SPU-22: The Unity of Science from the Big Bang to the Brontosaurus and Beyond

1. SPU-22: The Unity of Science from the Big Bang to the Brontosaurus and Beyond Lecture 19 14 April 2014 Science Center Lecture Hall A

2. Outline of Week’s Lectures Main Theme: Search for molecular basis of heredity. [As with many, but not all, of our detective stories, we now know answer. However, it helps one’s perspective -- my rationalization -- to follow trail that leads to pot of gold at rainbow’s end.] Story In Two Parts: 1. What is key molecule (or molecules) and how do we know? 2. How does it (or they) “work” and how do we know?

3. Today’s Topics Story of molecular basis of heredity via main contributors (early ones today): Friedrich Miescher (chemist) Phoebus Levene (chemist) Frederick Griffith (medical officer) Oswald Avery (biologist – chemist) Erwin Chargaff (chemist)

4. Discovery Of DNA Principal: Friedrich Miescher (another “loner”) Family contained distinguished scientists; went into chemistry because uncle’s conviction: last remaining questions on tissue development could be solved via chemistry

5. Brief Scene Setting Went to work in laboratory of Hoppe-Seyler, who had done fundamental work on hemoglobin Chose to work on chemical composition of cells (Note: Robert Hooke isolated cells with microscope and coined term “cell;” see next slide), another step towards accepting his uncle’s advice Hoppe-Seyler suggested leukocytes (white blood cells; see next slide plus one) Miescher set out to study cell nucleus At time living things thought to have only 3 components: fats (lipids), sugars & starches, and proteins

6. Collection of Cork Cells (Robert Hooke, 1665)

7. Lymphocyte (~1.5x10-3 cm)(20-40% Of All Leukocytes)

8. Developed Own Techniques Miescher invented protocols: • Had used bandages from clinic daily • Separated cells via soaking in (9:1 water to sodium sulfate) solution; several days cells settled • Found 5 protein-like entities (via solubility properties). Also found other substance, unlike any known protein

9. Serendipity Again New material from nuclei had unexpected properties: precipitated by acidifying solution and re-dissolved by making solution more alkaline (“basic”) Prolonged exposure of cells to very dilute HCl produced residue in test tube resembling nuclei of cells

10. Chemists’ Black Magic Tried to stain new nucleus material with iodine, which causes proteins to turn yellow Conclusion: Mystery substance is not protein Note: Little was then known about cell nucleus, despite its discovery by van Leeuwenhoek in 1719 and description in orchid by Robert Brown in 1831

11. More Black Magic Further study implied need for increased purity of mystery substance Developed complicated protocols involving, e.g., warm alcohol; acid in pig’s stomach, which contained enzyme pepsin; ether;… (see next slide) Extracted nucleus material sank to bottom of test tube as fine, white granules

13. Final Black Magic Wanted to determine atomic constitution of precipitate Used chemical tricks of day to isolate % of each element Weighed reaction products and compared with initial weight

14. Surprising Results In addition to C, O, H, and N-- all expected -- was large amount of unexpected element: P, almost nonexistent in then known organic materials. Further, new molecule big: wouldn’t diffuse through parchment; had molecular weight of (at least) 500 Fundamentally new type of molecule had been discovered; he called it “nuclein,” based on origin in nucleus (now “DNA”)

15. Contemporary Speculations In 1866, Ernst Haeckel suggested cell nucleus might be responsible for transmission of hereditary traits Miescher confident his new molecule would prove of equal stature to proteins in cell Scrapped initial idea that nuclein was solely responsible for diversity of species, although he speculated nuclein might provide sufficient variations, in analogy with words and letters (see later). So close…

16. Aftermath I Agony of publication permission Struggle with broadening of base of applicability (salmon in Rhine) Not self promoter; died young Fundamental contribution dropped from sight, despite uncle’s publication of collected works with introduction: “The appreciation of Miescher and his works will not diminish with time, instead it will grow, and the facts he has found and the ideas he has postulated are seeds which will bear fruit in the future.” That future was ~75 years in making

17. Aftermath II Why isn’t Miescher, discoverer of what is now called DNA, household name like Mendel, Watson, and Crick?? No good answer. Perhaps gap of ~75 years between discovery and understanding of importance was just too long

18. Friedrich Miescher (1844-1895) In next image, from earlier time, which person is Friedrich? How can you be sure?

19. Friedrich Miescher (1844-1895)

20. Phoebus Levene Superb chemist, Levene made important (+ & -) contributions to understanding Miescher’s “nuclein,” now (almost) universally called deoxyribonucleic acid, “DNA” for short Miescher determined relative amounts of five elements in DNA, but not chemical structure. Enter Levene (and others preceding), with more modern chemistry techniques

21. Chemical Structure of DNA: 1910-1930 Version Levene characterized chemical structure of DNA; contained four bases: adenine, guanine, thymine, cytosine (see next slide), as well as deoxyribose, and phosphate group (see next slide plus two and three)

22. Structure Of DNA Bases

23. Sugars in DNA & RNA

24. Phosphate Group

25. Downside In addition to determining chemical constituents of DNA, Levene also claimed that components were linked together in order phosphate-sugar-base to form units. He labeled each of these units one nucleotide, and concluded DNA molecule was string of such nucleotide units linked together through phosphate groups, which formed backbone of DNA

26. Downside (Concluded) He apparently formulated concept, called “tetranucleotide hypothesis,” in 1909. This hypothesis proposed that DNA was made up of equal amounts of adenine, guanine, cytosine, and thymine, four of them constituting each tetranucleotide (see next slide), unit of DNA molecule. This hypothesis was apparently not challenged for ~30 years. Led to conclusion: DNA could not carry genetic information. Thus, biologists thought protein was basis of heredity

27. Tetranucleotide

28. Phoebus Levene (1869-1940)

29. Genetic Material: What Exactly Is It? New progress started from unlikely source: Medical officer of British Ministry of Health, Frederick Griffith His specialty: Pneumonia His concern: Significance in spread of disease of different pneumococcal types

30. Exhaustive Study of Pneumococcal Types Griffith studied varieties of four types of lobar pneumonia; examined for his 1928 report on 278 cases, especially studying sputum samples Also conducted large number of experiments on mice of various strains of pneumococcal types under wide variety of conditions and pre-treatments of various strains of pneumococci

31. Surprising Discovery Some colonies of pneumococci had rough surfaces (“R” form); these pneumococci were generally not virulent. No particular surprise here Some colonies had smooth surfaces (“S” form) and were virulent Big surprise: R forms convertible to S forms when mixed with killed (via heating) S forms. What was“Transforming Principle”?

32. Cartoon Of Griffith’s Finding

33. Added Comments Conversions were brought about by different protocols involving inoculations of mice in different parts of their bodies Medical black magic or some fundamental property that could and should be pinned down?

34. Frederick Griffith (1879-1941)

35. Approach To Solving This Mystery - In Part Griffith’s results were subject of comment and inference, but little or no new experimentation, mostly near repetition (see next slide) Enter Avery, MacLeod, and McCarty They decided to seek and to study chemical nature of substance(s) inducing transformation (=conversion) of pneumococcal types

36. Preliminaries To Solution Griffith’s results had been confirmed by others in early 1930s, including inducing this strange transformation in vitro. In addition, by middle 1930s, transformation of virus in rabbit from benign to virulent form was effected

37. How Did Avery Et Al. Proceed? Approach: Isolate, purify, and test chemical agent responsible for transformation in vitro, to better control Procedure: S form of bacteria treated in manner reminiscent in general arcana of Miescher protocol. Result: 25 mg of agent from 75 l of culture.

38. Simplified View Of Experiment Experiment complicated in details. Simplified into following two cartoons, which suppress potential problems. Published 1944 paper, though, very thorough in discussion of procedures

39. Avery et al. Experiment: I

40. Avery Et Al. Experiment: II

41. Earlier Views Griffith suggested (1928) that some specific protein might serve as pabulum to enable R form to transform Dobzhansky (1941) stated that “if this transformation is described as a genetic mutation -- and it is difficult to avoid so describing it -- we are dealing with authentic cases of induction of specific mutations by specific treatments.” Other explanations were provided through preceding ~15 years, but not backed up by experiments, as noted earlier in slides (far) above

42. Penultimate Comment Detailed chemical basis for actions of DNA in this study were wholly unknown. But this 1944 paper through its careful experimental basis and detailed discussion considered landmark in biology: it seemed to establish with little doubt relevance of DNA, as opposed to proteins, to secret of heredity. Not so viewed at time

43. Final Comment Possible “biological activity is not inherent property of nucleic acid but is due to minute amounts of some other substance absorbed to it or so intimately associated with it as to escape attention” – cautious conclusion of Avery et al. paper Cautious and no Nobel: People weren’t convinced. My guess: too complicated; many people still enthralled with protein possibilities although wall weakening

44. Oswald Avery (1877-1955)

45. What Is Life? 1944 book by Erwin Schrodinger (inventor of quantum mechanics) Argued for aperiodic code script for heredity (vs. crystal periodicity) Noted number of parts to code can be small and account for life’s diversity (analogy with letters/words/books; not aware of Miescher’s similar idea)

46. Another Major Advance After learning about results of Avery et al. on likely role of DNA in heredity, Erwin Chargaff turned around his research program on proverbial dime and directed his attention to chemical attributes of DNA. He considered lack of specific methods for characterization of nucleic acids and related molecules to have hindered progress. He sought to change that condition. Inspired by Schrodinger’s book

47. Major Advance (Cont’d) Chargaff and his colleagues used new methods, comprising in essence two steps: 1. Separating DNA mixtures into individual components using new technique of paper chromatography (see next slide) 2. Identifying separated purines and pyrimidines (see next slide plus two) through their characteristic absorption lines in ultraviolet spectra, along with estimates of their quantity through amount of absorption

48. Chargaff Chromatogram Results

49. Major Advance (Concluded) Details of this work involved much of arcana of contemporary chemistry, omitted here Bottom lines are simplicity incarnate: 1. In DNA, number of guanine components is equal to number of cytosine components, and similarly for adenine and thymine 2. Different species have different numbers of these components in their DNA (see next two slides) 3. Tetranucleotide hypothesis is dead

50. Purine-Pyrimidine Ratios