1. History for the Discovery of DNA
Chapter 16
The Molecular Basis of Inheritance

2. Next Unit:. Chapter 16: DNA: History, Structure & Replication
Next Unit: **Chapter 16: DNA: History, Structure & Replication **Chapter 17: Genetic Expression (protein synthesis) Chapter 18: Viruses & Bacteria (selected parts) Chapter 19: Regulation (selected parts) **Chapter 20: Genetic Engineering & Biotechnology

3. Overview of Chapter 16: TOPIC Pgs. History & Discovery of DNA 293-296
as Genetic Material
Structure of DNA
DNA Replication

4. Introductory Questions (#1)
What was the significance of Griffith’s Experiment in 1928?
Give three reasons why Neurospora was in genetic studies to discover the “one gene, one enzyme” principle?
What did James Sumner purify in 1926?
How was Avery, MacLoed, and McCarty work different from Griffith’s?
Matching:
Garrod A. Urease
Griffith B. T2 Bacteriophage
Beadle & Tatum C. Alkaptonuria
Sumner D. Neurospora
Hershey & Chase E. Transformation Principle

5. Key Questions Explored in this Next unit:
What are Genes made of?
How do Genes work?
How can information be stored, retrieved, and modified over time?
What keeps this molecule so stable?
Why is DNA and not protein responsible for the inheritance of genetic traits?

6. Key Discoveries
Miescher (isolated “nuclein” from soiled bandages) 1869
Garrod (Proteins & inborn errors)
Sutton (Chromosome structure)
Morgan (Gene mapping)
Sumner (Purified Urease, showed it to be an enzyme) 1926
Griffith’s Experiment (Transforming Principle) 1928
Avery, McCarty, and Macleod
Chargaff (Base pairing & species specific) 1947
Hershey and Chase
Pauling, Wilkins, and Franklin ’s
Watson and Crick

7. Discovery of DNA
1868: Miescher first isolated deoxyribonucleic acid, or DNA, from cell nuclei

8. Fredrick Griffith (1928)
First suggestion that about what genes are made of.
Worked with:
1) Two strains of Pneumococcus bacteria:
Smooth strain (S) Virulent (harmful)
Rough strain (R) Non-Virulent
2) Mice-were injected with these strains of bacteria and watched to see if the survived.
3) Four separate experiments were done:
-injected with rough strain (Lived)
-injected with smooth strain (Died)
-injected with smooth strain that was heat killed (Lived)
-injected with rough strain & heat killed smooth (????)

9. Griffith’s Experiment-1928

10. Conclusion of Griffith’s Experiment
Somehow the heat killed smooth bacteria changed the rough cells to a virulent form.
These genetically converted strains were called “Transformations”
Something (a chemical) must have been transferred from the dead bacteria to the living cells which caused the transformation
Griffith called this chemical a “Transformation Principle”

11. Avery, MacLeod, and McCarty (1944)
Chemically identified Griffith’s transformation principle as DNA
Separated internal contents of the S cells into these fractions:
(lipids, proteins, polysaccharides, and nucleic acids)
They tested each fraction to see if it can cause transformation to occur in R cells to become S cells.
Only the nucleic acids caused the transformation
This was the first concrete evidence that DNA is the genetic material.
Some were not completely convinced because they were not sure if this was true for eukaryotes.

12. Next Breakthrough came from the use of Viruses
Viruses provided some of the earliest evidence that genes are made of DNA
Molecular biology studies how DNA serves as the molecular basis of heredity
Only composed of DNA and a protein shell

13. Various Types of Viruses

14. T2 Bacteriophage

15. Phage reproductive cycle
Phage attaches to bacterial cell.
Phage injects DNA.
Phage DNA directs host cell to make more phage DNA and protein parts. New phages assemble.
Cell lyses and releases new phages.
Figure 10.1C

16. A Typical Bacteriophage

17. Alfred Hershey & Martha Chase (1952)
Worked with T-2 Bacteriophages
Infected Escherchia coli (E. coli) = Host cell
Used Radioactive Isotopes:
(S35) Sulfur-35
(P32) Phosphorus-32
Why did they use these particular isotopes?
*Sulfur is found in proteins and not in DNA
*Phosphorus is found in DNA but not in protein

18. Labeling of Virus Structures

19. Details of the Hershey & Chase Experiment

20. The Hershey-Chase Experiment1
Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells. 2
Agitate in a blender to separate phages outside the bacteria from the cells and their contents. 3
Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube. 4
Measure the radioactivity in the pellet and liquid.
Radioactive protein
Empty protein shell
Radioactivity in liquid
Phage
Bacterium
Phage DNA
DNA
Batch 1 Radioactive protein
Centrifuge
Pellet
Radioactive DNA
Batch 2 Radioactive DNA
Centrifuge
Radioactivity in pellet
Figure 10.1B
Pellet

21. Video clip of Hershey Chase Experiment
Key findings: the phage DNA entered in the host cell and when these cells were returned to the culture medium the infection ran its course producing E.coli and other bacteriophages with the radioactive phosphorus. (pg. 298)

22. DNA is a Double-Stranded Helix
James Watson and Francis Crick worked out the three-dimensional structure of DNA, based on work by Rosalind Franklin
Figure 10.3A, B

23. Rosalind Franklin’s Image (pg. 297)
and Media 

24. DNA and RNA are polymers of Nucleotides
DNA is a nucleic acid, made of long chains of nucleotides
Phosphate group
Nitrogenous base
Nitrogenous base (A, G, C, or T)
Sugar
Phosphate group
Nucleotide
Thymine (T)
Sugar (deoxyribose)
DNA nucleotide
Polynucleotide
Sugar-phosphate backbone
Figure 10.2A

25. DNA has four kinds of bases, A, T, C, and G
Thymine (T)
Cytosine (C)
Adenine (A)
Guanine (G)
Pyrimidines
Purines
Figure 10.2B

26. DNA Maintains a Uniform Diameter
See pg. 298

27. DNA Bonding Purines: ‘A’ & ‘G’ Pyrimidines: ‘C’ & ‘T’ (Chargaff rules)
‘A’ H+ bonds (2) with ‘T’ and ‘C’ H+ bonds (3) with ‘G’
Van der Waals attractions between the stacked pairs

28. Nitrogenous base (A, G, C, or U)
RNA is also a nucleic acid
RNA has a slightly different sugar
RNA has U instead of T
Nitrogenous base (A, G, C, or U)
Phosphate group
Uracil (U)
Sugar (ribose)
Figure 10.2C, D

29. Hydrogen bonds between bases hold the strands together
Each base pairs with a complementary partner
A pairs with T
G pairs with C

30. DNA Structure Chargaff ratio of nucleotide bases (A=T; C=G)
Watson & Crick (Wilkins, Franklin)
The Double Helix
√ nucleotides: nitrogenous base (thymine, adenine, cytosine, guanine); sugar deoxyribose; phosphate group

31. Partial chemical structure
Three representations of DNA
Hydrogen bond
Ribbon model
Partial chemical structure
Computer model
Figure 10.3D

32. Each strand of the double helix is oriented in the opposite direction
5 end
3 end P P P P P P P P
3 end
5 end
Figure 10.5B

33. DNA Replication: History & Discovery
First model suggested by Watson & Crick
Three models were proposed:
-Semiconservative (half old & half new)
-Conservative (old strands remain together)
-Dispersive (random mixture)
Heavy isotopic nitrogen (N-15) was used to label the nitrogenous bases in the DNA
Density gradient centrifugation was used
DNA was mixed with Cesium chloride (CsCl)

34. Three Proposed Models of DNA Replication

35. Meselson & Stahl’s Experiment

36. Meselson-Stahl Experiment

37. Meselson & Stahl Experiment (Pg. 300)
Grew E. coli on a medium containing isotopic Nitrogen (15N) in the form of NH4Cl
Nitrogenous bases incorporated the isotopic nitrogen
DNA was extracted from the cells
Density gradient centrifugation was used on the DNA to determine the banding region of the heavy isotopic nitrogen.
The rest of the bacteria was then grown on a medium containing normal nitrogen and allowed to grow.

38. Meselson & Stahl Experiment cont’d.
The newly synthesized strands of DNA were expected to have the lighter normal nitrogen in their bases.
The older original strands were labeled with the heavier isotopic nitrogen.
Two generations were grown in order to rule out the conservative and dispersion models.

39. Introductory Questions (#1)
What was the significance of Griffith’s Experiment in 1928?
How was Avery, MacLoed, and McCarty work different from Griffith’s?
How was the dispersive model & conservative models ruled out as the way in which DNA replicates?
Matching:
Meselson & Stahl A. X-ray diffraction
Griffith B. T2 Bacteriophage
Franklin & Wilkins C. Semiconservative model
Chargaff D. Base pairing: C-G & T-A
Hershey & Chase E. Transformation Principle

40. Introductory Questions #2
Briefly explain what density gradient centrifugation is and what it is used for.
Name the organism used by Meselson & Stahl to label the DNA.
Name all of the enzymes required for DNA replication to occur and what purpose they serve.
In what direction is the newly synthesized strand made? What end of the old strand do the nucleotides add to?
What direction is the new strand growing? (towards or away from the replication fork)
How long (# nucleotides) are the Okasaki fragments? How long are the RNA primers?

41. The structure of DNA consists of two polynucleotide strands wrapped around each other in a double helix
1 chocolate coat,
Blind (PRA)
Figure 10.3C
Twist

42. DNA replication depends on specific base pairing
In DNA replication, the strands separate
Enzymes use each strand as a template to assemble the new strands A A
Nucleotides
Parental molecule of DNA
Both parental strands serve as templates
Two identical daughter molecules of DNA
Figure 10.4A

43. Untwisting and replication of DNA
Figure 10.4B

44. Anti-parallel Structure of DNA

45. Antiparallel nature 5’ end corresponds to the Phosphate end
3’ end corresponds to the –OH sugar
Replication runs in BOTH directions
• One strand runs 5’ to 3’ while the other runs 3’ to 5’
• Nucleotides are added on the 3’ end of the newly synthesized strand
The new DNA strand forms and grows in the ’  3’ direction only

46. How a Nucleotides adds to the old Strand
5’ end
3’ end
5’ end

47. Building New Strands of DNA
Each nucleotide it a triphosphate:
(GTP, TTP, CTP, and ATP)
Nucleotides only add to the 3’ end of the growing strand (never on the 5’ end)
Two phosphates are released (exergonic) and the energy released drives the polymerization process.

48. Origin of replication (“bubbles”): beginning of replication (pg. 301)

49. Key Enzymes Required for DNA Replication (pg. 303-304)
Helicase - catalyzes the untwisting of the DNA at the replication fork
DNA Polymerase - catalyzes the elongation of new DNA and adds new nucleotides on the 3’ end the growing strand.
SSBP’s - single stranded binding proteins, prevents the double helix from reforming
Topoisomerase – Breaks the DNA strands and prevents excessive coiling
RNA primase – synthesizes the RNA primers and starts the replication first by laying down a few nucleotides initially.
**DNA primase will get replaced by DNA polymerase

50. RNA Primers
Initiates the Replication process and begins the building of the newly formed strands.
Laid down by RNA primase
Consists of 5 to 14 nucleotides
Synthesized at the point where replication begins
Will be laid down on both template strands of the DNA

51. Overall direction of replication3
DNA polymerase molecule
How DNA daughter strands are synthesized
5 end 5
Daughter strand synthesized continuously
Parental DNA 5 3
Daughter strand synthesized in pieces 3 P 5
The daughter strands are identical to the parent molecule 5 P 3
DNA ligase
Overall direction of replication
Figure 10.5C

52. Laying Down RNA Primers

53. DNA Replication-New strand Development
Leading strand: synthesis is toward the replication fork
(only in a 5’ to 3’ direction from the 3’ to 5’ master strand)
-Continuous
Lagging strand: synthesis is away from the replication fork
-Only short pieces are made called “Okazaki fragments”
- Okazaki fragments are 100 to 2000 nucleotides long
-Each piece requires a separate RNA primer
-DNA ligase joins the small segments together
(must wait for 3’ end to open; again in a 5’ to 3’ direction)
View video clip:

54. DNA Replication Fork

55. Video Clip of DNA Replication

56. Prokaryotic vs Eukaryotic Replication
Prokaryotes
Circular DNA (no free ends)
Contains 4 x 106 base pairs (1.35 mm)
Only one origination point
Eukaryotes
-Have free ends
-Contains 3 x 109 base pairs (haploid cells) = 1 meter
-Lagging strand is not completely replicated
-Small pieces of DNA are lost with every cell cycle
-End caps (Telomeres) protect and help to retain the genetic information

57. Issues with Replication
Prokaryotes: (ex. E. coli)
Have one singular loop of DNA
E. coli has approx. 4.6 million Nucleotide base pairs
Rate for replication: 500 nucleotides per second
Eukaryotes w/Chromosomes:
Each chromosome is one DNA molecule
Humans (46) has approx. billion base pairs
Rate for replication: 50 per second (humans)
Errors:
Rate is one every 10 billion nucleotides copied
Proofreading is achieved by DNA polymerase (pg. 305)

58. Telomeres Short, non-coding pieces of DNA
Contains repeated sequences (ie. TTGGGG 20 times)
Can lengthen with an enzyme called Telomerase
Lengthening telomeres will allow more replications to occur.
Telomerase is found in cells that have an unlimited number of cell cycles (commonly observed in cancer cells)
Artificially giving cells telemerase can induce cells to become cancerous
Shortening of these telomeres may contribute to cell aging and Apotosis (programmed cell death)
Ex. A 70 yr old person’s cells divide approx X vs an infant which will divide 80-90X

59. Telomeres

60. Chapter 17

61. James Sumner (1926) Isolated the enzyme “Urease”
First to identify an enzyme as a protein
First to crystallize an enzyme
Awarded the Nobel prize in 1946 in chemistry for his crystallization of an enzyme.

62. Archibald Garrod ( )
Studied a rare genetic disorder: Alkaptonuria
Thought to be a recessive disorder
Tyrosine is not broken down properly into carbon dioxide and water.
An Intermediate substance: “Homogentisic acid” accumulates in the urine turning it BLACK when exposed to air.
An enzyme was thought to be lacking
A genetic mutation was thought to be the cause “An Inborn Error of Metabolism”

63. Metabolic Pathway for the breakdown of Tyrosine↓
Hydroxyphenylpyruvate
Homogentisic acid
Alkaptonuria Maleyacetoacetate
(Inactive enzyme) (active ↓ enzyme)
CO2 & H2O

64. Garrod’s Conclusion
A mutation in a specific gene is associated with the absence of a specific enzyme.
Led to the idea of:
“One gene, One Enzyme”
Not validated until Beadle & Tatum’s work in the 1940’s with Neurospora (breadmold)

65. George Beadle & EdwardTatum (1940’s)
Discovered the “One Gene, One Enzyme” Principle
Analyzed mutations that interfered with a known metabolic pathway
Organism they chose to work with: Neurospora (breadmold)
-Grows easily
-Grows as a haploid: (no homologs)
-Mutants are easily identified: Dominant allele won’t be expressed
Neurospora can grow easily in only: salt, sugar, & Biotin

66. George Beadle & EdwardTatum (1940’s) cont’d
Mutants-are unable to make certain organic molecules: amino acids, lipids, etc.
These substances are added to the media which will allow mutants to grow successfully
Exposed the haploid spores to x rays & UV to induce mutations
Haploid spores were crossed, grown in a variety of media to determine what kind of mutation was occurring
**They examined the effect of the mutation instead of identifying the enzyme.

67. Beadle & Tatum’s Conclusion
“One Gene affects One Enzyme”
Later  Revised
“One Gene affects One Protein”
“One Gene affects One Polypeptide Chain”

68. Suggestions on how to Review
Make a List of all Bold Terms (See summaries)
Make a list of key people & generate a timeline
Answer all MC questions at end of each chapter
Review all your Quizzes from textbook website
Review all the MC Questions from your study guides
Look at all the key figures & diagrams discussed
Review all Tables from the four chapters
Re-Look at the Powerpoint Pres. From my website. Think back to what was emphasized
Anticipate questions to be asked
Make an outline of all chapters & connect the concepts discussed