Procaryotic chromosomeProcaryotic chromosome((Escherichia coli)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

Negatively supercoiled

50-100 domains/loops (50-100kb in size)

The ends constrained by protein membrane scaffold

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.

Chromatin structureChromatin 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

HistonesHistonespositively chargedpositively 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

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.

Digestion of chromation by micrococcal nuclease

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)

/ Wedge-shaped disk

/ 1.8 turns, left-handed

Beads on a string

The 30 nm fiber

Higher ordered

Left-handed helix

Six-nucleosomes per turn

Back

From DNA to chromosome

Eucaryotic chromosomeEucaryotic chromosome

Cell cycle

Mitotic chromosome

Sister chromatids Section

Telomere

1. Specialized DNA sequences which form the ends of the linear DNA of the eukaryotic chromosome2. Contains up to hundreds copies of a short repeated sequ

ence (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

Human telomere structure

Interphase chromosomesInterphase 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

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

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

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.

CpG island

CpG island methylation

Histione variants and modificationHistione 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.

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.

Genome complexityGenome complexity

Genome

all DNA sequences in a cell

Gene

a stretch of continuous DNA sequence

encoding a protein or RNA

Genome sizes of model systems

Model systems size(bases) genes

Escherichia coli (Bacterium)  4.6 Million 3,000Sacchromyces cerevisiae (Yeast) 15 Million Drosophila melanogaster (Fruit fly) 120 Million 13,647 Anapheles gambiae (Mosquito) 280 Million 13,600Gallus gallus (Chicken) 120 Million Mouse 3 Billion 30,000Xenopus laevis (Frog) 3 BillionHuman 3 Billion 31,000Largest human chromosome (chr 1) 250 MillionSmallest human chromosome (Y) 50 Million 

Coding region <5% in human genome

Noncoding DNA introns

tandemly repeated sequences

e.g. satellite DNA

or interspersed repeats

e.g. Alu element

Uninterrupted genes in genomes

E. coli 100%

Yeast 96%

Drosophila 17%

Human 6%

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

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.

Dispersed repetitive DNA:

Human Alu elements 300bp, 300000 –500000 copies of 80-90% identityL1 element

Alu and L1: almost 10% of human genome.Transposition, as selfish or parasitic DNA, role in ev

olution

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

DNA fingerprinting

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

Essay questionsEssay 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?