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Chapter 16-Molecular Genetics
• You must know:
• The structure of DNA
• The history of the discovery of DNA gained from the work of
• Replication
• Replication, transcription and translation
• Differences between bacterial vs. eukaryotic chromosomes
• How DNA packaging affects gene expression
Life’s Operating Instructions
• In 1953, James Watson and Francis
Crick introduced an elegant doublehelical model for the structure of
deoxyribonucleic acid, or DNA
•
• DNA, the substance of inheritance,
is the most celebrated molecule of
our time
•
• Hereditary information is encoded in
DNA and reproduced in all cells of
the body
•
• This DNA program directs the
development of biochemical,
anatomical, physiological, and (to
some extent) behavioral traits
•
•
How do we know DNA the genetic
material?
• Scientific Inquiry
• Early in the 20th century
• ID of inheritance was a
major challenge
• Morgan showed that genes
are located on
chromosomes
• DNA and protein are
candidates for the genetic
material
• Bacterial and viruses
helped with this
Evidence that DNA can Transform
Bacteria
• Griffith in 1928 showed
the genetic role
• 2 strains of bacterium
• One pathogenic, other
was not
• Mixed heat-killed
remains with pathogenic
with the living, living
became pathogenic
• Transformation
Figure 16.2
EXPERIMENT
Living S cells
(control)
Living R cells
(control)
Heat-killed
S cells
(control)
Mixture of
heat-killed
S cells and
living R cells
RESULTS
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Living S cells
More evidence
• In 1944, Oswald Avery, Maclyn McCarty, and
Colin MacLeod announced that the
transforming substance was DNA
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• Their conclusion was based on experimental
evidence that only DNA worked in
transforming harmless bacteria into
pathogenic bacteria
•
• Many biologists remained skeptical, mainly
because little was known about DNA
• More evidence for DNA as the genetic material
came from studies of viruses that infect
bacteria
•
• Such viruses, called bacteriophages (or
phages), are widely used in molecular
genetics research
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Figure 16.3
Phage
head
Tail
sheath
Tail fiber
Bacterial
cell
100 nm
DNA
Do you need more?
• In 1952, Alfred Hershey and
Martha Chase performed
experiments showing that DNA
is the genetic material of a
phage known as T2
•
• To determine this, they designed
an experiment showing that only
one of the two components of
T2 (DNA or protein) enters an E.
coli cell during infection
•
• They concluded that the injected
DNA of the phage provides the
genetic information
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Additional Evidence
• It was known that DNA is a polymer of nucleotides, each
consisting of a nitrogenous base, a sugar, and a
phosphate group
•
• In 1950, Erwin Chargaff reported that DNA composition
varies from one species to the next
•
• This evidence of diversity made DNA a more credible
candidate for the genetic material
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•
Chargaff’s Rules
• Base composition of
DNA varies between
species
• A and T are equal in
number, G and C are
equal
• Basis for these rules
was not understood
until the discovery of
the double helix
Figure 16.5
Sugar–phosphate
backbone
Nitrogenous bases
5 end
Thymine (T)
Adenine (A)
Cytosine (C)
Phosphate
Guanine (G)
Sugar
(deoxyribose)
DNA
nucleotide
3 end
Nitrogenous base
The Race to discover the structure of
DNA
• DNA was accepted as
the genetic matrial
• But how?
• Maurice Wilkins and
Rosalind Franklin were
using x-ray
crystallography
• Rosalind produced the
famous plate 51
Figure 16.6
(a) Rosalind Franklin
(b) Franklin’s X-ray diffraction
photograph of DNA
Franklin’s work
• Enabled Watson to
deduce the helical
structure of the molecule
• Width and spacing was
calculated
• The pattern in the photo
confirmed that DNA is
double helix
• Thought that Maurice
Wilkins “borrowed”
Rosie’s plate to show
Watson- returned it
Figure 16.7
C
5 end
G
C
Hydrogen bond
G
C
G
C
G
3 end
A
T
3.4 nm
A
T
C
G
C
G
A
T
1 nm
C
A
G
C
G
A
G
A
T
3 end
T
A
T
G
C
T
C
C
G
T
A
(a) Key features of
DNA structure
0.34 nm
5 end
(b) Partial chemical structure
(c) Space-filling
model
The proof
• Watson and Crick built models of a double helix to
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conform to the X-rays and chemistry of DNA
Franklin had concluded that there were two outer sugarphosphate backbones, with the nitrogenous bases paired
in the molecule’s interior
Watson built a model in which the backbones were
antiparallel (their subunits run in opposite directions
At first, Watson and Crick thought the bases paired like
with like (A with A, and so on), but such pairings did not
result in a uniform width
Instead, pairing a purine with a pyrimidine resulted in a
uniform width consistent with the X-ray data
Figure 16.UN01
Purine  purine: too wide
Pyrimidine  pyrimidine: too narrow
Purine  pyrimidine: width
consistent with X-ray data
And there is more
• Watson and Crick reasoned that the pairing was more
specific, dictated by the base structures
• They determined that adenine (A) paired only with
thymine (T), and guanine (G) paired only with cytosine (C)
• The Watson-Crick model explains Chargaff’s rules: in any
organism the amount of A = T, and the amount of G = C
Figure 16.8
Sugar
Sugar
Adenine (A)
Thymine (T)
Sugar
Sugar
Guanine (G)
Cytosine (C)
Form fits function
• Relationship between
structure and function
• Copying mechanism
for genetic material
Base pairing to a Template Strand
• Each strand acts as a template for building a new strand
in replication
• Parent molecule unwinds
• Two daughter strands built
• Base pairing rules
Figure 16.9-1
A
T
C
G
T
A
A
T
G
C
(a) Parent molecule
Figure 16.9-2
A
T
A
T
C
G
C
G
T
A
T
A
A
T
A
T
G
C
G
C
(a) Parent molecule
(b) Separation of
strands
Figure 16.9-3
A
T
A
T
A
T
A
T
C
G
C
G
C
G
C
G
T
A
T
A
T
A
T
A
A
T
A
T
A
T
A
T
G
C
G
C
G
C
G
C
(a) Parent molecule
(b) Separation of
strands
(c) “Daughter” DNA molecules,
each consisting of one
parental strand and one
new strand
Semiconservative Model
• Watson and Crick’s
semiconservative model
• predicts that when a double
helix replicates, each
daughter molecule will have
one old strand (derived or
“conserved” from the parent
molecule) and one newly
made strand
Meselson and Stahl
• Supported the
semiconservative
model
• Labeled nucleotides of
old strands with heavy
isotopes of nitrogen
• New nucleotides
labeled with a lighter
isotope
Figure 16.11
EXPERIMENT
1 Bacteria
cultured in
medium with
15N (heavy
isotope)
2 Bacteria
transferred to
medium with
14N (lighter
isotope)
RESULTS
3 DNA sample
centrifuged
after first
replication
CONCLUSION
Predictions:
First replication
Conservative
model
Semiconservative
model
Dispersive
model
4 DNA sample
centrifuged
after second
replication
Less
dense
More
dense
Second replication
Structure of DNA
Prokaryotic DNA
One double stranded
circular DNA molecule
Small amount of protein
Eukaryotic DNA
• Linear DNA
• Protein packaged as
chromatin
• 4 levels of packaging
Nucleosome- packaging- 1
• 10-nm fiber, basic unit
• DNA histone form
beads on a string
• 8 histone molecules
with the aa tail
projecting outward
• Histone H1 (different
from the rest) binds
DNA to the next
histone
Why do we need all this packaging?
• 1. more tightly packaged, less accessible to transcription
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enzymes
2. reduces gene expression
3. Interphase- chromatin is the highly extended form or
euchromatin which can undergo transcription
4. More condensed is heterochromatin- generally not
transcribed
5. Barr bodies are heterochromatin
Packaging 2- 30nm fiber
• String of nucleosomes
coils to form chromatin
fiber that is 30 nm
• Interphase is when this
occurs
• Histone 3 &4
Packaging -3
• 300 nm fiber
• Looped domains
• Formed to a scaffold of
non histone proteins
Packaging -4
• 1,400nm
• Maximally folding
compact chromosome
• Metaphase
• 2 chromatids
Chromosome with Euchromatin and
Heterochromatin
DNA Replication
• S phase
• Mitosis/Meiosis
• Fast and accurate
• More than a dozen
enzymes are involved
6 points of DNA Replication
• 1. Begins at sites-
Origins of Replication
• Bubbles will eventually
fuse
Replication Fork or bubble
• 2. Initiation proteins bind
• Separate strands
• DNA replication proceeds in
both directions along the DNA
strand until molecule is copied
• DNA strand is short RNA called
primer, enzymes primase does
this
• Enzymes
• Helicase
• SS binding proteins
• Topoisomerase
• Relieves strand by breaking,
swiveling and rewind the DNA
DNA Polymerase
• 3. Catalyzes
elongation of new DNA
at the replication Fork
• Bacterial uses different
polymerases, III and I
• Adds DNA nucleotide to
primer which is RNA,
done by dATP, exergonic
reaction
Adding nucleotides
• 4. DNA polymerase
adds nucleotides to the
strand one at a time in
the 5’-3’ direction
Bonding of A-T, C-G
• Basically purine to
pyrimidine
• Keeps the symmetry to
the molecule
Antiparallel elongation
• 5 Leading and Lagging
strands
• 5. leading toward fork is
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continuous
Lagging 3’-5’-away from
fork is discontinuous
Antiparallel
A-T
C-G
Purine to pyrimidine
Okazaki Fragments
• 6.Lagging is in pieces
• Starts as discontinous
• Sealed by DNA ligase
• Forms continuous strand
DNA Accuracy
• Dependent on the
specificity of base pairing
• A-T, C-G
• Mismatch Repair
• Enzymes that cut out
nuclease
• New sequence is DNA poly
and DNA ligase
• Cancer cells accumulate
errors
• Nucleotide excision repair
system
Of importance
• DNA repair enzymes
on skin after exposure
to UV rays
• Thymine bases, or
thymine dimers cause
DNA to buckle,
interferes with
replication
• Xeroderma
pigmentosum
Telomeres- ase-Blackburn
• Replicating the ends
• TTAGGG- does not code
for anything noncoding
repeating sequence
• 100-1000x repeated
• Telomerase
• Enzyme that stimulates
ends to lengthen in germ
cells
• Zygotes only have
maximum
• Somatic cells do not have
this at birth
Figure 16.18
DNA pol III
Parental DNA
5
3
5
3
3
5
5
Connecting
protein
3
Helicase
3
DNA
pol III 5
Leading strand
3
5
Lagging strand
Lagging
strand
template
Bozeman Links
• Structure of DNA
• https://www.youtube.com/results?search_query=bozeman
+dna+structure
• Replication
• https://www.youtube.com/results?search_query=bozeman
+dna+replication