A brief history of understandings of inheritance, genetic materials and DNA structure

1. A brief history of understandings of inheritance, genetic materials and DNA structure Lau Kwok Chi The Chinese University of Hong Kong

2. How are characteristic of humans inherited? Early Greeks’ ideas Plato (429-347BC) proposed a theory of pangenesisto explain heredity Semen of both man and woman was drawn from all parts of the body, carried via blood, to the testes and ovaries. The semen contained miniature parts of the body (pangenes). He based this theory to the observation that all parts of the body showed hereditary differences. This theory persisted in the 19th century.

3. Anaxagoras argued that women had no semen. There was a homunculus (a tiny, fully formed human) in the semen. Both theories suggested that acquired characteristics could be passed on.

4. Aristotle He rejected both theories. For the homunculus theory, it just failed to explain how the mother contributed to the heredity. For the pangenesis, it cannot explain why a soldier who loses an arm would produce a normal child, why a man with premature grey hair would pass on it to his child though he was black hair when he gave birth to his child, why a child would look like his grandparents.

5. Aristotle’s theory of inheritance The menstrual blood of females contains hereditary information. The menstrual blood and semen mix up in the womb, producing the embryo. What is being transmitted is not ‘materials’,but ‘information’ for developing a characteristic in the embryo – remarkably similar to the modern concept of genes! He also believed that acquired characteristics could be passed on.

6. In the 17th century, Harvey proposed that eggsmust be produced by women, but it was just a hypothesis. Leeuwenhoek invented microscope and found sperms in semen. He claimed he saw homunculus in the sperms! Another group of scientists thought the homunculus was in the hypothetical egg, which was triggered to develop by a sperm.

7. 1759 – Caspar Friedrich Wolff observed how the embryo of chicken developedwith a microscope. He found the body parts form gradually, thus rejecting the homunculus theory. 1827 – Karl Ernst von Baer discovered the mammalian egg

8. In 1800s, the idea of human inheritance became clear: Man produces sperms and woman produces eggs, which carry the genetic information from the man and the woman to their child. But people had no ideas about what the genetic material is.

9. What is the genetic material in the egg and sperm? • 1830s – Robert Brown discovered cell nucleus •  1840s – Karl Wilhelm von Nägeli observed cell division of pollen grains and found chromosomes •  1882 - Walther Flemming reported his discovery of chromosomes and mitosis. • 1887 - Edouard-Joseph-Louis-Marie van Beneden discovered the formation of haploid cells during cell division of sperm and ova (meiosis). What is the invention that made the above discoveries possible?

10. 1860s – Gregor Mendel worked out the how characters were inherited with discrete “factors”. At that time, Mendel had no ideas about what the “factors’ were. • At the time, inheritance was thought to be a continuous blending of the parents’ characteristics. • but his work had been ignored for 40 years and rediscovered until 1900s.

11. 1902 – Boveri and Sutton independently found the behaviours of the chromosomes during meiosis closely similar to that ofMendel’s factors. • They suggested that chromosome is the genetic material – the Sutton-Boveri chromosome theory of inheritance. Sutton Boveri

12. Chromosomes 染色体

13. Mendel’s factors

14. Can you think how Mendel’s factors behave like the chromosomes during meiosis? But the chromosome theory were not widely accepted at that time!

15. Morgen provided another strong evidence to show that chromosome is the genetic material. • He accidentally found mutant trait in fruit flies, white eye rather than red eye, which appeared associated with sex – sex-linked. • At that time, sex determination was understood to be associated with the X and Y chromosomes. • He did a series of breeding crosses on this sex-linked trait to see if the mutant allele was located on the X chromosome. Morgan and sex linkage http://www.nature.com/scitable/topicpage/Thomas-Hunt-Morgan-and-Sex-Linkage-452

16. Hypothesis : If the mutant allele recessive white eye is located on X chromosome… • Prediction: When a male red eye fruit fly is crossed with a white eye female, …. • Results: As predicted – all male offspring were white eyed. • Conclusion: The results support(not prove) that the white eye allele is located on X chromosome Why can’t it be proved?

17. The final consensus • Finally, after about 100 years of efforts, most scientists agreed that the material carrying hereditary information is located on the chromosomes. From this history of how scientists understood inheritance and genetic materials, have you got some new ideas about science?

18. What is the genetic material in the chromosomes ? Why did scientists believe that? Do you think scientists will guess something before having any experimental evidence? Protein DNA

19. The problem followed:What is the genetic material in the chromosomes ? • Chromosomes are made of proteins and nucleic acid. • Most scientists at that time believed that proteins were the genetic materials. Why did scientists believe that? Do you think scientists will guess something before having any experimental evidence?

20. In1928, Griffith was investigating pneumonia, but accidentally shed important light on genetic materials. What can you conclude from this experiment?

21. He suspected that the genetic material of the deadly S bacteria has passed to the harmless R bacteria, making it deadly. • As bacteria only has DNA but not chromosome, Griffith concluded that DNA is the genetic material.

22. Avery, MacLeod, and McCarty, 1944 • Inspired by Griffith”s experiment, they did further experiments in order to find out the genetic materials. • They repeated Griffith’s experiment, but using enzymes to destroy the proteins, fats, carbohydrates, RNA and DNA of the bacterial extract one by one. • The transformation stopped when DNA was destroyed, proving that DNA is the genetic material being passed along.

24. The results provided stronger evidence than Griffith’s. • But other scientists at the time still insisted that proteins should be the genetic materials. Why?

25. 1952-Hershey and Chase studied the role of DNA and protein in virus • They labeled the viral DNA withradioactive phosphate and the viral protein withradioactive sulphur. • This experiment was only possible when the methods of radioactive labeling were developed at the time.

26. Hershey- Chase experiment(1952)

27. After allowing the virus to infect the bacteria, they found the bacterial cells contained mainly radioactive phosphate, indicating that the DNA of the virus was transferred to the bacteria. Even the progeny of the virus made from the bacteria contained the radioactive DNA, convincingly indicating that DNA is the genetic material. In addition, as only DNA of the virus went into the bacteria but progeny virus with both DNA and proteins came out, it implied that DNA can direct the production of proteins.

28. Compared to Griffith and Avery et al.’s findings, Hershey and Chase’s experiment gave the strongest support to the hypothesis that DNA is the genetic material. Why? • Griffith’s finding : Something from the deadly bacteria was passed to the harmless bacteria, making it deadly. It was likely the DNA. • Avery et al.’s finding: It was the DNA that passed from the deadly bacteria to the harmless bacteria, making it deadly. • Hershey and Chase’s finding: Virus reproduces by DNA, which passed from one generation to another.

29. And the next problem scientists at the time faced was : What is the structure of DNA and how can it carry genetic information? The chemical constituents of DNA had been known for long, but the spatial arrangement of those constituents was very difficult to determine.

30. What was known about DNA at that time? Levene (1919)

31. Q: What was already known by that time? • DNA is made up of subunits called nucleotides. • Each nucleotide is made up of a sugar, a phosphate and a base. • There are 4 different bases in a DNA molecule: Adenine, cytosine, guanine, thymine 4. At that time, people generally thought that all 4 types of DNA nucleotides were present in roughly equal amounts - DNA was a simple polymer in which the 4 nucleotides merely repeated. (for example, GACT, GACT,….. GACT,……GACT) Do you think scientists will guess something without evidence?

32. Questions about DNA structure • helix? • 2 chains or 3 chains? • Bases inside or outside?

33. In 1949, Chargaff analyzed the base composition of the DNA from different organisms Were the results different from what other scientists expected? What patterns can you find from the results?

34. The Chargaff’s rule • In the organisms he studied, amount of A is always equal to T, and G to C A = T , and, G = C • An equal amount of purines(A and G) and pyrimidines(C and T ) • The ratio of (A+T):(G+C) varies considerably from one species to another • Implication: These bases may code for the hereditary information.

35. X-ray crystallography

37. King’s College Maurice Wilkins Rosalind Franklin Cambridge James Watson Francis Crick

38. Is DNA a helix? • At the 1950s, the most successful method for studying the structure of protein and other complex molecule was X-ray diffraction. • Linus Pauling and Robert Corey showed that the haemoglobin, a kind of protein molecules, was arranged like a helix. • Inspired by it, most scientists thought DNA, another complex molecule as proteins, would be helix as well.

39. Pauling and Corey proposed a structure of DNA:3 chains of helical DNA with the phosphates near the axis and the bases on the outside. • Most scientists thought Pauling’s 3 chain model was bad as the chemical bonds seemed unable to hold the molecule stably and the DNA was not an acid! Why do we say a model/theory is bad/good but not right/wrong?

40. In 1952, Rosalind Franklin took an excellent X-ray diffraction photo of DNA

41. Franklin at KCL analyzed this picture carefully and proposed a double-stranded helical structure for DNA, with the 2 twisted legs of the ladder made of phosphates and sugars and the rungs made of the nitrogen bases. • She suggested that the bases were inside the helixes and phosphates outside, and even worked out the size of the DNA molecule. Did Franklin see the DNA directly from the photo? If not, how did she come to her conclusion?

42. What she did not know was how the bases fit together. • Due to personal conflicts with Wilkins, Franklin resigned from KCL. • Her paper was published together with Watson and Crick’s in 1953.

43. Watson and Crick were also working on DNA structure at that time.

44. Maurice Wilkins was Franklin’s supervisor at KCL and also an expert in X-ray diffraction. • He showed Franklin’s X-ray picture to Watson after Franklin left the lab without her permission(?). • Watson also knew more about Franklin’s findings from a ‘confidential’ report Franklin sent to her funding organization. • Watson and Crick immediately recognized that DNA is a helix as shown by the clear “X” in the picture.

45. Watson and Crick claimed that DNA was double-stranded, because of a vague intuitionthat “important biological objects come in pairs.” • But they did not mention having seen Franklin’s photo in their article. Were Franklin, Wilkins, Watson competing or collaborating? Do you think Watson and Wilkins were unethical?

46. The last puzzle! • The remaining problem was how the 4 bases of different shapes and sizes could fit together to form successive rungs of the same width in a helix.

48. Inspired by Pauling, they used the methods of model construction. • With pieces of paper, cardboard, and metal, cut to the shapes of the various constituents of DNA, they tried out different 3 dimensional models with their textbook chemistry knowledge about chemical bonding. • They have tried C-C, A-A pairings, but each rung was different in width. • They used Chargaff’s rule to pair up the bases, A-T, G-C, but also failed!

49. Luck coming! • A chemist, Jerry Donohue, visited Watson’s lab and corrected one mistake of the textbook for him – the bases should be in ‘keto’ form rather than ‘enol’ form.

50. This immediately made Watson able to make the A-T, G-C pairing, which were of the same width when in antiparallel direction. • This A-T, G-C pairing also satisfied the Chargaff’s rules in which A is always equal to T, and C equal to G.