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DNA: The Molecular Basis of Inheritance

DNA: The Molecular Basis of Inheritance. Hbio Ms. Pagodin. 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!. Nuclear Composition?. 1868- Johann Miescher

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DNA: The Molecular Basis of Inheritance

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  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

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