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Molecular Genetics
DNA as the Model of Inheritance
Components of DNA
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Nucleotides
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Sugar
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ADENINE
GUANINE
CYTOSINE
THYMINE
DEOXYRIBOSE
Phosphate Groups
Adenine and Guanine are PURINES
Cytosine and Thymine are
PYRIMIDINES
LE 16-5
Sugar–phosphate
backbone
Nitrogenous
bases
5 end
Thymine (T)
Adenine (A)
Cytosine (C)
Phosphate
Sugar (deoxyribose)
3 end
DNA nucleotide
Guanine (G)
Antiparallel
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Complimentary Arrangement
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Arrangement
Adenine always pairs with Thymine
Cytosine always pairs with Guanine
5’ end and 3’ end
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3’ end has a terminal –OH
5’ has a terminal Phosphate Group
LE 16-7
5 end
Hydrogen bond
3 end
1 nm
3.4 nm
3 end
0.34 nm
Key features of DNA structure
5 end
Partial chemical structure
Space-filling model
Watson and Crick
 Presented
the double helical
model in April 1953 (1 page
paper in Nature).
 Transcended the scientific world
 Won Nobel Prize in 1962 along
with Maurice Wilkins
 Their model explained
Chargoff’s rules.
What about Rosalind??
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Around 1950, Wilkins and his colleague, Rosalind
Franklin, used a technique called x-ray
crystallography to try to determine the structure of
DNA.
Watson and Crick used their data to develop their
theory. Rosalind found the sugar-phosphate
backbone
But, before the Nobel Prize was awarded, Rosalind
died, and therefore did not receive the Nobel
recognition.
But, would the committee have recognized
Rosalind’s contributions if she had lived? She was a
woman you know…
LE 16-6
Rosalind Franklin
Franklin’s X-ray diffraction
photograph of DNA
Watson & Crick – part 2
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In their 2nd paper, Watson and Crick
proposed the semiconservative
model of DNA replication.
It states that when DNA replicates,
each of the 2 daughter molecules
will have 1 old strand of from the
parent, and 1 newly formed strand.
LE 16-9_1
The parent molecule has
two complementary
strands of DNA. Each base
is paired by hydrogen
bonding with its specific
partner, A with T and G with
C.
LE 16-9_2
The parent molecule has
two complementary
strands of DNA. Each base
is paired by hydrogen
bonding with its specific
partner, A with T and G with
C.
The first step in replication
is separation of the two
DNA strands.
LE 16-9_3
The parent molecule has
two complementary
strands of DNA. Each base
is paired by hydrogen
bonding with its specific
partner, A with T and G
with C.
The first step in replication
is separation of the two
DNA strands.
Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
LE 16-9_4
The parent molecule has
two complementary
strands of DNA. Each base
is paired by hydrogen
bonding with its specific
partner, A with T and G
with C.
The first step in replication
is separation of the two
DNA strands.
Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
The nucleotides are
connected to form the
sugar-phosphate backbones of the new strands.
Each “daughter” DNA
molecule consists of one
parental strand and one
new strand.
If not Semiconservative,
then what?
 Conservative
– parent
emerges from the
replication intact.
 Dispersive – all 4 strands
formed are a mixture of old
and new.
LE 16-10
Parent cell
Conservative
model. The two
parental strands
reassociate after
acting as
templates for
new strands,
thus restoring
the parental
double helix.
Semiconservative
model. The two
strands of the
parental
molecule
separate, and
each functions as
a template for
synthesis of a
new, complementary strand.
Dispersive model.
Each strand of
both daughter
molecules
contains
a mixture of
old and newly
synthesized
DNA.
First
replication
Second
replication
Meselson and Stahl
 In
late 1950’s Matthew
Melelson and Franklin Stahl
devised experiments to test the
3 models of replication.
 Their findings supported
Watson and Crick that
replication was
semiconservative.
How did they do it?
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Cultured E. coli bacteria for several
generations in a medium containing
a heavy isotope of nitrogen (15N)
The bacteria incorporated the
isotope into their DNA
Then, the bacteria was transferred
to a medium containing a lighter
isotope of nitrogen (14N)
Then they…
 Observed
the bacteria. Any
new DNA that the bacteria
synthesized would be lighter
than the “old” DNA (made in
the 15N medium).
 Would centrifuge and extract
DNA strands and mass them.
Well, what did they
find?
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The 1st replication in the 14N
medium produced a band of hybrid
(15N-14N) DNA.
This eliminated the conservative
model.
The 2nd replication produced both
light and hybrid DNA.
This eliminated dispersive and
supported semi-conservative.
LE 16-11
Bacteria
cultured in
medium
containing
15N
Bacteria
transferred to
medium
containing
14N
DNA sample
centrifuged
after 20 min
(after first
replication)
DNA sample
centrifuged
after 40 min
(after second
replication)
First replication
Conservative
model
Semiconservative
model
Dispersive
model
Less
dense
More
dense
Second replication
Hershey and Chase
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In 1952, Alfred Hershey and Martha
Chase performed experiments
showing that DNA is the genetic
material of a phage (virus) called
T2.
T2 infects E. coli
Knew T2 could make a normal cell
produce viruses, but wanted to
know what it a protein or DNA?
LE 16-3
Phage
head
Tail
Tail fiber
Bacterial
cell
100 nm
DNA
How they did it…
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2 Batches of Broth
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One contained T2 with E. coli in the
presence of radioactive sulfur.
The other contained T2 with E. coli in
the presence of radioactive
phosphorus.
Radioactive Sulfur is only
incorporated into the protein coat of
phage.
Radioactive Phosphorus is only
incorporated into the DNA of the
phage.
Next they…
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Agitated each broth in a blender
separate phages outside the
bacteria from the cells in their
contents.
Then they centrifuged the mixture
so that the bacteria formed a pellet
at the bottom of the test tube.
Measured the radioactivity of the
pellet and the liquid.
Well, what did they
find?
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In the broth with the sulfur,
radioactivity was only measured in
the liquid, which indicated that the
protein was not incorporated into
the bacterium.
In the phosphorus broth,
radioavtivity was measured in the
pellet (bacteria), which indicated
that the viral DNA was incorporated
into the bacterium.
LE 16-4
Phage
Radioactive
protein
Empty
protein shell
Radioactivity
(phage protein)
in liquid
Bacterial cell
Batch 1:
Sulfur (35S)
DNA
Phage
DNA
Centrifuge
Pellet (bacterial
cells and contents)
Radioactive
DNA
Batch 2:
Phosphorus (32P)
Centrifuge
Pellet
Radioactivity
(phage DNA)
in pellet
DNA Replication
 It
takes a human cell
only a few hours to
complete replication.
 Use proteins and
enzymes
Origins of Replication
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Special site where replication begins
Specific sequence of nucleotides
2 parental strands will separate to
form replication bubbles.
At the end of each replication
bubble is a replication fork
The bubbles expand laterally, and
DNA replication proceeds in both
directions until replication is
complete.
DNA Polymerases
 Elongation
of new DNA at a
replication fork is catalyzed by
DNA Polymerases.
 Polymerase adds the new base
pairs to growing DNA strand.
 Rate of expansion is 500
nucleotides per second in
bacteria and 50 per second in
human cells.
Energy for Replication
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Comes from nucleoside triphosphates,
which are nucleotides with 3
phosphates.
Similar to ATP; only ATP has ribose, and
NTP has deoxyribose as sugar.
As each monomer is added to growing
DNA strand, it loses 2 phosphate groups
as a molecule of pyrophosphate.
Hydrolysis of pryophosphate into 2
phosphates is the exergonic reaction
that releases the energy for
polymerization.
Antiparallel
Arrangement of DNA
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The sugar-phosphate backbones run
in opposite directions.
The 5 carbons of one deoxyribose
sugar are numbered 1’ to 5’.
One strand has the first phosphate
attached at 5’.
The other strand does not have a
phosphate attached at the 3’ end.
5’ to 3’
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Replication always proceeds in a 5’
to 3’ direction.
DNA polymerase only adds
nucleotides to the 3’ end.
This creates 2 newly forming
strands, the leading strand and the
lagging strand.
Leading Strand
Parental
Strand runs 5’
to 3’
Continually elongates…
1 continuous strand
“Top” strand
Lagging Strand
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Parental Strand runs 3’ to 5’
But still grows 5’ to 3’
Grows in small segments, called
Okazaki fragments.
DNA Ligase connects the Okazaki
fragments.
Priming DNA Synthesis
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None of DNA polymerases actually
initiate the synthesis of a
polynucleotide.
They only add nucleotides to the
existing chain.
The start of the new DNA chain is
called the primer, which is a short
stretch of RNA.
Only 1 primer is required to begin
replication of new DNA strand.
Primase is the enzyme which makes
the primer.
Other Proteins Involved
in Replication
 Helicase
– enzyme that
untwists the original DNA at the
replication fork.
 Other proteins called single
strand binding protein line up
along the unpaired DNA, and
hold the strands apart so that
replication can proceed.
Damaged DNA
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Sometimes nucleotides are
mispaired in the DNA molecule.
Must undergo mismatch repair
Sometimes mismatches are caused
by chemicals, radioactive emissions,
X-rays, UV light
Damaged part is removed by
nuclease.
Repair is carried out by a DNA
polymerase and ligase
End of DNA molecule
replication
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The ends of DNA contain nucleotide
sequences called telomeres.
Telomeres do not contain genes.
Telomerase – enzyme which
synthesizes telomers.
From Gene to Protein
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One Gene, One Polypeptide
Hypothesis
Each gene will code for one enzyme
or one polypeptide.
Work of Garrod and Beadle and
Tatum led to this important
discovery.
Garrod
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In 1902, a British physician named
Archibald Garrod noticed that a
disorder called alkaptonuria seemed
to run in families.
Suggested that the disease was
result of Mendelian inheritance.
Disease was caused by the lack of
an enzyme which breaks down an
acid found in urine.
Without the enzyme, the acid
oxidized rapidly when exposed to
air, and turned the urine BLACK!
Beadle and Tatum
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In 1941, confirmed Garrod’s
suspicions that DNA sequences code
for enzymes.
They were Americans! Conducted
their experiments at Stanford
University.
Studied genetic mutations.
What did they do?
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Exposed bread mold to x-rays
expecting them to damage some of
the DNA.
Mutations
Concluded that mutations affected
the growth rate of mutants and
their ability to metabolize sugars.
Mutations
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What is a mutation?
What is it caused by?
Name several different types of
mutations.