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11 The History of DNA Mark Mayo Cypress College Mark Mayo Cypress College Last update 9/16/13.

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Presentation on theme: "11 The History of DNA Mark Mayo Cypress College Mark Mayo Cypress College Last update 9/16/13."— Presentation transcript:

1 11 The History of DNA Mark Mayo Cypress College Mark Mayo Cypress College Last update 9/16/13

2 2 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.

3 3 Transformation Frederick Griffith (1923)

4 4 Transformation II Avery, McCarty, Macleod (1944) Repeated Griffiths 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 Griffiths 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 Griffith

5 5 Transformation II Avery, McCarty, Macleod (1944)

6 6 DNA or Protein as Active Agent Alfred Hershey and Martha Chase * (1950s) Used radioactive labels on bacteriophage components to decide if DNA or protein was the transforming factor Label viral protein with S 35 * Label viral DNA with P 32 * Allow infection Wash viral particles (with blender!) Check for label after subsequent infection into new bacteria Found only P 32 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 S 35 * Label viral DNA with P 32 * Allow infection Wash viral particles (with blender!) Check for label after subsequent infection into new bacteria Found only P 32 Hence DNA is the transforming factor

7 7 DNA or Protein as Active Agent Alfred Hershey and Martha Chase

8 8 Chargaffs Rules Erwin 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)

9 9 Chargaffs Rules Erwin Chargaff (1949)

10 10 Alpha Helix Linus Pauling ( ) 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…

11 11 Alpha Helix Linus Pauling ( )

12 12 X Ray Diffraction Maurice 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

13 13 X Ray Diffraction Maurice Wilkins and Rosalyn Franklin

14 14 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.

15 15 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

16 16 Discontinuous DNA Replication Reiji 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

17 17 Discontinuous DNA Replication Reiji Okazaki *


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