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11
The History of DNA The History of DNA
Mark Mayo
Cypress College
Mark Mayo
Cypress College
Last update 9/16/13
22
Transformation Frederick Griffith (1923)
Transformation Frederick Griffith (1923)
• Used healthy mice• Mice were injected with either R(rough) strain of Streptococcus
pneumoniae. The mice live and their immune system kills R bacteria. No live bacteria
• Mice injected with the S (smooth) strain of Streptococcus pneumoniae. The mice die. The dead mice have live S bacteria.
• Mice injected with heat-killed S strain. Mice live with no live bacteria found in mice.
• Mice injected with mixture of live R strain and heat-killed S strain. Mice die and live S strain bacteria are found in the dead mice.
• Heat does not destroy the active factor that is responsible for heredity (DNA).
• It is said that the bacteria were transformed by this active agent.
• Used healthy mice• Mice were injected with either R(rough) strain of Streptococcus
pneumoniae. The mice live and their immune system kills R bacteria. No live bacteria
• Mice injected with the S (smooth) strain of Streptococcus pneumoniae. The mice die. The dead mice have live S bacteria.
• Mice injected with heat-killed S strain. Mice live with no live bacteria found in mice.
• Mice injected with mixture of live R strain and heat-killed S strain. Mice die and live S strain bacteria are found in the dead mice.
• Heat does not destroy the active factor that is responsible for heredity (DNA).
• It is said that the bacteria were transformed by this active agent.
33
Transformation Frederick Griffith (1923)
Transformation Frederick Griffith (1923)
44
Transformation II Avery, McCarty, Macleod (1944)
Transformation II Avery, McCarty, Macleod (1944)
• Repeated Griffith’s work, but knew that DNA was the substance of transformation
• Separated classes molecules from s cell debris
• Tested each fraction for transforming ability, one at a time
• Only DNA transformed r cells into s cells• To provide r cells with s DNA is to provide r
cells with s genes
• Repeated Griffith’s work, but knew that DNA was the substance of transformation
• Separated classes molecules from s cell debris
• Tested each fraction for transforming ability, one at a time
• Only DNA transformed r cells into s cells• To provide r cells with s DNA is to provide r
cells with s genes
Think re-mix of GriffithThink re-mix of Griffith
55
Transformation II Avery, McCarty, Macleod (1944)
Transformation II Avery, McCarty, Macleod (1944)
66
DNA or Protein as Active Agent Alfred Hershey and Martha Chase*
(1950’s)
DNA or Protein as Active Agent Alfred Hershey and Martha Chase*
(1950’s) • Used radioactive labels on bacteriophage
components to decide if DNA or protein was the transforming factor
• Label viral protein with S35 *• Label viral DNA with P32 *• Allow infection• Wash viral particles (with blender!)• Check for label after subsequent infection into new
bacteria• Found only P32
• Hence DNA is the transforming factor
• Used radioactive labels on bacteriophage components to decide if DNA or protein was the transforming factor
• Label viral protein with S35 *• Label viral DNA with P32 *• Allow infection• Wash viral particles (with blender!)• Check for label after subsequent infection into new
bacteria• Found only P32
• Hence DNA is the transforming factor
77
DNA or Protein as Active Agent Alfred Hershey and Martha Chase
DNA or Protein as Active Agent Alfred Hershey and Martha Chase
88
Chargaff’s RulesErwin Chargaff (1949)
Chargaff’s RulesErwin Chargaff (1949)
• He studied the relative amounts of each nucleic acid base in a great variety of plant and animal species
• Roughly found that A=T and G=C, but not exactly due to errors in the technology!
• Purines are exactly equal to pyrimidines• His methodology for the time was good, but now we
get exact amounts• He could not make the connection (Watson and
Crick used his data however)
• He studied the relative amounts of each nucleic acid base in a great variety of plant and animal species
• Roughly found that A=T and G=C, but not exactly due to errors in the technology!
• Purines are exactly equal to pyrimidines• His methodology for the time was good, but now we
get exact amounts• He could not make the connection (Watson and
Crick used his data however)
99
Chargaff’s Rules Erwin Chargaff (1949)
Chargaff’s Rules Erwin Chargaff (1949)
1010
Alpha HelixLinus Pauling (1948-1950)
Alpha HelixLinus Pauling (1948-1950)
• Worked with proteins and determined that collagen has a helical arrangement for its polypeptides
• He called the helix an alpha helix• He suspected that DNA might also have a helical
arrangement, but could not get it to compute with a single strand
• Pauling suggested DNA had a triple helix, but had no proof
• Watson and Crick heard his idea about a helix…
• Worked with proteins and determined that collagen has a helical arrangement for its polypeptides
• He called the helix an alpha helix• He suspected that DNA might also have a helical
arrangement, but could not get it to compute with a single strand
• Pauling suggested DNA had a triple helix, but had no proof
• Watson and Crick heard his idea about a helix…
1111
Alpha HelixLinus Pauling (1948-1950)
Alpha HelixLinus Pauling (1948-1950)
1212
X Ray DiffractionMaurice Wilkins and Rosalyn Franklin
X Ray DiffractionMaurice Wilkins and Rosalyn Franklin
• Used Xray diffraction to study proteins and other molecules• DNA was very difficult to crystallize and a tough candidate for
Xray diffraction• Rosalyn Franklin was a very talented graduate student in the lab
of Wilkins• She was successful at crystallizing DNA in two forms A and B• The forms on an X was seen indicating some kind of helix• She could measure the distances between repeating units on
the molecule• She could also measure the diameter of the molecule• Wilkins sent her unpublished data to Watson and Crick without
her permission• She died before the Nobel prize or she might have shared it
• Used Xray diffraction to study proteins and other molecules• DNA was very difficult to crystallize and a tough candidate for
Xray diffraction• Rosalyn Franklin was a very talented graduate student in the lab
of Wilkins• She was successful at crystallizing DNA in two forms A and B• The forms on an X was seen indicating some kind of helix• She could measure the distances between repeating units on
the molecule• She could also measure the diameter of the molecule• Wilkins sent her unpublished data to Watson and Crick without
her permission• She died before the Nobel prize or she might have shared it
1313
X Ray DiffractionMaurice Wilkins and Rosalyn Franklin
X Ray DiffractionMaurice Wilkins and Rosalyn Franklin
1414
Semi-conservative DNA Replication Matthew Meselsohn and Frank Stahl
Semi-conservative DNA Replication Matthew Meselsohn and Frank Stahl
• They used two isotopes of Nitrogen – 14N and 15N• 15N is heavier than 14N• They grew bacteria for several generations in heavy
15N (all DNA would be heavy!)• Abruptly changed the medium to lighter 14N for one
or two generations• Used density-gradient ultracentrifugation to separate
the DNA strands by weight• After one generation all DNA was medium between
heavy and light (thus SEMI-CONSERVATIVE)• After two generation DNA had two bands: medium
and light.
• They used two isotopes of Nitrogen – 14N and 15N• 15N is heavier than 14N• They grew bacteria for several generations in heavy
15N (all DNA would be heavy!)• Abruptly changed the medium to lighter 14N for one
or two generations• Used density-gradient ultracentrifugation to separate
the DNA strands by weight• After one generation all DNA was medium between
heavy and light (thus SEMI-CONSERVATIVE)• After two generation DNA had two bands: medium
and light.
1515
Semi-conservative DNA Replication Matthew Meselsohn and Frank Stahl
Semi-conservative DNA Replication Matthew Meselsohn and Frank Stahl
Half light and half heavy = medium weight
This one would be one heavy and one light band
1616
Discontinuous DNA ReplicationReiji Okazaki
Discontinuous DNA ReplicationReiji Okazaki
• He knew about DNA polymerase• It moves from 5’ to 3’ only on the leading strand• He searched for a second polymerase that worked in the
reverse direction• After unsuccessfully searching he used his brilliance• Found numerous small fragments and also long
segments as DNA was replicated • Finally decided that the short segments were from the
lagging strand *• DNA polymerase worked on both sides continuously on
the leading strand and in several places on the lagging strand
• DNA ligase connects the small portions (now called Okazaki fragments) * on the lagging strand
• He knew about DNA polymerase• It moves from 5’ to 3’ only on the leading strand• He searched for a second polymerase that worked in the
reverse direction• After unsuccessfully searching he used his brilliance• Found numerous small fragments and also long
segments as DNA was replicated • Finally decided that the short segments were from the
lagging strand *• DNA polymerase worked on both sides continuously on
the leading strand and in several places on the lagging strand
• DNA ligase connects the small portions (now called Okazaki fragments) * on the lagging strand
1717
Discontinuous DNA ReplicationReiji Okazaki
Discontinuous DNA ReplicationReiji Okazaki
**