2. The DNA Story Discovering the material for heredity Accel Bio 2014

3. “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. 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. 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 really…think about info on the next slide and about protein structure) It was generally believed that PROTEINS would prove to be the carriers of genetic information. WHY? (multiple reasons really…think about info on the next slide and about protein structure)

4. The Genetic Material had to: Carry information from one generation to the next Carry information from one generation to the next Somehow “code” for the heritable traits of an organism Somehow “code” for the heritable traits of an organism Be easily and accurately copied Be easily and accurately copied (but able to change as well, to allow for mutations) How are proteins appropriate for these functions? Why did so many scientists who were knowledgeable about protein structure suspect they were the genetic material? How are proteins appropriate for these functions? Why did so many scientists who were knowledgeable about protein structure suspect they were the genetic material?

5. Time Line of Discovery Frederick Griffith (1928) discovers that bacteria can change from one form to another (a process called transformation). Frederick Griffith (1928) discovers that bacteria can change from one form to another (a process called transformation). Oswald Avery & colleagues (1944) follow up Griffith’s earlier discovery and conclude that the transforming factor is DNA. Oswald Avery & colleagues (1944) follow up Griffith’s 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. 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. 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. 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. James Watson & Francis Crick (1953) publish the three dimensional structure and composition of DNA.

6. 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. 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? How did he accomplish this?

7. Griffith’s Experiment Griffith was attempting to develop a vaccine for Streptococcus pneumoniae (a type of bacteria). 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. 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. The term for the ability of an organism to cause disease is virulence. Such a disease-causing organism could also be called pathogenic.

8. More on Streptococcus… Strain #1: S strain 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.

9. Still More on Streptococcus… Strain #2: R strain 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).

10. Griffith (and later Avery & his colleagues) performed the following experiment… Live S strain injected into a mouse yields a dead mouse. Live S strain injected into a mouse yields a dead mouse. Live R strain injected into a mouse yields a live 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. 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? BUT… what if you heat-kill the S strain and add it to the live R strain?

11. Griffith’s Results Heat-killed S strain Live R strain mixed w/ Heat-killed S strain Living R strainLiving S strain

12. “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!

13. Avery, McLeod, & McCarthy’s Experiments Build on Griffith’s Avery and his colleagues repeated Griffith’s experiment but added an additional step. Avery and his colleagues repeated Griffith’s experiment but added an additional step. First they added a protein-destroying enzyme to the heat-killed S strain (then mixed with live R-strain). Mice still died. First they added a protein-destroying enzyme to the heat-killed S strain (then mixed with live R-strain). Mice still died. They repeated the experiment but the second time added a DNA-destroying enzyme to the heat-killed S strain (then mixed with live R-strain). The mice didn’t die! They repeated the experiment but the second time added a DNA-destroying enzyme to the heat-killed S strain (then mixed with live R-strain). The mice didn’t die! The “transforming factor” had to be DNA!! The “transforming factor” had to be DNA!!

14. Alfred Hershey & Martha Chase Even after Avery’s experiments, scientists were still skeptical about the possibility that DNA was the “stuff of heredity” in more complex organisms (other than bacteria) Even after Avery’s experiments, scientists were still skeptical about the possibility that DNA was the “stuff of heredity” in more complex organisms (other than bacteria) In 1952, Alfred Hershey & Martha Chase performed an elegant series of experiments which proved that DNA was the genetic material – using a household blender! In 1952, Alfred Hershey & Martha Chase performed an elegant series of experiments which proved that DNA was the genetic material – using a household blender!

15. Hershey and Chase used ???  Hershey and Chase used the T2_____________, a virus which infects and kills bacterial cells. bacteriophage 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

16. Bacteriophage Micrograph

17. Bacteriophages on surface of bacterial cell

18. 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 _______________. infect more cells lyse

19. 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. 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

20. The Hershey-Chase Experiment Use a batch of virus with radioactive capsid proteins (labeled with 35 S). Use a batch of virus with radioactive capsid proteins (labeled with 35 S). Use a second batch of virus with radioactive DNA (labeled with 32 P). Use a second batch of virus with radioactive DNA (labeled with 32 P). Allow each batch to infect bacteria (E. coli), then remove the viruses on the outside of the bacterial cells by agitating the cells…in a blender. Allow each batch to infect bacteria (E. coli), then remove the viruses on the outside of the bacterial cells by agitating the cells…in 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) 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 35 S (radioactive protein) or 32 P (radioactive DNA). Collect the E. coli bacteria and see whether they contain 35 S (radioactive protein) or 32 P (radioactive DNA).

21. Hershey-Chase Experiment

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

23. What next?? The Hershey-Chase experiment was strong evidence that DNA was genetic material in viruses The Hershey-Chase experiment was strong evidence that DNA was genetic material in viruses Fact that cells double amt DNA in cell prior to mitosis & then distribute equally to daughter cells was circumstantial evidence that DNA was genetic material in Eukaryotes as well Fact that cells double amt DNA in cell prior to mitosis & then distribute equally to daughter cells was circumstantial evidence that DNA was genetic material in Eukaryotes as well Given these suspicions, what do you think was the next question that came to mind to scientists? Given these suspicions, what do you think was the next question that came to mind to scientists? What would be your first step to figuring this out? What would be your first step to figuring this out? (remember proteins…what is the key to understanding their function?) How does DNA work? Figuring out the STRUCTURE…so you can learn something about FUNCTION!

24. DNA Structure and The Big Race

25. What did we know about DNA in the 1940’s? We knew that it was composed of chains of four __________ – containing four different _______________. We knew that it was composed of chains of four __________ – containing four different _______________. We knew Chargaff’s Rule. We knew Chargaff’s Rule. nucleotides nitrogenous bases

26. What is a Nucleotide? One nucleotide has three parts – a sugar (deoxyribose), a phosphate (PO 4 - ), and a nitrogenous base. One nucleotide has three parts – a sugar (deoxyribose), a phosphate (PO 4 - ), and a nitrogenous base. There are four different nitrogenous bases that appear in DNA: There are four different nitrogenous bases that appear in DNA: –Adenine ( ) –Thymine ( ) –Cytosine ( ) –Guanine ( ) A T C G

27. The Four 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)

28. http://www.sci.sdsu.edu/classes/bio100/Lectures/Lect08/Image196.gif Chargaff’s Rule 1949 – Austrian Biochemist Erwin Chargaff noticed that in every analysis of DNA that he performed, the amount of adenine always equaled the amount of thymine and the amount of cytosine equaled the amount of guanine. 1949 – Austrian Biochemist Erwin Chargaff noticed that in every analysis of DNA that he performed, the amount of adenine always equaled the amount of thymine and the amount of cytosine equaled the amount of guanine. In other words: 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! 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 Chargaff’s Rule. This is called Chargaff’s Rule. A = T and G = C A + G = T + C http://img0.liveinternet.ru/images/attach/c/0//47/349/47349945_Erwin_Chargaff.jpghttp://www.nature.com/scitable/content/24234/sadava_11_7_FULL.gif

29. The Big Race (early 1950’s) Once DNA was proven to be the genetic material, an increasing number of scientists became passionate about trying to discover its structure… 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: 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 Pauling Linus Pauling Maurice Wilkins

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

31. James Watson & Francis Crick Chargaff’s Rule: In a sample of DNA, adenine & thymine occur in equal amounts, as do cytosine and guanine. Chargaff’s Rule: In a sample of DNA, adenine & thymine occur in equal amounts, as do cytosine and guanine. Rosalind Franklin’s data indicated that the DNA molecule was an anti- parallel double-stranded molecule. Rosalind Franklin’s data indicated that the DNA molecule was an anti- parallel double-stranded molecule. What Watson & Crick knew…

32. What they finally figured out… DNA is a two-stranded (double) helix -- like a twisted ladder. 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 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. The rungs are composed of nitrogenous base pairs, which stick together by hydrogen bonding. *These pairs consist of either A & T or G & C.

33. DNA strands are Anti-parallel 5’ 3’ 5’ 3’

34. Three Representations of DNA

35. More Representations of DNA Note that the pictures are not oriented the same with respect to the 5’ & 3’ ends.

36. The reason for Chargaff’s Rule: Base-Pair Hydrogen Bonding Adenine (a purine) always pairs up with Thymine (a pyrimadine). Adenine (a purine) always pairs up with Thymine (a pyrimadine). Guanine (a purine) always pairs up with Cytosine (a pyrimadine). Guanine (a purine) always pairs up with Cytosine (a pyrimadine). Diameter of DNA helix is constant because A-T & G-C base pairs have equal width Diameter of DNA helix is constant because A-T & G-C base pairs have equal width

37. 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 Franklin’s contribution to the discovery of the double helix.

38. 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.

39. 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 Watson’s subjective depiction of the other scientists, especially his unflattering portrayal of Franklin, who was no longer alive to defend herself.

40. That Other Nucleic Acid: RNA

41. What is RNA? Is single-stranded Is single-stranded Uses ribose sugar instead of deoxyribose sugar Uses ribose sugar instead of deoxyribose sugar Has four different nitrogenous bases: Has four different nitrogenous bases: –Adenine –Cytosine –Guanine –Uracil instead of Thymine Ribonucleic acid is a polynucleotide similar to DNA with a few key differences. RNA: An RNA nucleotide

42. DNA, with helix dimensions

43. More DNA, with 3’ & 5’ ends