Download Slide 3 - Pulse 2012

DNA melting curve
Percent hyperchromicity
100
50
0
50
70
90
Temperature oC
Tm is the temperature at the midpoint of the transition•
1
Percent hyperchromicity
Tm is dependent on the G-C content of the DNA
E. coli DNA is
50% G-C
50
60
70
80
Temperature oC
Average base composition (G-C content) can be
determined from the melting temperature of DNA
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 Alu sequences
c). Chromosome structure - packaging of genomic DNA
i). Nucleosomes
ii). Histones
iii). Nucleofilament structure
iv). Telomeres, aging, and cancer
3
Complexity of chromosomal DNA:
DNA reassociation (renaturation)
•
•
•
•
•
4
The complexity of a DNA is a function of how many base
pairs it has.
In low complexity = few hundred
High complexity = millions of base pairs.
Low complexity sequences are able to find each other
much faster during a renaturation reaction, than are high
complexity sequences, since the low complexity
sequences have to make fewer collisions to find a basepairing partner.
Thus, the rate of a renaturation reaction is a function of
the complexity of the DNA. Or in other words, the
complexity of a DNA can be determined by measuring its
second-order reassociation rate constant (k2).
DNA reassociation (renaturation)
Double-stranded DNA
Denatured,
single-stranded
DNA
k2
5
Slower, rate-limiting,
second-order process of
finding complementary
sequences to nucleate
base-pairing
Faster,
zippering
reaction to
form long
molecules
of doublestranded
DNA
Type of DNA
% of Genome
Includes most genes 1
Interspersed throughout genome
between and within genes; includes Alu
sequences 2and VNTRs or mini (micro) satellites
Highly repeated, low complexity sequences
usually located in centromeres
and telomeres
0
fast ~10%
Features
~75%
1. Single-copy (unique)
~15%
2. Repetitive
a- Interspersed
~10%
b- Satellite (tandem)
2
50
100
6
Alu sequences are intermediate
about 300 bp in length
~15%
and are repeated about
300,000 times in the
genome. They can be
slow (single-copy)
found adjacent to or
~75%
within genes in introns
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 repeats are sequences that
are repeated many times and scattered
throughout the genome.
• In contrast, tandem repeats are
sequences that are repeated many times
adjacent to each other. They are usually
found in the centromeres and telomeres
of chromosomes (the sequence above
TTAGGG comprises human teleomeric
DNA
7
Classes of repetitive DNA
Interspersed (dispersed) repeats (e.g., Alu sequences)
GCTGAGG
GCTGAGG
GCTGAGG
Tandem repeats (e.g., microsatellites)
TTAGGGTTAGGGTTAGGGTTAGGG
8
Human Genome Project
• Knowing the complete sequence of the human
genome will:
 allow medical researchers to more easily find
disease-causing genes.
 understand how differences in our DNA
sequences from individual to individual may
affect our predisposition to diseases and our
ability to metabolize drugs.
• Because the human genome has ~3 billion bp
of DNA and there are 23 pairs of chromosomes
in diploid human cells, the average metaphase
chromosome has ~130 million bp DNA.
9
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
reptiles
is in single-copy DNA.
birds
The human genome is thought
mammals
to contain ~30,000 to 40,000 genes.
10
104
105
106
107
108
109
1010
1011
Gene Structure
•
•
•
•
•
11
Most genes in the human genome are called "split genes"
because they are composed of "exons" separated by
"introns."
The exons are the regions of genes that encode
information that ends up in mRNA.
The transcribed region of a gene (double-ended arrow)
starts at the +1 nucleotide at the 5' end of the first exon
and includes all of the exons and introns (initiation of
transcription is regulated by the promoter region of a
gene, which is upstream of the +1 site).
RNA processing (the subject of a another lecture) then
removes the intron sequences, "splicing" together the
exon sequences to produce the mature mRNA.
The translated region of the mRNA (the region that
encodes the protein) is indicated in blue. Note that there
are untranslated regions at the 5' and 3‘ ends of mRNAs
that are encoded by exon sequence but are not directly
translated.
Gene structure
promoter
region
exons (filled and unfilled boxed regions)
+1
introns (between exons)
transcribed region
mRNA structure
5’
12
3’
translated region
The (exon-intron-exon)n structure of various
genes
introns can be very long, while exons are usually
relatively short.
•
•
•
•
•
13
Next figure shows examples of the wide variety of gene
structures seen in the human genome. Some (very few) genes
do not have introns. One example is the histone genes, which
encode the small DNA-binding proteins, histones H1, H2A, H2B,
H3, and H4.
Shown here is a histone gene that is only 400 base pairs (bp) in
length and is composed of only one exon.
The beta-globin gene has three exons and two introns.
The hypoxanthine-guanine phosphoribosyl transferase (HGPRT
or HPRT) gene has nine exons and is over 100-times larger than
the histone gene, yet has an mRNA that is only about 3-times
larger than the histone mRNA (total exon length is 1,263 bp).
This is due to the fact that introns can be very long, while exons
are usually relatively short.
An extreme example of this is the factor VIII gene which has
numerous exons (the blue boxes and blue vertical lines).
The (exon-intron-exon)n structure of various genes
introns can be very long, while exons are usually relatively short.
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
14
Properties of the human genome
Nuclear genome
the haploid human genome has ~3 X 109 bp of DNA•
single-copy DNA comprises ~75% of the human genome•
the human genome contains ~20,000 to 25,000 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 genome
circular genome of ~17,000 bp•
contains <40 genes•
15