1. Procaryotic chromosome (Escherichia coli) Nucleoid 1. High concentration of DNA (single closed circular, 4.6Mb) and the proteins associated with DNA. 2. DNA concentration can be up to 30-50 mg/ml 3. Continuous replication (more than one copy of genome/cell) 4. Attachment to cell membrane

2. Negatively supercoiled 50-100 domains/loops (50-100kb in size) The ends constrained by protein membrane scaffold

3. DNA-binding proteins essential for DNA packing to nucleoid HU –small, dimeric & basic, non-specific binding H-NS –monomeric, neutral Specific DNA binding proteins RNA polymerases etc.

4. Chromatin structure Chromatin The highly ordered DNA-protein (nucleoprotein) complex which makes up the eukaryotic chromosomes. >50% of the mass is protein For solving the packing problem The length of a chromosomal DNA is at cm level, but the diameter of nucleus is 1-10 μm. The DNA concentration in nucleus is about 200 mg/ml

5. Histones positively charged Core histones 1. 10-20 kDa 2. located in the nucleosome core octamer core (H2A)2(H2B)2(H3)2 (H4)2 3. highly conserved in their sequences

6. H1 histone 1. Larger size (23 kDa) 2. Located outside of nucleosome core, bind to DNA more loosely 3. Less conserved in its sequence 4. Stabilize the point at which DNA enters and leaves the nucleosome core. 5. Stabilize the DNA between the nucleosome cores by C- tail of H1.

7. Digestion of chromation by micrococcal nuclease

8. Nucleosome the basic structural subunit of chromatin, consisting of ~200 bp of DNA and an octamer of histone proteins. DNA + Histone octamer (Nucleosome core 146bp) +H1 Chromatosome (166bp) + linker DNA (~55 bp) Nucleosome (~200bp of DNA)

9. / Wedge-shaped disk / 1.8 turns, left-handed

10. Beads on a string The 30 nm fiber Higher ordered Left-handed helix Six-nucleosomes per turn Back

11. From DNA to chromosome

12. Eucaryotic chromosome Cell cycle

14. Mitotic chromosome Sister chromatids Section

15. Telomere 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

16. Human telomere structure

17. Interphase chromosomes Chromatin = Heterochromatin + Euchromatin Heterochromatin Still highly condensed Transcriptionally inactive Repeated satellite DNA close to the centromeres Whole chromosome in case, e.g. one X chromosome in mammals

18. Euchromatin - More diffuse - Consisting of 30nm fiber (inactive), beads-on-a- string, and active transcrption region depleted of nucleosome. - Only a portion (~10%) of euchromatin is transcriptionally active

19. DNase I hypersensitivity - DNase I cuts the backbone of DNA unless protected by bound protein. - Short regions of DNase I hypersensitivity 30nm fiber is interrupted by the binding of a sequence-specific regulatory protein - Longer regions of DNase I hypersensitivity where transcription is taking place

20. CpG methylation - Methylation of C-5 in the cytosine base of 5’-CG-3’ sequences in mammalian cells - Signaling the appropriate level of chromosomalpacking 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 surrounding the promoters of housekeeping genes.

21. CpG island CpG island methylation

22. Histione variants and modification Short-term changes in chromosome packing modulated by chemical modification of histone proteins - Actively transcribed chromatin: via acetylation of lysine residues in the N-terminal regions of the core histones. - Condensation of chromosomes at mitosis by the phosphorylation of histone H1.

23. Long-term differences in chromatin condensation: associated with changes due to stages in development and different tissue types: Utilization of alternative histone variants, H5 replacing H1 in some very inactive chromatin.

24. Genome complexity Genome all DNA sequences in a cell Gene a stretch of continuous DNA sequence encoding a protein or RNA

25. Genome sizes of model systems Model systems size(bases) genes Escherichia coli (Bacterium) 4.6 Million 3,000 Sacchromyces cerevisiae (Yeast) 15 Million Drosophila melanogaster (Fruit fly) 120 Million 13,647 Anapheles gambiae (Mosquito) 280 Million 13,600 Gallus gallus (Chicken) 120 Million Mouse 3 Billion 30,000 Xenopus laevis (Frog) 3 Billion Human 3 Billion 31,000 Largest human chromosome (chr 1) 250 Million Smallest human chromosome (Y) 50 Million

26. Coding region <5% in human genome Noncoding DNA introns tandemly repeated sequences e.g. satellite DNA or interspersed repeats e.g. Alu element

27. Uninterrupted genes in genomes E. coli 100% Yeast 96% Drosophila 17% Human 6%

28. Genome complexity Unique sequence one to a few copies Moderately repetitive < 1 million copies Highly repetitive > 1 million copies Reassociation kinetics The kinetics of DNA reassociation reveal DNA classes differing in repetition frequency

29. Moderately repetitive (10-100/1000 level) Tandem gene clusters rDNA: 45S precursor (18,5.8 & 28S) –10- 10000 copies (human: 44kb with ca. 5x40 copies); histone genes: >100.

30. Dispersed repetitive DNA: Human Alu elements 300bp, 300000 –500000 copies of 80-90% identity L1 element Alu and L1: almost 10% of human genome. Transposition, as selfish or parasitic DNA, role in evolution

31. Satellite DNA - Highly repetitive in eukaryotic genome - very short (2 to 20-30bp, mini- or micro-) - 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 - Repeat number in some arrays hypervariable between individuals, DNA fingerprinting

32. DNA fingerprinting

33. Summary 1. Prokaryotic chromosome: closed-circular DNA, 50-100kb loops, negatively supercoiled, HU & H-NS 2. Eukaryotic chromatin: Histones (octamer)+146bp DNA > Nucleosome core + H1 >chromatosome + Linker DNA (10--55- 200+) > beads on string > 30nm fiber > fiber loop (to 100bp) +nuclear matrix > chromosome 3. Jargons: centromere, kinetochore, telomere, hetero or euchromatin, CpG island and methylation 4. Genome complexity: noncoding DNA, unique sequence, tandem/dispersed repetitive DNA, satellite DNA

34. Essay questions 1. What are the main biological macromolecules? How about their functions? 2. How to analyze an unknown protein and DNA fragments? 3. How about the chromosome structure? and genome complexity?