Download PHAR2811 Dale`s lecture 3 Review of DNA Structure Another

Review of DNA Structure
PHAR2811 Dale’s lecture 3
Genome Structure
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• DNA is a biopolymer made up of
nucleotides:
– the sugar; deoxyribose,
– the phosphate,
– the base: adenine, thymine, guanine or
cytosine.
Do not remove this notice
DNA as the store of genetic
information
Some useless statistics to drive
home the point:
The removal of the OH at position 2’
The formation of thymine from uracil
Two strands gives a template for repair
Two copies of the information
The information carrying face is buried in
the middle of the two strands
• E. coli has one single circular
chromosome containing one long DNA
molecule, 1.3 mm in length. The bacterium
it has to fit in is a cylinder of diameter ~1
um and length 3 um. In other words the
bacterial dimensions seem to be 1/1000 th
of the length of the DNA (mm um).
Some useless statistics to drive
home the point:
Another useless fact:
• The full human genome contains 2 metres
of DNA (this is all 46 chromosomes worth!) in
each cell.
• The 2 metres of DNA has to be packaged
into a nucleus with a diameter of ~6 um.
This makes packing the family station
wagon to go camping look like a breeze!!
• There are about 1013 cells in your average
human (some have more, some less!!).
• The distance from the earth to the sun is
1.5 X 1011 m.
• This means there is enough DNA in the
average human to stretch from the earth to
the sun and back about 50 times!!
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How is this amazing packaging
achieved?
Chromosomes!!
• Chromosomes!!
• Geneticists for years have predicted the
existence of chromosomes; both from
microscopy and from the observation that
certain genes did not inherit in the
standard Mendelian pattern.
Prokaryotes
Prokaryotes
• The genome of prokaryotes is extremely
efficient.
• There are 4.6 million base pairs in your
average E. coli
• If the average bacterial protein has a
molecular weight of ~40,000 D how many
different proteins does the average E. coli
make?
• To do this calculation you need to know:
• The average mol. Wt. of an amino acid
~100
• This means the average protein has 400
amino acids
• Which means 1200 bases + promoter and
terminator sequences ~1500 bp.
• 4.6 X 106/1500 = ~3000 different proteins.
Prokaryotes versus Eukaryotes
Prokaryotes versus Eukaryotes
• Prokaryotes have no room for redundant
sequences.
• Their survival depends on rapid
proliferation when nutrients are available
• Complex multi-cellular eukaryotes depend
for survival on quick responses, adjusting
to changes in the environment.
• E. coli can divide every 20 min if
conditions are optimal
• The human cell takes 18 to 24 h to go
through the cell cycle once.
• The human genome only has about 2%
coding regions.
• The gene density is much lower!!
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Chromosome Characteristics
What do these life forms have in
common?
• Chromosomes vary in number between
species. The chromosome number is a
combination of the haploid number (n) X
the number of sets. Algae and fungi are
haploid; most animals and plants are
diploid. The number of pairs of
chromosomes in different species’
genomes is bizarre.
Chromosome Characteristics
Species
E. coli
Genome size in # Haploid
Megabases (Mb) chromosomes
4.6
1 circular
yeast
13
cow
human
39
3 000
alligator
23
16
carp
salamander
16
52
90 000
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Centromere Characteristics
Chromosome Characteristics
• Chromosomes vary in size within a
species. Within the human genome there is a
four fold difference in the size of the
chromosomes.
• Centromere: the region of the chromosome
where the spindle fibres attach. Repetitive
satellite DNA is often found around the
centromere.
• Telomere: ends of the chromosome,
containing a distinct repeating sequence, which
enables the ends of the chromosome to
replicate.
Centromere Characteristics
• The relative position of the centromere is
constant, which means that the ratio of the
lengths of the two arms is constant for
each chromosome. This ratio is an
important parameter for chromosome
identification, and also, the ratio of lengths
of the two arms allows classification of
chromosomes into several basic
morphologic types:
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Chromosome Banding
The Human Genome
• Chromosomes can be stained with special
dyes which give a consistent and unique
pattern like a bar-code for each
chromosome; so much so that the bands
have been numbered.
• The most common stain used is a Giesma
stain. This stain, when applied after mild
proteolytic treatment (trypsin) gives light
(G-light) and dark (G-dark) bands.
The ps and qs of chromosomes
• There are 2 arms on the chromosome
denoted p and q
• For most chromosomes the short arm is
the petit or p arm
• The longer arm is the q or queue arm
• Numbering is done from the centromere
along one of the arms
Chromosome Banding
• When viewed at the lowest resolution only a few
bands appear. These are numbered p1, p2, p3
etc counting from the centromere.
• If the stained chromosomes are viewed at higher
resolution many sub-bands are revealed. So the
labelling then goes p11, p12, p13.
• So if your DNA marker may be given a position
on the chromosome with a set of numbers like
17p23. This means the locus is on chromosome
17 on the short p arm in sub-band 23.
Some terms:
• The general material which makes up the
chromosomes is called chromatin This
is composed of DNA and protein.
• Heterochromatin contains DNA which
is more tightly packaged or condensed
and probably is transcriptionally inert.
• Euchromatin contains most active
genes; those actively transcribed.
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Chromosome packaging at the
molecular level.
• Each chromosome contains a single
molecule of DNA
• This DNA is wound around small proteins
called histones
• These proteins have lots of lysine and
arginine residues, making them very
positively charged at pH 7 (and high pIs
~12)
Histone Octamer
Histones
• There are 5 major histone variants: H1,
H2A, H2B, H3 and H4.
• Two molecules each of H2A + H2B + H3 +
H4 make up an octamer which the DNA
wraps around with 1.7 turns.
• This structure is known as a nucleosome.
• Each nucleosome has an H1 associated
and a linker section of DNA, like beads on
a thread.
Histones
• The major force holding the association of
histones to DNA is electrostatic.
• To separate the histones from the DNA,
chromatin is treated with high ionic
strength solutions. The high salt reduces
the electrostatic interactions and the
protein dissociates from the DNA.
Higher order Packaging
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Figure 11.28
A model for chromosome structure,
human chromosome 4. The 2-nm
DNA helix is wound twice around
histone octamers to form 10-nm
nucleosomes, each of which contains
160 bp (80 per turn). These
nucleosomes are then wound in
solenoid fashion with six
nucleosomes per turn to form a 30nm filament. In this model, the 30-nm
filament forms long DNA loops, each
containing about 60,000 bp, which
are attached at their base to the
nuclear matrix. Eighteen of these
loops are then wound radially around
the circumference of a single turn to
form a miniband unit of a
chromosome. Approximately 106 of
these minibands occur in each
chromatid of human chromosome 4
at mitosis.
Chromosomes at interphase
The cell cycle
At interphase (G1, S and
G2) the chromosomes
look like a plate of
spaghetti, entangled and
dispersed throughout the
nucleus
At M phase the newly
replicated daughter
chromatids condense
and line up.
The role of histones
• Shield the negative charges of the
phosphates
• Allow bending and DNA wrapping
• Restrict access to transcription
• The interaction between the histones and
the DNA is dynamic and non-base
sequence specific
Histone Remodeling
• Influences the DNA accessibility for
transcription
• Can be one of the first events when
switching on a set of genes.
• Nucleosome remodeling complexes
• Nucleosome positioning
• Histone modification
Histone modifications
• Phosphorylation; serine residues
• Methylation, adding –CH3
• Acetylation, lysine residues
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Histone modifications
• Phosphorylation; serine residues
• Methylation, adding –CH3
• Acetylation, lysine residues
Acetylation
• Transferring an acetyl group to the amino
side chain of lysine residues
• Histone acetyl Transferases (HATs)
• Histone deacetylases (HDAs)
Lysine
Acetylated Lysine
NH
O
NH
O
C
CH
H2
C
H2
C
H2
C
H2
C
+NH3
C
CH
O
H2
C
H2
C
H2
C
H2
C
NH
C
CH3
NH
NH
C
C
O
O
What effect would acetylation
have on DNA accessibility?
• It neutralises the positive charge of the
lysine side chain
• The histone will not have as much affinity
for the DNA phosphates (negative)
• The nucleosome packing will be looser
• DNA more accessible for transcription
• Deacetylases will pack it up again!
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