Institute of Medical Genetics Qiji Liu 2010

1. Medical Genetics Institute of Medical Genetics Qiji Liu 2010

2. Cytogenetics • Human chromosome • Chromosomal aberration • Chromosome disease

3. History of Cytogenetics Dark Ages Painter TS. Studies in mammalian spermatogenesis. II. The spermatogenesis of man. J Exp Zool. 1923; 37: 291-336

4. Dark Ages Hypotonic Period Hsu TC. Mammalian chromosomes in vitro. I. The karyotype of man. J Hered. 1952; 43: 167-172 Tjio JH, Levan A. The chromosome number of man. Am J Obstet Gynecol. 1956; 130: 723-724

5. T. C. Hsu (徐道觉,April 17,1917-July,9,2003)

6. Tjio & Levan (1956) • Showed the normal human chromosome number to be 46.

7. Dark Ages Hypotonic Period Trisomy Period Lejeune J, et al. Etude des chromosomes somatiques de neuf enfants mongoliens. G. R. Acad. Sciences. 1959; 248: 1721-1722 Ford CE, et al. A sex chromosomal anomaly in a case of gonadal dysgenesis (Turner's syndrome). Lancet. 1959; 1: 711-713 JACOB PA, et al. A case of human intersexuality having a possible XXY sex determining mechanism. Nature. 1959; 183: 302-303.

8. Jérôme Lejeune (1959)

9. Dark Ages Hypotonic Period Trisomy Period Banding Era Caspersson T, et al. Differential banding of alkylating fluorochromes in human chromosomes. Exp Cell Res. 1970; 60: 315-319

10. Dark Ages Hypotonic Period Trisomy Period Banding Era Molecular Era Pardue ML, et al. Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc. Natl. Acad. Sci. USA. 1969; 64: 600 - 604 Pinkel D, et al. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA. 1986; 83: 2934-2938.

11. History of Cytogenetics • 1910-1926 Morgan, The Theory of the Gene, --beginning of cytogenetics • 1923 Painter, 2n=48, XX or XY • 1952 Hsu T C, hypo-osmotic preparing • 1956 Tjio & Levan, 2n=46,—beginning of human cytogenetics • 1959 Lejeune, Trisomy-21 • 1960 Denver system • 1970 Caspersson & Q-banding • FISH(1986), microdissection technique(1989), connect to cytogenetics — molecular cytogenetics

12. Chapter 2 Chromosomes

13. Genetic materials Chromosomes Genes DNA Chromatin Molecular level Cellular level

14. Thinking About Human Chromosomes • The human genome consists of 3 x 109 nucleotide pairs • One continuous strand of this many nucleotides would be 2 meters in length • The nucleus of a typical human cell is about 5-8 micrometers in diameter • How does the DNA fit into the nucleus?

15. 2.1.1 Human Chromatin • Definition • Components • Structure • Compaction

16. 2.1.1 Human Chromatin -Definition • Chromatin is the protein-DNA complex in which genetic material exist in interphase nucleus • During metaphase, chromatin ‘condenses’ into chromosomes

17. 2.1.1 Human Chromatin -Definition Euchromatin • Lightly stained • Loose structure • Genetically active Chromatin Heterochromatin • Deeply stained • Compact structure • Genetically silent

18. 2.1.1 Human Chromatin -Definition

19. Constitutive Heterochromatin • Always highly condensed • Enriched at centromeres and telomeres • Y chromatin Heterochromatin Facultative Heterochromatin • Specifically inactivated-specific times • Switch between two states • X chromatin 2.1.1 Human Chromatin -Definition

20. 2.1.1 Human Chromatin -Components • Chromatin is the protein-DNA complex in which genetic material exist in interphase nucleus • Components-DNA , associated proteins, and small amount of RNA

21. 2.1.1 Human Chromatin -Components • DNA: 24 different kinds of molecules • Two types of proteins • Histones Small, well-defined, highly conserved basic • Non-histone chromosomal proteins • Large number • Diverse • Mostly not well characterized

22. 2.1.1 Human Chromatin -Components Histones • Small proteins: • Arginine or lysine rich: positively charged • Interact with negatively charged DNA • Can be extensively modified(-make them less positively charged • Five types: H1, H2A, H2B, H3, H4

23. 2.1.1 Human Chromatin -Structure Nucleosomes: fundamental subunits of chromatin Electronic Microscope Evidences Nuclease digestion

24. 2.1.1 Human Chromatin -Structure EM of chromatin shows presence of nucleosomes as “beads on a string”

26. Nucleosome Structure • Contains ~ 200 bp of DNA • DNA wrapped around a core of eight basic proteins called histones – histone octamer • Linker DNA- linking nucleosome together • H1 binds to linker DNA

27. Four proteins: H2A, H2B, H3, and H4 H3 and H4 are arginine rich and highly conserved H2A and H2B are slightly enriched in lysine Both arginine and lysine are basic amino acids making the histone proteins both basic and positively charged The octomer is made of two copies of each protein The Histone Octomer

28. A fifth protein, H1, is part of the nucleosome, but resides outside the octomer H1 varies between tissue and organisms and seems to stick to the 19 bases attached to the end of the core sequence The Fifth Histone, H1 Ausio J

30. Histone H1 plays an important role in organizing the chromatin fiber

32. A Portion of the Nucleosome Looking from the top and showing the DNA wrapped around the Histones

35. 2.1.1 Human Chromatin -Compaction • Human DNA’s total length is ~2 meters! • This must be packaged into a nucleus that is about 5 micrometers in diameter • This represents a compression of more than 100,000 times ! • It is made possible by wrapping the DNA around nucleosomes and then packing these in helical filaments

36. 2.1.1 Human Chromatin -Compaction • Packing ratio is the length of the DNA divided by the length into which it’s packaged • Smallest human chromosome (21) has 4x107 bp of DNA • Equivalent to 14mm of extended DNA • In most condensed state the chromosome is about 2mm long • Packing ratio = 14000/2 = 7000

37. 2.1.1 Human Chromatin -Compaction • There are many levels of DNA compaction • First level of DNA compaction is to form nucleosome • Nucleosomes become helically arranged into a 30nM chromatin fiber (solenoid) • The fiber is super coiled by attachment to a non-histone protein scaffold (not completely understood)

39. 30 nm fiber

41. T G A Histone octomer C GC TA Histone proteins GC CG TA AT AT CG 2 nm GC TA Packaging DNA DNA Helix

42. Histone proteins 2 nm Packaging DNA T G A Histone octomer C GC TA GC CG TA AT AT CG DNA Helix GC TA

43. 11 nm Histone proteins Nucleosome 2 nm Packaging DNA T G A Histone octomer C GC TA GC CG TA AT AT CG DNA Helix GC TA

44. Histone H1 GC CG TA AT AT CG GC TA Packaging DNA

45. GC CG TA AT AT CG GC TA Packaging DNA Histone H1

46. 11 nm GC CG TA AT 30 nm 200 nm AT CG GC TA Protein scaffold Packaging DNA “Beads on a string” Looped Domains Tight helical fiber

47. 11 nm Nucleosomes 30 nm 700 nm 200 nm T Looped Domains Tight helical fiber G C A 2 nm Protein scaffold DNA Helix Packaging DNA Metaphase Chromosome

48. 2 nm – DNA double helix 10 nm – Nucleosome (10 nm fiber) 30 nm – 30 nm Fiber 300 nm – Loops I 700 nm – Loops II 1400 nm – chromosome

50. 2.1.2 Metaphase Chromosomes Interphase - replication Cell cycle Prophase - condensed Metaphase Dividing stages Anaphase - seperated Telophase