11 the history of dna mark mayo cypress college mark mayo cypress college last update 9/16/13

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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)


• 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.

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44

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

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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)


• 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…

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1212

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



1414

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



Half light and half heavy = medium weight

This one would be one heavy and one light band

1616

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



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11 the history of dna mark mayo cypress college mark mayo cypress college last update 9/16/13

Documents

live bacteria mice

healthy mice mice

live s bacteria

live s strain bacteria

s cells

dead mice

molecules dna

heredity dna