Discovering the material for heredity: DNA

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Discovering the material for heredity DNA

• Scientific Discovery from a
• Historical Perspective

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Time Line

• Frederick Griffith (1928) discovers that bacteria
  can change from one form to another (a process
  called transformation).
• Oswald Avery colleagues (1944) follow up
  Griffiths earlier discovery and conclude that
  the transforming factor is DNA.
• Rosalind Franklin Maurice Wilkins (1950)
  provide evidence that DNA is in the form of a
  double helix.
• Erwin Chargaff (1951) publishes that the
  nitrogenous bases of DNA occur in a ratio, with
  equal amounts of adenine and thymine, and equal
  amounts of cytosine and guanine.
• Alfred Hershey Martha Chase (1952) conduct
  experiments which further prove that DNA was the
  hereditary material, sufficient to code for the
  growth of a new organism.
• James Watson Francis Crick (1953) publish the
  three dimensional structure and composition of
  DNA.

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Ancient History

• By the 1920's chromosomes were suspected to be
  the carriers of genetic information based on
  observations of mitosis through a light
  microscope.
• Biochemical studies of chromosome composition
  demonstrated that they were composed of 30-50
  nucleic acid and 50-70 protein.
• It was generally believed that PROTEINS would
  prove to be the carriers of genetic information.
  WHY? (multiple reasons reallythink about info
  on this slide and about protein structure)

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Genetic Transformation Discovered

• Fred Griffith unwittingly discovered
  transformation. He showed that some active
  genetic substance could be transferred from dead
  bacteria capable of causing disease to live
  harmless bacteria making these live bacteria
  dangerous.
• How did he accomplish this?

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Griffiths Experiment

• Griffith was attempting to develop a vaccine for
  Streptococcus pneumoniae (a type of bacteria).
• There were two strains of Streptococcus, one of
  which was harmless to people. The other strain
  caused pneumonia.
• The term for the ability of an organism to cause
  disease is virulence. Such a disease-causing
  organism could also be called pathogenic.

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More on Streptococcus

• Strain 1 S strain
• Was called S strain because it formed smooth
  colonies on a petri dish culture.
• Had a polysaccharide coat that protected it from
  attack by the immune system.
• Was virulent.

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Still More on Streptococcus

• Strain 2 R strain
• Was called R strain because it formed rough
  colonies on a petri dish culture.
• Did not have the polysaccharide coat that
  protected it from attack by the immune system.
• Was avirulent (harmless).

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Griffith (and later Avery his colleagues)
performed the following experiment

• Live S strain injected into a mouse yields a dead
  mouse.
• Live R strain injected into a mouse yields a live
  mouse.
• Heat-killed S strain injected into a mouse also
  yields a live mouse.
• BUT what if you heat-kill the S strain and add
  it to the live R strain?

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Griffiths Results
Live R strain mixed w/ Heat-killed S strain
Heat-killed S strain
Living R strain
Living S strain
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Something (we now know this to be DNA) from the
virulent S strain had been able to transform the
harmless R strain into a mouse killer!
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Avery, McLeod, McCarthys Experiments Build on
Griffiths

• Avery and his colleagues repeated Griffiths
  experiment but added an additional step.
• First they added a protein-destroying enzyme to
  the heat-killed S strain. Mice still died.
• They repeated the experiment but the second time
  added a DNA-destroying enzyme to the heat-killed
  S strain. The mice didnt die!
• The tranforming factor had to be DNA!!

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Alfred Hershey Martha Chase

• Even after Averys experiments, scientists were
  still skeptical about the possibility that DNA
  was the stuff of heredity.
• In 1952, Alfred Hershey Martha Chase performed
  an elegant series of experiments which proved
  that DNA was the genetic material using a
  household blender!

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Hershey and Chase used ???
bacteriophage

• Hershey and Chase used the T2_____________, a
  virus which infects and kills bacterial cells.

• Virus Structure
• Protein coat or ______
• Nucleic acid (____________)
• H C knew T2 phages were made of DNA and
  protein, but they had no proof as to whether
  protein or DNA was the genetic material of the
  viruses.

capsid
DNA or RNA
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Virus Life Cycle (in brief)

• Viruses infect living cells and then multiply
  inside these cells, producing millions of copies
  of the virus which then explode (____) the cell,
  releasing these copies to go out and
  _______________.

lyse
infect more cells
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Experimental Predictions

• If the virus carried the instructions for making
  copies of itself (its genetic material) in the
  form of _______, then the virus would have to
  inject its _______ into the bacteria.
• If the virus carried the instructions for making
  copies of itself (its genetic material) in the
  form of _____, then the virus would have to
  inject its _____ into the bacteria.

protein
protein
DNA
DNA
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The Hershey-Chase Experiment

• Use a batch of virus with radioactive capsid
  proteins (labeled with 35S).
• Use a second batch of virus with radioactive DNA
  (labeled with 32P).
• Allow each batch to infect bacteria (E. coli),
  then remove the viruses on the outside of the
  bacterial cells by agitating the cellsin a
  blender.
• Spin the bacterial culture tubes in centrifuge so
  the dense bacterial cells sink to the bottom
  (forming a pellet) the lighter viruses stay in
  the upper liquid portion (called the supernatant)
• Collect the E. coli bacteria and see whether they
  contain 35S (radioactive protein) or 32P
  (radioactive DNA).

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Hershey-Chase Experiment One Picture
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Hershey-Chase Experiment Another Picture
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Hershey-Chase Results

• E. coli bacteria in the pellet contained
  virtually no 35S.
• The offspring of the virus (its progeny)
  contained lots of 32P-labelled DNA.
• Conclusion DNA carried the genetic information!!

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What next??

• The Hershey-Chase experiment was strong evidence
  that DNA was genetic material
• Given this finding, what do you think was the
  next question that came to mind to scientists?
  What does this finding make you want to know?
• What would be your first step to figuring this
  out?
• (remember proteinswhat is the key to
  understanding their function?)

What I wonder How does DNA work? How does it
dictate what goes on in a cell?
Figuring out the STRUCTUREso you can learn
something about FUNCTION!
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What did we knowabout DNA in the 1940s?

• We knew that it was composed of chains of four
  __________ containing four different
  _______________
• Adenine ( )
• Thymine ( )
• Cytosine ( )
• Guanine ( )

nucleotides
nitrogenous bases
A
T
C
G
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What is a nucleotide?
2
1
3
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Nitrogenous Bases

• The bases with one ring are pyrimidines.
• Cytosine
• Thymine

• The bases with two rings are purines.
• Adenine
• Guanine

Angels are pure in heart. (heart reminds you of
two rings)
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Chargaffs Rule

• 1949 Erwin Chargaff noticed that in every
  analysis of DNA that he performed, the amount of
  adenine present always equaled the amount of
  thymine and that the amount of cytosine always
  equaled the amount of guanine.
• In other words
• There could be different overall amount of C G
  and A T but these pairs of bases were always
  present in equal ratios!
• This is called Chargaffs Rule.

A T and G C
(A G) / (T C) 1
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The Big Race (early 1950s)
Linus Pauling

• Once DNA was proven to be the genetic material,
  an increasing number of scientists became
  passionate about trying to discover its
  structure
• The most significant scientists who were racing
  each other to be the first to discover the
  structure of DNA were
• Linus Pauling (CalTech)
• Rosalind Franklin Maurice Wilkins
• (Kings College, London)
• James D. Watson Francis Crick
• (Cambridge University)

James Watson Francis Crick
Maurice Wilkins
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Rosalind Franklin Maurice Wilkins

• Maurice Wilkins and Rosalind Franklin, a talented
  X-ray crystallographer, developed X-ray
  diffraction images of DNA.
• Franklins uniquely sharp images indicated that
  DNA existed in the form of two twisted strands.

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James Watson Francis Crick
What Watson Crick knew

• Chargaffs Rule In a sample of DNA, adenine
  thymine occur in equal amounts, as do cytosine
  and guanine.
• Rosalind Franklins data indicated that the DNA
  molecule was a double-stranded molecule.

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What they finally figured out

• DNA is a two-stranded (double) helix -- like a
  twisted ladder.
• The backbone of the molecule is composed of the
  phosphates and deoxyribose sugars of the
  nucleotides (covalently bonded).
• The rungs are composed of nitrogenous base pairs,
  which stick together by hydrogen bonding. These
  pairs consist of either A T or G C.

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Three Representations of DNA
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Base-Pair Hydrogen Bonding

• A - T pair forms two H-bonds
• G - C pair forms three H-bonds
• Diameter of DNA helix is constant because AT
  GC base pairs have equal width

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And the winner is
1953 Watson Crick publish their findings in
the journal Nature. 1962 The Nobel Prize for
Medicine Physiology was awarded to Crick,
Watson, Wilkins. Franklin died in 1958 of
ovarian cancer, at age 38. Because the Nobel
Prize is not awarded posthumously, and because no
more than three individuals can share the prize,
we can only speculate about whether the committee
would have recognized Franklins contribution to
the discovery of the double helix.
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Structure Shows Action
"It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic
material."
-Watson and Crick in the scientific paper that
was published in Nature, April 25, 1953.
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Adding fuel to the fire
1968 Watson publishes The Double Helix, his
(not-so-modest) account of his participation in
the race to find the structure of DNA and the
role Crick, Wilkins, Franklin played as well.
The book is widely read and popular, despite
criticism from those involved of Watsons
subjective depiction of the other scientists,
especially his unflattering portrayal of
Franklin, who was no longer alive to defend
herself.
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More Representations of DNA
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More Representations of DNA
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More Representations of DNA
Note that the pictures are not oriented the same
with respect to the 5 3 ends.