1. Mice live + Mice die a.b.c.d. Live Virulent Strain of S. pneumoniae Live Nonvirulent Strain of S. pneumoniae Heat-killed Virulent Strain of S. pneumoniae Mixture of Heat-Killed Virulent and Live Nonvirulent Strains of S. pneumoniae Mice die Their lungs contain live pathogenic strain of S. pneumoniae Polysaccharide coat Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1

2. + + Result: When the experiment is done, only 32 P makes it into the cell in any significant quantity. Hypothesis: DNA is the genetic material in bacteriophage. Prediction: The phage life cycle requires reprogramming the cell to make phage proteins. The information for this must be introduced into the cell during infection. Test: DNA can be specifically labeled using radioactive phosphate ( 32 P), and protein can be specifically labeled using radioactive sulfur ( 35 S). Phage are grown on either 35 S or 32 P, then used to infect cells in two experiments. The phage heads remain attached to the outside of the cell and can be removed by brief agitation in a blender. The cell suspension can be collected by centrifugation, leaving the phage heads in the supernatant. Phage grown in radioactive 35 S, which is incorporated into phage coat Virus infect bacteria Blender separates phage coat from bacteria Centrifuge forms bacterial pellet 35 S in supernatant 35 S-Labeled Bacteriophages Phage grown in radioactive 32 P. which is incorporated into phage DNA Virus infect bacteria Blender separates phage coat from bacteria Centrifuge forms bacterial pellet 32 P in bacteria pellet 32 P-Labeled Bacteriophages Conclusion: Thus, DNA must be the molecule that is used to reprogram the cell. Further Experiments: How does this experiment complement or extend the work of Avery on the identity of the transforming principle? SCIENTIFIC THINKING 2

3. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Purines Pyrimidines Adenine Guanine NH 2 C C N N N C H N C CH O H H OC NC H N C H C H O O C N C H N C H3CH3C C H H O O C N C H N C H C H C C N N N C H N C CH H Nitrogenous Base 4´4´ 5´5´ 1´1´ 3´3´2´2´ 2 8 76 39 4 5 1 O P O–O– –O–O Phosphate group Sugar Nitrogenous base O CH 2 N N O N NH 2 OH in RNA Cytosine (both DNA and RNA) Thymine (DNA only) Uracil (RNA only) OH H in DNA 3

4. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Base CH 2 O 5´5´ 3´3´ O P O OH CH 2 –O–OO C Base O PO 4 Phosphodiester bond 4

5. 5

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7. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´5´ 3´3´ P P P P OH 5-carbon sugar Nitrogenous base Phosphate group Phosphodiester bond O O O O 4´4´ 5´5´ 1´1´ 3´3´ 2´2´ 4´4´ 5´5´ 1´1´ 3´3´ 2´2´ 4´4´ 5´5´ 1´1´ 3´3´ 2´2´ 4´4´ 5´5´ 1´1´ 3´3´ 2´2´ 7

8. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2 nm 3´3´ 3´3´5´5´ 0.34 nm 5´5´ C C C G G G G G T T T T A A A 3.4 nm Minor groove Major groove Major groove Minor groove 8

9. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A H Sugar T G C N H N O H CH 3 H H N N N H N N N H H H N O H H H N NH N N H N N Hydrogen bond Hydrogen bond 9

10. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ConservativeSemiconservativeDispersive 10

11. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Samples are centrifuged E. coli 0 1 2 0 rounds1 round2 rounds Bottom 15 N medium 14 N medium E. coli cells grown in 15 N medium Cells shifted to 14 N medium and allowed to grow DNA Samples taken at three time points and suspended in cesium chloride solution Rounds of replication Top 0 min 0 rounds 20 min 1 round 40 min 2 rounds From M. Meselson and F.W. Stahl/PNAS 44(1958):671 11

12. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. P P P P P P P P P P P Pyrophosphate 3´ 5´ New StrandTemplate Strand O HO OH O O O O O O O O O O C C T T T A A A G G A P P P P P P P P P P P P P P 3´ 5´ New StrandTemplate Strand O HO OH O O O O O O O O O C C T T A A A G G A Sugar– phosphate backbone DNA polymerase III T O P 12

13. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´5´ 3´3´ 5´5´ 5´5´ 5´5´ 3´3´ 3´3´ RNA polymerase makes primerDNA polymerase extends primer 13

14. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replisome Origin Termination Origin Termination 14

15. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Supercoiling Replisomes No Supercoiling Replisomes DNA gyrase 15

16. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA primer Open helix and replicate First RNA primer Open helix and replicate further Lagging strand (discontinuous) Second RNA primer Leading strand (continuous) RNA primer 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 16

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18. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ 3´ Primase RNA primer Okazaki fragment made by DNA polymerase III Leading strand (continuous) DNA polymerase I Lagging strand (discontinuous) DNA ligase 18

19. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Leading strand (no problem) Lagging strand (problem at the end) Last primer Replication first round Shortened template Primer removal Replication second round Origin 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ Removed primer cannot be replaced Leading strand Lagging strand 19

20. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. G GGGGG TT TTTT G T T G G GGG T TT T CCCCCAAAA CCCCCAAAA Telomere extended by telomerase Template RNA is part of enzyme Telomerase Now ready to synthesize next repeat 5´ 3´ 5´ 3´ 5´ 3´ Synthesis by telomerase Telomerase moves and continues to extend telomere 20

21. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. T A T A AA AA T A T A T T T T Thymine dimer cleaved Photolyase Helix distorted by thymine dimer Thymine dimer DNA with adjacent thymines UV light Visible light Photolyase binds to damaged DNA 21

22. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Damaged or incorrect base Uvr A,B,C complex binds damaged DNA DNA polymerase Excision of damaged strand Resynthesis by DNA polymerase Excision repair enzymes recognize damaged DNA 22