Genomic DNA 2
Transcript of Genomic DNA 2
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2. Genomic DNA, Genes , Chromatin
a). Complexity of chromosomal DNAi). DNA reassociation
ii).Repetitive DNA and Alu sequences
iii). Genome size and complexity of genomic DNA
b). Gene structure
i). Introns and exonsii). 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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DNA reassociation (renaturation)
Double-stranded DNA
Denatured,
single-stranded
DNA
Slower, rate-limiting,
second-order process of
finding complementary
sequences to nucleate
base-pairing
k2
Faster,
zippering
reaction to
form long
molecules
of double-
stranded
DNA
a). Complexity of chromosomal DNA
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DNA reassociation kinetics (for a single DNA species)
Cot1/2= 1/k2 k2 = second-order rate constant
Co= DNA concentrationt1/2= time for half reaction
Cot1/2
50
100
0
%D
NAreassociated
log Cot
Ideal second-order DNA reassociation curve (Cot curve)
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10-5 10010-4 10-3 10-1 10410310210-6 10-2 101
101 106102 103 105 1010109108100 104 107Complexity expressed as base-pairs (bp)
Cot
1 2 3 4 5
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
Cot1/2
There is a direct
relationship between
Cot1/2and complexity
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Cot1/2
DNA reassociation kinetics for a mixture of DNA species
Cot1/2= 1/k2 k2 = second-order rate constantCo= DNA concentrationt1/2= time for half reaction
50
100
0
%D
NAreassociated
I I I I I I I I I
log Cot
fast (repeated)
intermediate
(repeated)
slow (single-copy)
human genomic DNA
Kinetic fractions:
fastintermediate
slowCot1/2
Cot1/2
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k2>>>>>>>>>> k2
110,000
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Type of DNA % of Genome Features
Single-copy (unique) ~75% Includes most genes 1
Repetitive
Interspersed ~15% Interspersed throughout genome betweenand within genes; includes Alu sequences 2
and VNTRs or mini (micro) satellites
Satellite (tandem) ~10% Highly repeated, low complexity sequences
usually located in centromeresand telomeres
2Alu sequences are
about 300 bp in length
and are repeated about
300,000 times in thegenome. 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 inthe repetitive DNA fraction
50
100
0
I I I I I I I I I
fast ~10%
intermediate
~15%
slow (single-copy)
~75%
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Classes of repetitive DNA
Interspersed (dispersed) repeats (e.g., Alu sequences)
TTAGGGTTAGGGTTAGGGTTAGGG
Tandem repeats (e.g., microsatellites)
GCTGAGG GCTGAGGGCTGAGG
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viruses
plasmids
bacteria
fungi
plants
algae
insects
mollusks
reptiles
birds
mammals
Genome sizes in nucleotide pairs (base-pairs)
104 108105 106 107 10111010109
The size of the human
genome is ~ 3 X 109bp;
almost all of its complexityis in single-copy DNA.
The human genome is thought
to contain ~30,000 to 40,000 genes.
bony fish
amphibians
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5 3
promoterregion
exons (filled and unfilled boxed regions)
introns (between exons)
transcribed region
translated region
mRNA structure
+1
b). Gene structure
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(exon-intron-exon)nstructure of various genes
-globin
HGPRT
(HPRT)
total = 1,660 bp; exons = 990 bp
histone
factor VIII
total = 400 bp; exon = 400 bp
total = 42,830 bp; exons = 1263 bp
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 109bp 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 genomegenes are composed of from 1 to >75 exons
genes vary in length from 2,300,000 bp
Alu sequences are present throughout the genome
Mitochondrial
circular genome of ~17,000 bp
contains
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Familial hypercholesterolemia
autosomal dominant
LDL receptor deficiency
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LDL receptor gene
Alu repeats present within introns
Alu repeats in exons
4
4
4
5
5
5 6
6
6
Alu Alu
AluAlu
X
4 6
Alu
unequal
crossing over
one product has a
deleted exon 5(the other product is not shown)
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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 108bp DNA.
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Nucleosome structure
Nucleosome core (left)
146 bp DNA; 1 3/4 turns of DNA
DNA is negatively supercoiledtwo each: H2A, H2B, H3, H4 (histone octomer)
Nucleosome (right)
~200 bp DNA; 2 turns of DNA plus spacer
also includes H1 histone
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Hi t (H1 H2A H2B H3 H4)
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Histones (H1, H2A, H2B, H3, H4)
small proteins
arginine or lysine rich: positively charged
interact with negatively charged DNAcan be extensively modified - modifications in
general make them less positively chargedphosphorylation
poly(ADP) ribosylation
methylationacetylation
hypoacetylation
by histone deacetylase (facilitated by Rb)
tight nucleosomesassoc with transcriptional repression
hyperacetylation
by histone acetylase (facilitated by TFs)
loose nucleosomes
assoc with transcriptional activation
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Nucleofilament structure
C d i d d d i
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Condensation and decondensation
of a chromosome in the cell cycle
T l t ti
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Telomeres and aging
Metaphase chromosome
centromere telomeretelomere
telomere structureyoung
senescent
Telomeres are protective
caps on chromosome
ends consisting of short
5-8 bp tandemly repeated
GC-rich DNA sequences,
that prevent chromosomesfrom fusing and causing
karyotypic rearrangements.
(TTAGGG)many
(TTAGGG)few
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
12 kb