Section D - Prokaryotic and Eukaryotic Chromosome Structure

1. Section D - Prokaryotic and Eukaryotic Chromosome Structure

2. Contents D1 Prokaryotic chromosome structure The Escherichia. coli chromosome, DNA domains, Supercoling of the genome, DNA-binding proteins D2 Chromatin Structure Chromatin, Histones, Nucleosomes, The role of H1, Linker DNA, The 30 nm fiber, Highter order structure D3 Eukaryotic Chromosomal Structure The mitotic chromosome, The centromere, Telomeres, Interphase chromosome, Heterochromatin, Euchromatin, DNase Ⅰ hypersensitivity, CpG methylation, Histone variants and modification D4 Genome complexity Noncoding DNA, Reasociation Kinetics, Unique sequence DNA, Tandem gene clusters, Dispersed repetitive DNA, Satellite DNA, Genetic polymorphism D5 The flow of genetic information The central dogma, Prokaryotic gene expression, Eukaryotic gene expression

3. D1 Prokaryotic chromosome structure — The Escherichia. coli chromosome • A single closed-circular DNA, 4.6Mp • The DNA packaged into a region known as Nucleoid that contains high concentration of DNA (up to 30-50 mg/ml) as well as all proteins associated with DNA. • Continuous replication (no cell cycle)

4. D1 Prokaryotic chromosome structure — DNA domains • Observed under electron microscope • 50-100 domains or loops per E. coli chromosome, with 50-100 kb/loop • The ends of loops are constrained by binding to a structure which probably consists of proteins attach to part of the cell membrane.

5. D1 Prokaryotic chromosome structure — Supercoling of the genome • E. coli chromosome as a whole is negatively supercoiled. • Individual domains may be supercoiled independently (topological independent). • Direct biochemical evidence is lacking for different level of supercoiling in different domains.

6. D1 Prokaryotic chromosome structure — DNA-binding proteins • Histone-likeproteins 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 proteinsimportant for organization of DNA domains (RNA polymerases, IHF etc).

7. DNA 10nm "beads-on-a-string" fibre metaphase chromosome DNA compaction 30nm fibre nucleosome D2 Chromatin Structure — Chromatin • Fig. 1: The major structures in DNA compaction; DNA, the nucleosome, the 10nm "beads-on-a-string" fibre, the 30nm fibre and the metaphase chromosome.

8. Mitosis 有丝分裂 Interphase间期 Chromosome (condensed) Chromatin (diffused)

10. D2 Chromatin Structure — Histones • 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.

11. Top view Histone octamer Side view

12. D2 Chromatin Structure — Nucleosomes • The nucleosome core is the basic unit of chromosome structure, consisting of a protein octamer containing two each of core histones. • With 146 bp of DNA wrapped 1.8 times in a left-handed fashin around it. • The wrapped of DNA into nucleosomes accounts for virually all of the negative supercoiling in eukaryotic DNA.

13. linker histone H1 and H5 family D2 Chromatin Structure — 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.

15. D2 Chromatin Structure — Linker DNA • The additional DNA required to make up the 200 bp nucleosomal repeat, ~55 bp

16. D2 Chromatin Structure — The 30 nm fiber • Higher ordered • Left-handed helix • Six nucloesomes per turn

17. D2 Chromatin Structure — Highter order structure • On the largest scale, chromosomal DNA is organized into loops of up to 100 kb in the foem of the 30nm fiber

18. D3 Eukaryotic Chromosomal Structure — The mitotic chromosome Telomere Sister chromatid Centromere Loops of 30nm fiber Mitotic chromosome at metaphase Nuclear matrix Chromatid

19. Mitotic chromosome

20. D3 Eukaryotic Chromosomal Structure — The centromere • The region where two chromatids are joined. • The sites of attachment to the mitotic spindle via kinetochore. • Centromere DNA. • Chromosomal components: • Chromatid • Centromere/Primary Constriction • Short arm • Long arm

21. D3 Eukaryotic Chromosomal Structure — Telomeres • 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.

22. Human chromosomes (grey) capped by telomeres (white).

23. D3 Eukaryotic Chromosomal Structure — Interphase chromosome • In interphase, the chromosomes adopt a much more diffuse structure, although the chromosomal loops remain attached to the nuclear matrix. • Cannot be visualized individually.

24. D3 Eukaryotic Chromosomal Structure — 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)

25. D3 Eukaryotic Chromosomal Structure — Euchromatin • Euchromatin: 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.

26. Plant cells Animal cells • The nucleus of a human cell showing the location of heterochromatin.

27. D3 Eukaryotic Chromosomal Structure — DNase Ⅰ hypersensitivity • Active regions of chromatin, or regions where the 30nm fiber is interrupted by the binding of a specific protein to the DNA, or by ongoing transcription, are characterized by hypersensitivity to deoxyribonuclease Ⅰ(DNase Ⅰ ).

28. D3 Eukaryotic Chromosomal Structure — CpG methylation • Methylation of C-5 in the cytosine （胞嘧啶）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 （渐成说） and may also to RNA silencing.

30. D3 Eukaryotic Chromosomal Structure — Histone variants and modification • The major mechanisms for the condensing and decondensing of chromatin operate directly through the histone proteins which carry out the packaging. • Histones undergo posttranslational modifications which alter their interaction with DNA and nuclear proteins. • The H3 and H4 histones have long tails can be covalently modified. Modifications of the tail include methylation, acetylation, phosphorylation, etc. • The core of the histones (H2A and H3) can also be modified. • Histone modifications act in diverse biological processes such as gene regulation, DNA repair and chromosome condensation (mitosis).

31. D4 Genome complexity — Noncoding DNA • 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 (e.g. satellite DNA) or interspersed repeats (e.g. Alu element) etc.

32. D4 Genome complexity — Reasociation Kinetics（复性动力学）

33. D4 Genome complexity — Unique sequence DNA • The slowest to reassociate （复性最慢） • 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.

34. D4 Genome complexity — Tandem gene clusters • Tandem gene clusters: • moderately repetitive DNA consists of a number of types of repeated sequence. • 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.

35. D4 Genome complexity — Dispersed repetitive DNA • Moderately repetitive (x100- x1000 copies) • 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

36. D4 Genome complexity — Satellite DNA • 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.

37. D4 Genome complexity — Genetic polymorphism 1. Single-nucleotide polymorphism 2. Simple sequence length polymorphism 3. Restriction fragment length polymorphism 4. Single strand conformation polymorphism

38. D5 The flow of genetic information — The central dogma

40. Promoter Terminator DNA Transcribed region 3‘ 5‘ 5‘ 3‘ Transcription RNA polymerase RBS mRNA AUG stop AUG stop 5‘ pop OH 3‘ RBS Ribosomes, aminoacyl-tRNAs Translation Proteins D5 The flow of genetic information — Prokaryotic gene expression

41. Promoter DNA Transcribed region 3‘ 5‘ 5‘ 3‘ Transcription RNA polymerase Ⅱ EXONS Pre-mRNA AUG stop INTRON 5‘ pop Poly( A) site Splicing, capping, polyadenylation RNA processing AUG stop mRNA AAA…3‘ 5‘ MeGppp Poly( A) tail CAP Ribosomes, aminoacyl-tRNAs Translation Proteins D5 The flow of genetic information — Eukaryotic gene expression

42. Cellular organelles you should further understand Cytoplasm－－－cellular organelles 类囊体 Chloroplast Mitochondrion The endoplasmic reticulum (ER) is responsible for the production of the protein and lipid components of most of the cell's organelles. 池 Golgi Apparatus。The Golgi is principally responsible for directing molecular traffic in the cell

43. Cellular organelles（continue） 肌动蛋白 Lysomoes（溶酶体），which contains hydrolytic enzymes Cytoskeleton(细胞骨架) The vacuole is used only in plant cells. It is responsible for maintaining the shape and structure of the cell. The ribosome plays a key role in the synthesis of proteins.

44. Multiple choice questions 1．Which of the following is common to both E. coli and eukaryotic chromosomes? A the DNA is circular. B the DNA is packaged into nucleosomes. C the DNA is contained in the nucleus. D the DNA is negatively supercoiled. 2．A complex of 166 bp of DNA with the histone octamer plus histone HI is known as a . . . A nucleosome core. B solenoid. C 30 nm fiber. D chromatosome.

45. 3．In what region of the interphase chromosome does transcription take place? A the telomere. B the centromere. C euchromatin. D heterochromatin. 4． Which statement about CpG islands and methylation is not true? A CpG islands are particularly resistant to DNase I. B CpG methylation is responsible for the mutation of CpG to TpG in eukaryotes. C CpG islands occur around the promoters of active genes. D CpG methylation is associated with inactive chromatin.

46. 5．Which of the following is an example of highly-repetitive DNA? A Alu element. B histone gene cluster. C DNA minisatellites. D dispersed repetitive DNA.

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