1. Chapter 5 Chromosomes

2. Chromosomes
In order to compact DNA molecules to fit inside a cell, specific proteins interact with DNA
Forms a condensed nucleoprotein complex called Chromatin
Chromatin structure varies greatly between and within the 3 domains of living organisms

3. Bacterial Chromatin E. coli DNA is a closed circle Length ≈ 1,600 μm
Fits into a cell 0.5 μm in diameter x 1μm long
DNA forms a condensed nucleoprotein complex called the nucleoid

4. Electron micrograph of released E. coli DNA reveals chromatin loops
Mostly supercoiled DNA loops with some relaxed loops (caused by nicks)
Each loop appears to be insulated from the others
Current model proposes supercoiled loops attached to a protein core
E. coli estimated to have 400 loops
Each loop is topologically independent

5. Macromolecular crowding and DNA binding proteins are critical contributors to DNA compaction
MukB protein
Organizes and compacts DNA
H-NS (histone-like nucleoid structuring) protein
Forms bridges across DNA segments
DNA bending proteins
Three nucleoid associated proteins bend DNA

6. Eukaryotic Chromatin Highly condensed structures called chromosomes
Germ cells (reproductive cells) have a characteristic number of chromosomes (n) also called the haploid number
Somatic cells are diploid (2n)

7. Mitosis
Mitosis – a type of nuclear division that produces two daughter cells with the same number of chromosomes as the parent cell
Eukaryotic cells spend only a small fraction of their life cycle in mitosis
Remainder is spent in interphase

8. Interphase chromatin exists in two forms
Euchromatin – less condensed
Actively transcribed
Heterochromatin – more condensed
Tends to be located near the nuclear membrane
Not actively transcribed

9. Mitosis
a liver cell nucleus

10. Interphase is broken into 3 phases
S phase: DNA replication and histone synthesis
Bracketed by 2 gap phases G1 and G2
G1→S→G2→M(mitosis)
S, G2 and M tend to be relatively uniform in length for a given cell type
G1 is quite variable

11. The cell cycle of a typical mammalian cell growing in tissue culture with a generation time of 24 hours

12. Four stages of mitosis
Prophase
Metaphase
Anaphase
Telophase

13. Sister chromatids held together by cohesin Nucleolus disappears
Prophase
Chromatin condenses
Sister chromatids held together by cohesin
Nucleolus disappears
A mitotic spindle made of microtubules begins to form
Chromosome behavior during mitosis in an organism with two pairs of chromosomes

14. Metaphase
Spindle fibers attach to the kinetochore
(a region of the centromere)
Chromosomes move toward the cell center line

15. Anaphase
Cohesin is cleaved
Sister chromatids (now chromosomes) move toward opposite poles

16. Telophase
Spindles disappear
Nuclear membrane forms
Nucleoli re-form
Chromosomes de-condense
The cell divides - cytokinesis

17. Meiosis Used to reduce the chromosome number from 2n to n
Meiosis produces gametes with a haploid chromosome number
Requires 2 successive nuclear divisions

18. Meiosis: First Meiotic Division
Prophase I
Chromatin begins to condense
Homologous chromosomes pair up (synapsis)
Fully paired chromosomes: bivalents or tetrads
Crossing over occurs and generates chiasmata: cross connections between homologous chromosomes

19. Metaphase I
Spindle fibers from one pole connect with one chromosome in a homologous pair
Each chromosome moves into the metaphase plate

20. Anaphase I
Homologous chromosomes are pulled apart
Each pole has a haploid number of chromosomes

21. Telophase I
The single Spindle dissasembles and two new spindles form
Seamless transition between Telophase I and Prophase II
No chromosome replication occurs between the 1st and 2nd meiotic divisions

22. Meiosis: Second Meiotic Division
Prophase II
Chromosomes begin moving to the midpoint
Metaphase II
Chromosomes align
Anaphase II
Centromeres split, chromatids (now considered chromosomes) move to opposite poles
Telophase II
Chromosomes decondense, nuclear membrane re-forms

23. Karyotype
Cytogenetics: study of the physical appearance of chromosomes
Examined during metaphase when chromosomes are most condensed
Centromere position determines the chromatid arm lengths
Shorter: p arm (petite)
Longer: q arm

24. Chromosomes can be stained to reveal reproducible band patterns
A karyotype of a normal human male. (a) The chromosomes as seen in the cell by microscopy

25. Each chromosome is assigned a number Largest assigned 1 Short arm - p
Human chromosome 1
Each chromosome is assigned a number
Largest assigned 1
Short arm - p
Long arm - q
Regions and bands are numbered from the centromere outward

26. Chromosome pairs are arranged in decreasing order of size
Produces an arrangement called a karyotype
Human karyotype: 23 pairs of chromosomes
22 pairs of autosomes
1 pair of sex chromosomes

27. Fluorescence in situ hybridization (FISH)
Uses fluorescent dyes bound to specific DNA probes
Probes are hybridized to the chromosomes
Chromosome painting
combines multiple dyes and specific DNA probes to uniquely label each chromosome

28. The fluorescent in situ hybridization (FISH) technique
Spectral karyotyping of human chromosomes. (b) The same chromosomes rearranged so that homologous chromosomes are shown as pairs.

29. The Nucleosome Multiple levels of chromatin packing
First level of organization: interactions between DNA and histones
Chromatin contains 5 major classes of histones
Each histone has a high percentage of the basic amino acids lysine and arginine

31. Histones are electrostatically attracted to the negatively charged phosphates of DNA
Attraction can be broken by high salt concentration
Purified DNA and purified histones mixed together will reconstitute chromatin
Extremely well conserved proteins

32. Uncondensed chromatin from interphase nuclei resemble beads on a string
Bead is a nucleoprotein complex called a nucleosome
DNA wound around 8 histone protein core
Treatment with micrococcal nuclease cleaves DNA in the linker region to release free nucleosomes

33. ”Beads on a string” structure of chromatin
Electron micrograph of chromatin. The beadlike nucleosome particles have diameters of approximately 11 nm
”Beads on a string” structure of chromatin

34. Free nucleosomes can be further digested to the nucleosome core particle made up of
164bp DNA
Octameric protein complex: 2 copies each of H2A, H2B, H3 and H4

35. X-ray crystallography reveals the atomic structure of nucleosome core particles
Histone octamer has two-fold symmetry
DNA is negatively supercoiled in the nucleosome
A-T rich minor grooves tend to contact the histone octamer

36. Nature of H1 interaction and the core particle is not known
When the fifth histone (H1) is present the particle is called the chromatosome
166bp DNA wrapped around octameric histone core held in place by H1

37. The next level of chromatin organization is not yet resolved
30nm fiber

38. Condensins and topoisomerase II help stabilize condensed chromosomes
Topo II - disentangles DNA from different chromosomes
Condensin I - shows DNA dependent ATPase activity that can in vitro introduce positive supercoils into closed circular DNA
Condensin II - activity not established
All localized along the chromatid long axis

39. Scaffold model
Predicts that non-histone proteins form a central scaffold along the long axis
Histone depleted chromosomes have DNA loops attached to a protein scaffold

40. The Centromere The Centromere
Chromosomal site of microtubule attachment
Last point of attachment between sister chromatids
Centromere DNA consists of repetitive DNA sequence
Proteins assemble to form the microtubule attachment site (Kinetochore)

41. A chromatin fiber in the centromere may fold into a cylindrical coil

42. The Telomere Telomeres: the chromosome ends
X-ray induced deletion experiments generate stable chromosomes with internal deletions, but never end deletions

43. Chromosome end has 3’ overhang
Single strand ranging from 100’s bp in yeast to 1,000’s bp in vertebrates
Folds back to form a loop
Telomeres are located at the ends of the sister chromatids. Each telomere has a G-rich 3′-overhang

44. Structure of telomere t- and D-loops

45. Werner syndrome helicase
Protein involved in telomerase formation
Defective in Werner syndrome
A genetic disease associated with premature aging

46. Eukaryotic chromosomes lose DNA from their ends during replication
Require the enzyme telomerase
Failure to replace telomeres is potentially responsible for finite number of divisions of cells in culture
Most cancer cells have telomerase