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Chapter 6
6.1
Genome size varies a lot
•DNA content is highly variable
•C-value paradox
no consistent relationship
between genome size and
organism complexity
Chapter 6
6.1
Genome size varies a lot
virus
virus
bacteria
yeast
pufferfish
salamander
(HIV)
smallpox
E.coli
S. cerevisiae
T. rubripes
A. means
9 kb
267 kb
4,600 kb
13,000 kb
400,000 kb
90,000,000 kb
virus
virus
bacteria
yeast
pufferfish
salamander
(HIV)
9 kb
smallpox
267 kb
E.coli
4,600 kb
S. cerevisiae
13,000 kb
Takifugu rubripes
400,000 kb
Amphiuma means 90,000,000 kb
mammals
birds
reptiles
amphibians
fish
fish
Vertebrates
fish
Vertebrates
Chordates
Arthropods
Echinoderms
Invertebrate
Chordates
deuterostomes
Annelids
Higher
Invertebrates
Mollusks
protostomes
Nematods
Flatworms
Cnidaria
Porifera
© 2006 Jones and Bartlett Publishers
Chapter 6
6.1
Genome size varies a lot
C-value paradox
“There is no consistent relationship
between the DNA content (C-value) and
the metabolic, developmental, or
behavioral complexity of the organism”
pg. 205
Much of the “additional” DNA has
functions besides coding for protein
Chapter 6
6.2
DNA is a linear polymer of four nucleotides
Each nucleotide has three “parts”
base - purine (A,G) or pyrimidine (C,T)
carbons and nitrogens are given numbers: 1, 2, 3, …
Fig. 6.2. Chemical structures of adenine, thymine, guanine, and cytosine
© 2006 Jones and Bartlett Publishers
Chapter 6
6.2
DNA is a linear polymer of four nucleotides
Each nucleotide has three “parts”
base - purine (A,G) or pyrimidine (C,T)
sugar - deoxyribose (H instead of OH at C2)
Fig. 6.3. A typical nucleotide showing the three main components, the difference
between DNA and RNA, and the distinction between a nucleoside and a nucleotide.
© 2006 Jones and Bartlett Publishers
Chapter 6
6.2
DNA is a linear polymer of four nucleotides
Each nucleotide has three “parts”
base - purine (A,G) or pyrimidine (C,T)
sugar - deoxyribose (H instead of OH at C2)
phosphate group - phosphate group
Fig. 6.3. A typical nucleotide showing the three main components, the difference
between DNA and RNA, and the distinction between a nucleoside and a nucleotide.
© 2006 Jones and Bartlett Publishers
Chapter 6
6.2
DNA is a linear polymer of four nucleotides
polymer (many parts)
linear
like beads on a string
pearls on a necklace
letters in a sentence
connected from phosphate group on 5’ carbon
to 3’ carbon of another nucleotide
phosphate
sugar
base
sugar-phosphate backbone
5’ phosphate
base
base
base
base
3’ hydroxyl
Fig. 6.4. Polynucleotide
strand structure
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Chapter 6
6.3
DNA is a double stranded helix
held together by hydrogen bonds
Fig. 6.5A. Three dimensional
structure of the double helix
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Fig. 6.5B. Computer model of DNA helix. [Courtesy of Antony M. Dean]
© 2006 Jones and Bartlett Publishers
A
T
Fig. 6.6A,B. A-T base pair in DNA. [(B) Courtesy of Antony M. Dean].
© 2006 Jones and Bartlett Publishers
G
C
Fig. 6.6C,D. G-C base pair model. [(D) Courtesy of Antony M. Dean].
© 2006 Jones and Bartlett Publishers
antiparallel
Fig. 6.7. DNA segment showing
the antiparallel orientation of
the complementary strands
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Chapter 6
6.4
DNA replication is semiconservative
Fig. 6.8. Watson-Crick
model of DNA
replication
© 2006 Jones and Bartlett Publishers
Chapter 6
6.4
DNA replication is semiconservative
How would you “prove” this?
Hints:
ultracentrifugation
N15 and N14
Chapter 6
DNA replication is semiconservative
CsCl gradient
less dense
centrifugation
Cs+
more dense
gradient
6.4
diffusion
Cs+
Chapter 6
6.4
DNA replication is semiconservative
Grow bacteria in media with only N15
(all DNA will be “heavy”
Then transfer them to media with only N14
(new DNA will be “light”)
If replication is semiconservative,
what would results look like?
DNA from bacteria grown with N15
Switch to growth with N14
Fig. 6.9. Predictions of semiconservative DNA replication
© 2006 Jones and Bartlett Publishers
Chapter 6
6.4
DNA replication is semiconservative
Grow bacteria in media with only N15
(all DNA will be “heavy”)
Then transfer them to media with only N14
(new DNA will be “light”)
If replication is not semiconservative,
what would results look like?
DNA from bacteria grown with N15
Switch to growth with N14
Chapter 6
6.4
So, which is it?
0 and 1.9
mixed
0 and 4.1
mixed
Fig. 6.10. The Meselson-Stahl experiment on DNA
replication [Photo courtesy of M. Meselson]
© 2006 Jones and Bartlett Publishers
Most prokaryotes and viruses have circular DNA
replication begins at the replication origin
may proceed in one or both directions
θ (theta) replication
Fig. 6.12. Unidirectional and bidirectional DNA replication
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Some viruses have a different kind of replication
rolling-circle replication
Fig. 6.13. Rolling-circle replication
© 2006 Jones and Bartlett Publishers
In eukaryotes
DNA is linear (not circular)
replicates bidirectionally
e.g., Drosophila
DNA synthesis is 10 - 100 nucleotides/sec
largest chromosome is 7 x 107 nucleotides
… … … 8 days
multiple replication origins
~ 8500 / chromosome
In eukaryotes
replication origins are about 40,000 nucleotides apart
Fig. 6.14B. Replication DNA of Drosophila melanogaster
© 2006 Jones and Bartlett Publishers
Chapter 6
6.5
Many proteins participate in DNA Replication
1.
2.
3.
4.
5.
helicase (s)
SSB
gyrase
primers
DNA polymerase(s)
Chapter 6
6.5
Many proteins participate in DNA Replication
1. helicase
•undwinding of the double-stranded DNA
(ATP hydrolysis)
•different helicases for different roles
replication, recombination, repair
strands need to be stabilized once unwound
Chapter 6
6.5
Many proteins participate in DNA Replication
1. helicase (s)
single-stranded binding protein
2. SSB
strong affinity for ss DNA
stabilize templates for replication
Chapter 6
6.5
Many proteins participate in DNA Replication
1. helicase (s)
2. SSB
3. gyrase
cuts double-stranded DNA
swivels DNA
reattaches strands
relieve stress caused by unwinding
aka., topoisomerase II
2
3
1
2
Fig. 6.15. Role of some key proteins in DNA replication
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2
3
1
2
Fig. 6.15. Role of some key proteins in DNA replication
© 2006 Jones and Bartlett Publishers
Chapter 6
6.5
Many proteins participate in DNA Replication
helicase (s)
SSB
gyrase
primers
DNA polymerase(s)
In most organisms, DNA Polymerase
can’t initiate synthesis, only elongate
an existing strand.
Chapter 6
6.5
Many proteins participate in DNA Replication
helicase (s)
SSB
gyrase
primers
a special RNA polymerase makes a
short RNA complimentary to the DNA
Chapter 6
6.5
Many proteins participate in DNA Replication
RNA differs from DNA in two ways:
sugar is ribose
bases are A, C, G and U
Fig. 6.17. Differences between DNA and RNA
© 2006 Jones and Bartlett Publishers
Chapter 6
6.5
Many proteins participate in DNA Replication
helicase (s)
SSB
gyrase
primers
a special RNA polymerase makes a
short RNA complimentary to the DNA
2-5 nucleotides in bacteria
15-20 nucleotides in eukaryotes
RNA polymerase
Fig. 6.18. Priming of DNA synthesis with an RNA segment
© 2006 Jones and Bartlett Publishers
Chapter 6
6.5
Many proteins participate in DNA Replication
helicase (s)
SSB
gyrase
primers
DNA polymerase(s)
elongates the primer at the 3” end
(can add nucleotides to 3’ end of existing D/RNA)
Chapter 6
6.5
Many proteins participate in DNA Replication
helicase (s)
SSB
gyrase
primers
DNA polymerase(s)
several types
In prokaryotes
DNA polymerase I
(Pol I)
DNA polymerase III
(Pol III)
Eukaryotes
polymerase delta (replication)
DNA polymerase
RNA polymerase
Fig. 6.18. Priming of DNA synthesis with an RNA segment
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Fig. 6.19. Structure of new DNA strand
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Fig. 6.20. Addition of nucleotides to the 3'-OH terminus of a growing strand
© 2006 Jones and Bartlett Publishers
Fig. 6.16. DNA gyrase introduces a double-stranded break
© 2006 Jones and Bartlett Publishers
Chapter 6
6.5
Many proteins participate in DNA Replication
helicase (s)
SSB
gyrase
primers
DNA polymerase(s)
Fig. 6.15. Role of some key proteins in DNA replication
Fig. 6.15. Role of some key proteins in DNA replication
Fig. 6.22. Short fragments in the replication fork
Chapter 6
6.5
Many proteins participate in DNA Replication
lagging strands
remove RNA
replace with DNA
join fragments
Replication Protein A (RPA)
polymerase delta
DNA ligase
Fig. 6.23A,B. Joining of adjacent precursor fragments
Fig. 6.23C. Joining of adjacent precursor fragments
Fig. 6.15. Role of some key proteins in DNA replication
Chapter 6
6.5
Many proteins participate in DNA Replication
helicase (s)
SSB
gyrase
primers
DNA polymerase(s)
several types
Polymerases can also cut nucleic acids
(exonuclease activity)
cut off last nucleotide put on
used as “spell-checker”
Fig. 6.21. The 3'-to-5' exonuclease activity of the proofreading function
© 2006 Jones and Bartlett Publishers
Chapter 6
6.6 - 6.8
Practical applications of our
knowledge of DNA structure
gel electrophoresis
nucleic acid hybridization
restriction enzymes
Southern blots
PCR
DNA sequencing
Chapter 6
6.6 - 6.8
Practical applications of our
knowledge of DNA structure
add sample
pp. 38-39
Chapter 6
6.6 - 6.8
Practical applications of our
knowledge of DNA structure
Put in figure 2.4
or equivalent
p. 224. Fragments of DNA in the wells of an ararose gel. [Courtesy of National
Cancer Institute]
© 2006 Jones and Bartlett Publishers
p. 224. Fluorescent dye has been added to make DNA bands visible
under ultraviolet light. [Courtesy of James Gathany/Centers for Disease Control]
© 2006 Jones and Bartlett Publishers
isolate molecule based on “size”
Chapter 6
6.6 - 6.8
Practical applications of our
knowledge of DNA structure
gel electrophoresis
nucleic acid hybridization
denaturation
(increase temp.
strands separate)
renaturation
(decrease temp.
strands anneal)
Fig. 6.24. Nucleic acid hybridization
Chapter 6
6.6 - 6.8
Practical applications of our
knowledge of DNA structure
gel electrophoresis
nucleic acid hybridization
restriction enzymes
EcoRI
HindIII
PvuII
GAATTC
CTTAAG
AAGCTT
TTCGAA
CAGCTG
GTCGAC
palindrome
Table 6.2. Some restriction
endonucleases, their sources, and their
cleavage sites
© 2006 Jones and Bartlett Publishers
cut DNA in specific places
Fig. 6.25. Mechanism of DNA cleavage by the restriction enzyme BamHI
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Fig. 6.26. Restriction maps of  DNA for two restriction enzymes, EcoRI and BamHI
© 2006 Jones and Bartlett Publishers
Chapter 6
6.6 - 6.8
Practical applications of our
knowledge of DNA structure
gel electrophoresis
nucleic acid hybridization
restriction enzymes
Southern blots
Fig. 6.27. Southern blot
Chapter 6
6.6 - 6.8
Practical applications of our
knowledge of DNA structure
Gel electrophoresis
Nucleic acid Hybridization
Restriction enzymes
Southern blots
PCR
Fig. 6.28. Polymerase chain reaction
Thermus aquaticus
Taq polymerase
p. 232, Hot Springs at Yellowstone National Park.
[Courtesy of Monte Later/Yellowstone National Park/NPS]
© 2006 Jones and Bartlett Publishers
Chapter 6
PCR
E coli:
3000 bp fragment is 0.06% of cells DNA
Do PCR with oligo’s for that fragment - (25 rounds)
99.95% of DNA in tube would be the amplified sequence
Good for sequences less than 5000 bp
Easily automated
Chapter 6
6.6 - 6.8
Practical applications of our
knowledge of DNA structure
Gel electrophoresis
Nucleic acid Hybridization
Restriction enzymes
Southern blots
PCR
DNA sequencing
Fig. 6.29. Structures of normal deoxyribose and the dideoxyribose
sugar used in DNA sequencing
© 2006 Jones and Bartlett Publishers
Fig. 6.30. Dideoxy
method of DNA
sequencing.
template, primer (P32), polymerase
and normal nucleotides and
+
ddG
+
ddA
+
ddT
+
ddC
all tubes will have DNA
fragments that end with
the base corresponding to
the ddX in the tube
Fig. 6.30. Dideoxy
method of DNA
sequencing.
© 2006 Jones and Bartlett Publishers
G
A
T
C
20 +
Fig. 6.31. Florescence pattern trace obtained from a DNA sequencing gel
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Fig. 6.32. AIDS drugs
© 2006 Jones and Bartlett Publishers
Table 6.1. Genome size of some representative
viral, bacterial, and eukaryotic genomes
© 2006 Jones and Bartlett Publishers