2' Genomic DNA, Genes , Chromatin

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2. Genomic DNA, Genes , Chromatin
a). Complexity of chromosomal DNA i). DNA
reassociation ii). Repetitive DNA and Alu
sequences iii). Genome size and complexity of
genomic DNA b). Gene structure i). Introns and
exons ii). Properties of the human genome
iii). Mutations caused by repetitive
sequences c). Chromosome structure - packaging
of genomic DNA i). Nucleosomes ii).
Histones iii). Nucleofilament structure and
chromosome condensation iv). Telomeres and
aging
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a). Complexity of chromosomal DNA
DNA reassociation (renaturation)
Double-stranded DNA
Denatured, single-stranded DNA
Faster, zippering reaction to form
long molecules of double- stranded DNA
k2
Slower, rate-limiting, second-order process
of finding complementary sequences to
nucleate base-pairing
3
DNA reassociation kinetics (for a single DNA
species)
Cot1/2 1 / k2 k2 second-order rate
constant Co DNA concentration t1/2 time
for half reaction
log Cot
0
DNA reassociated
50
100
Cot1/2
Ideal second-order DNA reassociation curve (Cot
curve)
4
Complexity expressed as base-pairs (bp)
101
106
102
103
105
1010
109
108
100
104
107
1
2
3
4
5
Cot1/2
10-5
100
10-4
10-3
10-1
104
103
102
10-6
10-2
101
Cot
There is a direct relationship between Cot1/2
and complexity
1 poly(dT)-poly(dA) 2 purified human
satellite DNA 3 T4 bacteriophage DNA 4 E.
coli genomic DNA 5 purified human single-copy
DNA
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DNA reassociation kinetics for a mixture of DNA
species
Cot1/2 1 / k2 k2 second-order rate
constant Co DNA concentration t1/2 time
for half reaction
Kinetic fractions fast intermediate slow
0
fast (repeated)
intermediate (repeated)
Cot1/2
DNA reassociated
50
Cot1/2
slow (single-copy)
Cot1/2
100
I I I I I I I
I I
log Cot
human genomic DNA
6
1
10,000
k2 gtgtgtgtgtgtgtgtgtgt k2
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Type of DNA of Genome
Features
Single-copy (unique) 75 Includes most
genes 1 Repetitive Interspersed 15
Interspersed throughout genome between
and within genes includes Alu sequences 2
and VNTRs or mini (micro) satellites
Satellite (tandem) 10 Highly repeated,
low complexity sequences usually
located in centromeres and
telomeres 2 Alu sequences are
about 300 bp in length and are
repeated about 300,000 times in
the genome. They can be found
adjacent to or within genes in
introns or nontranslated regions. 1
Some genes are repeated a few times to
thousands-fold and thus would be in the
repetitive DNA fraction
0
fast 10
intermediate 15
50
slow (single-copy) 75
100
I I I I I I I
I I
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Classes of repetitive DNA
Interspersed (dispersed) repeats (e.g., Alu
sequences)
GCTGAGG
GCTGAGG
GCTGAGG
Tandem repeats (e.g., microsatellites)
TTAGGGTTAGGGTTAGGGTTAGGG
9
(No Transcript)
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b). Gene structure
promoter region
exons (filled and unfilled boxed regions)
1
introns (between exons)
transcribed region
mRNA structure
5
3
translated region
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(exon-intron-exon)n structure of various genes
histone
total 400 bp exon 400 bp
b-globin
total 1,660 bp exons 990 bp
HGPRT (HPRT)
total 42,830 bp exons 1263 bp
factor VIII
total 186,000 bp exons 9,000 bp
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• Properties of the human genome
• Nuclear
• the haploid human genome has 3 X 109 bp of DNA
• single-copy DNA comprises 75 of the human
  genome
• the human genome contains 30,000 to 40,000
  genes
• most genes are single-copy in the haploid genome
• genes are composed of from 1 to gt75 exons
• genes vary in length from lt100 to gt2,300,000 bp
• Alu sequences are present throughout the genome
• Mitochondrial
• circular genome of 17,000 bp
• contains lt40 genes

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• Familial hypercholesterolemia
• autosomal dominant
• LDL receptor deficiency

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LDL receptor gene
Alu repeats present within introns
4
5
6
Alu repeats in exons
unequal crossing over
4
5
6
Alu
Alu
X
Alu
Alu
4
5
6
one product has a deleted exon
5 (the other product is not shown)
Alu
4
6
15
EM of protein-depleted human metaphase chromosome
There are 23 pairs of chromosomes in diploid
human cells. The average metaphase chromosome has
1.3 X 108 bp DNA.
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Chromatin structure
EM of chromatin shows presence of nucleosomes as
beads on a string
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Nucleosome structure

• Nucleosome core (left)
• 146 bp DNA 1 3/4 turns of DNA
• DNA is negatively supercoiled
• two each H2A, H2B, H3, H4 (histone octomer)
• Nucleosome (right)
• 200 bp DNA 2 turns of DNA plus spacer
• also includes H1 histone

18
(No Transcript)
19
(No Transcript)
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• Histones (H1, H2A, H2B, H3, H4)
• small proteins
• arginine or lysine rich positively charged
• interact with negatively charged DNA
• can be extensively modified - modifications in
• general make them less positively charged
• phosphorylation
• poly(ADP) ribosylation
• methylation
• acetylation
• hypoacetylation
• by histone deacetylase (facilitated by Rb)
• tight nucleosomes
• assoc with transcriptional repression
• hyperacetylation
• by histone acetylase (facilitated by TFs)
• loose nucleosomes
• assoc with transcriptional activation

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Nucleofilament structure
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Condensation and decondensation of a chromosome
in the cell cycle
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Telomeres are protective caps on
chromosome ends consisting of short 5-8 bp
tandemly repeated GC-rich DNA sequences, that
prevent chromosomes from fusing and
causing karyotypic rearrangements.
Telomeres and aging
Metaphase chromosome
lt1 to gt12 kb
telomere structure
(TTAGGG)many
young
(TTAGGG)few
senescent

• telomerase (an enzyme) is required to maintain
  telomere length in
• germline cells
• most differentiated somatic cells have decreased
  levels of telomerase
• and therefore their chromosomes shorten with
  each cell division