Download Procaryotic chromosome

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
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 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
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
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 chromosome
Cell cycle
Mitotic chromosome
Sister chromatids
Section
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
Human telomere structure
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
Euchromatin
- More diffuse
- Consisting of 30nm fiber (inactive), beads-on-astring, 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 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 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)
Sacchromyces cerevisiae (Yeast)
Drosophila melanogaster (Fruit fly)
Anapheles gambiae (Mosquito)
Gallus gallus (Chicken)
Mouse
Xenopus laevis (Frog)
Human
Largest human chromosome (chr 1)
Smallest human chromosome (Y)
4.6 Million
15 Million
120 Million
280 Million
120 Million
3 Billion
3 Billion
3 Billion
250 Million
50 Million
3,000
13,647
13,600
30,000
31,000
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
Yeast
Drosophila
Human
100%
96%
17%
6%
Genome complexity
Reassociation kinetics
Unique sequence
one to a few copies
Moderately repetitive
< 1 million copies
Highly repetitive
> 1 million copies
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) –1010000 copies
(human: 44kb with ca. 5x40 copies);
histone genes: >100.
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
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--55200+) > 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 questions
1.
2.
3.
What are the main biological
macromolecules? How about their
functions?
How to analyze an unknown protein and
DNA fragments?
How about the chromosome structure?
and genome complexity?