Structure (chapter 10, pages 266 – 278) and Replication of DNA (chapter 12, pages 318 – 334)

1. Structure (chapter 10, pages 266 – 278)and Replication of DNA (chapter 12, pages 318 – 334)

2. Dr. Ravi Palanivelu Rpalaniv@ag.arizona.edu http://ag.arizona.edu/research/ravilab/

5. Transmission genetics • Previous discussions focused on the individual. • Phenotype caused by an individual genotype • Transmission of genes of an individual organism to the next generation • Offspring produced by crossing two individuals

6. Molecular genetics • Focus will now shift to genes • How are they encoded (Lecture 9) • How are they replicated (Lecture 9) • How are they expressed (genotype --> phenotype; (Lectures 10, 11) • How do we study them (Lectures 12, 13)

7. What is the hereditary/genetic material? • Until the mid-20th century, most scientists believed that proteins must be the hereditary (genetic) material

8. Why did they believe this? • Nuclei from male and female reproductive cells fuse during reproduction • Nuclei contain chromatin • Chromatin made-up of nuclein (DNA) and protein • Understood chromosome movements, and that chromosomes were the vehicles of inheritance • Chromosomes made-up of nuclein (DNA) and protein

9. How did they come to this conclusion? • The basic chemistry (the parts) of DNA had been worked-out, but not the structure (how the parts fit together) • Thought it was a very simple repeating molecule • Thought DNA was structural in chromosomes (scaffolding) • Proteins are much more complex • Could account for the complexity of cells

10. Miescher isolates DNA from nuclei Important Dates in Determining the Structure of DNA

11. Experiments that led to determining the genetic material • Frederick Griffith, 1928 • British physician/bacteriololgist • Streptococcus pneumoniae • Pneumonia in humans • Septicemia in mice (lethal)

12. Frederick Griffith Streptococcus pneumoniae Used two strains IIIS = smooth,virulent • Enclosed in a polysaccharide capsule • The colonies have a smooth appearance IIR = rough, non-virulent • Polysaccharide coat is missing • Colonies have a rough appearance

13. Frederick Griffith Injected mice with 1. Live IIIS strain only 2. Live IIR strain only 3. Heat-killed IIIS strain 4. Heat-killed IIIS strain + live IIR strain

14. Frederick Griffith • Injected mice with 1. Live IIIS strain • Mice died • IIIS strain recovered from blood

15. Frederick Griffith • Injected mice with 1. Live IIIS strain • Mice died • IIIS strain recovered from blood 2. Live IIR strain • Mice lived • Nothing recovered in blood

16. Frederick Griffith • Injected mice with 1. Live IIIS strain • Mice died • IIIS strain recovered from blood 2. Live IIR strain • Mice lived • Nothing recovered in blood 3. Heat-killed IIIS strain • Mice lived • Nothing recovered in blood

17. Frederick Griffith • Injected mice with • Heat-killed IIIS strain + live IIR strain • Expect the mice to live • Recover nothing from the blood

18. Frederick Griffith • Injected mice with 4. Heat-killed IIIS strain + live IIR strain • Found mice died • Live IIIS strain recovered from blood

20. Frederick Griffith • Found • Something in the dead (heat-killed) IIIS strain transformedsome of the live IIR cells into live IIIS cells • The idea was that this transforming principle is the genetic material

21. Generalized Bacterial/Prokaryotic Cell

23. Experiments toward determining the genetic material • Oswald Avery, Colin MacLeod, and Maclyn McCarty, 1944 • Wanted to find the transforming (principle) agent from Griffith’s experiment • The transforming agent must be the genetic (hereditary) material

24. Avery, MacLeod and McCarty • Oswald Avery, Colin MacLeod, and Maclyn McCarty, 1944 • Separated debris from dead S cells into classes of molecules (DNA, RNA, proteins, lipids, polysaccharides) • Added each separately to live R cells to see what happens

27. Avery, MacLeod and McCarty • Oswald Avery, Colin MacLeod, and Maclyn McCarty, 1944 • DNA was shown to be the transforming agent • By using proteases and nucleases they were able to show that there were no contaminating proteins that could really be the transforming agent • Most people assumed that the transforming agent and the genetic material were one and the same, thus DNA was the genetic material

28. Practice problems (8, structure and replication of DNA) is due next Monday (4/5/01)

29. http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/micro04.swf::DNA%20Replication%20Forkhttp://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/micro04.swf::DNA%20Replication%20Fork

30. Experiments toward determining the genetic material • Alfred Hershey and Martha Chase, 1952 • The experiment that convinced all the others, most of whom were virologists, that DNA is the genetic material

31. Hershey and Chase • Alfred Hershey and Martha Chase, 1952 • Worked with T2 virus • A bacteriophage of E. coli

32. T2 virus is made-up of • A protein coat • Surrounding a DNA molecule

33. Alfred Hershey and Martha Chase, 1952 • Knew that viruses inject something into the bacteria, then the bacteria reproduce new viruses • Hypothesis: whatever is injected into the bacteria is the genetic material

34. T2 virus is made-up of • A protein coat • (contains sulfur) • Surrounding a DNA molecule • (contains phosphorous)

35. Hershey and Chase • T2 virus is made-up of a protein coat, surrounding a DNA molecule • Proteins contain sulfur • Labeled the proteins with radioactive 35S • DNA contains phosphorous • Labeled DNA with radioactive 32P

36. Hershey and Chase • Allowed the virus enough time to infect the bacteria • Sheared-off the viruses from the bacteria in a Warring blender • Looked to see whether the radioactivity in the bacteria was 35S (protein) or 32P (DNA)

39. Hershey and Chase • Hershey and Chase found that the radioactive DNA was injected into the bacteria, and passed to the phage progeny

40. Structure of DNA • Once it was determined that DNA was the genetic material, the race was on to elucidate the structure of DNA. • It was felt that by understanding the structure it would explain much about inheritance and function of DNA

41. What I appreciated was that genetics was the key part of biology – and that one had to explain genetics in structural terms. Francis Crick

42. Structure of DNA Players (College of Physicians and Surgeons, Columbia University, New York) • Erwin Chargaff (biochemist) • Worked with nucleic acids • Found the fundamental ratio of bases

44. Structure of DNA Players (Cambridge University) • Cavendish Laboratory (Sir Lord Rutherford, found neutrons and electrons) • Sir Lawrence Bragg (physicist) • X-ray crystallography • Max Perutz (chemist) • Director of Watson and Crick’s unit • Nobel prize for structure of hemoglobin

45. X-ray crystallography Crystallized substance

46. Players (Kings College – London) Maurice Wilkins (physicist) X-ray studies of DNA Rosalind Franklin (chemist turned crystallographer) Structure of DNA

48. Structure of DNA Players (California Institute of Technology, Pasadena) • Linus Pauling (Chemist) • Structure of chemical bonds • One of three people to receive two Nobel Prizes [Marie Curie (Physics, Chemistry); John Burden (Physics)] • Chemistry – chemical bonds – 1954 • Peace – campaign against above ground atomic testing - 1962

49. Structure of DNA Players (Cambridge University, the Cavendish Laboratory) • Francis Crick (physicist turned biologist) • Ph.D. candidate • Mid-thirties • James Watson (biologist, American) • Young postdoc • Early 20s • University of Chicago • Quiz Kids • University of Indiana • Studied bacteriophage with Luria