Heterochromatin (异染色质)
Highly condensed
Transcriptionally inactive
Can 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 structure
Euchromatin (常染色质) : chromatin other than heterochromatin.
More diffused and not visible
The region where transcription 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.

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 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 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)

54. D3 Eukaryotic chromosome structure
Euchromatin

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

56. Transcription & nucleosome formation
Supplementary
Transcription & nucleosome formation

57. D3-4 Histone variants and modification
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. D3 Eukaryotic chromosome structure
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.

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.

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

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

62. Range of genome size in different phyla
D4 Genome Complexity
Range 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 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
D4 Genome Complexity
D4-2 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-annealing
5. Measure & plotting the re-annealing process will yield kinetics

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

66. D4-3: non-coding sequence
D4 Genome Complexity
D4-3: non-coding sequence
DNA sequence that does not code for protein or RNA, including
Introns (unique sequence) in 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.

67. D4-4 Unique sequence DNA (单一序列DNA) The slowest to reassociate
D4 Genome Complexity
D4-4 Unique sequence DNA
(单一序列DNA)
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. D4-5 Repetitive DNA (重复序列DNA)
D4 Genome Complexity
D4-5 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 Complexity
Dispersed repetitive DNA (离散重复序列）
Moderately repetitive (x100- x1000 copies)
Scattered throughout the genome
Human Alu elements: 300bp, – copies of 80-90% identity
Human L1 element
Alu + L1= ~ 10% of human genome.
Functions of these repetitive DNA：largely unknown

70. Satellite DNA (卫星DNA, simple sequence) :
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 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?

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

72. Drosophila satellite DNA repeat (several million copies)
D4 Genome Complexity
5’ – ATAAACTATAAACTATAAACT – 3’
3’ – TATTTGATATTTGATATTTGA – 5’ n
ACAAACT, 1.1x107 bp, 25% genome
ATAAACT, 3.6x106 bp, 8% genome
ACAAATT, 3.6x106 bp, 8% genome
AATATAG, cryptic
Satellites comprise more than 40% of the genome
Drosophila satellite DNA repeat
(several million copies)

73. Genes in Drosophila genome
Supplementary
Genes in Drosophila genome

74. 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 > 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 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 structure
Finish all the critical thinking exercise