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
a). Complexity of chromosomal DNA
DNA reassociation (renaturation)
Double-stranded DNA

Denatured,
single-stranded
DNA
Faster,
zippering
reaction to
k2 form long
Slower, rate-limiting, molecules
second-order process of of double-
finding complementary stranded
sequences to nucleate DNA
base-pairing
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)
 Complexity expressed as base-pairs (bp)
100 101 102 103 104 105 106 107 108 109 1010

1 2 3 4 5

Cot1/2

10-6 10-5 10-4 10-3 10-2 10-1 100 101 102 103 104
Cot There is a direct
1 = poly(dT)-poly(dA) relationship between
2 = purified human satellite DNA Cot1/2 and complexity
3 = T4 bacteriophage DNA
4 = E. coli genomic DNA
5 = purified human single-copy DNA
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

0 Kinetic fractions:
% DNA reassociated

fast (repeated)
fast
intermediate intermediate
(repeated) slow
Cot1/2
50
Cot1/2
slow (single-copy)
Cot1/2
100
I I I I I I I I I
log Cot
human genomic DNA
10,000 1

k2 >>>>>>>>>> k2
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
0 and telomeres
fast ~10%

intermediate 2 Alu sequences are

~15% about 300 bp in length
50 and are repeated about
300,000 times in the
slow (single-copy) genome. They can be
~75% found adjacent to or
within genes in introns
100 or nontranslated regions.
I I I I I I I I I
1 Some genes are repeated a few times to thousands-fold and thus would be in
the repetitive DNA fraction
 Classes of repetitive DNA

Interspersed (dispersed) repeats (e.g., Alu sequences)

GCTGAGG GCTGAGG GCTGAGG

Tandem repeats (e.g., microsatellites)

TTAGGGTTAGGGTTAGGGTTAGGG
 Genome sizes in nucleotide pairs (base-pairs)

plasmids
viruses
bacteria
fungi
plants
algae
insects
mollusks
bony fish
The size of the human
genome is ~ 3 X 109 bp; amphibians
almost all of its complexity
is in single-copy DNA. reptiles
birds
The human genome is thought
to contain ~30,000 to 40,000 genes. mammals

104 105 106 107 108 109 1010 1011

b). Gene structure

promoter exons (filled and unfilled boxed regions)

region

+1
introns (between exons)

transcribed region

mRNA structure

5’ 3’

translated region
 (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
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 >75 exons
• genes vary in length from <100 to >2,300,000 bp
• Alu sequences are present throughout the genome

Mitochondrial

• circular genome of ~17,000 bp

• contains <40 genes
Familial hypercholesterolemia
• autosomal dominant
• LDL receptor deficiency
LDL receptor gene

Alu repeats present within introns

4 5 6
Alu repeats in exons
unequal 4 5 6
crossing over Alu Alu

X
Alu Alu
4 5 6

one product has a

deleted exon 5
Alu (the other product is not shown)
4 6
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.
 Chromatin structure

EM of chromatin shows presence of

nucleosomes as “beads on a string”
 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
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
Nucleofilament structure
Condensation and decondensation
of a chromosome in the cell cycle
 Telomeres are protective
Telomeres and aging “caps” on chromosome
ends consisting of short
Metaphase chromosome 5-8 bp tandemly repeated
GC-rich DNA sequences,
that prevent chromosomes
from fusing and causing
karyotypic rearrangements.
telomere centromere telomere

<1 to >12 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