DNA: The Molecular Basis of Inheritance

1. DNA: The Molecular Basis of Inheritance Hbio Ms. Pagodin

2. Do Now: • Happy Pi Day! • Grab your clickers • Talk to your classmates and find out who read the same article as you! • In your article group, discuss the experiment and conclusion!

3. Nuclear Composition? • 1868- Johann Miescher • Collected pus & fish sperm • Isolated and identified acidic compound with nitrogen and phosphorus…. Today we know it as Deoxyribonucleic acid

4. Molecule of Heredity • Is it Proteins or Nucleic Acid??

5. What makes up proteins? • Nucleotides • Amino acids • Monosaccharides

6. What makes up nucleic acids? • Nucleotides • Amino acids • Monosaccharides

7. How many amino acids are there? • 4 • 16 • 20 • 60

8. How many different DNA nucleotides are there? • 4 • 16 • 20 • 60

9. Identifying the Genetic Material • 1928 Fredrick Griffith (English Bacteriologist) • Trying to find a vaccine for pneumonia • Vaccine: prepared from killed/weakened microorganisms introduced into the body to produce immunity • Griffith worked with 2 strains of Streptococcus pneumoniae bacteria • S strain • Polysaccharide Capsule • “Smooth” edged colonies • Virulent – able to cause disease • R strain • No Capsule • “Rough” edged colonies • Nonvirulent - does not cause disease

10. Griffith’s Experiment • Griffith’s Conclusion: Something had passed from heat killed bacteria to the nonvirulent R strain making them virulent… he called this the “transforming principal” • Griffith did not know what it was, but many scientists thought it was proteins

11. Today we know… • Transformation – cells take up foreign genetic material, changing their own genes (used for genetic engineering) • Heat killed S bacteria – enzymes were denatured therefore the DNA could not be copied • Proteins are denatured at 600C and DNA is denatured at 900C • DNA of heat killed S bacteria survived and transformed DNA of R bacteria

12. Virulent strains • Have a capsule • Cause disease • Do not have a capsule • Do not cause disease • A&B • C&D

13. Transformation is the addition of genes to another organisms genome • True • False

14. The Search for what caused the Transformation… • 1944 – Oswald Avery, MacLeod, & McCarty (American Bacteriologists) • Experiment: • Added protease to “R and heat-killed S” mixture Result  Mice died • Added DNAase to “R and heat-killed S” mixture Result  Mice Lived • Conclusion: • DNA, not protein, is the transforming factor in Griffith’s experiment

15. More Evidence that DNA is the Genetic Material… • 1952 – Alfred Hershey & Martha Chase (NY) • Used T2 bacteriophages (phage) – virus that infects bacteria • Composed of nucleic acid surrounded by a protein coat • Viruses infect specific host • Viruses are not living • Not composed of cells • Cannot reproduce on their own • Do not grow and develop

16. Background Info on Viruses

17. Which type of virus is chicken pox? • Lytic • lysogenic

18. Which type of virus is the flu? • Lytic • lysogenic

19. Hershey & Chase Experiment • Experiment: • Grew T2 w/radioactive Sulfur 35S (protein coat takes in 35S) • Grew another group of T2 w/ radioactive Phosphorus 32P (DNA takes in the 32P) • 35S-labeled and 32P–labeled phages were used to infect E.Coli bacteria • Separated phages from bacteria using a blender and a centrifuge… the bacterial cells at bottom and viral parts at the top • Results: • 35S-labels still in viral parts • 32P-labels mostly in the bacterial cells, and new phages also contained 32P DNA • Conclusion: • Viral DNA (not protein) enters bacteria and carries instructions on how to make more phages • Without a doubt, DNA is the hereditary material!

20. Hershey & Chase Experiment

21. A bacteriophage • Is a virus that infects bacteria • Is a virulent bacteria • Cannot be used for experiments

22. Structure of DNA? • Linus Pauling • Nobel prize for deducing structure of protein Collagen • If protein structure could be determined and modeled, why not DNA?

23. Structure of DNA • By 1950’s most scientists were convinced that • Chromosomes carry genetic material • Genes are on chromosomes • Genes are made of DNA • Basic Structure of DNA • Composed of nucleotides • Nucleotides made of 3 parts deoxyribose, phosphate, N base • 2 types nitrogen bases: • Purines – double ring of C and N • Adenine • Guanine • Pyrimidines – single ring of C and N • Cytosine • Thymine

24. Discovering DNA’s Structure • Erwin Chargaff (NYC) • 1947 – DNA composition varies among different species • 1949 -Chargaff’s Rules- Discovered regularity of ratios: • # Adenines = # Thymines • (ie. Humans A =30%, T=30%) • # Guanines = # Cytosines • (ie. Humans G = 20%, C = 20%) • 1952 Rosalind Franklin & Maurice Wilkins (England) • Developed X-ray crystallography photographs of DNA • Suggested “helix” shape of 2-3 chains of nucleotides

25. April 25th, 1953 • James Watson & Francis Crick (England) • Built the 1st accurate 3D (tin and wire) model of DNA • “Double Helix” – spiral staircase • Purine is always linked by h-bond to a pyrimidine • 2 strands of DNA are complimentary to each other • 2 strands are anti-parallel • 5’(phosphate end) 3’(deoxyribose end) • 1962 Awarded the Nobel Prize

26. More on DNA • Ex. If the sequence of bases on one strand is AATGCGCAT, than the complimentary strand will be: ________________ • Human DNA has 3 billion base pairs.. Less than 1% of our DNA makes us different from one another!

27. Which seems most likely?

28. Models of DNA Synthesis • Semiconservative • ea/ daughter molecule will have 1 new strand and 1old strand • Conservative • Parent molecule reforms • Dispersive • All 4 strands have a combination of old and new strands

29. Assignment: • Propose an experiment to determine how DNA replication occurs

30. 1950’s Meselson & Stahl • Cultured Ecoli on medium labeled w/ 15N nt • Transferred EColi to medium labeled w/ 14N nt • Centrifuge after each replication and analyze

31. Origin of Replication • Prokaryotic Cell – single origin of replication where proteins separate the 2 strands and create a replication bubble, replication proceeds in both directions from the replication fork • Eukaryotic Cells – hundreds or thousands of replication bubbles form to speed up the copying process, replication proceeds in both directions from the replication fork

32. http://sites.fas.harvard.edu/~biotext/animations/replication1.swfhttp://sites.fas.harvard.edu/~biotext/animations/replication1.swf

33. DNA Replication • Watson and Crick proposed that the complimentary strand of DNA serves as a template for which the other strand is built…experiments confirmed this 5 years later • DNA Replication: Process of Synthesizing new molecules of DNA • Helicases catalyze the breaking of H-bonds (driven by ATP) and opens up the double helix forming replication forks (point at which DNA separates into single strands) • Topoisomerase temporarily bind to relieve strain ahead of replication fork • Single-strand binding protein – binds to unpaired DNA strands until they serve as templates for new complimentary strand

34. Elongation • DNA pol adds 50 nt/sec in Euk cells • Each nt is a nucleoside triphosphate • At the replication fork, DNA Polymerase III continuously adds complimentary nucleotides to exposed bases on 3’ end of new strand, this is called the leading strand • DNA polymerase III must work away from the replication fork on the other strand, the lagging strand, to follow the 5’-3’ direction creating short segments of DNA called Okazaki fragments. DNA Ligase joins the Okazaki fragments together. • Process continues until all DNA has been copied, end result is 2 new molecules of DNA each identical to the original and composed of one new and one old strand

35. Priming DNA Synthesis • DNA pol can not initiate – only add nt to 3’ end of existing chain • Primer – short chain (5-10nt) of RNA • Primase – enzyme starts RNA chain from scratch • Leading strand – 1 primer needed • Lagging strand – 1 primer needed for ea/Okazaki fragment • DNA pol I replaces RNA nt of primers w/DNA versions

36. DNA Synthesis http://www.dnai.org/a/index.html

37. Proofreading • DNA polymerase only moves to the next nucleotide if the previous nucleotide was a correct match • If mismatched, DNA Polymerase backs up, removes the mismatched nucleotide(s) and replaces it with the correct one(s). • Repair enzymes can recognize and repair damaged sites too • Only 1 error per 1 billion nucleotides!

38. DNA Replication & Aging • Every time DNA is copied, DNA polymerase cannot complete replication on the ends • Eukaryotic DNA has a non-coding, repeating nucleotide sequence on the ends called telomeres that protects genes from being eroded over successive replications • It is believed that telomeres are directly related to the aging process